Antioxidant, antitumor, and anti-inflammatory activity of natural products obtained from the brown alga Cystoseira usneoides

! ! ! ! UNIVERSITY OF SEVILLE FACULTY OF PHARMACY DEPARTMENT OF PHARMACOLOGY ! ! ! Antioxidant, antitumor, and anti-inflammatory activity of natural products obtained from the brown alga Cystoseira usneoides Thesis submitted in fulfillment of the requirements for the award of the degree of DOCTOR in PHARMACY (Ph.D.) Hanaa Zbakh Seville, June 2019 ! VIRGINIA MOTILVA SÁNCHEZ, Catedrática de Universidad del Departamento de Farmacología de la Universidad de Sevilla, y EVA ZUBÍA MENDOZA, Catedrática de Universidad del Departamento de Química Orgánica de la Universidad de Cádiz, CERTIFICAN: Que la presente Tesis Doctoral titulada “Antioxidant, antitumor, and antiinflammatory activity of natural products obtained from the brown alga Cystoseira usneoides”, ha sido realizada bajo su dirección por HANAA ZBAKH para aspirar al grado de Doctor en Farmacia. Sevilla, 24 de junio de 2019 Fdo.: Virginia Motilva Sánchez Fdo.: Eva Zubía Mendoza ! MARÍA CONCEPCIÓN PÉREZ GUERRERO, Directora del Departamento de Farmacología de la Universidad de Sevilla, CERTIFICA: Que la presente Tesis Doctoral titulada “Antioxidant, antitumor, and antiinflammatory activity of natural products obtained from the brown alga Cystoseira usneoides”, realizada por HANAA ZBAKH para aspirar al grado de Doctor en Farmacia, ha sido dirigida por la Dra. Virginia Motilva Sánchez y la Dra.!Eva Zubía Mendoza, cumpliendo los requisitos para este tipo de trabajo. Sevilla, 24 de junio de 2019 Fdo.:María Concepción Pérez Guerrero ! ACKNOWLEDGMENTS With passion and curiosity about science, I traveled from Morocco to Spain to pursue my Ph.D at Faculty of Pharmacy-University of Seville in collaboration with the University of Cadiz. My research life in my new lab and new working atmosphere was new and different for me, but very exciting. During this long journey, there was happy and sadness, excitement and depression, encourage and disappointment, progress and retrogress, success and failure, gain and loss, all these feelings allowed me continue my destiny without despair or frustration till I've reached my goals and achieved my greatest dream. First and foremost, I have been lucky to have two supervisors who are real supervisors with high sense of responsibility and whose personalities and areas of expertise and supervising styles harmoniously complement each other. Virginia Motilva, I am very grateful for believing in me, supporting, and inspiring me throughout my studies. I am amazed by your energy, drive, and dedication to, not only science, but also family. I am so grateful that I get to be part of your second family, the research family. Eva Zubia, my brilliant co-supervisor, full of energy, thank you for leading me to the fantastic world of chemistry. Thanks for all the professional meetings and discussions and time you took out of your busy schedules to revise my manuscript and thesis. Our discussions were a great pleasure, and it was wonderful to be inspired by your critical-thinking and deep knowledge. Special mention goes to my Professor Hassane Riadi, who has been a truly dedicated mentor. I would like to express my special appreciation and to thank you for encouraging my research and for allowing me to grow as a research scientist. Your advice on both research as well as on my career have been invaluable. Elena Talero, you are such a kind and caring person, thank you for your tremendous support. Thank you for your patience and for teaching me everything about the in vivo experiment. Your knowledge and laboratory skills have been such a great asset. My sincere thanks to Carolina De los Reyes from the Department of Organic Chemistry, Faculty of Marine and Environmental Sciences, University of Cádiz, for her great help and her contributions to the phytochemical work. Thank you, Javier Avila, for supporting and helping me, especially with the cell experiments. You have a fantastic enthusiasm, never let anyone take it away! To my colleagues, my co-authors and my friends from FARMOLAP group: Antonio, Azahara, Jose Antonio, Jesus, Bui: I couldn't have asked for a better team to work with. I miss you all and I hope to have the opportunity to meet you in the future. Many thanks to all professors and members of the department of Pharmacology, you have jointly created an open and friendly work environment, to talk about research, to have lunch together and for sharing with me the fun of lab work. I am so grateful to Carmen Claro, for making work at the Lab so much easier with all your support and help, you are truly valued. I am also very grateful to all technicians at CITIUS, especially to Modesto who was always so helpful and provided me the technical support to analyze Flow cytometry throughout my dissertation. I truly appreciate my lovely friends, Hanaa, Houda, Ghizlane and Mustapha, for cheering me on throughout this journey. I wouldn’t have made it without you. A big thanks to all my colleagues and professors of the Department of Biology at University of Abdelmalek Essaadi in Morocco, for being like a friend and for giving me considerate advice both in career and life. ﺎﻤھ ،ﻲﺑأو ﻲﻣأ ﻲﺒﻠﻗ ﻲﻓ سﺎﻨﻟا ﻰﻠﻏﻷ ﻲﺒﺣو ﻲﻧﺎﻨﺘﻣا ،يﺮﻜﺷ ىﺪﻤﻟ ﺮﯿﺒﻌﺘﻟا ﻦﻋ تﺎﻤﻠﻜﻟا ﺰﺠﻌﺗ ﻢﻜﻘﺣ ﻲﻔﺗ ﻻ ﺮﻜﺸﻟا تﺎﻤﻠﻛ ﻞﻛ ،ﻲﻣﻼﺣأ ﻖﯿﻘﺤﺘﻟ ﻲﺘﻤﯾﺰﻋ ﺐﺒﺳو ﻲﺗﻮﻗ ﺮﺳ ،ةﺎﯿﺤﻟا ﻲﻓ يﺪﻨﺳ... ،ﻲﺧأ ﺎﻤﺋاد ﻲﺗﺪﻧﺎﺴﻣو ﻲﻤﻋد ﻰﻠﻋ ﻢﻜﻟ اﺮﻜﺷ ،ةﺎﯿﺤﻟا هﺬھ ﻲﻓ يﺰﻨﻛ ﻢﺘﻧأ ،تاﺰﯾﺰﻌﻟا ﻲﺗاﻮﺧأ ...ﻢﻜﺒﺣأ ،اﺪﺑأو ! ! ii! H2AX!! ! ! H2A histone family member X H460 Human lung cancer cell line HeLa Human cervical cancer cell line Hep-G2 Human liver cancer cell line HIF-1!! ! ! Hypoxia-inducible factor 1 HL-60 Human promyelocytic leukemia cell line HPLC High Performance Liquid Chromatography HRMS High Resolution Mas Spectrometry Hs683!! ! ! Human Neuronal Glioma cell line HT-1080 Human fibrosarcoma cell line HT-29 Human colorectal adenocarcinoma cell line IC50 50% inhibitory concentration IL Interleukine iNOS Inductible nitric oxide synthase IPP!! ! ! Isopentenyl diphosphate J coupling constant JNK C-Jun NH2-terminal kinase Jurkat!! ! ! Immortalized line of human T lymphocyte cell line K562!! ! ! Human immortalised myelogenous leukemia cell line KB!! ! ! Human epidermoid carcinoma cell line L-1210 Mouse lymphocytic leukemia cell line L929 Mouse fibroblast cell line LOVO Human colon adenocarcinoma cell line LPS Lipopolysaccharide LS174 Human colon cancer cell line m In NMR: multiplet MAPK Mitogen-activated protein kinase MBA-MD468 Human triple-negative breast cancer cell line MCF-7 Human adenocarcinoma cell line MCP-1!!! ! Monocyte chemoattractant protein-1 MDA-MB-231 Human breast carcinoma cell line MIA!! ! ! Human pancreatic cancer cell line MM144!! ! Human myeloma cell line MMP Matrix metallopeptidase MRC-5 Human fetal lung fibroblast MS Mass Spectrometry mTOR Mammalian target of rapamycin MTT! 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide mult. Multiplicity m/z Mass-charge relationship NCI-H187 Human lung cancer cell line NCI-H460 Non-small-cell lung cancer cell line NF-κB Nuclear factor-kappa B NMR Nuclear Magnetic Resonance NO Nitric oxide NOS Nitric oxide synthases NPs Natural products OE21 Human oesophageal squamous cell carcinoma P-388 Murine lymphocytic leukemia cell P38 Mitogen-activated protein kinase P53 Tumor protein 53 PA-1 Human ovarian teratocarcinoma cell line PARP!! ! ! Poly ADP ribose polymerase PC-3 Human prostate cancer cell lines PG Prostaglandin PI Propidium Iodide ! iii! PI3K Phosphatidyl inositol-3 kinase PMA Phorbol myristate acetate pRb!! ! ! Retinoblastoma protein PS Phosphatidylserine Raf Rapidly accelerated fibrosarcoma Ras Rat sarcoma Raw 264.7 Mouse leukaemic monocyte macrophage cell line RBL-2H3!! ! Basophilic leukemia cell line Ref-1 Redox factor-1 RNase Ribonuclease RONS Reactive oxygen and nitrogen species ROS Reactive oxygen species s In NMR: singlet S17!! ! ! Clonal stromal cell line SDS Sodium dodecyl sulfate SDS-PAGE SDS-polyacrylamide gel electrophoresis SF-268!! ! ! Human tumor glioblastoma cell line SH-SY5Y Human neuroblastoma cell line SJ-G2!! ! ! Human glioblastoma cell line SKMEL-28!! ! Human melanoma cell line SRB Sulforhodamine B STAT-3!! ! Signal transducer and activator of transcription 3 t in NMR: triplet T47D-T.R Human breast cancer cell line TBARS!!! ! Thiobarbituric acid reactive substances TBS-T Tris-buffered saline Tween TCA Trichloroacetic acid TGF- ! !! ! ! Transforming growth factor beta! THP-1 Human monocytic leukemia cell line TNBC!! ! ! Triple-negative breast cancer TNF-α Tumor necrosis factor alpha Tris Tris(hydroxymethyl)aminomethane Tris-HCl Tris (hydroxymethyl)aminomethane hydrochloride Trolox 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid U373!! ! ! Human glioblastoma astrocytoma cell line U87!! ! ! Human primary glioblastoma cell line U937!! ! ! Pro-monocytic, human myeloid leukaemia cell line V79!! ! ! Chinese hamster lung fibroblast cell line VEGF!! ! ! Vascular endothelial growth factor Vero African green monkey kidney normal cell line WHCO1!! ! Oesophageal cancer cell line γH2AX Phosphorylation of the histone H2AX ! ! iv! LIST OF FIGURES Figure 1.1. (A) Microalgae species from the Mediterranean coast of Morocco. (B) Macroalgae species during low tide at the Mediterranean coast of Morocco. Figure 1.2. Green alga Ulva lactuca (A), red alga Asparagopsis armata (B) and brow alga Cystoseira tamariscifolia (C), during low tide at the Mediterranean coast of Morocco. Figure 1.3. Biosynthesis of terpenoids and carotenoids. Figure 1.4. Examples of of algal terpenoids: (A) three terpenoids with regular isoprenoid carbon skeleton (the C5 units of the skeleton are depicted in red); (B) a diterpenoid with rearranged carbon skeleton; (C) a meroterpenoid. Figure 1.5. Antioxidant terpenoids from the brown alga Sargassum thunbergii. Figure 1.6. Antioxidant merterpenoids from the brown algae Sargassum siliquastrum (Ss-1 to Ss- 14) and Stypopodium zonale (Ss-15). Figure 1.7. Antioxidant meroterpenoids isolated from the brown alga Cystoseira abies-marina. Figure 1.8. Antioxidant meroditerpenoids isolated from the brown alga Taonia atomaria. Figure 1.9. Antioxidant diterpenoids isolated from the brown alga Dictyota dichotoma. Figure 1.10. Antioxidant aryl-terpenoid isolated from the red alga Hypnea musciformis. Figure 1.11. Cytotoxic terpenoids isolated from the green algae Caulerpa taxifolia (Ct-1), Ulva fasciata (Uf-1), and Tydemania expeditionis (Te-1 – Te-4) and cytotoxic derivatives prepared from Te-3 (Te-3a, Te-3b, Te-3c). Figure 1.12. Cytotoxic monoterpenoids from the red algae Plocamium corallorhiza (Pcr-1), P. suhrii (Ps-1 – Ps-7), P. cornutum (Pco-1 – Pco-5), P. cartilagineum (Pca-1 ‒ Pca-3), and Pterocladiella capillacea (Ptc-1). Figure 1.13. Cytotoxic sesquiterpenes from the red algae Laurencia microcladia (Lmi-1–Lmi-6), L. tristicha (Lt-1), L. catarinensis (Lc-1 ! Lc-6), L. obtusa (Lo-1 – Lo-5), L. okamurai (Lok-1, Lok-2) and L. pacifica (Lp-1, Lp-2). Figure 1.14. Cytotoxic diterpenoids isolated from the red algae Laurencia obtusa (Lo-6) and Laurencia filiformis (Lf-1 and Lf-2). Figure 1.15. Antitumor diterpenoids isolated from the red alga Sphaerococcus coronopifolius (Sc- 1–Sc-9). Figure 1.16a. Cytotoxic meroditerpenes isolated from the red alga Callophycus serratus. Figure 1.16b. Cytotoxic meroditerpenes isolated from the red alga Callophycus serratus. Figure 1.17. Cytotoxic meroditerpenes isolated from the red alga Callophycus sp. Figure 1.18. Cytotoxic triterpenoids isolated from the red algae Laurencia mariannensis (Lm-1, Lm-2), L. viridis (Lv-1 – Lv-10) and L. catarinensis (Lc-7) together with the antitumor derivatives Lv-4a, Lv-4b, Lv-4c, and Lv-4d prepared from Lv-4. ! v! Figure 1.19. Antitumor bis-prenylated compounds isolated from the brown algae Perithalia capillaris (Pc-1–Pc-3) and Sporochnus comosus (Pc-1, Pc-2, Sco-1 – Sco-5). Figure 1.20. Cytotoxic meroterpenoids isolated from the brown algae Sargassum fallax (St-1, St-2 and St-3), S. siliquastrum (Ss-16 ! Ss-18) and S. macrocarpum (Sm-1). Figure 1.21. Cytotoxic terpenoids isolated from the brown algae Stypopodium flabelliforme (Sf-1– Sf-6) and Stypopodium zonale (Sz-1). Figure 1.22. Cytotoxic meroterpenoids from the brown algae Cystoseira abies-marina (Cab-1 – Cab-3) and C. tamariscifolia (Ct-1). Figure 1.23. Cytotoxic meroterpenoids isolated from the brown alga Homoeostrichus formosana. Figure 1.24. Cytotoxic diterpenoids isolated from the brown algae Dictyota sp. (Dsp-1) and Dictyota dichotoma (Dd-1). Figure 1.25. Cytotoxic diterpenoids isolated from the brown alga Stoechospermum marginatum (Sma-1, Sma-1a, Sma-2a, and Sma-3a). Figure 1.26. Anti-inflammatory sesquiterpenoids isolated from the red algae Laurencia dendroidea (Ld-1 ! Ld-3), L. snackeyi (Ls-1 ! Ls-4), and L. tristicha (Lt-1 ! Lt-4) Figure 1.27. Anti-inflammatory diterpene from the red alga Laurencia grandulifera. Figure 1.28. Anti-inflammatory meroterpenoids isolated from the brown algae Perithalia capillaris (Pc-1, Pc-2), Homoeostrichus formosana (Hf-1) and Sargassum siliquastrum (Ss-19) and S. micracanthum (St-3). Figure 1.29. Anti-inflammatory diterpenoids isolated from the brown alga Dictyota plectens. Figure 1.30. Brown alga Cystoseira usneoides. Figure 3.1. Effect of the extract of C. usneoides on the ABTS+• radical compared to Trolox. Figure 3.2. Effect of the acetone/methanol extract of C. usneoides on HT-29 cells proliferation. Figure 3.3. Effects of C. usneoides extract on the apoptosis of HT-29 cells. Figure 3.4. Effects of the extract of C. usneoides extract on HT-29 cell cycle distribution. Figure 3.5. Effect of C. usneoides extract on TNF-α production in THP-1 macrophages stimulated with LPS. Figure 4.1. Examples of meroterpenoids isolated from algae of the genus Cystoseira and a tetraprenyltoluquinol as potential biosynthetic precursor. Figure 4.2. Chemical structures of the meroterpenoids 1-12 isolated from C. usneoides collected in the Moroccan coasts of the Gibraltar Strait. Figure 4.3. Interconversion between the ABTS and its radical. Figure 4.4. Chemical structure of Trolox®. Figure 5.1. Chemical structures of the meroterpenoids 1-8 from C. usneoides subjected to anticancer studies ! vi! Figure 5.2. Effect of AMTs 1-8 at different concentrations on the viability of both HT-29 colon cancer cells and normal colonic epithelial cells CCD 841 CoN after 72 h of treatment. Figure 5.3. Apoptosis rates by flow cytometry of HT-29 colon cancer cells treated with the AMTs 1-8 for 24 h. Figure 5.4. Flow cytometry analysis of cell cycle arrest in colon cancer cells HT-29 treated for 24 h with the AMTs 1-8. Figure 5.5. Effects of AMTs 1-8 on migration and invasion of HT-29 cells. Figure 5.6. Effect of AMTs 1-8 on the activation of ERK by measuring the expression levels of the phosphorylated form (p-ERK) in HT-29 cells. Figure 5.7. Effect of AMTs 1-8 on the activation of JNK by measuring expression levels of the phosphorylated form (p-JNK) in HT-29 cells. Figure 5.8. Effect of AMTs 1-8 on the activation of AKT by measuring expression levels of the phosphorylated form (p-AKT) in HT-29 cells. Figure 5.9. Chemical structures of AMTs previously reported to possess antitumor activity against HT-29 cells. Figure 5.10. Chemical structures of the AMTs tuberatolide B (Sm-1) and sargachromanol E (Ss- 10) previously reported to cause apoptosis in colon cancer cells HCT-116 and HL-60, respectively. Figure 6.1. Chemical structures of the meroterpenes from C. usneoides subjected to antiinflammatory and anticancer studies. Figure 6.2. The AMTs 1-8 inhibit LPS-induced expression of TNF-α, IL-6, and IL-1β in THP-1 macrophaghes. Figure 6.3. Effect of AMTs 1-8 on LPS-induced COX-2 and iNOS protein expression in THP-1 macrophages. Figure 6.4. Effects of different concentrations of AMTs 1-8 on the viability of human lung cancer cell line A549 and the human fetal lung fibroblastic MRC-5 cells. Figure 6.5. Effects of AMTs 1-8 on cell cycle distribution of A549 cells. Figure 6.6. Chemical structures of algal terpenoids and AMTs which inhibit the production of proinflammatory mediators in LPS-stimulated RAW264.7 macrophages. Figure 6.7. Chemical structures of algal terpenoids and AMTs with anticancer activity against A549 cells. Figure 7.1. Chemical structure of the meroditerpene 11-hydroxy-1’-O-methylamentadione (AMT- E, 2). Figure 7.2. 11-hydroxy-1′-O-methylamentadione (AMT-E) protect mice against dextran sodium sulphate (DSS)-induced colitis. Figure 7.3. 11-hydroxy-1′-O-methylamentadione (AMT-E) administration attenuates microscopic colon damage induced by dextran sulfate sodium (DSS). ! vii! Figure 7.4. 11-hydroxy-1′-O-methylamentadione (AMT-E) administration reduces leukocyte infiltration. Figure 7.5. Effects of 11-hydroxy-1′-O-methylamentadione (AMT-E) administration on cytokine levels of colonic tissue in DSS-induced colitis. Figure 7.6. 11-hydroxy-1′-O-methylamentadione (AMT-E) administration reduces colonic protein levels of COX-2 and iNOS enzymes in DSS-induced colitis. Figure 7.7. Chemical structures of marine meroterpenoids tested on inflammatory bowel disease (IBD) experimental models: bolinaquinone (BQ) (Busserolles et al., 2005) and zonarol (ZN) (Yamada et al., 2014). Figure 8.1. Schematic representation indicating the interdependence between oxidative stress, inflammation, and cancer. Figure 8.2. Biological properties identified in this thesis for the meroterpenoids of the alga C. usneoides. ! viii! LIST OF TABLES Table 3.1. Antioxidant activity of extracts of algae of the genus Cystoseira in the ABTS assay. Table 3.2. Antioxidant activity of extracts of algae of the genus Cystoseira in the DPPH assay. Table 4.1. 13C NMR data of cystodiones A-F (7-12) in CD3OD. Table 4.2. 1H NMR data of cystodiones A-F (7-12) in CD3OD. Table 4.3. HRMS data of cystodiones A-F (7-12). Table 4.4. Antioxidant activities of compounds 1−10 in the ABTS Assay. Table. 5.1. IC50 values (µg/mL) obtained for AMTs 1-8 against the colon cancer cells HT-29 and the normal colon cells CCD 841 CoN after 72h of treatment. Table 6.1. Inhibitory effects of AMTs 1-8 on the production of TNF-α, IL-6, and IL-1ß in LPS- stimulated THP-1 macrophages. Table 6.2. Inhibitory effects of AMTs 1-8 on the production of COX-2 and iNOS in LPS- stimulated THP-1 macrophages. Table 6.3. IC50 values (µg/mL) of AMTs 1-8 against the normal lung cells MRC-5 and the lung cancer cells A549, after 72 h of treatment. Table 7.1. Effects of AMT-E (1, 10, and 20 mg/kg p.o.) on colonic length in dextran sodium sulphate (DSS)-treated mice. Table 7.2. Effects of AMT-E on histological score in the dextran sodium sulphate (DSS) model. ! ix# Abstract Macroalgae are an inexhaustible source of natural products that display a variety of pharmacological and biological activities. Among marine algae, the genus Cystoseira has proven to produce a wide variety of secondary metabolites that could benefit human health. However, studies on the natural products of the species Cystoseira usneoides and their respective biological properties are scarce. Hence, in this thesis, the extract of the brown alga Cystoseira usneoides and the natural products isolated therefrom were tested for antioxidant, anticancer, and antiinflammatory activities. The extract of C. usneoides exhibited potent radical-scavenging and inhibited the proliferation of HT-29 cancer cells by inducing apoptotis and G2/M arrest of the cell cycle. The extract was also found to inhibit the production of TNF-α in LPS-stimulated THP-1 human macrophages. The chemical study of the extract yielded six known algal meroterpenoids (AMTs): usneoidone Z (1), 11-hydroxy-1´-O-methylamentadione (2), cystomexicone B (3), cystomexicone A (4), 6-cis-amentadione-1′-methyl ether (5), and amentadione-1′-methyl ether (6), together with six new compounds: cystodiones A-F (7-12). AMTs 1-10 showed radical-scavenging activity in the ABTS assay. The most active were compounds 5, 6, 7, and 8, which exhibited antioxidant capacity equal or superior to that of the Trolox standard. AMTs 1-8 displayed significant cytotoxic activity against HT-29 human colon cancer cells, whereas lower cytotoxicity was observed against non-tumor cells CCD 841 CoN. Flow cytometry analysis revealed that AMTs 2, 3, and 5 caused apoptosis in HT-29 cells and compounds 1, 2, 3, 4, 5, and 7 induced cell cycle arrest in G2/M phase. Furthermore, AMTs 1-8 also inhibited the migration and invasion of colon cancer cells. Interestingly, exposure of HT-29 cells to different concentrations of AMTs 1-8 correlated with the down-regulation of p-ERK, p-JNK, and p-AKT pathways. Regarding to the anticancer effect of AMTs 1-8 against A549 human lung cancer cells, the cytotoxicity of all compounds was selective for A549 cells when compared to the non-tumor MRC-5 cells. Moreover, treatment of A549 cancer cells with AMTs 1-8 also led to cell cycle arrest in S and G2/M phases. In the in vitro anti-inflammatory assays, AMTs 1-8 showed significant activity as inhibitors of the production of the pro-inflammatory mediators TNF-α, IL-1β, IL-6, COX-2 and iNOS in LPS- stimulated THP-1 human macrophages. The in vivo study clearly demonstrated that the compound 11-hydroxy-1´-O-methylamentadione (AMT-E, 2) significantly ameliorated DSS colitis (seven days), including the reduction of weight loss, disease activity, macroscopic and microscopic colonic injury. The levels of TNF-α, IL-1β, IL-10, COX-2 and iNOS expression were also significantly decreased in AMT-E treated DSS colitis. Taken together, these findings strengthen the potential of the natural products isolated from C. usneoides as leads for novel antioxidant, anticancer, and anti-inflammatory agents. Keywords: Cystoseira usneoides; marine natural products; meroterpenes; biological activities; antioxidant; anticancer; anti-inflammatory; cytokines; DSS-colitis 1. General Introduction_____________________________________________________________________________ " " 6 Oxidative stress is involved in the activation of multiple transcription factors, which lead to the differential expression of over 500 different genes, including those of growth factors, cytokines, chemokines, cell cycle regulatory molecules, and anti-inflammatory molecules (Imbesi et al., 2013; Reuter et al., 2010). These mediator players activate complex mechanisms and signalling pathways that characterise inflammation (Imbesi et al., 2013; Federico et al., 2007; Reuter et al., 2010). Chronic inflammation contributes to tumour development at all three phases: initiation, promotion, and progression (Gonda et al., 2009). Thus, inflammatory cells liberate a number of reactive oxygen species leading to DNA damage and reducing DNA repair. On the other hand, inflammation also favors carcinogenesis by further recruiting inflammatory cells to the site of damage and activating and producing many mediators, including transcription factors (NF-κB, STAT-3, and HIF-1), chemokines (MCP-1, IL-8, CXC chemokine receptor), and cytokines (TNF-!, IL-6, IL-10, TGF-!). These promoters influence the whole tumour and impact on the different stages of tumorigenesis such as initiation, promotion, and progression, as well as on the angiogenesis (Landskron et al., 2014; Multhoff et al., 2012; Reuter et al., 2010; Coussens and Werb, 2002). Therefore, discovering natural drugs that target redox-sensitive pathways, and transcription factors offers great promise for cancer prevention and therapy. In this context, bioactive terpenoids from marine algae could be used as scaffolds in the design of novel compounds for pharmacological purposes. Herein, we provide a comprehensive overview of the terpenoids and meroterpenoids with antioxidant, antitumor, and anti-inflammatory activities that have been described from macroalgae during the last twelve years (2007-2018). 1.3.1. Antioxidant properties of terpenoids and meroterpenoids isolated from macroalgae Oxidative stress is defined as an imbalance between the production and accumulation of oxygen reactive species (ROS) in cells and tissues and the ability of a biological system to detoxify these reactive products (Pizzino et al., 2017). ROS are products of the normal cellular metabolism (Birben, et al., 2012) and play important physiological roles, especially in the maintenance of cell homeostasis and in the regulation of functions such as proliferation, signal transduction, gene expression, and activation of _____________________________________________________________________1. General Introduction " " 7 receptors (Dröge, 2002). However, when the production of ROS exceeds the cellular protective mechanisms (oxidative stress) the excessive ROS levels can cause damage to DNA, lipids, and proteins (Wu et al., 2013). The cellular antioxidant defense mechanisms include enzymatic (enzymes that reduce lipid peroxidation) and non-enzymatic pathways (e.g. glutation, L-arginine, coenzyme Q) (Kumar and Pandey, 2015; López-Alarcón and Denicola, 2013). In addition to these endogenous antioxidants, a variety of exogenous antioxidants can be obtained through the diet and nutritional supplements (e.g. vitamins C and E, flavonoids, carotenoids) (Pizzino et al., 2017; Nimse and Palb, 2015). It has been noted that although marine macroalgae are exposed to intense light and oxygen concentrations they do no show oxidative damages in their structural components, suggesting the presence of metabolites with antioxidative defense functions (Jiménez- Escrig et al., 2001). In this line, the extracts from a great variety of macroalgae and some pure compounds have been shown to possess significant antioxidant properties in different assays (Balboa et al., 2013; Farvin and Jacobsen, 2013). Herein we present the terpenoids and meroterpenoids with antioxidant activity described from macroalgae during the last twelve years (2007-2018). Most of the algal NPs with antioxidant properties are meroterpenoids containing a phenolic ring and have been isolated from brown algae. Thus, sargahydroquinoic acid (St- 1), sargaquinoic acid (St-2), sargachromenol (St-3) and sargathunbergol (St-4) (Figure 1.5) from Sargassum thunbergii showed the capacity to scavenge the DPPH free radical with ED50 values in the range 20-38 µg/mL, which were similar to those of the reference compounds BHT (42 µg/mL) and α-tocopherol (23 µg/mL) (Seo et al., 2007). Figure 1.5. Antioxidant meroterpenoids from the brown alga Sargassum thunbergii. 1. General Introduction_____________________________________________________________________________ " " 8 The chemical study of S. siliquastrum led to the isolation of twenty meroterpenoids most of which showed radical scavenging activity in the DPPH assay (EC50 values in the range 0.10-23.23 µg/mL) (Jung et al., 2008). The more active compounds were the known compounds sargahydroquinoic acid (St-1, Figure 1.5), isonahocol D1 (Ss-1) and isonahocol D2 (Ss-2) (Figure 1.6), together with the new compounds Ss-3, Ss-4, Ss-5, Ss-6, and Ss-7 (Figure 1.6), that showed ED50 values in the range 0.10-0.33 µg/mL. Another study of this species yielded the new chromene mojabanchromanol (Ss-8, Figure 1.6), which at 500 µg/mL caused 84.08% decrease of TBARS formation and 96.07% radical elimination in the DPPH assay (Cho et al., 2008). Figure 1.6. Antioxidant meroterpenoids from the brown algae"Sargassum siliquastrum (Ss- 1 to Ss-14) and Stypopodium zonale (Ss-15). _____________________________________________________________________1. General Introduction " " 9 A third report on S. siliquastrum described six chromanols, the known sargachromanols D (Ss-9), E (Ss-10), and K (Ss-11) and the new Ss-12, Ss-13, and Ss-14 (Figure 1.6) whose antioxidant effects were evaluated in different assays (Lee and Seo 2011). At 5 µg/mL all the compounds decreased by more than 67.2% the intracellular generation of ROS in HT1080 cells and increased the intracellular level of glutathione. The most active was sargachromanol E (Ss-10), which caused 87.2% decrease of ROS levels. In addition, at 50 µg/mL all the compounds inhibited the lipid peroxidation, in particular the new compounds Ss-12 and Ss-13, which caused 43.2 and 38.9% inhibition, respectively. The related compound sargaol (Ss-15, Figure 1.6), isolated from Stypopodium zonale, was also described to scavenge 86% of the DPPH radical at 50 µg/mL (Penicooke et al., 2013). The meroterpenes cystoazorol A (Cab-1) and cystoazorol B (Cab-2) (Figure 1.7), isolated from Cystoseira abies-marina, were described to cause 29% and 30% scavenging of the DPPH radical at 500 µg/mL, respectively; these are values rather lower than those observed for the reference compounds quercetin and trolox (78% and 75 % scavenging, respectively) at the same concentration (Gouveia et al., 2013b). Figure 1.7. Antioxidant meroterpenoids isolated from the brown alga Cystoseira abies-marina. After a screening of the extracts of thirteen species of algae from the Aegean Sea using chemiluminescence (CL) and DPPH assays, the extract of the brown alga Taonia atomaria showed the highest levels of antioxidant activity (Nahas et al., 2007). The isolation guided by radical scavenging activity led to the obtention of six polycyclic meroditerpenes active in the CL and DPPH assays. The highest levels of activity were observed for taondiol (Ta-1) and isoepitaondiol (Ta-2) (Figure 1.8), which were more active than the reference compounds trolox and ascorbic acid. 1. General Introduction_____________________________________________________________________________ " " 10 " Figure 1.8. Antioxidant meroditerpenoids isolated from the brown alga Taonia atomaria. A few terpenoids lacking a phenolic ring have ben reported to possess antioxidant activity, such as the diterpenes amijiol acetate (Dd-1) and amijiol-7,10-diacetate (Dd-2) (Figure 1.9) isolated from Dictyota dichotoma (Ayyad et al., 2011). These compounds, at 100 µM, caused 83.35 and 80.24% inhibition of the ABTS radical, respectively, while another seven diterpenes isolated in the same study were less active. Figure 1.9. Antioxidant diterpenoids isolated from the brown alga Dictyota dichotoma. The only example of antioxidant meroterpenoids from red algae has been found in the arylterpenoids isolated from Hypnea musciformis (Chakraborty et al., 2016). Among the isolated compounds, Hm-1 (Figure 1.10) showed IC50 = 25.05 µM in the DPPH assay and IC50 = 350.7 µM in the Fe2+ ion chelating activity assay, which were similar to those shown by gallic acid (IC50 32.3 µM and 646.6 µM, respectively). Figure 1.10. Antioxidant arylterpenoid isolated from the red alga Hypnea musciformis. _____________________________________________________________________1. General Introduction " " 11 1.3.2. Antitumor properties of terpenoids and meroterpenoids isolated from macroalgae Cancer is a worldwide leading cause of mortality (Torre et al., 2015). In spite of the therapeutic progresses achieved during the last decades, the fail of various types of chemotherapies, mainly because of side effects or drugs resistance, evidence the need and importance of the research for new molecules with anticancer activity, more effective and with smaller adverse effects. NPs have great potential in cancer therapy (Cragg and Pezzuto, 2015; Cragg et al., 2009) as demonstrates the fact that 77% of the anti-cancer drugs (excluding biologicals and vaccines) developed from 1930 to 2014 are derived from NPs (Newman and Cragg, 2016). Therefore, the search for novel therapeutic agents from NPs that target specific, novel, or so far not investigated molecular signaling pathways, in order to arrest the growth and metastasis of tumors, continues to be an active research area (Harvey et al., 2015). Although historically plant-derived compounds have played a prominent role in the discovery and development of anticancer drugs (Seca and Pinto, 2018), more recently marine NPs have also led to new anticancer drugs and several compounds are currently in clinical trials (Ruiz-Torres et al., 2017; Newman and Cragg, 2014). Marine macroalgae have been shown to contain a variety of compounds with interesting antitumor properties, including polysaccharides, polyphenols, and small molecules such as terpenoids and steroids, among others (Murphy et al., 2014). Herein we present the structures and antitumor properties of the terpenoids and meroterpenoids reported from macroalgae during the last twelve years (2007-2018). The data are organized by class of algae, and within each class, by type of terpenoid, from mono- to triterpenes. Green algae During the last years only two sesquiterpenes from green algae, caulerpenyne (Cp- 1) from Caulerpa taxifolia and Uf-1 from Ulva fasciata (Figure 1.11), have been studied for their antitumor properties. In particular, research on the mechanism of action of caulerpenyne (Cp-1), which is cytotoxic for several cancer cell lines and inhibits the polymerization of tubulin, have shown that Cp-1 neither interact to colchicine, taxol, and vinka-alkaloid binding domain, not bind covalently to tubulin (Bourdron et al., 2009). 1. General Introduction_____________________________________________________________________________ " " 12 Figure 1.11. Cytotoxic terpenoids isolated from the green algae Caulerpa taxifolia (Ct-1), Ulva fasciata (Uf-1), and Tydemania expeditionis (Te-1 – Te-4) and cytotoxic derivatives prepared from Te-3 (Te-3a, Te-3b, Te-3c). More recently, the novel guaiane sesquiterpene Uf-1 has shown cytotoxic activity against triple-negative breast cancer (TNBC) cell line MDA MB-231 with IC50 =17.35 µM at 24h of treatment (Lakshmi et al., 2018). More advanced studies showed that Uf-1 was capable of inducing apoptosis and cell cycle arrest in G1 phase, via regulation of EGFR/PI3K/Akt pathway, in human TNBC cells (Lakshmi et al., 2018). On the other hand, four triterpenoid disulfates, one of lanostane-type (Te-1) and three of cycloartane type (Te-2, Te-3 and Te-4) isolated from Tydemania expeditionis, along with the monosulfated derivatives Te-3a and Te-3b, and the desulfated Te-3c prepared from Te-3 (Figure 1.11), were tested against a panel of 12 tumor cell lines including breast (BT-549, DU4475, MDA-MB-468, MDA-MB-231), colon (HCT116), lung (NCI-H446, SHP-77), prostate (PC-3, LnCaP-FGC, Du145), ovarian (A2780/DDPS), and leukemia (CCRF-CEM) cancer cell lines. Compounds Te-1, Te-2, Te-3, and Te-4 displayed antitumor effects with mean IC50 values ranging from 31 to 38 µM, while the derivatives Te-3a, Te-3b and Te-3c were more active with IC50 values from 6.0 to 11 µM (Jiang et al., 2008). _____________________________________________________________________1. General Introduction " " 13 Red algae Several species of red algae of the genus Plocamium, Laurencia, Sphaerococcus, and Callophycus have been source of a variety of terpenoids with interesting antitumor properties. Thus, the studies of three Plocamium species from the South African coasts have led to the isolation of several polyhalogenated monoterpenes cytotoxic against WHCO1 esophageal cancer cell line (Antunes et al., 2011; Mann et al., 2007). The highest activity levels were observed for Pcr-1 (Figure 1.12) (IC50 = 7.5 µM), isolated from Plocamium corallorhiza (Mann et al., 2007), and for Ps-1, Ps-2, Ps-3, Ps-4, Ps-5, and Ps-6 (Figure 1.12) (IC50 = 6.6–9.9 µM), isolated from P. suhrii, which were more active than the known anticancer drug cisplatin (IC50 = 13 µM) (Antunes et al., 2011). However, the monoterpenoids isolated from P. cornutum were less active and only compounds Pco-1, Pco-2, Pco-3 (Figure 1.12) displayed IC50 values lower than 50 µM (17.8, 47.3, and 40.2 µM, respectively) (Antunes et al., 2011). Figure 1.12. Cytotoxic monoterpenoids from the red algae Plocamium corallorhiza (Pcr-1), P. suhrii (Ps-1 – Ps-7),"P. cornutum (Pco-1 – Pco-3), P. cartilagineum (Pca-1 ‒ Pca-3), and Pterocladiella capillacea (Ptc-1). Later, the monoterpene Pca-1, isolated from P. cartilagineum, together with Pca-2, Ps-4 and Pco-1, obtained by synthesis, were assayed against a panel of 9 cancer cell lines, including colon (H116), lung (H125), breast (MCF-7, MDA-MB-231), prostate (LNCap), 1. General Introduction_____________________________________________________________________________ " " 14 ovary (OVC-5), glioblastoma (U251N), pancreas (PANC-1), and liver (HepG2) tumors, and showed mean IC50 values of 1.3, 15, 3.6, and 1.3 µg/mL, respectively (Vogel et al., 2014). More recently, the species P. cartilagineum has also yielded the monoterpene Pca-3, whose configuration at C-3 and C-4 has not been assigned. This compound was cytotoxic against the NCI-H460 tumor cell line with IC50= 4 µg/mL (Sabry et al., 2017). On the other hand, a chemical report on the red macroalga Pterocladiella capillacea described the new halogenated monoterpene mertensene (Ptc-1, Figure 1.12), which exhibited antiproliferative activity against the human colorectal adenocarcinoma cell lines HT29 and LS174 with IC50 of 56.50 µg/mL and 49.77 µg/mL, respectively. The compound Ptc-1 induced G2/M cell cycle arrest in HT29 cells through a negative modulation of phosphorylated forms of p53, retinoblastoma protein (Rb), cdc2, and chkp2, and also reduced the expression level of cyclin-dependent kinases CDK2 and CDK4. Moreover, the cytotoxicity of Ptc-1 was associated with the induction of cell apoptosis via modulation of ERK-1/-2, AKT and NF-κB pathways (Tarhouni-Jabberi et al., 2017). Red algae of the genus Laurencia have been a prolific source of bioactive sesqui-, di-, and triterpenoids. Among the sesquiterpenes, the new aromatic derivatives Lmi-1 and Lmi-2, along with the known metabolites dibromophenol (Lmi-3), (+)-α- isobromocuparene (Lmi-4) and (-)-α-bromocuparene (Lmi-5) (Figure 1.13), isolated from Laurencia microcladia, exhibited weak inhibitory effect against the human cancer cell lines HT-29, MCF7, PC3, HeLa, and A431 with IC50 values in the range 75.2-287.3 µM (Kladi et al., 2007). On the other hand, the chamigrane-type sesquiterpene elatol (Lmi-6) (Figure 1.13) also obtained from L. microcladia was reported to show significant cytotoxic activity against the murine cell lines B16F10 (melanoma) and L929 (fibroblast), and against the human tumor cell lines MCF7 (breast), DU145 (prostate), and A549 (lung), with IC50 values between 1.1 and 10.1 µM (Campos et al., 2012). Further studies showed that elatol (Lmi-6) exhibited cytotoxicity against murine melanoma B16F10 cells (IC50 10.1 µM) by promoting the activation of the apoptotic process and inducing cell cycle arrest in the G1/S phase, via the reduction of cyclin-D1, cyclin-E, cyclin-A, cdk-4, cdk-2, and pRb expression. In an in vivo experiment, treatments of C57Bl6 mice bearing B16F10 cells with elatol (Lmi-6) at 3, 10, and 30-mg/kg p.o. caused 50.4, 41.3, and 61.2% reduction of tumor _____________________________________________________________________1. General Introduction " " 15 volume, respectively. Higher reductions of 51.8, 61.4, and 71,4% were observed in 1, 3, and 10 mg/kg i.p.-treated mice, respectively, while cisplatin at 4 mg/kg (i.p.) caused 85% reduction of tumor growth (Campos et al., 2012). Figure 1.13. Cytotoxic sesquiterpenes from the red algae Laurencia microcladia (Lmi-1 –Lmi-6), L. tristicha (Lt-1), L. catarinensis (Lc-1 ! Lc-6), L. obtusa (Lo-1 – Lo-5), L. okamurai (Lok-1, Lok-2) and L. pacifica (Lp-1, Lp-2). Among the nineteen sesquiterpenoids isolated from Laurencia tristicha, the new compound debromoepiaplysinol (Lt-1, Figure 1.13) showed selective cytotoxicity against the HeLa cell line with IC50 of 15.5 µM (Sun et al., 2007). The in vitro cytotoxicity of seven new and seven known halogenated sesquiterpenoids isolated from Laurencia catarinensis was evaluated against the human tumor cell lines HT-29 (colon carcinoma), MCF7 (breast carcinoma), and A431 1. General Introduction_____________________________________________________________________________ " " 22 Figure 1.18. Cytotoxic triterpenoids isolated from the red algae Laurencia mariannensis (Lm-1, Lm-2), L. viridis (Lv-1 – Lv-10) and L. catarinensis (Lc-7) together with the antitumor derivatives Lv-4a, Lv-4b, Lv-4c, and Lv-4d prepared from Lv-4. _____________________________________________________________________1. General Introduction " " 23 significant cytotoxic activity against the same panel of cancer cell lines (IC50 = 2.0-30.0 µM) (Cen-Pacheco et al., 2011b). The highest inhibitory activity was observed for Lv-5, Lv-6, and Lv-8 against the Jurkat leukemic cells, with IC50 values of 3.5, 2.0, and 2.5 µM, respectively. In addition, secodehydrothyrsiferol (Lv-8)"at" 20 µM induced apoptosis, as inferred from the appearance of a sub-G1/G0 subpopulation in cell cycle analysis (Cen- Pacheco et al., 2011b). The last report of this series on L. viridis described two new triterpenoids, saiyacenols A (Lv-9) and B (Lv-10) (Figure 1.18), that inhibited the proliferation of the same four cell lines with IC50 values in the range 2.7-27.5 µM. Again, the highest effect was observed against Jurkat cells and it was caused by compound Lv-10 (IC50 2.7 µM) (Cen-Pacheco et al., 2012). "On the other hand, studies on the mechanism of action of the cytotoxic triterpene thyrsiferol (Lc-7, Figure 1.18), obtained from Laurencia catarinensis, showed that Lc-7 at 3µM caused 66% inibition of the hypoxia-inducible factor-1 (HIF-1) activation in T47D human breast cancer cells (Mahdi et al., 2011). Moreover, thyrsiferol suppressed mitocondrial respiration by selectively inhibiting ETC complex I (IC50 3 µM)." Brown algae A few studies have investigated the antitumor potential of terpenoids and meroterpenoids isolated from brown macroalgae. The new bis-prenylated quinone Pc-1 (Figure 1.19) isolated from Perithalia capillaris showed potent antiproliferative activity against HL60 cells with IC50 = 0.34 µM (Sansom et al., 2007), while the phenol Pc-2 and the new chromane Pc-3 were less active (IC50 = 2.7 and 5.6 µM, respectively). The compounds Pc-1 and Pc-2 were later isolated from Sporochnus comosus together with four new bis-prenylated phenols, comosusols A (Sco-1), B (Sco-2), C (Sco-3), and D (Sco-4), and the bis-prenylated cyclohexenone comosone A (Sco-5) (Figure 1.19). All these compounds showed cytotoxic activity against the human tumor cell lines MCF-7 (breast), SF-268 (CNS), H460 (lung), and HT-29 (colon) with GI50 values ranging from 5 to 59 µM (Ovenden et al., 2011). The most active was comosusol B (Sco-2) with IC50 values of 5, 6, 6, and 6 µM, respectively. 1. General Introduction_____________________________________________________________________________ " " 24 Figure 1.19. Antitumor bis-prenylated compounds isolated from the brown algae Perithalia capillaris (Pc-1 – Pc-3) and Sporochnus comosus (Pc-1, Pc-2, Sco-1 – Sco-5). The meroditerpenoids sargaquinoic acid (St-1), sargahydroquinoic acid (St-2), and sargaquinone (St-3) (Figure 1.20), isolated from the seaweed Sargassum fallax, proved to have moderate antitumor activity against P388 murine leukaemia cell line (IC50 = 17, 14 and 32 µM, respectively) (Reddy and Urban 2009). The antitumor activity of the chromane sargachromanol E (Ss-10) (Figure 1.20) isolated from Sargassum siliquastrum was investigated against the HL-60 cell line (Heo et al., 2011). The meroditerpene Ss-10 inhibited tumor cell proliferation (68.9% at 25 µM) and induced apoptosis by mechanisms involving the downregulation of Bcl-xL, upregulation of Bax, activation of caspase-3, and cleavage of poly-ADP-ribose-polymerase (PARP). Another chemical investigation of S. siliquastrum yielded the known meroterpenoid sargachromanol J (Ss-16), and the new sargachromanols Q (Ss-17) and R (Ss-18) (Figure 1.20), which were described to inhibit the proliferation of a panel of human cancer cell lines including AGS (gastric adenocarcinoma), HT-29 (colon adenocarcinoma), HT-1080 (fibrosarcoma), and MCF-7 (breast adenocarcinoma) (Lee et al., 2014). Sargachromanol R (Ss-18) displayed the strongest activity against AGS, HT-29, and HT- 1080 cell lines with IC50 values of 6.5, 3.4, and 13.9 µg/mL, respectively. Sargachromanol J (Ss-16) was active againts the four cells (IC50 27.8-31.3 µg/mL) and sargachromanol Q (Ss-17) was only active against AGS (IC50 42.8 µg/mL). _____________________________________________________________________1. General Introduction " " 25 Figure 1.20. Cytotoxic meroterpenoids isolated from the brown algae Sargassum fallax (St-1, St-2, and St-3), S. siliquastrum (Ss-10, Ss-16, Ss-17, and Ss-18) and S. macrocarpum (Sm-1). The meroterpenoid tuberatolide B (Sm-1), isolated from Sargassum macrocarpum, has been shown to inhibit tumor growth on breast, lung, colon, prostate, and cervical cancer cells by inducing apoptosis (Choi et al., 2017). Tuberatolide B (Sm-1)"enhanced DNA damage by inducing γH2AX foci formation and the phosphorylation of DNA damagerelated proteins such as Chk2 and H2AX. Furthermore, Sm-1"selectively inhibited STAT3 activation, which resulted in a reduction in cyclin D1, MMP-9, survivin, VEGF, and IL-6. The activity of six meroditerpenoids isolated from Stypopodium flabelliforme, epitaondiol (Sf-1), epitaondiol diacetate (Sf-2), epitaondiol monoacetate (Sf-3), stypotriol triacetate (Sf-4), 14-ketostypodiol diacetate (Sf-5), and stypodiol (Sf-6) (Figure 1.21) as inhibitors of cell proliferation was investigated against a panel of human (Caco-2 and SHSY5Y), and non-human (RBL-2H3 and RAW.267) cancer cell lines, as well as against the non-cancer cells V79 (Pereira et al., 2011). Overall, all compounds showed significant activity against the tested cancer cell lines. The compounds Sf-1, Sf-3 and Sf-4 displayed nearly 100% inhibition of RAW.267 cells at all the tested concentrations (12.5, 25, 50 µM for Sf-1 and Sf-4; 6.25, 12.5 and 25 µM for Sf-3). The cells SH-SY5Y were the most susceptible and except for Sf-2 all compounds caused nearly 100% inhibition of SH-SY5Y 1. General Introduction_____________________________________________________________________________ " " 26 at the highest tested dose (50 µM). For Caco-2 and RBL-2H3 cell lines, all meroterpenes showed a concentration-dependent inhibitory effect, with Sf-1, Sf-3, and Sf-4 being the most actives. On the other hand, Sf-2, Sf-5, and Sf-6 had no effect on the non-cancer cell line. A related compound, zonaquinone acetate (Sz-1) isolated from Stypopodium zonale (Figure 24) was described to inhibit the proliferation of MCF-7 (breast) and HT-29 (colon) cancer cell lines with IC50 values of 20.4 µM and 17.7 µM respectively, which were comparable to the values shown by the standard drugs tamoxifen (17.3 µM for MCF-7) and fluorouracil (29.3 µM for HT-29) (Penicooke et al., 2013); however, no activity was observed against HepG2 cells. " Figure 1.21. Cytotoxic terpenoids isolated from the brown algae Stypopodium flabelliforme (Sf-1 – Sf-6) and Stypopodium zonale (Sz-1). The new meroditerpene cystoazorol A (Cab-1), along with the new meronorsesquiterpenes cystoazorones A (Cab-2) and B (Cab-3) (Figure 1.22), were isolated from Cystoseira abies-marina and tested for their cytotoxicity toward tumor (HeLa) and non-tumor (Vero) cell lines (Gouveia et al., 2013b). The most active compound was cystoazorol A (Cab-1), with IC50 values of 10.2 µg/mL against cells in lag phase and 2.8 µg/mL against cells in log phase. The compound was less cytotoxic against non-tumor cells, displaying selectivity index (SI = IC50Vero/ IC50HeLa) of 1.64 and 2.46 for cells in _____________________________________________________________________1. General Introduction " " 27 lag and log phases, respectively, which are superior to those shown by taxol (1.5 and 0.5, respectively). Cystoazorones Cab-2 and Cab-3 were also cytotoxic (IC50 of 14.3-32.0 µg/mL) against HeLa, but less selective. More recently, the compound Ct-1 (Figure 1.22), a derivative of the meroditerpenoid cystoketal isolated from"Cystoseira tamariscifolia, was shown to display a significant inhibition of HepG2 cells (IC50 14.77 µg/ml), and less activity towards the nontumor S17 cells (IC50 48.46 µg/ml, SI = 3.28) (Vizetto-Duarte et al., 2016). Figure 1.22. Cytotoxic meroterpenoids from the brown algae Cystoseira abies-marina (Cab-1 – Cab-3) and C. tamariscifolia (Ct-1). Among the NPs isolated from Homoeostrichus formosana, the known compound methylfarnesylquinone (Hf-1) together with the new chromene derivative Hf-2 and its acetyl derivative Hf-2a (Figure 1.23),"displayed moderate to weak cytotoxicity on HepG2 (liver), A549 (lung) and MDA-MB-231 (breast) cancer cell lines, with IC50 values ranging from 20.2 to 43.1 µM (Fang et al., 2015)."" Figure 1.23. Cytotoxic meroterpenoids isolated from the brown alga Homoeostrichus formosana. 1. General Introduction_____________________________________________________________________________ " " 28 Brown algae belonging to the family Dictyotaceae represent a rich source of biologically active terpenoids. The new compound 8α,11-dihydroxypachydictyol A (Dsp- 1), isolated from an unidentified species of the genus Dictyota (Figure 1.24) showed cytotoxicity against the lung cancer cell line NCI-H187 (IC50 5 µg/ml) and moderate activity against KB oral carcinoma cell (IC50 14.1 µg/ml) (Jongaramruong and Kongkam 2007). The related compound pachydictyol A (Dd-1), isolated from Dictyota dichotoma, (Figure 1.24), exhibited cytotoxicity against a panel of 12 human tumor cell lines with a mean IC50 of 23.6 µM (Abou-El-Wafa et al., 2013). Figure 1.24. Cytotoxic diterpenoids isolated from the brown algae Dictyota sp. (Dsp-1) and Dictyota dichotoma (Dd-1). A chemical study of Stoechospermum marginatum led to the isolation of ten terpenoids that were used to synthetize seven derivatives. The compounds were assayed against the cancer cell lines U937 (histocytic lymphoma), THP-1 (acute monocytic leukemia), COLO205 (colon adenocarcinoma), B16F10 (mouse melanocarcinoma), and HL60 (promyelocytic leukemia). Although many compounds were active against most of the cell lines, the highest levels of cytotoxicity were described for the natural spatane diterpenoid Sma-1, and the semi synthetic derivatives Sma-1a, Sma-2a, and Sma-3a (Figure 1.25). These exibited cytotoxic activity against U937 cells with IC50 values of 5.60, 8.80, 7.52, and 9.58 µg/mL, respectively, and against B16F10 with IC50 values of 3.45, 4.11, 3.62 and 3.28 µg/mL, respectively, which are comparable to those of the standard drug etoposide (IC50 = 2.27 and 4.12 µg/mL against U937 and B16F10, respectively) (Chinnababu et al., 2015). The apoptotic mechanism of the compound Sma-1 was investigated on B16F10 cancer cells, and the results indicated that Sma-1 induced intrinsic mitochondrial apoptosis pathway by generating ROS and deregulating the PI3K/Akt pathway (Velatooru et al., 2016). In addition, the in vivo antitumor activity of Sma-1 was _____________________________________________________________________1. General Introduction " " 29 investigated in C57BL/6 mice bearing B16F10 melanoma. At doses of 4, 10, and 15 mg/ kg (i.p.), the compound Sma-1 caused an effective inhibition of tumor volume (up to 70 %) in a dose dependent manner without apparent toxic effects. Figure 1.25. Cytotoxic diterpenoids isolated from the brown alga Stoechospermum marginatum (Sma-1, Sma-1a, Sma-2a, and Sma-3a). 1.3.3. Anti-inflammatory properties of terpenoids and meroterpenoids isolated from macroalgae Inflammation is a natural defense mechanism against exogenous and endogenous noxious stimuli (Okin et al., 2012). The purpose of inflammation is to protect the area of injury or infection by eliminating the injurious agent and removing the damaged tissue components (Khanna et al., 2014). During the process of inflammation, different cell types are recruited, including macrophages, which are involved in the production of various pro- and anti-inflammatory mediators such as the cytokines (tumor necrosis factor TNF-α and interleukins), the enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), nitric oxide (NO), the prostaglandins and leukotrienes (Duque and Descoteaux 2014). Thus, the inhibition of the pro-inflammatory mediators produced by macrophages is believed to be crucial for managing inflammatory diseases. Moreover, the expression of most of the pro-inflammatory cytokines is controlled through the activation of NF-κB; this is a key regulator of immune system and inflammatory response (Biswas, 2016), and therefore an important target for the discovery of anti-inflammatory compounds. Many investigations have been focused on the identification of anti-inflammatory agents from natural sources, mainly terrestrial plants, using a variety of in vitro and in vivo assays (Azab et al., 2016). These studies have also shown that many NPs with antiinflammatory effects, including terpenoids, target and modulate the NF-κB signaling pathway (Salminen et al., 2008; Folmer et al., 2008). 1. General Introduction_____________________________________________________________________________ " " 30 Macroalgae have been shown to possess different types of compounds with antiinflammatory potential, such as polyphenols, sulfated polysaccharides, terpenoids, and fatty acids (Fernando et al., 2016; Jaswir and Monsur 2011; Park et al., 2011). Herein we present the data reported during the last twelve years (2007-2018) on the terpenoids and meroterpenoids from macroalgae with anti-inflammatory properties, organized by class of algae and type of terpenoids. Red algae Among the five sesquiterpenoids isolated from Laurencia dendroidea, the new chamigrane-type sesquiterpenes debromoelatol (Ld-1) and obtusane (Ld-2), and the known compound Ld-3 (Figure 1.26) inhibited the release of the inflammatory mediator NO in LPS-stimulated RAW 264.7 macrophages (Machado et al., 2014). Debromoelatol Ld-2 (IC50 44.9 µM) was more active than the reference compound LNMNA (L- monomethylarginine, IC50 = 71.3 µM), while the IC50 values of Ld-1 and Ld-3 (69.1 µM and 74.6 µM, respectively) were similar to that of LNMMA. In addition, Ld-1 also caused moderate inhibition of the TNF-α levels (IC50 133.8 µM). Figure 1.26. Anti-inflammatory sesquiterpenoids isolated from the algae Laurencia dendroidea (Ld-1 ! Ld-3), L. snackeyi (Ls-1 ! Ls-4), and L. tristicha (Lt-1 ! Lt-4). _____________________________________________________________________1. General Introduction " " 31 The sesquiterpene 5β-hydroxypalisadin B (Ls-1), along with another three compounds, palisadin (Ls-2), palisol (Ls-3), and pacifigorgiol (Ls-4) (Figure 1.26) isolated from the red alga Laurencia snackeyi also showed in vitro inhibition of the NO production in LPS-stimulated RAW 264.7 cells (Wijesinghe et al., 2014a). The most active compound was Ls-1, which caused 90% inhibition at 50 µM (IC50=17.56 µM). This compound also decreased the LPS-induced release of prostaglandin E2 (PGE2, 37.4% inhibition at 50 µM), markedly suppressed the iNOS and COX-2 expression at 25-50 µM, and diminished the production of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 (38%, 52%, and 30%, respectively, at 20 µM). Furthermore, the compound Ls-1 was tested for its antiinflammatory effect on zebrafish embryo in an in vivo model (Wijesinghe et al., 2014b). At concentrations in the range 0.25-1 µg/mL the compound showed protective effect against stress-induced ROS formation and effectively inhibited the LPS-induced nitric oxide (NO) production in zebrafish embryos. Moreover, all the protective effects observed for Ls-1 were comparable to those caused by the reference compound dexamethasone (0.5 µg/mL). The chemical investigation of Laurencia tristicha led to the isolation of eighteen metabolites among which the new chamigrane sesquiterpene Lt-2 and the two known chamigranes Lt-3 and L-4 (Figure 1.26) caused at 20 µM significant inhibition of elastase release (33%, 60%, and 69%, respectively) (Chen et al., 2016). In addition, Lt-2 and Lt-3 at 20 µM significantly inhibited the N-formylmethionyl-leucyl-phenylalanine /cytochalasin B (fMLP/CB)-induced superoxide anion (O2−•) secretion (32% and 48%, respectively). Only one diterpene with anti-inflammatory properties has been described from red algae in the last years, neorogioltriol (Lg-1) (Figure 1.27) (Chatter et al., 2011). Figure 1.27. Anti-inflammatory diterpene from the red alga Laurencia glandulifera. 1. General Introduction_____________________________________________________________________________ " " 38 Chen, J.Y.; Huang, C.Y.; Lin, Y.S.; Hwang, T.L.; Wang, W.L.; Chiou, S.F.; Sheu, J.H. Halogenated sesquiterpenoids from the red alga Laurencia tristicha collected in Taiwan. J. Nat. Prod. 2016, 79, 2315–2323. Cheng, S.; Zhao, M.; Sun, Z.; Yuan, W.; Zhang, S.; Xiang, Z.; Cai, Y.; Dong, J.; Huang, K.; Yan, P. Diterpenes from a chinese collection of the brown alga Dictyota plectens. J. Nat. Prod. 2014, 77, 2685−2693. Chinnababu, B.; Purushotham Reddy, S.; Sankara Rao, P.; Loka Reddy, V.; Sudheer Kumar, B.; Rao, J.V.; Prakasham, R.S.; Suresh Babu, K. Isolation, semi-synthesis and bioevaluation of spatane derivatives from the brown algae Stoechospermum marginatum. Bioorg. Med. Chem. Lett. 2015, 25, 2479–2483. Cho, S.H.; Cho, J.Y.; Kang, S.E.; Hong, Y.K.; Ahn, D.H. Antioxidant activity of mojabanchromanol, a novel chromene, isolated from brown alga Sargassum siliquastrum. J. Environ. Biol. 2008, 29, 479–484. Choi, Y.K.; Kim, J.; Lee, K.M.; Choi, Y.J.; Ye, B.R.; Kim, M.S.; Ko, S.G.; Lee, S.H.; Kang, D.H.; Heo, S.J. Tuberatolide B suppresses cancer progression by promoting ROS-mediated inhibition of STAT3 signaling. Mar. Drugs 2017, 15, 55. Coussens, L.M.; Werb, Z. Inflammation and cancer. Nature 2002. 420, 860–867. Cragg, G.M.; Grothaus, P.; Newman, D.J. Impact of natural products on developing new anti-cancer agents. Chem. Rev. 2009, 109, 3012–3043. Cragg, G.M.; Pezzuto, J.M. Natural products as a vital source for the discovery of cancer chemotherapeutic and chemopreventive agents. Med. Princ. Pract. 2016, 25, 41–59. Dewick, P.M. The mevalonate and deoxyxylulose phosphate pathway. In Medicinal Natural Products: a biosynthetic approach, 2nd Ed. John Wiley and Sons: Chichester, 2002; pp.167-232. Draisma, S.G.A.; Ballesteros, E.; Rousseau, F.; Thibaut, T.J. DNA sequence data demonstrate the polyphyly of the genus Cystoseira and Sargassaceae genera (Phaeophyceae). J. Phycol. 2010, 46, 1329−1345. Dröge, D. Free radicals in the physiological control of cell function. Physiol. Rev. 2002, 82, 47–95. Duque, G.A.; Descoteaux, A. Macrophage cytokines: involvement in immunity and infectious diseases. Front. Immunol. 2014, 5, 491. Fadli, M.; Aracil, J.M.; Jeanty, G.; Banaigs, B.; Francisco, C. Novel meroterpenoids from Cystoseira mediterranea: use of the crown-gall bioassay as a primary screen for lipophilic antineoplastic agents. J. Nat. Prod. 1991, 54, 261−264. _____________________________________________________________________1. General Introduction " " 39 Fang, H.Y.; Chokkalingam, U.; Chiou, S.F.; Hwang, T.S.; Chen, S.L.; Wang, W.L.; Sheu, J.H. Bioactive chemical constituents from the brown alga Homoeostrichus formosana. Int. J. Mol. Sci. 2015, 16, 736–746. Farvin, K.H.S.; Jacobsen, C. Phenolic compounds and antioxidant activities of selected species of seaweeds from Danish coast. Food Chem. 2013, 138, 1670–1681. Faulkner, D.J. Marine Natural products: metabolites of marine algae and herbivorous marine molluscs. Nat. Prod. Rep. 1984, 1, 251–280. Federico, A.; Morgillo, F.; Tuccillo, C.; Ciardiello, F.; Loguercio, C. Chronic inflammation and oxidative stress in human carcinogenesis. Int. J. Cancer 2007, 121, 2381–2386. Fernando, I.P.S.; Nah, J.W.; Jeon, Y.J. Potential anti-inflammatory Natural products from marine algae. Environ. Toxicol. Pharmacol. 2016, 48, 22–30. Fisch, K.M.; Bohm, V.; Wright, A.D.; Konig, G.M. Antioxidative meroterpenoids from the brown alga Cystoseira crinita. J. Nat. Prod. 2003, 66, 968-975. Folmer, F.; Jaspars, M.; Dicato, M.; Diederich, M. Marine Natural products as targeted modulators of the transcription factor NF-κB. Biochem. Pharmacol. 2008, 75, 603–617. Foti, M.; Piattelli, M.; Amico, V.; Ruberto, G. Antioxidant activity of phenolic meroditerpenoids from marine algae. J. Photochem. Photobiol. B. 1994, 26, 159−164. Francisco, C.; Banaigs, B.; Teste, J.; Cave, A. Mediterraneols: a novel biologically active class of rearranged diterpenoid metabolites from Cystoseira mediterranea (Pheophyta). J. Org. Chem. 1986, 51, 1115−1120. Gonda, T.A.; Tu, S.; Wang, T.C. Chronic inflammation, the tumor microenvironment and carcinogenesis. Cell Cycle 2009, 8, 2005–2013. Gouveia, V.; Seca, A.M.L.; Barreto, M.C.; Pinto, D.C.G.A. Di- and sesquiterpenoids from Cystoseira genus: Structure, intra-molecular transformations and biological activity. Mini- Rev. Med. Chem. 2013a, 13, 1150−1159. Gouveia, V.L.M.; Seca, A.M.L.; Barreto, M.C.; Neto, A.I.; Kijjoa, A.; Silva, A.M.S. Cytotoxic meroterpenoids from the macroalga Cystoseira abies-marina. Phytochemistry Lett. 2013b, 6, 593–597. Graham, L.E.; Wilcox, L.W. Algae. Prentice Hall: Upper Saddle River, 2000; p. 640. Guiry, M.D. In Guiry, M.D. & Guiry G. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. http://www.algaebase.org; searched on 17 February 2019 Harvey, A.L.; Edrada-Eber, R.; Quinn, R.J. The re-emergence of Natural products for drug discovery in the genomics era. Nature Rev. Drug. Discov. 2015, 14, 111-129. 1. General Introduction_____________________________________________________________________________ " " 40 Heo, S.J.; Kim, K.N.; Yoon, W.J.; Oh, C.; Choi, Y.U.; Affan, A.; Lee, Y.J.; Lee, H.S.; Kang, D.H. Chromene induces apoptosis via caspase-3 activation in human leukemia HL- 60 cells. Food Chem. Toxicol. 2011, 49, 1998–2004 Huang, X.; Sun, Y.-L.; Salim, A.A.; Chen, Z.-S.; Capon, R.J. Parguerenes: Marine red alga bromoditerpenes as inhibitors of P-glycoprotein (ABCB1) in multidrug resistant human cancer cells. Biochem. Pharmacol. 2013, 85, 1257–1268. Hussain, E.; Wang, L.; Jiang, B.; Riaz, S.; Butt, G.Y.; Shi, D. A review of the components of brown seaweeds as potential candidates in cancer therapy. RSC Adv. 2016, 6, 12592–12610. Imbesi, S.; Musolino, C.; Allegra, A.; Saija, A.; Morabito, F.; Calapai, G.; Gangemi, S. Oxidative stress in oncohematologic diseases: an update. Expert Rev. Hematol. 2013, 6, 317–325. Jaswir, I.; Monsur, H.A. Anti-inflammatory compounds of macro algae origin: A review. J. Med. Plant Res. 2011, 5, 7146–7154. Ji, N.-Y.; Li, X.-M.; Xie, H.; Ding, J.; Li, K.; Ding, L.-P.; Wang, B.-G. Highly oxygenated triterpenoids from the marine red alga Laurencia mariannensis (Rhodomelaceae). Helv. Chim. Acta 2008, 91, 1940–1946. Jiang, R.W.; Lane, A.L.; Mylacraine, L.; Hardcastle, K.I.; Fairchild, C.R.; Aalbersberg, W.; Hay, M.E.; Kubanek, J. Structures and absolute configurations of sulfate-conjugated triterpenoids including an antifungal chemical defense of the green macroalga Tydemania expeditionis. J. Nat. Prod. 2008, 71, 1616–1619. Jiménez-Escrig, A.; Jiménez-Jiménez, I.; Pulido, R.; Saura-Calixto, F. Antioxidant activity of fresh and processed edible seaweeds. J. Sci. Food Agric. 2001, 81, 530–534. Jongaramruong, J.; Kongkam, N. Novel diterpenes with cytotoxic, anti-malarial and antituberculosis activities from a brown alga Dictyota sp. J Asian Nat. Prod. Res. 2007, 9, 743– 751. Jung, M.; Jang, K.H., Kim, B.; Lee, B.H, Choi, B.W.; Oh, K.B., Shin, J. Meroditerpenoids from the brown alga Sargassum siliquastrum. J. Nat. Prod. 2008, 71, 1714-1719. Kang, G.-J.; Han, S.-C.; Yoon, W.-J.; Koh, Y.-S.; Hyun, J.-W.; Kang, H.-K.; Cho, J.Y.; Yoo, E.-S. Sargaquinoic acid isolated from Sargassum siliquastrum inhibits lipopolysaccharideinduced nitric oxide production in macrophages via modulation of nuclear factor-κB and c- Jun N-terminal kinase pathways. Immunopharm. Immunotox. 2013, 35, 80-87. Khanna, R.D.; Karki, K.; Pande, D.; Negi, R.; Khanna, R.S. Inflammation, free radical damage, oxidative stress and cancer. Interdiscip. J. Microinflammation 2014, 1, 109. Kladi, M.; Vagias, C.; Papazafiri, P.; Furnari, G.; Serio, D.; Roussis, V. New sesquiterpenes from the red alga Laurencia microcladia. Tetrahedron 2007, 63, 7606–7611. _____________________________________________________________________1. General Introduction " " 41 Kumar, S.; Pandey, A.K. Free radicals: health implications and their mitigation by herbals. Br. J. Med. Med. Res. 2015, 7, 438–457. Kuzuyama, T. Mevalonate and non mevalonate pathways for the biosynthesis of isoprene units. Biosci. Biotechnol. Biochem. 2002, 66, 1619–1627. Lakshmi, P.T.; Vajravijayan, S.; Moumita, M.; Sakthivel, N.; Gunasekaran, K.; Krishna, R. A novel guaiane sesquiterpene derivative, guai-2-en-10α-ol, from Ulva fasciata Delile inhibits EGFR/PI3K/Akt signaling and induces cytotoxicity in triple-negative breast cancer cells. Mol. Cell Biochem. 2018, 438,123–139. Landskron, G.; De la Fuente, M.; Thuwajit, P.; Thuwajit, C.; Hermoso, M.A. Chronic inflammation and cytokines in the tumor microenvironment. J. Immunol. Res. 2014, 2014, 1–19. Lane, A.L.; Stout, E.P.; Hay, M.E.; Prusak, A.C.; Hardcastle, K.; Fairchild, C.R.; Franzblau, S.G.; Le Roch, K.; Prudhomme, J.; Aalbersberg, W.; Kubanek, J. Callophycoic Acids and Callophycols from the Fijian Red Alga Callophycus serratus. J. Org. Chem. 2007, 72, 7343–7351. Lane, A.L.; Stout, E.P.; Lin, A.S.; Prudhomme, J.; Le Roch, K.; Fairchild, C.R.; Franzblau, S.G.; Hay, M.E.; Aalbersberg, W.; Kubanek, J. Antimalarial bromophycolides J-Q from the Fijian red alga Callophycus serratus. J. Org. Chem. 2009, 74, 2736–2742. Leal, M.C.; Munro, M.H.G.; Blunt, J.W.; Puga, J.; Jesus, B.; Calado, R.; Rosa, R.; Madeira, C. Biogeography and biodiscovery hotspots of macroalgal marine Natural products. Nat. Prod. Rep. 2013, 30, 1380–1390. Lee, J.C.; Hou, M.F.; Huang, H.W.; Chang, F.R.; Yeh, C.C.; Tang, J.Y.; Chang, H.W. Marine algal Natural products with anti-oxidative, anti-inflammatory, and anti-cancer properties. Cancer Cell Int. 2013, 13, 1–7. Lee, J.I.; Park, B.J.; Kim, H.; Seo, Y. Isolation of Two New Meroterpenoids from Sargassum siliquastrum. Bull. Korean Chem. Soc. 2014, 35, 2867–2869. Lee, J.I.; Seo, Y. Chromanols from Sargassum siliquastrum and their antioxidant activity in HT1080 cells. Chem. Pharm. Bull. 2011, 59, 757–761. Lee, R.E. Basic characteristics of the algae. In Phycology. Fourth ed. Cambridge University Press: Cambridge, 2008; pp. 3–29. Lhullier, C.; Falkenberg, M.; Ioannou, E.; Quesada, A.; Papazafiri, P.; Horta, P.A.; Schenkel, E.P.; Vagias, C.; Roussis, V. Cytotoxic halogenated metabolites from the Brazilian red alga Laurencia catarinensis. J. Nat. Prod. 2010, 73, 27–32. Lin, A.S.; Stout, E.P.; Prudhomme, J.; Le Roch, K.; Fairchild, K.R.; Franzblau, S.G.; Aalbersberg, W.; Hay, M.E.; Kubanek, J. Bioactive bromophycolides R–U from the Fijian red alga Callophycus serratus. J. Nat. Prod. 2010, 73, 275–278. 1. General Introduction_____________________________________________________________________________ " " 42 López-Alarcón, C.; Denicola, A. Evaluating the antioxidant capacity of Natural products: A review on chemical and cellular-based assays. Anal. Chim. Acta 2013, 763, 1–10. Machado F.L.S.; Ventura T.L.B.; Gestinari, L.M.S.; Cassano, V.; Resende J.A.L.C.; Kaiser, C.R.; Lasunkaia, E.B.; Muzitano, M.F.; Soares A.R. Sesquiterpenes from the brazilian red alga Laurencia dendroidea. J. Agardh. Molecules 2014, 19, 3181–3192. Mahdi, F.; Falkenberg, M.; Ioannou, E.; Roussis, V.; Zhou, Y.D.; Nagle, D.G. Thyrsiferol inhibits mitochondrial respiration and HIF-1 activation. Phytochemistry Lett. 2011, 4, 75– 78. Mann, M.G.A.; Mkwananzi, H.B.; Antunes, E.M.; Whibley C.E.; Hendricks D-T.; Bolton, J.J.; Beukes, D.R. Halogenated monoterpene aldehydes from the South African marine alga Plocamium corallorhiza. J. Nat. Prod. 2007, 70, 596–599. Mesguiche, V.; Valls, R.; Piovetti, L.; Banaigs, B. Meroditerpenes from Cystoseira amentacea var. stricta collected off the Mediterranean coasts. Phytochemistry 1997, 45, 1489−1494. Multhoff, G.; Molls, M.; Radons, J. Chronic inflammation in cancer development. Front. Immunol. 2012, 2, 1–17. Murphy, C.; Hotchkiss, S.; Worthington, J.; McKeown, S.R. The potential of seaweed as source of drugs for use in cancer therapy. J. Appl. Phycol. 2014, 26, 2211–2264. Nahas, R.; Abatis, D.; Anagnostopoulou, M.A.; Kefalas, P.; Vagias, C.; Roussis, V. Radicalscavenging activity of Aegean Sea marine algae. Food Chem. 2007, 102, 577–581. Newman, D.J.; Cragg, G.M. Marine-sourced anti-cancer and cancer pain control agents in clinical and late preclinical development. Mar. Drugs 2014, 12, 255–278. Newman, D.J.; Cragg, G.M. Natural products as sources of new drugs from 1981 to 2014. J. Nat Prod. 2016, 79, 629-661. Nimse, S.B.; Palb, D. Free radicals, natural antioxidants, and their reaction mechanisms. RSC Adv. 2015, 5, 27986–28006. Norte, M., Sánchez, A., González, G. Claraenone, a new meroditerpene from brown alga. Tetrahedron Lett. 1993, 24, 3485–3486. Okin, D.; Medzhitov, R. Evolution of Inflammatory Diseases. Curr. Biol. 2012, 22, 733–740. Ovenden, S.P.; Nielson, J.L.; Liptrot, C.H.; Willis, R.H.; Wright, A.D.; Motti, C.A.; Tapiolas, D.M. Comosusols A-D and comosone A: cytotoxic compounds from the brown alga Sporochnus comosus. J. Nat. Prod. 2011, 74, 739–743. Park, H.Y.; Min, H.H.; Cheol, P.; Jin, C.Y., J.; Kim, G.Y., Choi, I.W.; Kim, N.D.; Nam, T.J.; Kown, T.K.; Choi, Y.H. Anti-inflammatory effects of fucoidan through inhibition of NF- _____________________________________________________________________1. General Introduction " " 43 κB, MAPK and Akt activation in lipopolysaccharide-induced BV2 microglia cells. Food Chem. Toxicol. 2011, 49, 1745–1752. Penicooke, N.; Walford, K.; Badal, S.; Delgoda, R.; Williams, L.A.; Joseph-Nathan, P.; Gordillo-Román, B.; Gallimore, W. Antiproliferative activity and absolute configuration of zonaquinone acetate from the Jamaican alga Stypopodium zonale. Phytochemistry 2013, 87, 96–101. Pereira, D.M.; Cheel J.; Areche, C.; San-Martin, A.; Rovirosa, J.; Silva, L.R.; Valentao, P.; Andrade, P.B. Anti-`proliferative activity of meroditerpenoids isolated from the brown alga Stypopodium flabelliforme against several cancer cell lines. Mar. Drugs 2011, 9, 852–862. Pereira, L.; A review of the nutrient composition of selected edible seaweeds. In Seaweed: ecology, nutritional composition and medicinal uses; Pomin, V.H., Ed.; Nova Sicence: New York, 2012; pp.15–49. Pizzino, G.; Irrera, N.; Cucinotta, M.; Pallio, G.; Mannino, F.; Arcoraci, V.; Squadrito, Altavilla, F.D.; Bitto, A. Oxidative Stress: Harms and benefits for human health. Oxid. Med. Cell Longev. 2017, 2017, 1–13. Reddy, P.; Urban, S. Meroditerpenoids from the southern Australian marine brown alga Sargassum fallax. Phytochemistry 2009, 70, 250–255. Reuter, S.; Gupta, S.C.; Chaturvedi, M.M.; Aggarwal, B.B. Oxidative stress, inflammation, and cancer: How are they linked? Free Radic. Biol. Med. 2010, 49, 1603–1616. Rodrigues, D.; Alves, C.; Horta, A.; Pinteus, S.; Silva, J.; Culioli, G.; Thomas, O.P.; R. Antitumor and antimicrobial ptential of bromoditerpenes isolated from the red alga, Sphaerococcus coronopifolius. Mar. Drugs 2015, 13, 713–726. Roohinejad, S.; Koubaa, M.; Barba, F.J.; Saljoughian, S.; Amid, M.; Greiner, R. Application of seaweeds to develop new food products with enhanced shelf-life, quality and healthrelated beneficial properties. Food Res. Int. 2017, 99, 1066–1083. Ruan, B.F.; Ge, W.W.; Lin, M.X.; Li, Q.S. A Review of the components of seaweeds as potential candidates in cancer therapy. Anticancer Agents Med. Chem. 2018, 18, 354–366. Ruiz-Torres, V.; Encinar, J.A.; Herranz-López, M.; Pérez-Sánchez, A.; Galiano, V.; Barrajón-Catalán, E.; Micol, V. An updated review on marine anticancer compounds: the use of virtual screening for the discovery of small-molecule cancer drugs. Molecules 2017, 22, article 1037. Sabry, O.M.; Goeger, D.E.; Valeriote, F.A.; Gerwick, W.H. Cytotoxic halogenated monoterpenes from Plocamium cartilagineum. Nat. Prod. Res. 2017, 31, 261–267 Salminen, A.; Lehtonen, M.; Suuronen, T.; Kaarniranta, K.; Huuskonen, J. Terpenoids: natural inhibitors of NF-kB signaling with anti-inflammatory and anticancer potential. Cell Mol. Life Sci. 2008, 65, 2979–2999. 1. General Introduction_____________________________________________________________________________ " " 44 Sansom, C.E.; Larsen, L.; Perry, N.B.; Berridge, M.V.; Chia, E.W.; Jacquie L. Harper, J.K.; Webb V.L. An Antiproliferative bis-prenylated quinone from the New Zealand brown alga Perithalia capillaris. J. Nat. Prod. 2007, 70, 2042–2044. Seca, A.M.L.; Pinto, D.C.G.A. Plant secondary metabolites as anticancer agents: successes in clinical trials and therapeutic application. Int. J. Mol. Sci. 2018, 19, 263; doi:10.3390/ijms19010263. Seo, Y.; Park, K.E.; Nam, T.J. Isolation of a new chromene from the brown alga Sargassum thunbergii. Bull. Korean Chem. Soc. 2007, 28, 1831–1833. Shannon, E.; Abu-Ghannam, N. Antibacterial derivatives of marine algae: An overview of pharmacological mechanisms and applications. Mar. Drugs 2016, 14, 1–23. Smyrniotopoulos, V.; Kiss, R.; Mathieu, V.; Vagias, C.; Roussis, V. Diterpenes with unprecedented skeletons from the red alga Sphaerococcus coronopifolius. Eur. J. Org. Chem. 2015, 13, 2848–2853. Smyrniotopoulos, V.; Vagias, C.; Bruyère, C.; Lamoral-Theys, D.; Kiss, R.; Roussis, V. Structure and in vitro antitumor activity evaluation of brominated diterpenes from the red alga Sphaerococcus coronopifolius. Bioorg. Med. Chem. 2010, 18, 1321–1330. Sonani, R.R.; Rastogi, R.P.; Madamwar, D. Natural antioxidants from algae. Algal Green Chem. 2017, 91–120; https://doi.org/10.1016/B978-0-444-63784-0.00005-9. Sun, J.; Shi, D.Y.; Li, S.; Wang, S.J.; Han, L.J.; Fan, X.; Yang, Y.C.; Shi, J.G. Chemical constituents of the red alga Laurencia tristicha. J. Asian Nat. Prod. Res. 2007, 9, 725–734. Tarhouni-Jabberi, S.; Zakraoui, O.; Ioannou, E.; Riahi-Chebbi, I.; Haoues, M.; Roussis, V.; Kharrat, R.; Essafi-Benkhadir, K. Mertensene, a Halogenated monoterpene, induces G2/M cell cycle arrest and caspase dependent apoptosis of human colon adenocarcinoma HT29 cell line through the modulation of ERK-1/-2, AKT and NF-κB signaling. Mar. Drugs 2017, 15, E221. Teasdale, M.E.; Shearer, T.L.; Engel, S.; Alexander, T.S.; Fairchild, C.R.; Prudhomme, J.; Torres, M.; Le Roch, K.; Aalbersberg, W.; Hay, M.E.; Kubanek, J. Bromophycoic acids: bioactive natural products from a Fijian red alga Callophycus sp. J. Org. Chem. 2012, 77, 8000–8006. Torre, L.A.; Siegel, R.L.; Ward, E.M.; Jemal, A. Global cancer incidence and mortality rates and trends—An Update. Cancer Epidemiol. Biomarkers Prev. 2015, 25, 16–27. Urones, J.G.; Araújo, M.E.M.; Brito Palma, F.M.S.; Basabe, P.; Marcos, I. S.; Moro, R. F.; Lithgow, A. M.; Pineda, J. Meroterpenes from Cystoseira usneoides II. Phytochemistry 1992a, 31, 2105−2109. Urones, J.G.; Basabe, P.; Marcos, I.S.; Pineda, J.; Lithgow, A.M.; Moro, R.F.; Brito Palma, F.M.S.; Araújo , M.E.M.; Graválos, M.D.G. Meroterpenes from Cystoseira usneoides. _____________________________________________________________________1. General Introduction " " 45 Phytochemistry 1992b, 31, 179−182. Valls, R.; Piovetti, L. The chemistry of the Cystoseiraceae (Fucales: Pheophyceae): Chemotaxonomic relationships. Biochem. Syst. Ecol. 1995, 23, 723−745. Van den Hoek, C.; Mann, D.G.; Jahns, H.M. Algae — An introduction to phycology. Cambridge University Press: Cambridge, 1995; p. 158. Velatooru, L.R.; Baggu, C.B.; Janapala, V.R. Spatane diterpinoid from the brown algae, Stoechospermum marginatum induces apoptosis via ROS induced mitochondrial mediated caspase dependent pathway in murine B16F10 melanoma cells. Mol. Carcinog. 2016, 55, 2222–2235. Vizetto-Duarte, C.; Custódio, L.; Acosta, G.; Lago, J.H.G.; Morais, T.R.; Bruno de Sousa, C.; Gangadhar, K.N.; Rodrigues, M.J.; Pereira, H.; Lima, R.T.; Vasconcelos, M.H.; Barreira, L.; Rauter, A.P.; Albericio, F.; Varela, J. Can macroalgae provide promising antitumoral compounds? A closer look at Cystoseira tamariscifolia as a source for antioxidant and anti-hepatocarcinoma compounds. Peer J. 2016, 4, 1704. Vogel, C.V.; Pietraszkiewicz, H.; Sabry, O.M.; Gerwick, W.H.; Valeriote, F.A. Enantioselective divergent syntheses of several polyhalogenated Plocamium monoterpenes and evaluation of their selectivity for solid tumors. Angew. Chem. Int. Ed. 2014, 53, 12205– 12209. Wells, M.L.; Potin, P.; Craigie, J.S.; Raven, J.A.; Merchant, S.S.; Helliwell, K.E.; Smith, A.G.; Camire, M.E.; Brawley, S.H. Algae as nutritional and functional food sources: revisiting our understanding. J. Appl. Phycol. 2017, 29, 949–982. Wijesinghe, W.A.J.P.; Kang, M.N.; Lee, W.W.; Lee, H.S.; Kamada, T.; Vairappan, C.S.; and Jeon, Y.J. 5β-Hydroxypalisadin B isolated from red alga Laurencia snackeyi attenuates inflammatory response in lipopolysaccharide-stimulated RAW 264.7 macrophages. Algae 2014a, 29, 333–341. Wijesinghe, W.A.; Kim, E.A.; Kang, M.C.; Lee, W.W.; Lee, H.S.; Vairappan, C.S.; Jeon, Y.J. Assessment of anti-inflammatory effect of 5β-hydroxypalisadin B isolated from red seaweed Laurencia snackeyi in zebrafish embryo in vivo model. Environ. Toxicol. Pharm. 2014b, 37, 110–117. Woolner, V.H.; Gordon, R.M.A.; Miller, J.H.; Lein, M.; Northcote, P.T.; Keyzers, R.A. Halogenated meroditerpenoids from a South Pacific collection of the red alga Callophycus serratus. J. Nat. Prod. 2018, 81, 2446–2454. Wu, J.Q.; Kosten, T.R.; Zhang, X.Y. Free radicals, antioxidant defense system, and schizophrenia. Prog. Neuropsychopharmacol. Biol. Psychiatry 2013, 46, 200–206. 1. General Introduction_____________________________________________________________________________ " " 46 Yang, E.-J.; Ham, Y.M.; Yang, K.-W.; Lee, N.H.; Hyun, C.-G. Sargachromenol from Sargassum micracanthum inhibits the lipopolysaccharide-induced production of inflammatory mediators in RAW 264.7 macrophages. Sci. World J. 2013, 712303. " Yoon, W.J.; Heo, S.J.; Han, S.C.; Lee, H.J.; Kang, G.J.; Kang, H.K.; Hyun, J.W.; Koh, Y.S.; Yoo, E.S. Anti-inflammatory effect of sargachromanol G isolated from Sargassum siliquastrum in RAW 264.7 Cells. Arch. Pharm. Res. 2012, 35, 1421–1430. Yu, X.Q.; He, W.F.; Liu, D.Q.; Feng, M.T.; Fang, Y.; Wang, B.; Feng, L.H.; Guo, Y.W.; Mao, S.C. A seco-laurane sesquiterpene and related laurane derivatives from the red alga Laurencia okamurai Yamada. Phytochemistry 2014, 103, 162–170. Zaleta-Pinet, D.A.; Holland, I.P.; Muñoz-Ochoa, M.; Murillo-Alvarez, J.I.; Sakoff, J.A.; van Altena, I.A.; McCluskey, A. Cytotoxic compounds from Laurencia pacifica. Org. Med. Chem. Lett. 2014, 4, 8. Zhao, M.; Cheng, S.; Yuan, W.; Dong, J.; Huang, K.; Sun, Z.; Yan, P. Further new xenicanes from a chinese collection of the brown alga Dictyota plectens. Chem. Pharm. Bull. 2015, 63, 1081–1086. 47 2. Objectives While acute inflammatory response has therapeutic benefits, chronic inflammation is associated with multitude of diseases. In this line, several epidemiological, pre-clinical, and clinical studies over the last years have established the close relationship between the process of chronic inflammation and cancer. The search for lead compounds from natural resources continues to be an important strategy for the discovery and development of new drugs. The marine environment is a rich source of functional compounds and promising bioactive molecules for a wide range of applications, including not only new therapeutics, but also cosmetics, and biotechnology. Among marine organisms, macroalgae have proven to be a valuable source of structurally diverse compounds exhibiting a variety of biological activities, including antioxidant, antitumor, and anti-inflammatory properties. Therefore, the search for novel therapeutic agents from macroalgae has aroused great interest among scientific groups and pharmaceutical industrialists. In this context, brown algae of the genus Cystoseira are characterized by containing a variety of natural products of the class of the meroterpenoids (AMTs) whose pharmacological potential has been scarcely explored. This thesis is aimed to study the antioxidant, anticancer, and anti-inflammatory activities of the AMTs produced by the brown alga Cystoseira usneoides, by using in vitro and in vivo assays of inflammation and cancer. This main objective comprises the following specific objectives: 1. To extract specimens of the brown alga C. usneoides collected in the Moroccan coasts of the Gibraltar Strait and to evaluate the antioxidant, anticancer, and antiinflammatory activities of the extract. 2. To isolate the natural products of the class of AMTs from the algal extract. 3. To evaluate the antioxidant properties of the isolated AMTs by using radical scavenging assays. 3. Bioactivity of the extract of C. usneoides_____________________________________________________ ! 54 in the literature (Skehan et al., 1990). Determination of TNF-α production THP-1 cells were plated at a density of 3 × 105 cells/mL in 24-well plates and incubated with PMA (0.2 µM) for 72 h in a humidified atmosphere of 5% CO2 at 37°C. The macrophages were then treated with 10 µg/mL of the algal extract for 24 h (the viability of cells was greater than 95% throughout the experiment), then stimulated with lipopolysaccharide (LPS, 1 µg/mL) and incubated for another 24 h. Dexamethasone was used as positive reference compound. The levels of TNF-α in the cultured medium (supernatant) were measured with enzyme-linked immunosorbent assay (ELISA) kits (Diaclone GEN-PROBE) according to the manufacturer’s protocols. The absorbance was determined at 450 nm using a microplate reader. To calculate the concentration of cytokines, a standard curve was constructed using serial dilutions of cytokine standards provided with the kit. 3.2.7. Statistical analysis The results were expressed as means ± standard deviations (S.D.). Differences between two groups were analyzed by the Student’s t-test. One-way analysis of variance (ANOVA) was used in multiple group comparisons. Difference with p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***) was considered statistically significant. 3.3. Results and Discussion 3.3.1. Antioxidant effects of the extract of C. usneoides The antioxidant capacity of the acetone/methanol extract of C. usneoides was determined by the ABTS+• radical cation decolorization assay. This is one of the most commonly employed methods for measuring antioxidant activity, which determines the radical scavenging activity of an extract, fraction or compound toward the radical cation ABTS+•. This method has been strongly recommended for analysing plant and algae extracts, because the maximum absorption of the radical cation at 734 nm prevents the color interference usually found when working with plant extracts (Awika et al., 2003). ______________________________________________________3. Bioactivity of the extract of C. usneoides ! 55 The extract of C. usneoides was assayed at concentrations between 3.125 and 200 µg/mL, causing decreases of the absorbance of the ABTS+• solution between 6.67 and 60.55%, which were lower than the decreases caused by the reference compound Trolox® (between 23 and 97.78% at concentrations between 3.125 and 25 µg/mL) (Figure 3.1). The reaction of the extract with the radical ABTS+• was dose-dependent, and the concentration that caused 50% decrease of the absorbance (IC50) was 94.05 ± 6.3 µg/mL. The Trolox standard was more active, showing IC50 = 7.36 ± 0.6 µg/mL. 0 20 40 60 80 100 120 3.125 6.25 12.5 25 50 100 200 % Inhibition of ABTS radical Concentration (µg/ml) Trolox Extract Figure 3.1. Effect of the extract of C. usneoides at different concentrations (3.125 – 200 µg/mL) on the ABTS+• radical compared to Trolox. The antioxidant activity measured for our acetone/methanol extract of C. usneoides is much higher than that reported in the same assay for the extracts of this species collected in the Portuguese coasts (Vizetto-Duarte et al., 2016) (Table 3.1). In that study, the hexane, diethyl ether, ethyl acetate, and methanol extracts obtained by sequential extraction of lyophilised samples of C. usneoides were tested in the ABTS assay, and only the hexane and the diethyl ether extracts displayed significant radical-scavenging activity. Moreover, the antioxidant activity of our extract of C. usneoides was also stronger than that reported for the extracts of C. humilis and C. tamariscifolia (Vizetto-Duarte et al- 2016) (Table 3.1). A number of recent reports, which are summarized in Table 3.2, have also described the antioxidant activities of extracts of C. usneoides and of other algae of the genus Cystoseira by using the DPPH assay (Tenorio-Rodriguez et al., 2017; Custodio et al., 2016; 3. Bioactivity of the extract of C. usneoides_____________________________________________________ ! 56 Çelenk et al., 2016; Vizzeto-Duarte et al., 2016; Güner et al., 2015; Mhadhebi et al., 2014; Airanthi et al., 2011; Zubia et al., 2009). The significant antioxidant activity detected in our study of the acetone/methanol extract of C. usneoides reinforces the potential of this marine genus of brown algae as a new and alternative source of antioxidant components, which could be suitable for exploitation in the food and cosmetic sectors. Table 3.1. Antioxidant activity of extracts of algae of the genus Cystoseira in the ABTS assay. Species (collection coast) Solvent of extraction Radical scavenging activit in the ABTS assay Reference C. usneoides (Morocco) acetone/methanol IC50 = 94.05 µg/mL Control:Trolox, IC50 = 7.36µg/mL Present study C. usneoides (Portugal)) hexane diethyl ether ethyl acetate methanol IC50 = 5.54 mg/mL IC50 = 600 µg/mL IC50 > 10 mg/mL IC50 > 10 mg/mL Control: BHT, IC50 = 110 µg/mL Vizetto-Duarte et al., 2016 C. humilis (Portugal) hexane diethyl ether ethyl acetate methanol IC50 >10 mg/mL IC50 = 8.85 mg/mL IC50 = 9.25 mg/mL IC50 > 10 mg/mL Control: BHT, IC50 = 110 µg/mL Vizetto-Duarte et al., 2016 C. tamariscifolia (Portugal) hexane diethyl ether ethyl acetate methanol IC50 = 520 µg/mL IC50 = 470 µg/mL IC50 = 250 µg/mL IC50 = 2.93 mg/mL Control: BHT, IC50 = 110 µg/mL Vizetto-Duarte et al., 2006 Table 3.2. Antioxidant activity of extracts of algae of the genus Cystoseira in the DPPH assay Species (collection coast) Solvent of extraction Radical scavenging activity in the DPPH assay Reference C. baccata (Portugal) methanol 21.6% inhibition at 1 mg/mL 90.4% inhibition at 10 mg/mL Custodio et al., 2016 C. barbata (Turkey) methanol 58.22% inhibition at 1 mg/mL Çelenk et al., 2016 C. compressa (Tunisia) water IC50 = 12 µg/mL Control: Trolox, IC50 = 90 µg/mL Mhadhebi et al., 2014 C. compressa (Turkey) methanol chloroform hexane IC50 = 15.94 mg/mL IC50 = 7.46 mg/mL IC50 = 5.0 mg/mL Güner et al., 2015 C. compressa (Turkey) methanol 65% inhibition at 1 mg/mL Çelenk et al., 2016 C. crinita (Tunisia) water IC50 = 20 µg/mL Control: Trolox, IC50 = 90 µg/mL Mhadhebi et al., 2014 ______________________________________________________3. Bioactivity of the extract of C. usneoides ! 57 C. crinita (Turkey) methanol 79.3% inhibition at 1 mg/mL Çelenk et al., 2016 C. hakodatensis (Japan) methanol 65.32 µg αtocopherol equivalent per mg extract Airanthi et al., 2011 C. humilis (Portugal) methanol 8.8 % inhibition at 1 mg/mL 41.1 % inhibition at 10 mg/mL Custodio et al., 2016 C. humilis (Portugal) hexane diethyl ether ethyl acetate metanol IC50 >10 mg/mL IC50 = 8.28 mg/mL IC50 = 5.04 mg/mL IC50 > 10 mg/mL Control: BHT, IC50 = 70 µg/mL Vizzeto-Duarte et al., 2016 C. nodicaulis (Portugal) methanol 39.6% inhibition at 1 mg/mL 95.1 % inhibition at 10 mg/mL Custodio et al., 2016 C. osmundacea (Baja California, Mexico) ethanol IC50 = 69 µg/mL Tenorio-Rodriguez et al., 2017 C. sedoides (Tunisia) chloroform ethyl acetate IC50 = 120 µg/mL IC50 = 121 µg/mL Control: Trolox, IC50 = 90 µg/mL Mhadhebi et al., 2011 C. sedoides (Tunisia) water IC50 = 75 mg/mL Control: Trolox, IC50 = 90 µg/mL Mhadhebi et al., 2014 C. tamariscifolia (Brittany) dichloromethane/ methanol EC50 = 490 µg/mL Control compounds: BHA, EC50 = 40 µg/mL BHT, EC50 = 60 µg/mL Ascorbic acid, EC50 = 60 µg/mL α-tocopherol, EC50 = 140 µg/mL Zubia et al., 2009 C. tamariscifolia (Portugal) hexane diethyl ether ethyl acetate metanol IC50 = 630 µg/mL IC50 = 300 µg/mL IC50 = 170 µg/mL IC50 = 1.08 mg/mL Control: BHT, IC50 = 70 µg/mL Vizetto-Duarte et al., 2016 C. tamariscifolia (Portugal) methanol 92% inhibition at 1 mg/mL Custodio et al., 2016 C. usneoides (Portugal) hexane diethyl ether ethyl acetate metanol IC50 = 4.37 mg/mL IC50 = 650 µg/mL IC50 = 7.73 mg/mL IC50 = 7.16 mg/mL Control:BHT, IC50 = 70 µg/mL Vizetto-Duarte et al., 2006 C. usneoides (Portugal) methanol 37.1 % inhibition at 1 mg/mL Custodio et al., 2016 3.3.2. Anticancer effects of the extract of C. usneoides Effect of the extract of C. usneoides on the viability of HT-29 cells The effect of the extract of C. usneoides (0, 6.25, 12.5, 25, 50 and 100 µg/mL) on the viability of HT-29 cells was determined by the SRB assay after 48 and 72 h of incubation (Figure 3.2). As shown in Figures 3.2a and 3.2b, the extract of C. usneoides 3. Bioactivity of the extract of C. usneoides_____________________________________________________ ! 58 decreased cell viability in a dose- and time-dependent manner and the IC50 values were 33.05 µg/mL and 25.28 µg/mL, after 48 and 72 h of incubation, respectively. 0 20 40 60 80 100 120 0 6 13 25 50 100 % Cell Viability Concentration (µg/mL) (a) 0 20 40 60 80 100 120 0 6 13 25 50 100 % Cell Viability Concentration (µg/mL) (b) Figure 3.2. Effect of the acetone/methanol extract of C. usneoides on HT-29 cells proliferation. Cells were treated with different concentrations of extract (6.25, 12.5, 25, 50, and 100 µg/mL) for 48 h (a) and 72 h (b). Cell viability was determined by SRB assay (n = 6). Induction of apoptosis in HT29 cells by treatment with the extract of C. usneoides In an attempt to explore the effects of the extract of C. usneoides on apoptosis, the HT-29 cells were treated with the extract and then analyzed by flow cytometry, using Annexin V-FITC as a marker of phosphatidylserine exposure and propidium iodide as a marker of dead cells. Figure 3.3 shows the results of the Annexin V-FITC binding and propidium iodide staining on HT-29 cells, after exposure for 24 h either to the extract (two different doses) or to curcumin as positive control. The treatment with the extract significantly increased the proportion of apoptotic cells. Thus, in the vehicle treated samples, 0.35 ± 0.01% of cells stained positive for Annexin V-FITC, while the treatment with the extract of C. usneoides at 30 µg/mL caused an increase up to 12.52 ± 3.12% of cells in late stage apoptosis and 5.32± 0.21% in early apoptosis (p<0.05). When HT-29 cells were treated with 60 µg/mL of extract for 24 h, the percentage of cells in late apoptosis was 17.71 ± 0.01%, although no significant difference with early stage apoptosis was observed. These results demonstrate the ability of the extract of C. usneoides to induce apoptosis, mainly late stage apoptosis in HT-29 cells. ______________________________________________________3. Bioactivity of the extract of C. usneoides ! 59 ! Curcumin (75 µg/mL) ! Negative control ! Extract (30 µg/mL) ! Extract (60 µg/mL) Figure 3.3. Effects of C. usneoides on the apoptosis of HT-29 cells. Cells were treated without and with different concentrations of C. usneoides extract (30 µg/mL and 60 µg/mL, for 24 h), then stained with annexin V-FITC, and evaluated by flow cytometry. Curcumin was used as positive reference compound at 75 µM. (E1: Necrotic, E2: Late apoptotic, E3: Live and E4: Early apoptotic). Effect of the extract of C. usneoides on cell cycle progression in HT-29 cells To investigate the possible mechanism(s) involved in the cytotoxic effect caused by the extract of C. usneoides on colon cancer cells HT-29, we examined whether such reduction was associated with cytostatic effects due to changes in cell cycle progression. Figure 3.4 shows the cell cycle distribution of HT-29 cells incubated for 24 h in the absence or in the presence of different concentrations of extract. In the absence of the extract, most HT-29 cells (about 70%) were in G0/G1 phase. However, after 24 h incubation with the algal extract (60 µg/mL) there was an increase in the percentage of cells in the G2/M phase, while the percentage of cells in the other phases decreased. These data markedly suggest that the extract of C. usneoides interferes with cell cycle progression. The present study has demonstrated for the first time that the extract of the alga C. usneoides inhibits the proliferation of colon cancer cells by arresting cell cycle progression at the G2/M phase and inducing apoptosis. As shown in Figure 3.2, the extract of C. 3. Bioactivity of the extract of C. usneoides_____________________________________________________ ! 60 usneoides inhibited cell growth in a dose- and time-dependent manner and this inhibition seems to be associated with apoptosis (Fig. 3.3) and accumulation of cells at the G2/M region (Figure 3.4). ! ! ! Extract (30 μg/mL) G2/M = 21.01% S = 5.57% G1 = 72.27% Apop = 0.86% ! G2/M = 19.25% S = 9.84% G1 = 70.32% Apop = 0.83% Negative Control Colchicine (0.2 µg/mL ) G2/M = 86.12%** S = 3.14% G1 = 5.86% Apop = 0.33% Extract (30 µg/mL ) Extract (60 µg/mL ) G2/M = 21.01% S = 5.57% G1 = 72.27% Apop = 0.86% G2/M = 50.87%* S = 13.87% G1 = 43.13% Apop = 0.86% Figure 3.4. Effects of the extract of C. usneoides on HT-29 cell cycle distribution. Cells were incubated with or without extract for the indicated concentrations (30 and 60 µg/mL) for 24 h. Then, the cells were fixed and stained with propidium iodide to analyze DNA content by FAC Scan flow cytometer. Colchicine was used as positive reference compound at 0.2 µg/mL; (*p < 0.05, **p < 0.01, ***p < 0.001). Previous reports have described the cytotoxicity of the hexane and the diethyl ether extracts of C. usneoides towards HepG2 cells (IC50 = 31.4 µg/mL and 52.0 µg/mL, respectively) (Vizetto-Duarte et al., 2016). In the present study, we have shown that the acetone/methanol extract of C. usneoides is cytotoxic for HT-29 cells with an IC50 of 25.28 µg/mL against HT-29 cells. This activity is comparable to that reported for aqueous extracts of C. crinita, C. sedoides, and C. compressa against the colon cancer cells HCT-15 (IC50 = 26.4, 10.5, 20.3 µg/mL, respectively) (Mhadhebi et al., 2014) and much better than those described for the hexane fraction of C. myrica against the colon cancer cells HT-29 and Caco-2 (IC50= 328.7 µg/mL and 254.92 µg/mL, respectively) (Khanavi et al., 2010) and for ______________________________________________________3. Bioactivity of the extract of C. usneoides ! 61 the chloroform and the ethyl acetate fractions of C. sedoides against HCT-15 (IC50 = 250.6 and 255.3 µg/mL, respectively) (Mhadhebi et al., 2011). Several recent researches have dealt with the anticancer properties of the extracts of other brown algae of the genus Cystoseira, showing that these extracts cause cell growth inhibition in various cancer cell lines. Thus, the dichloromethane/methanol extract of C. tamariscifolia from the Brittany coasts, at 100 µg/mL was effective inhibiting by 60-80% the viability of the tumor cell lines Daudi (lymphoma), Jurkat (leukemia), and K562 (leukemia) (Zubia et al., 2009). Another in vitro study showed that the hexane fraction obtained from the methanol extract of C. myrica was cytotoxic for the human breast carcinoma cells T47D, T47D-T.R and MBA-MD468 with IC50= 99.9, 143.15 and 56.50 µg/mL, respectively (Khanavi et al., 2010). Moreover, this fraction was described to induce apoptosis in T47D cells with a proportion of apoptotic cells of 2.71%. Similarly, the methanol extract of C. mediterranea was cytotoxic toward the breast cancer cells MCF-7, causing at 100 µg/mL 55% of cell growth inhibition (Taskin et al., 2010). The aqueous extracts and organic fractions derived from the methanol extracts of C. sedoides, C. compressa, and C. crinita have also shown antiproliferative properties against the cancer cell lines MCF-7 (IC50 from 17.9 to 130.0 µg/mL) and A-549 (IC50 from 33.0 to 110.0 µg/mL) (Mhadhebi et al., 2011, 2012, and 2014) while the hexane extract of C. tamariscifolia has shown cytotoxic effects on HeLa, SH-SY5Y, AGS, HCT-15 and HepG2 cells, being specially potent on these latter cells (IC50 = 2.31 µg/mL) through apoptosis induction (Vizetto-Duarte et al. 2016). A meroditerpene, demethoxy cystoketal chromane, was identified in the same study as the active compound in the extract of C. tamariscifolia. It is worth noting that along the studies on the antitumor potential of algae of the genus Cystoseira, a series of metabolites of the meroditerpene class have been isolated and identified as cytotoxic compounds in C. crinita (Fisch et al., 2003), C. myrica (Ayyad et al., 2003) and C. usneoides (Urones et al., 1992). These data suggest that the meroterpenes could also be responsible for the anticancer activity observed for the extract of C. usneoides tested in our study. 3. Bioactivity of the extract of C. usneoides_____________________________________________________ ! 62 3.3.3. Anti-inflammatory activity of the extract of C. usneoides The anti-inflammatory properties of the extract of C. usneoides was also investigated, in particular its activity as inhibitor of TNF-α. This is a potent proinflammatory cytokine mainly produced by monocytes and macrophages in immunologic and inflammatory responses (Iqbal et al., 2013). Assays were performed on the human THP-1 macrophages using lipopolysaccharide (LPS) to stimulate the TNF-α production. In order to rule out cytotoxic effects, the extract of C. usneoides was tested at a maximal concentration of 10 µg/mL, which did not affect THP-1 cell viability. To test the effects of the extract on TNF-α production, THP-1 macrophages were pretreated with the extract, then stimulated with LPS and finally analyzed to quantify TNF-α. As shown in Figure 3.5, the TNF-α production in THP-1 cells was increased about 4-fold, up to 282.05 ng/mL after LPS-stimulation. The pretreatment of cells with the extract of C. usneoides at 10 µg/mL significantly inhibited the production of TNF-α by 43% upon comparison with LPS- stimulated THP-1 control cells (Figure 3.5). 0 50 100 150 200 250 300 Control LPS Dexamethasone (1 µM) Extract (10 µg/mL ) TNF-α production (ng/mL ) ++" ***" **" Figure 3.5. Effect of C. usneoides extract on TNF-α production in THP-1 macrophages stimulated with LPS. The cells were treated at concentration of 10 µg/mL for 24 h (the viability of cells was greater than 95% throughout the experiment), and then stimulated with LPS (1 µg/mL) for another 24 h. TNF-α concentration in the supernatants was measured using the ELISA assay. Dexamethasone was used as positive reference compound at 1 µM. Data were analyzed by one-way ANOVA followed by Tukey test for comparison between groups, (++) p < 0.01 vs. control; **p < 0.01, and ***p < 0.001 vs. LPS.! Previous accounts on the anti-inflammatory properties of extracts of algae of the genus Cystoseira have focused only on the carrageenan-induced rat paw edema model. In ______________________________________________________3. Bioactivity of the extract of C. usneoides ! 63 particular, the chloroform and the ethyl acetate fractions of C. sedoides or C. compressa produced, at doses of 25 and 50 mg/kg i.p., marked inhibitions of edema (63.7-78.3% after 3h) when compared with the standard acetylsalicylic–lysine (ASL) (61.3-66.1% inhibition at 300 mg/kg i.p.) (Mhadhebi et al., 2011; Mhadhebi et al., 2012). A third report described the in vivo anti-inflammatory activity of the aqueous extracts of C. crinita, C. sedoides, and C. compressa, which at doses of 25 and 50 mg/kg i.p., exhibited significant antiinflammatory activity in a dose dependent manner (70.9-82.1% inhibition of edema after 3h) upon comparison with the reference drugs ASL (61.2% at 300 mg/Kg) and dexamethasone (74.3% at 1 mg/kg) (Mhadhebi et al., 2014). Therefore, the present study is the first description of the activity of an extract of algae of the genus Cystoseira as inhibitor of the production of proinflammatory cytokines, and the results suggest that this class of marine algae is worth of exploring in the antiinflammatory area. 3.4. Conclusion Marine algae have long been identified as valuable sources of metabolites with potent functional bioactivities. In this study, we have demonstrated that the extract of C. usneoides has a prominent radical-scavenging activity and an anti-proliferative effect on colon cancer cells by reducing cell viability through the apoptotic and cell cycle arrest pathways. Moreover, we have demonstrated for the first time that C. usneoides extract exhibits a significant activity as inhibitor of the production of the pro-inflammatory cytokine TNF-α in LPS-stimulated THP-1 human macrophages. The antioxidant activity together with the anticancer properties and the strong anti-inflammatory effects shown by the extract of C. usneoides make this alga an interesting candidate for further investigations. The prevalence of these properties in a single alga could be beneficial in terms of preventive and therapeutic purposes. 3.5. References Agregán, R.; Munekata, P.E.S.; Franco, D.; Carballo, J.; Barba, F.J.; Lorenzo, J.M. Antioxidant potential of extracts obtained from macro- (Ascophyllum nodosum, Fucus 4. Chemical study of C. usneoides_____________________________________________________________" " 70 samples were shade dried, cut into small pieces and powdered in a mixer grinder (IKA R A11 basic, Sigma-Aldrich). The obtained powder was cold-preserved (−12 °C). 4.2.3. Extraction and isolation of natural products The powder previously prepared of C. usneoides (150 g) was extracted with acetone/methanol (MeOH) (1:1, v/v, 400 mL) at room tª. The solution was filtered through paper and the residual solid material extracted five more times using the same procedure. The solutions were combined and the solvent evaporated at reduced pressure to yield a dark green oily extract (4.63 g). The compounds were isolated from the extract by using column chromatography (CC) performed on Merck silica gel 60 (70−230 mesh) and HPLC separations that were performed on a LaChrom-Hitachi apparatus equipped with LiChrospher Si-60 (Merck, 250 × 10 mm, 10 µm) and Luna Si (2) (Phenomenex, 250 × 4.6 mm, 5 µm) columns, using an RI-71 differential refractometer or L-7400 UV detector (Merck) working at 254 nm. First the extract was subjected to CC eluting with n-hexane/diethyl ether (Et2O) mixtures of increasing polarity (from 90:10 to 30:70, v/v), then Et2O, CHCl3/MeOH mixtures (95:5 and 80:20, v/v), and finally MeOH. The fractions eluted with n-hexane/Et2O (30:70, v/v) and Et2O were chromatographed over a silica gel column using n-hexane and n-hexane/ethyl acetate (EtOAc) mixtures (80:20 to 30:70, v/v) as eluents. Repeated HPLC separations of selected fractions using n-hexane/EtOAc (60:40, v/v) and n-hexane/isopropanol (90:10, v/v) yielded the compounds 4 (29.6 mg, 2.0 × 10−2 % dry wt), 3 (73.2 mg, 4.9 × 10−2 % dry wt), 11 (5.3 mg, 3.5 × 10−3 dry wt), 5 (86.6 mg, 5.8 × 10−2 % dry wt), and 6 (85.1 mg, 5.7 × 10−2 % dry wt). The fraction eluted with CHCl3/MeOH (95:5) in the first column chromatography of the extract was further separated over a silica gel column using nhexane/EtOAc mixtures (from 50:50 to 15:85, v/v) as eluents. Repeated separations of selected fractions by normal-phase HPLC using n-hexane/EtOAc (50:50, v/v) and nhexane/isopropanol (90:10, v/v) as eluents afforded the compounds 1 (86.9 mg, 5.8 × 10−2 % dry wt), 12 (3.5 mg, 2.3 × 10−3 % dry wt), 2 (88.7 mg, 5.9 × 10−2 % dry wt), 9 (2.9 mg, 1.9 × 10−3 dry wt), 7 (29.5 mg, 2.0 × 10−2 % dry wt), 8 (16.7 mg, 1.1 × 10−2 % dry wt), 10 (4.9 mg, 3.3 × 10−3 % dry wt), and additional amounts of 6 (46.1 mg, total yield 8.7 × 10−2 % dry wt). _____________________________________________________________4. Chemical study of C. usneoides " 71 4.2.4. NMR and HRMS analysis 1H and 13C NMR spectra were recorded on a Varian INOVA 600 or on an Agilent 500 using CD3OD or CDCl3 as solvent. Chemical shifts were referenced using the corresponding solvent signals [δH 3.30 and δC 49.0 for CD3OD, δH 7.26 and δC 77.0 for CDCl3]. COSY, HSQC, HMBC, and NOESY experiments were performed using standard Varian pulse sequences. High-resolution mass spectra (HRMS) were obtained on a Waters SYNAPT G2 spectrometer. 4.2.5. ABTS radical scavenging activity Antioxidant activity was determined by the ABTS (2,2′-azinobis(3- ethylbenzothiazoline-6-sulfonic acid)) free radical decolorization assay developed by Re et al., 1999 with slight modifications. ABTS was dissolved in H2O to a 2.9 mM concentration. The ABTS•+ radical cation was produced by reacting the ABTS stock solution with 0.98 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12− 18 h before use. For the study of the algal compounds the ABTS•+ solution was diluted with EtOH to an absorbance of 0.7 ± 0.02 at 734 nm. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as standard. After addition of 100 µL of ABTS•+ reagent to 90 µL of EtOH and 10 µL of the tested compound or Trolox standard (final concentration from 3.125 to 200 µM), the absorbance at 734 nm was taken 1 min after mixing and up to 6 min using a microtiter plate reader. Appropriate solvent blanks (controls) were run in each assay. All the determinations were carried out in triplicate. The percentage of inhibition of the absorbance was calculated by the following equation: % Inhibition = [(A0 − A1)/A0] × 100, where A0 expresses the absorbance of the control and A1 the absorbance of the tested compound. 4.2.6. Statistical analysis The results were expressed as means ± standard deviations (S.D.). Differences between two groups were analyzed by the Student’s t-test. One-way analysis of variance (ANOVA) was used in multiple group comparisons. Difference with p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***) was considered statistically significant. 4. Chemical study of C. usneoides_____________________________________________________________" " 72 4.3. Results and Discussion 4.3.1. Isolation and identification of the natural products of C. usneoides The separation of the acetone/methanol extract of C. usneoides by column chromatography (CC) over silica gel followed by repeated purification of selected fractions by CC and HPLC led to the isolation of twelve compounds. The analysis of each compound by spectroscopic techniques indicated that all the isolated metabolites were meroterpenoids and allowed to determining the chemical structures 1-12 shown in Figure 4.2. (De los Reyes et al., 2013). Figure 4.2. Chemical structures of the meroterpenoids 1-12 isolated from C. usneoides collected in the Moroccan coasts of the Gibraltar Strait. _____________________________________________________________4. Chemical study of C. usneoides " 73 From a structural point of view, all the isolated meroterpenoids consisted of a toluhydroquinone ring that possess O-methylated the phenolic group at C-1′ and bears at C- 2′ a terpenoid moiety. Two main groups of compounds were obtained: compounds 1, 2, 5, 6, 7, 8, 11, and 12 which possessed a regular diterpenoid moiety (twenty carbon atoms) derived from four isoprenoid C5 units linked head to tail, and compounds 3, 4, 9, and 10, which contained a truncated isoprenoid chain of fourteen carbon atoms, where only two isoprenoid C5 unis can be recognized. This C14 chain likely arises from a tetraprenyltoluquinol precursor by oxidative cleavage of a carbon-carbon double bond at C- 11,C-12 of the diterpenoid chain. Whithin each group of meroterpenoids, the structural differences among the compounds were found in the terpenoid residue. Thus, compounds 1, 5, and 7 possess a diterpenoid chain, which differ by the location of double bonds and/or the presence hydroxy groups in the chain, and compound 11 is characterized by containing an unusual tetrahydrofuran-3-one ring. Compounds 2, 6, 8, and 12 differ from 1, 5, 7, and 11, respectively, by the configuration of the double bond at C-6,C-7. Within the meroterpenoids with the C14 chain, compounds 4 and 9 differ by the presence of a hydroxy group and the location of a double bon in the terpenoid chain and compounds 3 and 10 are the 6E isomers of 4 and 9, respectively. A survey of literature revealed that the isolated compound 1 was usneoidone Z, a meroditerpene previously reported from C. usneoides collected in the Portuguese coasts (Urones et al., 1992a),1 while compound 2 was identified as 11-hydroxy-1′-O- methylamentadione, a meroterpene that has been previously cited as a metabolite of C. usneoides (Valls and Piovetti 1995). Compounds 3 and 4 were identified as cystomexicones B and A, respectively, previously described from C. abies-marina (Fernández et al., 1998). Compounds 5 and 6 were identified as 6-cis-amentadione-1′-methyl ether and amentadione-1′-methyl ether, respectively, two meroditerpenoids that had been previously """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 1"The structure assigned to usneoidone Z by Urones et al. (1992a) was not consistent with the spectroscopic data and was revised to 1 in De los Reyes et al., 2013." 4. Chemical study of C. usneoides_____________________________________________________________" " 74 reported from C. tamariscifolia (Amico et al. 1989). Compounds 7-12 resulted to be new meroterpenoids and were given the names cystodiones A-F. The structures of these new compounds were determined by spectroscopic means, mainly NMR and MS, in particular through the analysis of the 1H and 13C NMR spectra and the correlations observed in the two-dimensional NMR spectra. The NMR data of compounds 7-12 are listed in Tables 4.1 and 4.2. The molecular formulae obtained for each new compound by HRMS are shown in Table 4.3. Table 4.1. 13C NMR data of cystodiones A-F (7-12) in CD3OD. a At 150 MHz; b At 125 MHz; cValues may be interchanged Position 7a 8a 9a 10a 11a 12b δC, type δC, type δC, type δC, type δC, type δC, type 1 123.3, CH 123.3, CH 123.3, CH 123.3, CH 29.4, CH2 29.4, CH2 2 137.7, CH 137.8, CH 137.7, CH 137.7, CH 129.5, CH 129.5, CH 3 73.5, C 73.5, C 73.5, C 73.5, C 131.4, C 131.5, C 4 56.2, CH2 56.4, CH2 56.2, CH2 56.4, CH2 56.2, CH2 56.2, CH2 5 201.7, C 202.1, C 201.7, C 202.2 C 201.4, C 202.0, C 6 126.4, CH 125.9, CH 126.8, CH 126.1, CH 124.4, CH 123.7, CH 7 161.5, C 160.6, C 160.9, C 160.2, C 161.5, C 160.6, C 8 34.6, CH2 42.1, CH2 33.8, CH2 41.2, CH2 34.6, CH2 42.0, CH2 9 26.8, CH2 26.2, CH2 23.1, CH2 22.5, CH2 23.0, CH2 22.2, CH2 10 34.2, CH2 33.7, CH2 43.7, CH2 43.2, CH2 36.2, CH2 35.8, CH2 11 44.9, CH 45.0, CH 211.8, C 211.3, C 84.5, C 84.4, C 12 206.9, C 206.7, C 29.7, CH3 29.8, CH3 219.5, C 219.5, C 13 125.6, CH 125.6, CH 25.4, CH3 19.5, CH3 37.7, CH2 37.7, CH2 14 155.2, CH 155.2, CH 28.7, CH3 28.7, CH3 81.1, CH 81.1, CH 15 71.3, C 71.3, C 71.7, C 71.7, C 16 29.3, CH3 29.3, CH3 25.8c, CH3 25.7c, CH3 17 29.3, CH3 29.3, CH3 25.6c, CH3 25.6c, CH3 18 17.0, CH3 17.1, CH3 22.3, CH3 22.4, CH3 19 25.6, CH3 19.6, CH3 25.5, CH3 19.3, CH3 20 28.6, CH3 28.6, CH3 16.6, CH3 16.6, CH3 1’ 150.7, C 150.7, C 150.7, C 150.7, C 150.8, C 150.7, C 2’ 131.9, C 131.9, C 131.9, C 131.9, C 135.6, C 135.6, C 3’ 111.0, CH 111.0, CH 111.0, CH 111.0, CH 115.0, CH 114.9, CH 4’ 154.4, C 154.4, C 154.3, C 154.3, C 154.3, C 154.3, C 5’ 118.0, CH 118.0, CH 118.0, CH 118.0, CH 116.3, CH 116.3, CH 6’ 133.1, C 133.1, C 133.1, C 133.1, C 132.7, C 132.7, C 6’-CH3 16.2, CH3 16.2, CH3 16.1, CH3 16.1, CH3 16.4, CH3 16.4, CH3 -OCH3 61.4, CH3 61.4, CH3 61.4, CH3 61.4, CH3 60.9, CH3 61.0, CH3 _____________________________________________________________4. Chemical study of C. usneoides " 75 Table 4.2. 1H NMR data of cystodiones A-F (7-12) in CD3OD. a At 600 MHz; b At 500 MHz Position 7a 8a 9a 10a 11a 12b δH, m (J in Hz) δH, m (J in Hz) δH, m (J in Hz) δH, m (J in Hz) δH, m (J in Hz) δH, m (J in Hz) 1 6.76, d (16.1) 6.76, d (16.1) 6.76, d (16.1) 6.77, d (16.2) 3.33, d (7.4) 3.33, d (7.0) 2 6.29, d (16.1) 6.30, d (16.1) 6.29, d (16.1) 6.30, d (16.2) 5.42, br t (7.4) 5.44, br t (7.3) 3 4 2.79, d (14.2) 2.70, d (14.2) 2.80, d (13.9) 2.72, d (13.9) 2.80, d (14.4) 2.71, d (14.4) 2.81, d (14.2) 2.72, d (14.2) 3.09, s 3.11, s 5 6 6.22, br s 6.20, br s 6.25, br s 6.21, br s 6.21, br s 6.21, br s 7 8 2.55, m 2.51, m 2.11, t (7.5) 2.51, m 2.10, m 2.60, ddd (11.7, 9.1, 5.7) 2.54, m 2.15, t (6.6) 9 1.37, m 1.38, m 1.67, m 1.68, m 1.56, m 1.48, m 1.59, m 1.49, m 10 1.65, m 1.33, m 1.61, m 1.30, m 2.44, t (7.2) 2.43, t (7.2) 1.60, m 1.46, m 1.55, m 1.40, m 11 2.82, m 2.81, m 12 2.07, s 2.08, s 13 6.33, d (15.8) 6.34, d (15.9) 1.87, d (1.2) 206, d (1.2) 2.54, dd (18.2, 9.4) 2.43, dd (18.2, 6.6) 2.54, dd (18.3, 9.5) 2.41, dd (18.3, 6.6) 14 6.93, d (15.8) 6.93, dd (15.9, 1.2) 1.40, s 1.41, s 4.03, dd (9.4, 6.6) 4.00, dd (9.5, 6.2) 15 16 1.31, s 1.32, s 1.12, s 1.12, s 17 1.31, s 1.32, s 1.24, s 1.24, s 18 1.02, d (6.8) 1.04, d (7.0) 1.18, s 1.18, s 19 1.85, d (1.0) 2.05, d (1.1) 1.87, d (1.1) 2.08, d (1.5) 20 1.40, s 1.40, s 1.70, br s 1.71, br s 1’ 2’ 3’ 6.69, d (2.9) 6.70, d (3.1) 6.69, d (2.9) 6.70, (2.9) 6.43, d (2.9) 6.43, d (2.6) 4’ 5’ 6.50, d (2.9) 6.51, d (3.1) 6.50, d (2.9) 6.50, d (2.9) 6.44, d (2.9) 6.44, d (2.6) 6’ 6’-CH3 2.18, s 2.18, s 2.18, s 2.18, s 2.19, s 2.19, s -OCH3 3.60, s 3.60, s 3.60, s 3.60, s 3.64, s 3.64, s 4. Chemical study of C. usneoides_____________________________________________________________" " 76 Table 4.3. HRMS data of cystodiones A-F (7-12) Compound Molecular formula Observed ion,a m/z Calculated 7 C28H40O6 [M ! H], 471.2758 For C28H39O6, 471.2747 8 C28H40O6 [M ! H], 471.2751 For C28H39O6, 471.2747 9 C22H30O5 [M + Na], 397.1981 For C22H30O5Na, 397.1991 10 C22H30O6 [M + Na], 397.1983 For C22H30O5Na, 397.1991 11 C28H40O6 [M ! H], 471.2751 For C28H39O6, 471.2747 12 C28H40O6 [M + H], 473.2892 For C28H41O6, 473.2903 a Ionization technique: ESI(-) for 7, 8,and 11; ESI(+) for 9 and 10; APCI for 12. 4.3.2. Antioxidant activity of meroterpenoids isolated from C. usneoides The meroterpenoids 1−10 isolated from C. usneoides were tested for their antioxidant activity by using the ABTS assay, which determines the radical-scavenging activity of compounds toward the ABTS•+ radical cation (Re et al., 1999). In this method, the ABTS is chemically or enzymatically oxidized to its radical cation ABTS•+, which is intensely blue-green colored, and the antioxidant capacity of a compound is measured as its ability to reduce the color by reacting with the ABTS•+ radical (Figure. 4.3). Figure 4.3. Interconversion between the ABTS and its radical. The results are expressed relative to the activity of Trolox (Figure 4.4), a synthetic vitamin E analogue, measured under the same conditions. " Figure 4.4. Chemical structure of Trolox®. _____________________________________________________________4. Chemical study of C. usneoides " 77 All the tested meroterpenoids exhibited strong radical-scavenging activity, as shown in Table 4.4. The most active compounds were 5, 6, 7, and 8, which showed an antioxidant capacity equal or slightly superior to that of the Trolox standard. Compounds 2 and 3 were also strongly active, showing a potency of 78% that of Trolox, while compounds 1, 4, 9, and 10 displayed an antioxidant activity about 50−60% that of Trolox. Table 4.4. Antioxidant activities of compounds 1−10 in the ABTS assay. Compound EC50 (µM)b TEc Troloxa 25.9±0.5 1 1 43.1±3.1 0.60 2 33.1±5.1 0.78 3 33.3±2.3 0.78 4 51.6±4.8 0.50 5 26.3±2.3 0.98 6 24.5±1.6 1.06 7 22.5±2.1 1.15 8 24.4±0.9 1.06 9 55.9±9.9 0.46 10 44.7±1.1 0.58 aStandard compound: 6-hydroxy-2,5,7,8-tetramethylchroman- 2-carboxylic acid. bConcentration that caused a 50% reduction of the absorbance. Values are mean ± SD of three determinations. cTrolox equivalents: EC50(Trolox)/EC50(compound). From a structural point of view, except for compounds 7 and 8, the compounds possessing a 6E configuration (2, 3, 6, 10) were more active than the corresponding 6Z isomers (1, 4, 5, 9). On the other hand, compounds with a C20 side chain were in general more active than the corresponding analogues with a C14 side chain, as evidenced by the comparison of the activity of the meroditerpenes 5, 6, 7, and 8 with that of the short chain analogues 4, 3, 9, and 10, respectively. 4. Chemical study of C. usneoides_____________________________________________________________" " 78 After an initial account on the properties of three Cystoseira-derived meroditerpenes as singlet oxygen and peroxyl radical scavengers (Foti et al., 1994), the antioxidant activities of a series of metabolites obtained from C. crinita have more recently been described (Fisch et al., 2003). The meroditerpenes of C. crinita were found to be less active than Trolox in the ABTS assay, with the most active compound displaying an antioxidant activity about 40% that of the Trolox standard. Our study of C. usneoides has allowed the identification of new members of this class of algal meroterpenoids provided of more potent antioxidative properties. 4.4. Conclusion The chemical study herein described has shown than the brown alga C. usneoides contains a variety of meroterpenoids whose strcutures consist of a C20 or a C14 terpenoid moiety linked to an O-methyltoluhydroquinone. Six of the isolated meroterpenes are new natural products, the cystodiones A-F (7-12), while the compounds cystomexicone B (3), cystomexicone A (4), 6-cis-amentadione-1′-methyl ether (5), and amentadione-1′-methyl ether (6), are described for the first time in the species C. usneoides. In antioxidant assays the meroterpenes 5, 6, 7, and 8 show potent radical-scavenging properties, further supporting the potential of algae of the genus Cystoseira as source of new natural antioxidants. 4.5. References Amico, V. Marine brown algae of family Cystoseiraceae: chemistry and chemotaxonomy. Phytochemistry 1995, 39, 1257−1279. Amico, V.; Cunsolo, F.; Piattelli, M.; Ruberto, G. Five novel tetraprenylhydroquinols from the brown alga Cystoseira algeriensis. Phytochemistry 1984, 23, 2017−2020. Amico, V.; Piattelli, M.; Neri, P.; Ruberto, G."Metabolites of mixed biogenesis from Cystoseira tamariscifolia. Gazz. Chim. Ital. 1989, 119, 467−470. Danet, M.; Normand-Bayle, M.; Mahuteau, J.; D’Angelo, J.; Morgant, G.; Desmaële, D. Enantioselective synthesis of the originally proposed usneoidone structure: evidence for a structural revision. Eur. J. Org. Chem. 2004, 2004, 1911−1922. De los Reyes, C.; Ortega, M.; Zbakh, H.; Motilva, V.; Zubía, E. Cystoseira usneoides: A brown alga rich in antioxidant and anti-inflammatory meroditerpenoids. J. Nat. Prod. _____________________________________________________________4. Chemical study of C. usneoides " 79 2016, 79, 395−405. De los Reyes, C.; Zbakh, H.; Motilva, V.; Zubía, E. Antioxidant and anti-inflammatory meroterpenoids from the brown alga Cystoseira usneoides. J. Nat. Prod. 2013, 76, 621−629. Draisma, S.G.A.; Ballesteros, E.; Rousseau, F.; Thibaut, T.J. DNA sequence data demonstrate the polyphyly of the genus Cystoseira and Sargassaceae genera (Phaeophyceae). J. Phycol. 2010, 46, 1329−1345. Fernández, J.J.; Navarro, G.; Norte, M. Novel metabolites from the brown alga Cystoseira abies-marina. Nat. Prod. Lett. 1998, 12, 285−291. Fisch, K.M.; Böhm, V.; Wright, A.D.; König, G.M. Antioxidative meroterpenoids from the brown alga Cystoseira crinita. J. Nat. Prod. 2003, 66, 968−975. Foti, M.; Piattelli, M.; Amico, V.; Ruberto, G. J. Antioxidant activity of phenolic merodierpenoids from marine algae. Photochem. Photobiol. B. 1994, 26, 159−164. Francisco, C.; Banaigs, B.; Teste, J.; Cave, A. Mediterraneols: a novel biologically active class of rearranged diterpenoid metabolites from Cystoseira mediterranea (Pheophyta). J. Org. Chem. 1986a, 51, 1115−1120. Gouveia V.; Seca A.L.M.; Barreto M.C.; Pinto D.C.G.A. Di- and Sesquiterpenoids from Cystoseira Genus: Structure, intra-molecular, transformations and biological activity. Mini-Rev. Med. Chem. 2013a, 13, 1150−1159. Gouveia, V.L.M.; Seca, A.M.L.; Barreto, M.C.; Neto, A.I.; Kijjoa, A.; Silva, A.M.S. Cytotoxic meroterpenoids from the macroalga Cystoseira abies-marina. Phytochemistry Lett. 2013b, 6, 593–597. Mokrini, R.; Ben Mesaoud, M.; Daoudi, M.; Hellio, C.; Mare!chal, J.P.; El Hattab, M.; Ortalo-Magne, A.; Piovetti, L.; Culioli, G. J. Meroditerpenoids and derivatives from the brown alga Cystoseira baccata and their antifouling properties. J. Nat. Prod. 2008, 71, 1806−1811. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C."Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med. 1999, 26, 1231−1237. Urones, J. G.; Araújo, M.E.M.; Brito Palma, F.M.S.; Basabe, P.; Marcos, I.S.; Moro, R.F.; Lithgow, A.M.; Pineda, J. Meroterpenes from Cystoseira usenoides II. Phytochemistry 1992a, 31, 2105−2109. Urones, J.G.; Basabe, P.; Marcos, I.S.; Pineda, J.; Lithgow, A.M.; Moro, R.F.; Brito Palma, F. M.S.; Araújo, M.E.M.; Grávalos, M.D.G. Meroterpenes from Cystoseira usenoides. Phytochemistry 1992b, 31, 179−182. 5. Anticancer activity of AMTs on HT-29 cells___________________________________________________ ! 86 2018): SI= IC50 value of the compound against a normal cell line/IC50 of the same compound against a cancer cell line. 5.2.5. Flow cytometric analysis for apoptosis induction assay Quantitative assessment of apoptosis was performed by flow cytometry using an Annexin V-FITC Apoptosis Detection Kit (Vermes et al., 1995). Briefly, 1x106 HT-29 cells were seeded per well in 6-well plates and incubated for 24 h, followed by the addition of the AMTs dissolved in DMSO (final concentrations of 10, 20, and 30 µg/mL for compounds 1, 2, 5, 6, 7, and 8, and 30, 60, and 90 µg/ml for compounds 3 and 4, and less than 0.05 % DMSO) and incubation for 24 h. Curcumin (final concentrations of 50 and 75 µM) was used as a positive control. The cells were then washed, harvested with trypsin, and centrifuged at 1,500 rpm (5 min, 25 ºC). The pellet was resuspended in 195 µL of 1×Annexin buffer and then stained with 5 µL of Annexin V-FITC and 10 µL of PI for 10 min at room temperature, in the dark. To this mixture 200 µL of 1×Annexin buffer were added before analysis using a Cytomics FC500 flow cytometer (Beckman Coulter, Indianapolis, IN, USA). 10,000 cellular events in each sample were analyzed using DML program. 5.2.6. Cell cycle analysis by flow cytometry HT-29 cells (1x106 cells/well) were seeded in 6-well plates and incubated for 24 h, followed by treatment with AMTs dissolved in DMSO (final concentrations of 10, 20, and 30 µg/mL for compounds 1, 2, 5, 6, 7, and 8, and 30, 60, and 90 µg/mL for compounds 3 and 4, and always below 0.05 % DMSO) and 24 h of additional incubation (Nicoletti et al., 1991). Colchicine (final concentration of 0.2 µg/mL) was used as a positive control. The cells were then washed, harvested with trypsin, and centrifuged at 1,500 rpm (5 min, 25 ºC). The resulting pellet was fixed with ice-cold 70% ethanol (1 mL/106 cells) and the samples were stored at -4 ºC overnight. After fixation, the cells were washed with PBS, stained with PBS containing 5 mg/mL of RNase A, and incubated for 48 h at 4 °C. Subsequently, 50 µL of 0.1 mg/mL of IP was added and incubated for 1 h at 4 °C. The relative DNA content per cell was analyzed using a Cytomics FC500 flow cytometer (Beckman Coulter, Indianapolis, IN, USA). The data acquisition was performed with the DML program (DML, USA) and the analysis of the acquired data with the CXP Software (USA). _____________________________________________________________________5. Anticancer activity of AMTs on HT-29 cells! ! 87 5.2.7. Wound migration assay For cell migration assay, HT-29 cells were seeded in 6-well culture plates and allowed growing to 80–90% confluence for the experiment. After aspirating the medium, similar sized wounds were performed in the monolayer cells using a sterile micropipette tip. Wounded monolayer cells were washed three times with PBS to remove cell debris and then replaced with complete McCoy’s medium. HT-29 cells were treated with the AMTs (10 µg/mL for compounds 1, 2, 5, 6, 7, and 8, and 30 µg/mL for compounds 3 and 4) and incubated for 24h. For the image analysis of the effect of each treatment on cell migration, the wounded area was photographed immediately after scratching and after 24h. The level of cell migration was determined using NIH Image software (Image J 1.44g, Wayne Rasband, USA) and then expressed as a percentage of wound closure area using the equation: % of wound closure = [(At0h – At24h) / At0h] × 100%, where, At0h is the area of wound measured immediately after scratching and At24h is the area of wound measured 24 h after scratching. 5.2.8. Cell invasion assay The invasion activities were measured using Trevigen’s Cultrex® 96 Well Cell Invasion Assays, as previously described (Moghadamtousi et al., 2014). Briefly, about 50 µL of basement membrane extract (BME, 1X) coat was added to each well. After incubation for 4 h at 37° in a 5% CO2 atmosphere, the HT-29 cells at 50,000 cells/50µL in serum free McCoy’s medium were added per well to the top chamber containing the tested compound (10 µg/mL for 1, 2, 5, 6, 7, and 8, and 30 µg/mL for compounds 3 and 4). About 150 µL of McCoy’s medium were then added to the lower chamber containing 10% FBS and penicillin/streptomycin as chemoattractants. Cells were allowed to migrate to the lower chamber in a humidified atmosphere containing 5% CO2 at 37°C for 24 h. Afterwards, top and bottom chambers were aspirated and washed with washing buffer supplemented with the kit. About 100 µL of 1X Cell Dissociation Solution/Calcein-AM was added to each bottom chamber well and incubated for 1 h at 37 °C in CO2 incubator. The cells internalized calcein-AM and the intracellular esterases cleaved the AM moiety to generate free calcein. Fluorescence of the samples was determined at 485 nm excitation, 520 nm emission, using ELISA plate reader (BioTek Instruments, USA). The number of cells that 5. Anticancer activity of AMTs on HT-29 cells___________________________________________________ ! 88 had invaded through the BME coat was calculated using a standard curve. Results were expressed as percentage of invasion (%) compared to the control. 5.2.9. Preparation of cell lysates Cells were cultured to 80% confluence at 37 °C. After 24 h of treatment with the compounds (10 and 20 µg/mL for compounds 1, 2, 5, 6, 7, and 8, and 30 and 60 µg/mL for compounds 3 and 4), HT-29 cells were rinsed twice with ice-cold PBS, and then lysed immediately in lysis buffer (250 mM NaCl, 50 mM Tris (pH 7.5), 0.5 mM EDTA, 5 mM EGTA, 8 mM MgCl2, 1 mM PMSF, 0.01 mg/mL pepstatin A, 0.01 mg/mL leupeptin, 0.01 mg/mL aprotinin, 1% Triton X-100). After centrifugation at 12,000 g for 3 min at 4 °C to separate the cellular debris, the supernatant was collected and stored at −80 °C until use. The protein concentrations were determined using Bio-Rad Protein Assay (BioRad, Richmond, CA, USA), according to the manufacturer’s instructions. 5.2.10. Western blot analysis Equal protein content (50 µg) samples of cell lysates were separated on 10% SDS– polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Hybond-P, Amershan Biosciences, UK). The membranes were blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 buffer (pH 7.4) (TBST) for 1 h at room temperature and incubated with agitation with specific antibodies: anti-pERK1/2 (Cell Signaling; 1:1000), anti-pJNK (Cell Signaling; 1:1000), anti-pAKT (Cell Signaling; 1:1000), anti-β-actin (Sigma-Aldrich; 1:500). Membranes were incubated overnight at 4 °C with gentle shaking. The secondary antibody was a peroxidase-conjugated goat anti-mouse or -rabbit antibody (1:10,000; Dako Cytomation, USA). After washing the membrane three times in TBST buffer (10 min), the signals were detected using an enhanced chemiluminescence light-detecting kit (Super-Signal West Pico Chemiluminescent Substrate, Pierce, IL, USA), according to the manufacturer’s instructions and exposed to an X-ray film (GE Healthcare Ltd., Amersham, UK). The protein band densities were analyzed and quantified with a Scientific Imaging Systems (Biophotonics Image J Analysis Software, USA). β-actin was used to confirm the equal loading and transfer of proteins. _____________________________________________________________________5. Anticancer activity of AMTs on HT-29 cells! ! 89 5.2.11. Statistical analysis The results were expressed as means ± Standard Error (SE). Data were evaluated with GraphPad Prism® Version 5.00 software. Differences between two groups were analyzed by the Student’s t-test. Difference with P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) were considered statistically significant. 5.3. Results 5.3.1. The AMTs 1-8 inhibit cell proliferation in human colon adenocarcinoma cells HT- 29 The AMTs usneoidone Z (1), 11-hydroxy-1′-O-methylamentadione (2), cystomexicone B (3), cystomexicone A (4), 6-cis-amentadione-1′-methyl ether (5), amentadione-1′-methyl ether (6), cystodione A (7), and cystodione B (8) (Figure 5.1) isolated from the alga C. usneoides have been investigated for their anticancer activity. The ability of these compounds at different concentrations to inhibit the viability of cancer and non-cancer colon cells (HT-29 and CCD 841 CoN, respectively) was examined by the SRB assay. All compounds caused a dose-dependent decrease in cell survival for both, cancer and non-cancer cells, although at different extents (Figure 5.2). Usneoidone Z (1) and 6-cis-amentadione-1′-methyl ether (5) showed the strongest growth inhibitory activity against colon cancer cells HT-29 (IC50 8.81 and 7.83 µg/mL, respectively), while the effects of both compounds were much lower towards the normal colon cells CCD 841 CoN (IC50 46.41 and 40.97 µg/mL, respectively) (Table 5.1). The meroditerpenes 2, 6, 7, and 8 also induced strong decreases of the viability of HT-29 cells, greater than those observed for CCD 841 CoN cells (IC50, 9.14, 10.72, 14.00, and 9.14 µg/mL for HT-29 cells and IC50 21.41, 48.38, >50, and 31.88 µg/mL for CCD 841 CoN cells, respectively). Compounds 3 and 4 were the less cytotoxic towards both, the tumor and the normal cells. 5. Anticancer activity of AMTs on HT-29 cells___________________________________________________ ! 90 Figure 5.2. Effect of AMTs 1-8 at different concentrations on the viability of both HT-29 colon cancer cells and normal colonic epithelial cells CCD 841 CoN after 72 h of treatment. Results obtained by the SRB assay are reported as the percentage of viable cells (% cell viability). Data represent mean ± SE from three independent experiments. *p<0.05 and **p<0.01 compared with the untreated group. 0 20 40 60 80 100 0 10 20 30 40 50 % Cell viability Concentration (µg/ml) 2 CCD841CoN HT-29 ** ** ** 0 20 40 60 80 100 0 10 20 30 40 50 % Cell viability Concentration (µg/ml) 1 CCD841CoN HT-29 ** ** * 0 20 40 60 80 100 0 10 20 30 40 50 % Cell viability Concentration (µg/ml) 5 CCD841CoN HT-29 ** * 0 20 40 60 80 100 0 10 20 30 40 50 % Cell viability Concentration (µg/ml) 6 CCD841CoN HT-29 ** ** 0 20 40 60 80 100 0 10 20 30 40 50 % Cell viability Concentration (µg/ml) 7 CCD841CoN HT-29 ** 0 20 40 60 80 100 0 10 20 30 40 50 % Cell viability Concentration (µg/ml) 8 CCD841CoN HT-29 ** ** 0 20 40 60 80 100 0 10 20 30 40 50 % Cell viability Concentration (µg/ml) 4 CCD841CoN HT-29 ** *" 0 20 40 60 80 100 0 10 20 30 40 50 % Cell viability Concentration (µg/ml) 3 CCD841CoN HT-29 * _____________________________________________________________________5. Anticancer activity of AMTs on HT-29 cells! ! 91 Table. 5.1. IC50 values (µg/mL) obtained for AMTs 1-8 against the colon cancer cells HT-29 and the normal colon cells CCD 841 CoN after 72h of treatment (data are means ± SE of three experiments).!SI = IC50 value for normal cells/ IC50 value for cancer cells. Compound Cell lines Selectivity Index (SI) CCD 841 CoN HT-29 1 46.41±3.87 8.81±1.55 5.26 2 21.41±2.50 9.14±0.23 2.34 3 >50 34.34±1.81 >1.45 4 61.86±2.37 36.86±2.86 1.68 5 40.97±9.80 7.83±0.05 5.23 6 48.38±3.84 10.72±0.27 4.51 7 >50 14.00±1.33 >3.57 8 31.88±7.51 9,08±1.04 3.51 According to the literature, compounds with selectivity index (SI) values greater than 3 are considered highly selective (Bézivin et al., 2003), although other authors consider that compounds with a SI higher than or equal to 2.0 are also interesting (Suffness and Pezzuto, 1990). Treatments with the AMTs 1 and 5 afforded selectivity indexes higher that 5 (SI=5.26 and 5.23, respectively), indicating that these compounds are five times more toxic towards the cancer cells than towards the non-cancer cells (Table 5.1). The AMTs 6, 7, and 8 were also highly selective for cancer cells, affording SI higher than 3 (4.51, >3.57, and 3.51, respectively). However, the selectivity index observed for 2, 3, and 4 were more moderated (2.34, >1.45, and 1.68, respectively). 5.3.2. The AMTs 11-hydroxy-1′-O-methylamentadione (2), cystomexicone B (3), and 6- cis-amentadione-1′-methyl ether (5) induce apoptosis of HT-29 colon cancer cells Flow cytometry analysis by annexin V/PI staining was performed in order to investigate the induction of apoptosis in HT-29 cells by AMTs 1-8. Compounds 1, 2, 5, 6, 7, and 8 were tested at concentrations of 10, 20, and 30 µg/mL, and compounds 3 and 4 were 5. Anticancer activity of AMTs on HT-29 cells___________________________________________________ ! 92 tested at 30, 60, and 90 µg/mL. The percentages of viable, early apoptotic, late apoptotic, and necrotic cells after 24 h of treatment with the AMTs 1-8 are shown in Figure 5.3. Significant differences were observed between control and treated cells. As shown in Figure 5.3A, after 24 h 89.34% of vehicle alone-treated HT-29 cells were viable (Annexin V-PI-), 0.18% were early apoptotic cells (Annexin V+PI-) and 0.22% were late apoptotic (Annexin V+PI+). In contrast, HT-29 cells treated for 24 h with cystomexicone B (3), showed a progressive induction of the apoptosis process, with a significant increase at the dose of 90 µg/mL, both in the early (4.47%) and in the late (52.12%) apoptotic cells (total percentage of apoptotic cells, 56.6%). The AMT 2 also caused significant, although more moderated, apoptotic effect (57.7% of viable cells, 1.94 % early apoptotic, and 12.94 % late apoptotic). (Figure 5.3B). Compound 5 induced a percentage of apoptosis similar to 2, but the most prominent effect was necrosis (72.75%). In this line, although cell growth was not affected by the AMTs 1, 6, and 8 at concentrations up to 20 µg/mL, at the higher concentration of 30 µg/mL a strong necrotic effect was detected, with 31.22, 59.55 and 20.12 % of dead cells, respectively. After 24 h of treatment, the AMTs 4 and 7 did not induce neither apoptosis nor death of HT-29 cells. 5.3.3. Effects of the meroterpenoids 1-8 on cell cycle arrest in HT-29 cells In an attempt to explore the effects of the AMTs 1-8 on the cell cycle progression of colon carcinoma cells HT-29, the cell cycle was analyzed by flow cytometry. The effects of increasing concentrations of usneoidone Z (1) on HT-29 cell progression through G0/G1-, S- and G2/M-phases are shown in Figure 5.4A. This compound was the most active among the tested AMTs and increasing concentrations (10, 20, 30 µg/mL) resulted both in a significant cell cycle arrest in the G2/M (p<0.01) and in the reduction of the number of cells in the G0/G1 phase (p<0.01). The accumulation of cells at the G2/M phase was also significant with the AMTs 2, 3, 4, 5 and 7 (p<0.05) and it was correlated with a subsequent significant decrease of cells in the G0/G1-phase (Figure 5.4B). Compounds 6 and 8 showed the same tendency in cell cycle progression but the changes were no significant. _____________________________________________________________________5. Anticancer activity of AMTs on HT-29 cells! ! 93 Figure 5.3. Apoptosis rates by flow cytometry of HT-29 colon cancer cells treated with the AMTs 1-8 for 24 h. Negative control cells received no treatment. Positive control received 50 and 75 µM of curcumin. (A) Flow cytometry histograms show the percentage of HT-29 cells treated with 30, 60, and 90 µg/mL of 3 undergoing early and late apoptosis. (B) Bar charts illustrate the percentage of viable, necrotic, early, and late apoptosis cells treated for 24 h with 30 µg/mL of 1, 2, 5, 6, 7 and 8, and 90 µg/mL of 3 and 4. Data represent mean ± SE from three independent experiments. Significant differences from control group: *p<0.05, and **p<0.01. A)# B)# 25,53±5,25 52,12±0,05** 4,47±0,99* 18,67±1,15 4,61±0,27 1,39±0,16 9,05±0,2 1,15±0,10 0,51±0,11 3 (60µg/ml) 3 (90µg/ml) 3 (30µg/ml) 6,48±0,45 67,9±1,91** 2,34±0,36 ! 13,86±0,77 1,09±0,08 0,8±0,15 10,27±0,21 0,22±0,09 0,18±0,04 Curcumin (75 µM) Curcumin (50 µM) Control 0 10 20 30 40 50 60 70 80 90 100 Control Curcumin 1 2 3 4 5 6 7 8 Percentage Cell Population (%) Meroterpenoids Viable Necrosis Early apoptosis Late apoptosis ** * * ** ** ** 5. Anticancer activity of AMTs on HT-29 cells___________________________________________________ ! 94 Figure 5.4. Flow cytometry analysis of cell cycle arrest in colon cancer cells HT-29 treated for 24 h with the AMTs 1-8. Negative control cells received no treatment. Positive control received 0.2 µg/mL of colchicine. (A) Flow cytometry histograms showing HT-29 cells treated with 10, 20, and 30 µg/mL of compound 1. (B) Bar charts for HT-29 cells treated with 30 µg/mL of 1, 2, 5, 6, 7, and 8, and 90 µg/mL of 3, and 4. Data represent mean ± SE from three independent experiments. Significant differences to control group: *p<0.05, and **p<0.01. SUB-G0: 0,77±0,04 G0-G1: 74,73±1,25 S: 9,96±1,07 G2/M: 14,1±0,73 SUB-G0: 0,19±0,19 G0-G1: 9,92±2,46 S: 4,74±1,29 G2/M: 82,77±2,06 SUB-G0: 0±0 % G0-G1: 28,81±0,82** S: 13,17±1,77 % G2/M: 61,75±2,11** SUB-G0: 0,47±0,42 G0-G1: 68,29±0,7 S: 7,48±1,03 G2/M: 20,84±0,46 SUB-G0: 0,25±0,17 G0-G1: 58,24±4,05 S: 9,53±1,55 G2/M: 25,71±1,14 Control Colchicine 1 (20µg/ml) 1 (30µg/ml) 1 (10µg/ml) A)# B)# 0 10 20 30 40 50 60 70 80 90 100 Control Colchicine 1 2 3 4 5 6 7 8 Percentage Cell Population (%) Meroterpenoids SUB-G0 G0-G1 S G2/M ** ** * * ** * *** * * ** ** * ** _____________________________________________________________________5. Anticancer activity of AMTs on HT-29 cells! ! 95 5.3.4. Effects of the AMTs 1-8 on the migration and invasion of HT-29 cells Cell migration is a measure of the metastatic potential of cancer cells. To examine whether the AMTs 1-8 had any inhibitory effect on cell migration process, HT-29 cells were incubated for 24 h in the absence or presence of the compounds (10 µg/mL) in a wound-healing assay (Figure 5.5). Figure 5.5. Effects of AMTs 1-8 on migration and invasion of HT-29 cells. (A) Seeded colon cancer cells in 6-well plates were wounded and imaged (0h). Untreated HT-29 cells and cells treated with compound 8 (10 µg/mL) were incubated for 24 h. (B) Bar charts show the percentage of migrated HT-29 cells after treatment for 24 h with 1, 2, 5, 6, 7, and 8 at 10 µg/mL and with 3 and 4 at 30 µg/mL. (C) Anti-invasive activity of AMTs against HT-29 cells measured using a Cultrex assay kit. HT-29 cells were untreated or treated for 24 h with the AMTs (10 µg/mL for 1, 2, 5, 6, 7, and 8, and 30 µg/mL for 3, and 4). The data represent the means ± SE of three independent experiments. Significant differences to control group: *p<0.05 and **p<0.01. 0h 24h 0h 24h Control Compound 8 A)# B)# C)# 0 20 40 60 80 100 Control 1 2 3 4 5 6 7 8 Cell Invasion (%) Meroterpenoids * * ** ** * ** ** 0 20 40 60 80 100 120 Control 1 2 3 4 5 6 7 8 Cell Migration (%) Meroterpenoids * * * * * 5. Anticancer activity of AMTs on HT-29 cells___________________________________________________ ! 102 death, and metastasis of cancer cells (Go et al., 2011). In colorectal carcinoma, the MAPK pathway is aberrantly activated (Shan et al, 2009) and, therefore, the inhibition of this pathway is a potential therapeutic approach (Go et al., 2011). It has been shown that the ERK promotes growth, differentiation, and proliferation of cancer cells (Jiang et al., 2018). It has also been reported that transient ERK activation might be linked to cellular proliferation while strong and persistent activation may lead to programmed cell death (Park et al., 2014). ERK promote either intrinsic or extrinsic apoptotic pathways by induction of mitochondrial cytochrome c release or caspase-8 activation (Cagnol and Chambard, 2010). ERK pathway not only participates in the regulation of apoptosis, but also controls G2/M cell cycle phase. ERK has been reported to regulate cyclin B1 transcriptional induction and also controls the assembly of cyclin-CDK complex via the CDK translocation (Li et al., 2008; Abrieu et al., 1997), which play pivotal roles in regulating cell cycle progression. Moreover, ERK1/2 regulates focal adhesion and cytoskeletal reorganization via the phosphorylations of specific cytoskeletal and focal adhesion proteins, including paxillin, FAK, and myosin light chain kinase, which are crucial signalling components to control cell migration, invasion, and cell cycle progression (Wu et al., 2008; Kuphal, 2005). In this study, we have examined the effect of compounds 1-8 on the ERK pathway. A decreased protein expression of phosphorylated ERK was observed in HT-29 cells treated with compounds 1, 2, 5, 6, 7, and 8, which support the crucial role of ERK in the regulation of proliferation, cycle progression, and metastasis processes in colon cancer cells. Activation of JNK has been involved in the regulation of various cellular processes, including cell survival, proliferation, differentiation, and cell death (Tournier, 2013). However, few papers have been published regarding to the potential role of JNK in the cell cycle. Mingo-Sion et al., 2004 reported that the induced G2/M arrest may be due to the inability of some cells to sustain p21Cip1/Waf1, a JNK substrate, in the absence of JNK activity. When p21Cip1/Waf1 expression is increased in response to DNA damage, cyclin B/Cdk1 kinase activity is inhibited causing G2/M phases arrest (Bates et al., 1998). In this study, we have found that compounds 2, 3, 4, 5, and 7 inhibit cell proliferation through inducing a G2/M phase arrest in HT-29 cells via JNK pathway. Regarding to programed cell death, JNK play an active role in the regulation of both the intrinsic and extrinsic _____________________________________________________________________5. Anticancer activity of AMTs on HT-29 cells! ! 103 apoptotic pathways (Dhanasekaran and Reddy, 2017). These findings suggest that p-JNK may be involved in the activation of cell apoptosis after the treatment with compound 3. JNK plays also a crucial role in cell migration and invasion. The oncogenic functions of JNK are particularly based on its ability to phosphorylate c-Jun and to activate transcriptional factor Activator Protein-1 (AP-1). Matrix metalloproteinases (MMPs), a key role for degrading the basement membrane, have an AP-1 consensus sequence that regulates tumour progression by enhancing tumour-induced angiogenesis and destroying local tissue architecture and basement membranes to allow tumor invasion and metastasis (Cai et al., 2017). The AMTs showing anti-migration (5, 7, and 8) and anti-invasion (2, 3, 4, 5, 7, and 8) activities also decreased phosphorylation of JNK in colon cancer cells. suggesting that these compounds may have efficacy in the prevention of the metastasis of colon cancer cells. Alteration of the PI-3K/AKT pathway has been detected during tumor formation in numerous cancers, including colorectal cancer (Jung et al., 2017). Many studies have reported that PI3K activation stimulates the downstream target AKT, which plays various and important roles in regulating cell proliferation, cell cycle, apoptosis, and cell invasion (Shant et al., 2009; Hollborn, 2007; Chang et al., 2003). AKT pathway has been shown to be involved in the cell cycle progression by down-regulating Cdk1 and Cyclin B1 expression, both of which can ultimately lead to the arrest of G2/M transition (Hiraoka et al., 2016). On the other hand, AKT modulate apoptosis signaling by inducing expression of multiple pro-apoptotic members of the Bcl2-family of mitochondria-targeting proteins. These pro-apoptotic proteins translocate in the mitochondria leading to caspase activation thus leading to apoptosis (Agarwal et al., 2013). AKT is also known to regulate the expression of FAK (focal adhesion kinase) proteins mediating colorectal cancer metastasis. In response to extracellular pressure, AKT and FAK bind directly, thus phosphorylating AKT at three serine residues. The phosphorylation of the three serine residues consequently phosphorylate the tyrosine residue (Tyr397) thus activating it. It therefore induces cell adhesion by increasing the binding of integrins to matrix, which finally lead to increased metastasis (Wang and Basson, 2011). Hence, inhibiting AKT may be an important therapeutic target for regulating cell cycle progression, apoptosis, and preventing cancer metastasis. In the present study, we have found that the AMTs 1, 2, 3, 4, 7, and 8 reduce the 5. Anticancer activity of AMTs on HT-29 cells___________________________________________________ ! 104 protein levels of p-AKT in HT-29 cells, indicating the role of these AMTs in the downregulation of proliferation, cell cycle, apoptosis, and metastasis in colon cancer cells through the regulation of MAPK and AKT pathways. These results are in line with a previous study where the algal halogenated monoterpene mertensene was shown to induce G2/M cell cycle arrest and apoptosis in human colon adenocarcinoma HT-29, through!the modulation of ERK-1/-2 and AKT signaling (Tarhouni-Jabberi et al., 2017). Nonetheless, some studies on the mechanism of action of other algal terpenoids have also demonstrated the intervention of other signaling pathways. For example, the sesquiterpene guai-2-en- 10α-ol, from Ulva fasciata, was reported to induce apoptosis and cell cycle arrest in G1 phase of triple-negative breast cancer (TNBC) cell line (MDA MB-231) via regulation of EGFR/PI3 K/Akt pathway (Lakshmi et al., 2018) and laurinterol, from Laurencia okamurae, showed anticancer activity against melanoma cells (B16F1) through the p53- dependent pathway (Kim et al., 2008). 5.5. Conclusion In summary, the present study demonstrates for the first time that various AMTs obtained from the alga C. usneoides inhibit in vitro the proliferation of colon cancer cells, while being significantly less cytotoxic against normal cells, induce cell cycle arrest, and decrease migration and invasion of HT-29 cells. Moreover, with cystomexicone B (3), apoptosis in HT-29 cells was detected. Our results provide proof that the tested AMTs promote strong anticancer effects through down-regulation of signaling pathways by ERK, JNK, and/or AKT. On the basis of the activity observed for the AMTs 1, 2, 5, 7 and 8 in most of the assays performed in this study, these AMTs could be promising as agents for the prevention and treatment of colon cancer, although the potential of these NPs to act as chemopreventive and therapeutic agents for colorectal carcinomas needs to be previously evaluated in animal models. 5.6. References Abrieu, A.; Fisher, D.; Simon, M.N.; Doree, M.; Picard, A. MAPK inactivation is required for the G2 to M-phase transition of the first mitotic cell cycle. EMBO J. 1997, 16, 6407– 6413. _____________________________________________________________________5. Anticancer activity of AMTs on HT-29 cells! ! 105 Agarwal, E.; Brattain, M.G.; Chowdhury, S. Cell survival and metastasis regulation by akt signalling in colorectal cancer. Cell Signal. 2013, 25, 1711–1719. Amico, V. Marine brown algae of family Cystoseiraceae: Chemistry and chemotaxonomy. Phytochemistry 1995, 39, 1257−1279. Atanasov, A.G.; Waltenberger, B.; Pferschy-Wenzig, E.M.; Linder, T.; Wawrosch, C.; Uhrin, P.; Temml, V.; Wang, L.; Schwaiger, S.; Heiss, E.H.; Rollinger, J.M.; Schuster, D.; Breuss, J.M.; Bochkov, V.; Mihovilovic, M.D.; Kopp, B.; Bauer, R.; Dirsch, V.M.; Stuppner, H. Discovery and resupply of pharmacologically active plant-derived natural products: A review. Biotechnol. Adv. 2015, 33, 1582–1614. Bates, S.; Ryan, K.M.; Phillips, A.C.; Vousden, K.H.!Cell cycle arrest and DNA endoreduplication following p21Waf1/Cip1 expression. Oncogene 1998, 17, 1691–1703. Bézivin, C.; Tomasi, F.; Lohézie-Le, D.; Boustie, J. Cytotoxic activity of some lichen extracts on murine and human cancer cell lines. Phytomedicine 2003, 10, 499–503. Cagnol, S.; Chambard, J.C. ERK and cell death: mechanisms of ERK-induced cell death-- apoptosis, autophagy and senescence. FEBS J. 2010, 277, 2–21. Cai, J.; Du, S.; Wang, H.; Xin, B.; Wang, J.; Shen, W.; Wei, W.; Guo, Z.; Shen, X.; Tenascin-C induces migration and invasion through JNK/c-Jun signalling in pancreatic cancer. Oncotarget 2017, 8, 74406–74422. Campos, A.; Souza, C.B.; Lhullier, C.; Falkenberg, M.; Schenkel, E.P.; Ribeiro-do-Valle, R.M.; Siqueira, J.M. Anti-tumour effects of elatol, a marine derivative compound obtained from red algae Laurencia microcladia. J. Pharm. Pharmacol. 2012, 64, 1146-1154. Castedo, M.; Perfettini, J.L.; Roumier, T.; Andreau, K.; Medema, R.; Kroemer, G. Cell death by mitotic catastrophe: a molecular definition. Oncogene 2004, 23, 2825–2837. Cen-Pacheco, F.; Villa-Pulgarin, J.A.; Mollinedo, F.; Martín, M.N.; Fernández, J.J.; Daranas, A.H. New polyether triterpenoids from Laurencia viridis and their biological evaluation. Mar. Drugs 2011, 9, 2220–2235. Chang, F.; Lee, J.T.; Navolanic, P.M.; Steelman, L.S.; Shelton, J.G.; Blalock, W.L.; Franklin, R.A.; McCubrey, J.A. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy. Leukemia 2003, 17, 590–603. Chen, P.N.; Hsieh, Y.S.; Chiou, H.L.; Chu, S.C. Silibinin inhibits cell invasion through inactivation of both PI3K-Akt and MAPK signaling pathways. Chem. Biol. Interact. 2005, 156, 141–150. Choi, Y.K.; Kim, J.; Lee, K.M.; Choi, Y.J.; Ye, B.R.; Kim, M.S.; Ko, S.G.; Lee, S.H.; Kang, D.H.; Heo, S.J. Tuberatolide B Suppresses Cancer Progression by Promoting ROS- Mediated Inhibition of STAT3 Signaling. Mar. Drugs 2017, 15, pii: E55. 5. Anticancer activity of AMTs on HT-29 cells___________________________________________________ ! 106 Cragg, G.M.; Newman, D.J. Natural products: a continuing source of novel drug leads. Biochim. Biophys. Acta 2013, 1830, 3670–3695. De los Reyes, C.; Zbakh, H.; Motilva, V.; Zubía, E. Antioxidant and anti-inflammatory meroterpenoids from the brown alga Cystoseira usneoides. J. Nat. Prod. 2013, 76, 621−629. Dhanasekaran, D.N.; Reddy, E.P. JNK-signaling: A multiplexing hub in programmed cell death. Genes Cancer 2017, 8, 682–694. Dominguez-Brauer, C.T.; Thu, K.L.; Mason, J.M.; Blaser, H.; Bray, M.R.; Mak, T.W. Targeting mitosis in cancer: Emerging strategies. Mol. Cell. 2015, 60, 524–536. Ferlay, J.; Soerjomataram, I.; Dikshit, R. et al. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer 2015, 136, 359– 386. Go, H.; Hwang, H.J.; Kim, I.H.; Nam, T.J. Anoikis induction by glycoprotein from Laminaria japonica in HT-29 cells. Prev. Med. 2011, 1, 49−61. Gouveia, V.; Seca, A.M.L.; Barreto, M.C.; Pinto, D.C.G.A. Di- and sesquiterpenoids from Cystoseira genus: Structure, intra-molecular transformations and biological activity. Mini- Rev. Med. Chem. 2013a, 13, 1150−1159. Gouveia, V.L.M.; Seca, A.M.L.; Barreto, M.C.; Neto, A.I.; Kijjoa, A.; Silva, A.M.S. Cytotoxic meroterpenoids from the macroalga Cystoseira abies-marina. Phytochemistry Lett. 2013b, 6, 593–597. Greiner, A.K.; Papineni, R.V.; Umar, S. Chemoprevention in gastrointestinal physiology and disease. Natural products and microbiome. Am. J. Physiol Gastrointest. Liver Physiol. 2014, 307, 1–15. Heo, S.J.; Kim, K.N.; Yoon, W.J.; Oh, C.; Choi, Y.U.; Affan, A.; Lee, Y.J.; Lee, H.S.; Kang, D.H. Chromene induces apoptosis via caspase-3 activation in human leukemia HL- 60 cells. Food Chem. Toxicol. 2011, 49, 1998–2004. Hiraoka, D.; Aono, R.; Hanada, S.; Okumura, E.; Kishimoto, T. Two new competing pathways establish the threshold for cyclin-B–Cdk1 activation at the meiotic G2/M transition. J. Cell. Sci. 2016, 129, 3153–3166. Hollborn, M.; Stathopoulos, C.; Steffen, A.; Wiedemann, P.; Kohen, L.; Bringmann, A. Positive feedback regulation between MMP-9 and VEGF in human RPE cells. Invest. Ophthalmol. Vis. Sci. 2007, 48, 4360–4367. Hood, J.D.; Cheresh, D.A. Role of integrins in cell invasion and migration. Nat. Rev. Cancer 2002, 2, 91–100. Huang, M.; Lu, J.J.; Huang, M.Q.; Bao, J.L.; Chen, X.P.; Wang, Y.T. Terpenoids: Natural products for cancer therapy. Expert Opin. Investig. Drugs 2012, 21, 1801−1818. _____________________________________________________________________5. Anticancer activity of AMTs on HT-29 cells! ! 107 Jiang, H.; Zhu, Y.; Zhou, Z.; Xu, J.; Jin, S.; Xu, K.; Zhang, H.; Sun, Q.; Wang, J.; Xu J. PRMT5 promotes cell proliferation by inhibiting BTG2 expression via the ERK signaling pathway in hepatocellular carcinoma. Cancer Med. 2018, 7, 869–882. Jung, S.K.; Jeong, C.H. Dehydroglyasperin D inhibits the proliferation of HT-29 human colorectal cancer cells through direct interaction with phosphatidylinositol 3-kinase. J. Cancer Prev. 2016, 21, 26–31. Kajanne, R.; Miettinen, P.; Mehlem, A.; Leivonen, S.K.; Birrer, M.; et al. EGFR regulates MMP function in fibroblasts through MAPK and AP-1 pathways. J. Cell Physiol. 2007, 212, 489–497. Kavousipour, S.; Khademi, F.; Zamani, M.; Vakili, B.; Mokarram, P. Novel biotechnology approaches in colorectal cancer diagnosis and therapy. Biotechnol. Lett. 2017, 39, 785–803. Kim, M.M.; Mendis, E.; Kim, S.K. Laurencia okamurai extract containing laurinterol induces apoptosis in melanoma cells. J. Med. Chem. 2008, 11, 260–266. Kuphal, S.; Bauer, R.; Bosserhoff, A.K. Integrin signaling in malignant melanoma. Cancer Metastasis Rev. 2005, 24, 195−222. Lakshmi, P.T.; Vajravijayan, S.; Moumita, M.; Sakthivel, N.; Gunasekaran, K.; Krishna, R. A novel guaiane sesquiterpene derivative, guai-2-en-10α-ol, from Ulva fasciata Delile inhibits EGFR/PI3K/Akt signaling and induces cytotoxicity in triple-negative breast cancer cells. Mol. Cell Biochem. 2018, 438,123–139. Leal, M.C.; Munro, M.H.G.; Blunt, J.W.; Puga, J.; Jesus, B.; Calado, R.; Rosa, R.; Madeira, C. Biogeography and biodiscovery hotspots of macroalgal marine natural products. Nat. Prod. Rep. 2013, 30, 1380–1390. Lee, J.I.; Park, B.J.; Kim, H.; Seo, Y. Isolation of Two New Meroterpenoids from Sargassum siliquastrum. Bull. Korean Chem. Soc. 2014, 35, 2867–2869. Li, Z.; Li, J.; Mo, B.; Hu, C.; Liu, H.; Qi, H.; Wang, X.; Xu, J. Genistein induces G2/M cell cycle arrest via stable activation of ERK1/2 pathway in MDA-MB-231 breast cancer cells. Cell. Biol. Toxicol. 2008, 24, 401–409. Lisiak, N.; Paszel-Jaworska, A.; Totoń, E.; Rubiś, B.; Pakuła, M.; Bednarczyk-Cwynar, B.; Zaprutko, L.; Rybczyńska, M. Semisynthetic oleanane triterpenoids inhibit migration and invasion of human breast cancer cells through downregulated expression of the ITGB1/PTK2/PXN pathway. Chem. Biol. Interact. 2017, 268, 136–147. Mayer, R.J. Targeted therapy for advanced colorectal cancer–more is not always better. New Engl. J. Med. 2009, 360, 623–625. Mingo-Sion, A.M.; Marietta, P.M.; Koller, E.; Wolf, D.M.; Van Den Berg, C.L. Inhibition of JNK reduces G2/M transit independent of p53, leading to endoreduplication, decreased proliferation, and apoptosis in breast cancer cells. Oncogene 2004, 23, 596–604. 5. Anticancer activity of AMTs on HT-29 cells___________________________________________________ ! 108 Moghadamtousi, S.Z; Karimian, H.; Rouhollahi, E.; Paydar, A.; Fadaeinasab, M.; AbdulKadi, H. Annona muricata leaves induce G1 cell cycle arrest and apoptosis through mitochondria-mediated pathway in human HCT-116 and HT-29 colon cancer cells. J. Ethnopharmacol. 2014, 156, 277–289. Newman, D.J.; Cragg, G.M. Marine-sourced anti-cancer and cancer pain control agents in clinical and late preclinical development. Mar. Drugs 2014, 12, 255-278 Newman, D.J.; Cragg, G.M. Natural products as sources of new drugs from 1981 to 2014. J. Nat. Prod. 2016, 79, 629-661. Nicoletti, I.; Migliorati, G.; Pagliacci, M.; Grignani, F.; Riccardi, C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods. 1991, 139, 271–279. Palmer, N.; Kaldis, P. Chapter One - Regulation of the Embryonic Cell Cycle During Mammalian Preimplantation Development. Curr. Top. Dev. Biol. 2016, 120, 1–53. Park, J.I. Growth arrest signaling of the Raf/MEK/ERK pathway in cancer. Front. Biol. (Beijing) 2014, 9, 95–103. Penicooke, N.; Walford, K.; Badal, S.; Delgoda, R.; Williams, L.A.D.; Joseph-Nathan, P.; Gordillo-Román, B.; Gallimore, W. Antiproliferative activity and absolute configuration of zonaquinone acetate from the Jamaican alga Stypopodium zonale. Phytochemistry 2013, 87, 96–101. Rocha, D.H.A.; Seca, A.M.L.; Pinto, D.C.G.A. Seaweed secondary metabolites in vitro and in vivo anticancer activity. Mar. Drugs 2018, 16, 410. Ruiz-Torres, V.; Encinar, J.A.; Herranz-López, M.; Pérez-Sánchez, A.; Galiano, V.; Barrajón-Catalán, E.; Micol, V. An updated review on marine anticancer compounds: the use of virtual screening for the discovery of small-molecule cancer drugs. Molecules 2017, 22, article 1037; doi:10.3390/molecules22071037. Shan, J.Z.; Xuan, Y.Y.; Zheng, S.; Dong, Q.; Zhang, S.Z. Ursolic acid inhibits proliferation and induces apoptosis of HT-29 colon cancer cells by inhibiting the EGFR/MAPK pathway. J. Zhejiang Univ. Sci. B. 2009, 10, 668−674. Shant, J.; Cheng, K.; Marasa, B.S; Wang, J.Y.; Raufman, J.P. Akt-dependent NF-kappaB activation is required for bile acids to rescue colon cancer cells from stress-induced apoptosis. Exp. Cell Res. 2009, 315, 432–450. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M.R.J. New colorimetric cytotoxicity assay for anticancerdrug screening. J. Natl. Cancer Inst. 1990, 82, 1107−1112. Suffness, M.; Pezzuto, J.M. Assays related to cancer drug discovery. In: Methods in Plant Biochemistry: Assays for Bioactivity, Hostettmann, K. (Ed.), Academic Press: London, _____________________________________________________________________5. Anticancer activity of AMTs on HT-29 cells! ! 109 1990; pp. 71–133. Sunasee, S.N.; Davies-Coleman, M.T. Cytotoxic and antioxidant marine prenylated quinones and hydroquinones. Nat. Prod. Rep. 2012, 29, 513-535. Tarhouni-Jabberi, S.; Zakraoui, O.; Ioannou, E.; Riahi-Chebbi, I.; Haoues, M.; Roussis, V.; Kharrat, R.; Essafi-Benkhadir, K. Mertensene, a halogenated monoterpene, induces G2/M cell cycle arrest and caspase dependent apoptosis of human colon adenocarcinoma HT29 cell line through the modulation of ERK-1/-2, AKT and NF-κB signaling. Mar. Drugs 2017, 15, E221.!doi: 10.3390/md15070221. Tournier, K. The 2 Faces of JNK signaling in cancer. Genes & Cancer 2013, 4, 397–400. Turner, N.A.; Aley, P.K.; Hall, K.T.; Warburton, P.; Galloway, S.; Midgley, L.; O’regan, D.J.; Wood, I.C.; Ball, S.G.; Porter, K.E. Simvastatin inhibits TNF-α induced invasion of human cardiac myofibroblasts via both MMP-9-dependent and -independent mechanisms. J. Mol. Cell Cardio. 2007, 43, 168–176. Urones, J.G.; Basabe, P.; Marcos, I.S.; Pineda, J.; Lithgow, A.M.; Moro, R.F.; Brito Palma, F.M.S.; Araujo, M.E.M.; Gravalos, M.D.G. Meroterpenes from Cystoseira usneoides. Phytochemistry 1992, 31, 179−182. Valls, R.; Piovetti, L. The chemistry of the Cystoseiraceae (Fucales: Pheophyceae): Chemotaxonomic relationships. Biochem. Syst. Ecol. 1995, 23, 723−745. Velatooru, L.R.; Baggu, C.B.; Janapala, V.R. Spatane diterpinoid from the brown algae, Stoechospermum marginatum induces apoptosis via ROS induced mitochondrial mediated caspase dependent pathway in murine B16F10 melanoma cells. Mol. Carcinog. 2016, 55, 2222–2235. Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutellingsperger, C. Anovel assay for apoptosis flow cytometric detection of phosphatidyl serine expression on early apoptotic cells using fluorescein labeled annexinV. J. Immunol. Methods 1995, 184, 39–51. Vizetto-Duarte, C.; Custódio, L.; Acosta, G.; Lago, J.H.G.; Morais, T.R.; Bruno de Sousa, C.; Gangadhar, K.N.; Rodrigues, M.J.; Pereira, H.; Lima, R.T.; Vasconcelos, M.H.; Barreira, L.; Rauter, A.P.; Albericio, F.; Varela, J. Can macroalgae provide promising antitumoral compounds? A closer look at Cystoseira tamariscifolia as a source for antioxidant and anti-hepatocarcinoma compounds. Peer J. 2016, 4, 1704, doi 10.7717/peerj.1704. Wang, S.; Basson, M.D. AKT directly regulates focal adhesion kinase through association and serine phosphorylation: implication for pressure-induced colon cancer metastasis. Am. J. Physiol. Cell Physiol. 2011, 300, 657–670. Williams, G.H.; Stoeber, K. The cell cycle and cancer. J. Pathol. 2012, 226, 352–364. Wu, W.S.; Wu, J.R.; Hu, C.T. Signal cross talks for sustained MAPK activation and cell migration: the potential role of reactive oxygen species. Cancer Metastasis Rev. 2008, 27, 5. Anticancer activity of AMTs on HT-29 cells___________________________________________________ ! 110 303–314. Yang, V.W. The Cell Cycle. Physiology of the Gastrointestinal Tract (Fifth Edition).! Academic Press:!USA, 2012; chapter 15, p.451–471. 111 6. Meroterpenoids from the brown alga Cystoseira usneoides as potential anti-inflammatory and anticancer agents On the basis of the anti-inflammatory properties of the extract of C. usneoides, the pharmacological screening of the isolated AMTs was continued by testing in vitro the profile of anti-inflammatory activity in stimulated THP-1 human macrophages. Moreover, the promising anticancer activity observed for the AMTs against colon cancer cells encouraged us to perform further studies in new cell models, specifically in human lung adenocarcinoma A549 cells. Taken together, these results could evidence the potential preventive role of the AMTs in the chronic inflammation and tissue damage for cancer development, including lung cancer. Abstract: The anti-inflammatory and anticancer properties of eight meroterpenoids isolated from the brown seaweed Cystoseira usneoides have been evaluated. The algal meroterpenoids (AMTs) 1-8 were tested for their inhibitory effects on the production of the pro-inflammatory cytokines tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), and the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in LPS-stimulated THP-1 human macrophages. The anticancer effects were assessed by cytotoxicity assays against human lung adenocarcinoma A549 cells and normal lung fibroblastic MRC-5 cells, together with flow cytometry analysis of the effects of these AMTs on different phases of the cell cycle. The AMTs 1-8 significantly reduced the production of pro-inflammatory cytokines TNF-α, IL-6, and IL- 1β, and suppressed the COX-2 and iNOS expression in LPS-stimulated cells (p<0.05). The AMTs 1-8 displayed higher cytotoxic activities against A549 cancer cells than against MRC-5 normal lung cells. Cell cycle analyses indicated that most of the AMTs caused the arrest of A549 cells at the G2/M and S phases. The AMTs 2 and 5 stand out by combining significant anti-inflammatory and anticancer activities while 3 and 4 showed interesting selective anticancer effects. These findings suggest that the AMTs produced by C. usneoides may have therapeutic potential in inflammatory diseases and lung cancer. ____________________________________________6. Anti-inflammatory/Anticancer activity on A549 cells " 118 of 0.1 mg/mL of propidium iodide was added to the cells and were incubated for 1 h at 4°C. The relative DNA content per cell was analyzed using a Bekhman Coulter FC500 flow cytometer (Cytomics, MPL, USA). The data acquisition was performed with the DML program. The analysis of the acquired data was performed with the CXP cytometer. 6.2.5. Statistical Analysis The results are presented as the mean ± Standard Error (SE) of at least three independent experiments. Data were evaluated with GraphPad Prism® Version 5.00 software. Differences between two groups were analyzed by the Student’s t-test. Difference with P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) were considered statistically significant. 6.3. Results The algal meroterpenoids (AMTs) usneoidone Z (1), 11-hydroxy-1’-O- methylamentadione (2), cystomexicone B (3), cystomexicone A (4), 6-cis-amentadione-1′- methyl ether (5), amentadione-1′-methyl ether (6), cystodione A (7), and cystodione B (8) (Figure 5.1) isolated from the alga C. usneoides have been investigated for their antiinflammatory and anticancer activities. 6.3.1. Anti-inflammatory activity Effects of AMTs 1-8 on the viability of THP-1 cells The cytotoxic effect of AMTs 1-8 on LPS-stimulated THP-1 macrophages was determined at different concentrations and incubation times (0–100 µg/mL, 48 and 72 h) using the SRB assay. The results of this analysis demonstrated that none of the molecules affect cell viability at concentrations up to 10 µg/mL for AMTs 1, 2, 3, 4, 7, 8, and up to 6 µg/mL for AMTs 5 and 6 (data not shown). Therefore, in order to rule out cytotoxic effects, compounds 1, 2, 3, 4, 7, and 8 were tested on THP-1 cells at maximum concentration of 8 µg/mL while 5 and 6 were tested at maximum concentration of 4 µg/mL. ____________________________________________6. Anti-inflammatory/Anticancer activity on A549 cells " 119 Effects of AMTs 1-8 on TNF-α, IL-6, and IL-1ß expression in LPS-stimulated THP-1 macrophages To determine the effects of the AMTs 1-8 on the production of TNF-α, IL-6, and IL- 1ß, THP-1 macrophaghes were pretreated with the compounds and then stimulated with LPS, as the triggering factor to stimulate the cytokines production. The levels of proinflammatory cytokines in the cell supernatants were determined using the ELISA kits. Upon comparison with control cells, TNF-α, IL-6, and IL-1ß levels were significantly increased in LPS stimulated cells up to 282.06, 389.47 and 181.8 ng/mL, respectively (Table 6.1). However, LPS-stimulated THP-1 macrophages pre-treated with the AMTs 1-8 showed a significant reduction of the production of pro-inflammatory cytokines (Table 6.1, Figure 6.2). Table 6.1. Inhibitory effects of AMTs 1-8 on the production of TNF-α, IL-6, and IL-1ß in LPS-stimulated THP-1 macrophages. Cytokines production (ng/mL) TNF-α IL-6 IL-1β Control 77.69±7.6 59.07±1.7 53.35±4.2 Control + LPS 282.06±2.1+++ 389.48±20.1++ 181.81±9.6+++ Dexamethasonea 55.58±6.9*** 28.18±2.3*** 105.94±5.7** AMTs: 1b 75.85±15.5** 112.16±16.1*** 46.25±2.6*** 2b 101.15±19.5** 74.75±14.4*** 28.30±5.7*** 3b 120.91±17.5** 120.04±2.7** 117.67±11.4* 4b 163.1±13.9** 221.97±8.1** 141.83±9.5 5c 125.96±7.3*** 125.3±12.1*** 25.46±2.4*** 6c 161.48±10.6** 191.07±5** 70.78±3.4** 7b 159.86±7.8** 196.42±10.1** 100.26±7.5** 8b 133.82±24.1** 160.18±18** 100.73±7.5* a 1 µg/mL; b 8 µg/mL; c 4 µg/mL; Values are the mean ± SE of triplicate experiments; +++ p < 0.001 vs Control; *p < 0.05, **p < 0.01, ***p < 0.001 vs Control+LPS. ____________________________________________6. Anti-inflammatory/Anticancer activity on A549 cells " 120 0 50 100 150 200 250 300 Control Control + LPS DEX 1 2 3 4 5 6 7 8 TNF-α production (ng/mL) Compound + LPS +++ *** *** ** ** ** ** ** ** ** A 0 50 100 150 200 250 300 350 400 450 Control Control + LPS DEX 1 2 3 4 5 6 7 8 IL-6 production (ng/mL) Compound + LPS *** *** *** ** *** ** ** ** ** ++ B 0 50 100 150 200 250 Control Control + LPS DEX 1 2 3 4 5 6 7 8 IL-11β production (ng/mL) Compound + LPS +++ ** ** ** *** *** *** * * C Figure 6.2. AMTs 1-8 inhibit LPS-induced expression of TNF-α, IL-6, and IL-1β in THP-1 macrophaghes (A, B and C) respectively. Cells were pretreated for 1 h with the AMTs (1, 2, 3, 4, 7 and 8 at 8 µg/mL; 5 and 6 at 4 µg/mL), followed by 24 h treatment with LPS. TNF-α (A), IL-6 (B) and IL-1β (C) contents in the culture medium were determined by ELISA. Data are expressed as means ± SE from three independent experiments. Statistical significance is indicated (+++p < 0.001 vs Control; *p < 0.05, **p < 0.01, ***p < 0.001 vs Control+LPS). ____________________________________________6. Anti-inflammatory/Anticancer activity on A549 cells " 121 Regarding to TNF-α, although all compounds induced a significant reduction of the level of this cytokine in THP-1, the meroditerpenes 1 and 2, at 8 µg/mL, showed the higher suppressive effect causing 73% and 64% inhibition (Table 6.1 and Figure 6.2A). Compounds 3, 5, and 8 also induced more than 50% of inhibition (57%, 55%, and 52%, respectively), while compounds 4, 6, and 7 were less active, reducing the production of TNF-α by 42-43% (P<0,01). As shown in Table 6.1 and in Figure 6.2B, among the eight AMTs, compound 2 markedly inhibited LPS-induced IL-6 production in THP-1 macrophages by 81% and compounds 1, 3, and 5 caused strong inhibitions of 71%, 69% and 67%, respectively. The treatment of cells with compounds 4, 6, 7, and 8 also significantly inhibited the production of IL-6 upon comparison with LPS-stimulated THP-1 control cells, although to a lesser extent (43%, 51%, 50% and 59%, respectively). With regard to IL-1ß production, the pretreatment of cells with the AMTs 1-8 resulted in significant inhibition of this cytokine (Table 6.1 and Fig. 6.2C). The most marked effects were observed in the cells treated with compounds 2 (8 µg/mL) and 5 (4 µg/mL), which blocked the effect of 1 µg/mL LPS by 84% and 86%, respectively. Moreover, pretreatment with the AMTs 1 (8 µg/mL) and 6 (4 µg/mL) also strongly inhibited LPS-induced IL-1ß production by 74% and 61%, respectively. The AMTs 3, 7, and 8 displayed inhibitory activity, causing IL-β decreases of 50%, 44%, and 35%, respectively. Effects of AMTs 1-8 on the expression of COX-2 and iNOS proteins in LPS-stimulated THP-1 cells COX-2 is the key enzyme regulating the production of prostaglandins, which are the central mediators of inflammation. On the other hand, iNOS enzyme represent an important molecular target closely involved in inflammatory responses. Thus, the effect of the AMTs 1-8 on LPS-induced COX-2 and iNOS protein expression was investigated by western blot analysis. As shown in Figure 6.3 and in Table 6.2, the expression of COX-2 and iNOS proteins was markedly augmented upon LPS treatment in THP-1 macrophages. The pretreatment with the AMTs 2, 3, 4, 5, 6, and 7 significantly down-regulated ____________________________________________6. Anti-inflammatory/Anticancer activity on A549 cells " 122 the expression of COX-2, while no significant effect was observed for compounds 1 and 8. The more active compounds were 3, 5, 6, and 7 which decreased COX-2 levels by 53, 53, 64, and 58 % respectively. On the other hand, all AMTs 1-8 effectively suppressed LPS- induced iNOS expression, decreasing iNOS levels in the range 40-54 %. iNOS β-actin 130 kDa 45 kDa LPS - + + + + + + + + + 1 2 3 4 5 6 7 8 COX-2 β-actin 72 kDa 45 kDa LPS - + + + + + + + + + 1 2 3 4 5 6 7 8 0 20 40 60 80 100 120 Control Control + LPS 1 2 3 4 5 6 7 8 COX-2 and iNOS Production (%) Meroterpenoids COX-2 iNOS +++ ** ++ ** * *** ** ** ** ** ** ** ** ** ** Figure 6.3. Effect of AMTs 1-8 on LPS-induced COX-2 and iNOS protein expression in THP-1 macrophages. Cells were pretreated for 1 h with the AMTs (1, 2, 3, 4, 7 and 8 at 8 µg/mL; 5 and 6 at 4 µg/mL) and then stimulated with LPS (1 µg/mL). Cytosolic lysates from 24 h-stimulated cells were separated on 10% SDS-PAGE. COX-2, iNOS, and β-actin were detected by western blot analysis. Data are expressed as means ± SE from three independent experiments. Statistical significance is indicated (+++ p < 0.001 and ++ p < 0.01 vs Control; *p < 0.05, **p < 0.01, ***p < 0.001 vs Control+LPS). ____________________________________________6. Anti-inflammatory/Anticancer activity on A549 cells " 123 Table 6.2. Inhibitory effects of AMTs 1-8 on the production of COX-2 and iNOS in LPS-stimulated THP-1 macrophages. Percentage of production COX-2 iNOS Control 34.26±7.7 35.95±2.29 Control + LPS 100.00±0.2+++ 100.00±7.04++ AMTs: 1a 83.29±11.37 53.96±2.15** 2a 69.18±8.68* 46.06±1.68** 3a 46.65±11.3* 52.95±7.31** 4a 54.16±3.63** 45.07±6.37** 5b 46.48±4.46** 59.69±8.4** 6b 35.77±2.5** 57.12±6.3** 7a 41.98±0.61** 56.77±2.57** 8a 88.61±3.59 45.91±8.19** a8 µg/mL; b4 µg/mL. Values are the mean ± SE of triplicate experiments; +++ p < 0.001 vs Control; *p < 0.05, **p < 0.01, ***p < 0.001 vs Control+LPS. 6.3.2. Anticancer activity Cytotoxic effects and selectivity of the AMTs 1-8 The effects of the AMTs 1-8 on cell survival was investigated in the human lung cancer cell line A549. The results, shown in Figure 6.4, indicated that all the AMTs inhibited the A549 cell growth after 72-h incubation in a dose-dependent manner. The most active compounds were 1, 2, 5 and 6 with IC50 values of 8.68, 6.61, 4.56, and 6.19 µg/mL, respectively (Table 6.3). Since the AMTs 1-8 showed an interesting cytotoxic activity towards A549 cells, the compounds were similarly evaluated in a normal cell line (human fetal lung fibroblastic MRC-5 cells) to determine the selectivity index (SI). ____________________________________________6. Anti-inflammatory/Anticancer activity on A549 cells " 124 Figure 6.4. Effects of different concentrations of AMTs 1-8 on the viability of human lung cancer cell line A549 and the human fetal lung fibroblastic MRC-5 cells, using MTT assay and after 72 h of treatment. Results are expressed as percentage of viable cells (% cell viability). Data are mean ± SE from three independent experiments. *p<0.05, **p<0.01 and ***p<0.01 compared with the untreated group. 0 20 40 60 80 100 120 0 5 10 15 20 25 % Cell viability Concentration (µg/ml) 11 MRC-5 A549 0 20 40 60 80 100 120 0 2 4 6 8 10 12 14 % Cell viability Concentration (µg/ml) 12 MRC-5 A549 * * ** 1 2 0 20 40 60 80 100 120 0 10 20 30 40 50 % Cell viability Concentration (µg/ml) 10 MRC-5 A549 *** ** 3 0 20 40 60 80 100 120 0 10 20 30 40 50 % Cell viability Concentration (µg/ml) 9 ! MRC-5 A549 ** ** 4 0 20 40 60 80 100 120 0 2 4 6 8 10 12 14 % Cell viability Concentration (µg/ml) 7 MRC-5 A549 *** * 0 20 40 60 80 100 120 0 2 4 6 8 10 12 14 % Cell viability Concentration (µg/ml) 8 MRC-5 A549 *** 5 6 0 20 40 60 80 100 120 0 5 10 15 20 25 % Cell viability Concentration (µg/ml) 1 MRC-5 A549 * * 0 20 40 60 80 100 120 0 5 10 15 20 25 % Cell viability Concentration (µg/ml) 2 MRC-5 A549 ** 7 8 ____________________________________________6. Anti-inflammatory/Anticancer activity on A549 cells " 125 Table 6.3. IC50 values (µg/mL) of AMTs 1-8 against the normal lung cells MRC-5 and the lung cancer cells A549, after 72 h of treatment." Compound Cell lines Selectivity Index MRC-5 A549 SI 1 10.64±0.50 08.68±0.15 1.22 2 08.65±0.18 06.61±0.38 1.30 3 51.91±2.76 24.42±1.71 2.12 4 58.38±3.30 21.00±1.79 2.78 5 08.39±0.14 04.56±0.21 1.83 6 08.87±0.05 06.19±0.25 1.43 7 14.46±1.14 09.31±0.33 1.55 8 17.83±0.09 13,96±0.55 1.28 SI = IC50 values for normal cells/ IC50 values for cancer cells. Data are means ± SE from three independent experiments." It has been reported that compounds with SI value higher or equal to 2.0 are potentially selective (Suffness and Pezzuto, 1990). According to the data in Table 6.3, all AMTs showed selective cytotoxicity against the cancer cells; especially compounds 3 and 4 which at increasing concentrations maintained higher toxicity against A549 cancer cells than against MRC-5 non-malignant cells (Figure 6.4). It is worth noting that after treatment with 50 µg/mL of these two AMTs the cell viability was lower than 15% for cancer cells and higher than 55% for normal cells. On the other hand, 1 was the less selective compound; its SI was 1.22. In view of the high incidence of harmful side effects induced by most existing anticancer drugs, the selective in vitro cytotoxicity exhibited by of 3 and 4 suggest that these compounds could be promising and interesting anticancer agents. Effects of AMTs 1-8 on A549 cell cycle progression We next investigated whether the treatment with AMTs 1-8 caused any cell cyclerelated event which inhibit the viability of A549 cell line. Thus, the cells were treated with the AMTs (at concentrations of IC50 (Table 6.3)) or with colchicine (at 0.2 µg/mL) for 24 h, and then subjected to flow cytometry analysis to evaluate the distribution of cells in the ____________________________________________6. Anti-inflammatory/Anticancer activity on A549 cells " 126 different phases of the cell cycle (Figure 6.5). Figure 6.5. Effects of AMTs 1-8 on cell cycle distribution of A549 cells. Cells were incubated with IC50 doses of AMTs for 24 h. (A) Cells were harvested to measure the cell cycle distribution by flow cytometry. (B) Quantitative analysis of cell cycle distribution after treatment with AMTs. Data represent mean ± SE from three independent experiments. **p<0.01 and ***p<0.01 compared with the untreated group. 0 10 20 30 40 50 60 70 80 90 100 Control Colchicine 1 2 3 4 5 6 7 8 Percentage Cell Population (%) Compounds SUB-G0 G0-G1 S G2/M ** ** * ** ** *** *** * * * * * * * * ** ** ** ** ** ** ** ** * * Control Colchicine 202_00010650 513.PRO : CXP Institution: Run Date: 02-May-12, 14:18:08 Protocol: 202_00010650 513.PRO Sample ID: Well 26 Listmode Replay: New Protocol User ID: anajavi Analysis Date: 03-May-2012, 12:56:37 Acquisition Time/Events: 54.1s / 10000 (UNKNOWN) Settings File: 00Ciclo Celular de Javi.PRO, 18-Apr-2012, 11:34:20 Listmode File: 26_00010638 501.LMD Instrument SN: AL01002 Software Version: CXP v2.2 (F1)[Ungated] 26_00010638 501.LMD : FS Lin/SS Lin - ADC C FS Lin SS Lin 0 1023 0 1023 (F1)[Ungated] 26_00010638 501.LMD : FL3 Lin/AUX - ADC H FL3 Lin AUX 0 1023 0 1023 (F1)[H] 26_00010638 501.LMD : FL3 Lin - ADC Apop G1 S G2M FL3 Lin 0 1023 200 (F1)[H] 26_00010638 501.LMD : FL3 Lin/SS Lin - ADC J I FL3 Lin SS Lin 0 1023 0 1023 01_00010606 476.PRO : CXP Institution: Run Date: 02-May-12, 14:01:20 Protocol: 01_00010606 476.PRO Sample ID: Well 01 Listmode Replay: New Protocol User ID: anajavi Analysis Date: 03-May-2012, 12:36:36 Acquisition Time/Events: 93.8s / 10000 (UNKNOWN) Settings File: 00Ciclo Celular de Javi.PRO, 18-Apr-2012, 11:34:20 Listmode File: 03_00010635 498.LMD Instrument SN: AL01002 Software Version: CXP v2.2 (F1)[Ungated] 03_00010635 498.LMD : FS Lin/SS Lin - ADC C FS Lin SS Lin 0 1023 0 1023 (F1)[Ungated] 03_00010635 498.LMD : FL3 Lin/AUX - ADC H FL3 Lin AUX 0 1023 0 1023 (F1)[H] 03_00010635 498.LMD : FL3 Lin - ADC Apop G1 S G2M FL3 Lin 0 1023 200 (F1)[H] 03_00010635 498.LMD : FL3 Lin/SS Lin - ADC J I FL3 Lin SS Lin 0 1023 0 1023 78 01_00010606 476.PRO : CXP Institution: Run Date: 02-May-12, 13:18:37 Protocol: 01_00010606 476.PRO Sample ID: Well 19 Listmode Replay: New Protocol User ID: anajavi Analysis Date: 03-May-2012, 12:47:05 Acquisition Time/Events: 107.2s / 10000 (UNKNOWN) Settings File: 00Ciclo Celular de Javi.PRO, 18-Apr-2012, 11:34:20 Listmode File: 19_00010622 492.LMD Instrument SN: AL01002 Software Version: CXP v2.2 (F1)[Ungated] 19_00010622 492.LMD : FS Lin/SS Lin - ADC C FS Lin SS Lin 0 1023 0 1023 (F1)[Ungated] 19_00010622 492.LMD : FL3 Lin/AUX - ADC H FL3 Lin AUX 0 1023 0 1023 (F1)[H] 19_00010622 492.LMD : FL3 Lin - ADC G1 S G2M Apop FL3 Lin 0 1023 200 (F1)[H] 19_00010622 492.LMD : FL3 Lin/SS Lin - ADC J I FL3 Lin SS Lin 0 1023 0 1023 01_00010606 476.PRO : CXP Institution: Run Date: 02-May-12, 13:07:11 Protocol: 01_00010606 476.PRO Sample ID: Well 13 Listmode Replay: New Protocol User ID: anajavi Analysis Date: 03-May-2012, 12:43:42 Acquisition Time/Events: 151.7s / 10000 (UNKNOWN) Settings File: 00Ciclo Celular de Javi.PRO, 18-Apr-2012, 11:34:20 Listmode File: 13_00010617 487.LMD Instrument SN: AL01002 Software Version: CXP v2.2 (F1)[Ungated] 13_00010617 487.LMD : FS Lin/SS Lin - ADC C FS Lin SS Lin 0 1023 0 1023 (F1)[Ungated] 13_00010617 487.LMD : FL3 Lin/AUX - ADC H FL3 Lin AUX 0 1023 0 1023 (F1)[H] 13_00010617 487.LMD : FL3 Lin - ADC G2M S G1 Apop FL3 Lin 0 1023 200 (F1)[H] 13_00010617 487.LMD : FL3 Lin/SS Lin - ADC J I FL3 Lin SS Lin 0 1023 0 1023 4 01_00011212 602.PRO : CXP Institution: Run Date: 03-Jul-12, 11:23:34 Protocol: 01_00011212 602.PRO Sample ID: Well 02 Listmode Replay: New Protocol User ID: anajavi Analysis Date: 03-Jul-2012, 12:15:15 Acquisition Time/Events: 42.6s / 10000 (UNKNOWN) Settings File: 00Ciclo Celular de Javi.PRO, 04-Jun-2012, 12:01:05 Listmode File: 02_00011213 603.LMD Instrument SN: AL01002 Software Version: CXP v2.2 (F1)[Ungated] 02_00011213 603.LMD : FS Lin/SS Lin - ADC C FS Lin SS Lin 0 1023 0 1023 (F1)[Ungated] 02_00011213 603.LMD : FL3 Lin/AUX - ADC H FL3 Lin AUX 0 1023 0 1023 (F1)[H] 02_00011213 603.LMD : FL3 Lin - ADC G1 G2M S Apop FL3 Lin 0 1023 250 (F1)[H] 02_00011213 603.LMD : FL3 Lin/SS Lin - ADC J I FL3 Lin SS Lin 0 1023 0 1023 3 01_00011212 602.PRO : CXP Institution: Run Date: 03-Jul-12, 11:22:12 Protocol: 01_00011212 602.PRO Sample ID: Well 01 Listmode Replay: New Protocol User ID: anajavi Analysis Date: 03-Jul-2012, 12:14:48 Acquisition Time/Events: 47.6s / 10000 (UNKNOWN) Settings File: 00Ciclo Celular de Javi.PRO, 04-Jun-2012, 12:01:05 Listmode File: 01_00011212 602.LMD Instrument SN: AL01002 Software Version: CXP v2.2 (F1)[Ungated] 01_00011212 602.LMD : FS Lin/SS Lin - ADC C FS Lin SS Lin 0 1023 0 1023 (F1)[Ungated] 01_00011212 602.LMD : FL3 Lin/AUX - ADC H FL3 Lin AUX 0 1023 0 1023 (F1)[H] 01_00011212 602.LMD : FL3 Lin - ADC G1 G2M S Apop FL3 Lin 0 1023 250 (F1)[H] 01_00011212 602.LMD : FL3 Lin/SS Lin - ADC J I FL3 Lin SS Lin 0 1023 0 1023 12 01_00010606 476.PRO : CXP Institution: Run Date: 02-May-12, 13:03:56 Protocol: 01_00010606 476.PRO Sample ID: Well 12 Listmode Replay: New Protocol User ID: anajavi Analysis Date: 03-May-2012, 12:44:12 Acquisition Time/Events: 158.6s / 10000 (UNKNOWN) Settings File: 00Ciclo Celular de Javi.PRO, 18-Apr-2012, 11:34:20 Listmode File: 12_00010616 486.LMD Instrument SN: AL01002 Software Version: CXP v2.2 (F1)[Ungated] 12_00010616 486.LMD : FS Lin/SS Lin - ADC C FS Lin SS Lin 0 1023 0 1023 (F1)[Ungated] 12_00010616 486.LMD : FL3 Lin/AUX - ADC H FL3 Lin AUX 0 1023 0 1023 (F1)[H] 12_00010616 486.LMD : FL3 Lin - ADC G2M S G1 Apop FL3 Lin 0 1023 200 (F1)[H] 12_00010616 486.LMD : FL3 Lin/SS Lin - ADC J I FL3 Lin SS Lin 0 1023 0 1023 01_00010606 476.PRO : CXP Institution: Run Date: 02-May-12, 13:21:00 Protocol: 01_00010606 476.PRO Sample ID: Well 20 Listmode Replay: New Protocol User ID: anajavi Analysis Date: 03-May-2012, 12:47:40 Acquisition Time/Events: 91.7s / 10000 (UNKNOWN) Settings File: 00Ciclo Celular de Javi.PRO, 18-Apr-2012, 11:34:20 Listmode File: 20_00010623 493.LMD Instrument SN: AL01002 Software Version: CXP v2.2 (F1)[Ungated] 20_00010623 493.LMD : FS Lin/SS Lin - ADC C FS Lin SS Lin 0 1023 0 1023 (F1)[Ungated] 20_00010623 493.LMD : FL3 Lin/AUX - ADC H FL3 Lin AUX 0 1023 0 1023 (F1)[H] 20_00010623 493.LMD : FL3 Lin - ADC G2M G1 S Apop FL3 Lin 0 1023 200 (F1)[H] 20_00010623 493.LMD : FL3 Lin/SS Lin - ADC J I FL3 Lin SS Lin 0 1023 0 1023 56 202_00010650 513.PRO : CXP Institution: Run Date: 02-May-12, 13:25:23 Protocol: 202_00010650 513.PRO Sample ID: Well 22 Listmode Replay: New Protocol User ID: anajavi Analysis Date: 03-May-2012, 12:54:38 Acquisition Time/Events: 112.3s / 10000 (UNKNOWN) Settings File: 00Ciclo Celular de Javi.PRO, 18-Apr-2012, 11:34:20 Listmode File: 22_00010625 495.LMD Instrument SN: AL01002 Software Version: CXP v2.2 (F1)[Ungated] 22_00010625 495.LMD : FS Lin/SS Lin - ADC C FS Lin SS Lin 0 1023 0 1023 (F1)[Ungated] 22_00010625 495.LMD : FL3 Lin/AUX - ADC H FL3 Lin AUX 0 1023 0 1023 (F1)[H] 22_00010625 495.LMD : FL3 Lin - ADC G1 S G2M Apop FL3 Lin 0 1023 200 (F1)[H] 22_00010625 495.LMD : FL3 Lin/SS Lin - ADC J I FL3 Lin SS Lin 0 1023 0 1023 01_00010606 476.PRO : CXP Institution: Run Date: 02-May-12, 14:03:29 Protocol: 01_00010606 476.PRO Sample ID: Well 02 Listmode Replay: New Protocol User ID: anajavi Analysis Date: 03-May-2012, 12:38:46 Acquisition Time/Events: 51.1s / 10000 (UNKNOWN) Settings File: 00Ciclo Celular de Javi.PRO, 18-Apr-2012, 11:34:20 Listmode File: 06_00010636 499.LMD Instrument SN: AL01002 Software Version: CXP v2.2 (F1)[Ungated] 06_00010636 499.LMD : FS Lin/SS Lin - ADC C FS Lin SS Lin 0 1023 0 1023 (F1)[Ungated] 06_00010636 499.LMD : FL3 Lin/AUX - ADC H FL3 Lin AUX 0 1023 0 1023 (F1)[H] 06_00010636 499.LMD : FL3 Lin - ADC G1 G2M S Apop FL3 Lin 0 1023 200 (F1)[H] 06_00010636 499.LMD : FL3 Lin/SS Lin - ADC J I FL3 Lin SS Lin 0 1023 0 1023 A B ____________________________________________6. Anti-inflammatory/Anticancer activity on A549 cells " 127 The results showed that most of the assayed AMTs induced a significant accumulation of cells at the G2/M phase (20-40%) and at the S phase (10-18%) of the cell cycle, with a parallel depletion of the percentage of cells in G0/G1 phase (49-65%) (Figure 6.5). The AMTs 1, 2, 7 and 8 were the most active and caused a remarkably increase of cells at G2/M phase (31.15, 31.62, 40.19, and 32.50%, p<0.01, respectively) compared with the control group (14.06 %) and at S phase (17.75, 17.90, 13.69, and 16.73%, p<0.01, respectively) compared with the control group (4.99 %). In parallel, a decrease of the population at G0/G1 phase was observed in the cells treated with these compounds (64.52, 65.42, 49.3, and 61.3%, p<0.01, respectively) in comparison with the control group (71.4 %). 6.4. Discussion As a part of our ongoing project aimed to evaluate the biomedical potential of marine natural products, in the present study we have demonstrated the in vitro antiinflammatory and anticancer activities of eight meroterpenoids (AMTs 1-8) isolated from the brown alga C. usneoides. First we investigated the anti-inflammatory effects of the AMTs 1-8 and their molecular mechanisms in LPS-induced THP-1 macrophages. The production of proinflammatory mediators including TNF-α, IL-6, and IL-1β by macrophages exposed to endotoxins is well established (Li et al., 2016; Rios et al., 2016). TNF-α plays a major role in initiating and regulating the release of adhesion molecules and the expression of inflammatory mediators during inflammatory responses (McCoy et al., 2011). IL-6 is a multifunctional cytokine that plays a role in inflammatory responses through the stimulation of acute phase responses, hematopoiesis, and immune reactions (Tanaka et al., 2014). Moreover, inhibition of IL-6 signaling has been successfully translated into the clinic as a powerful anti-inflammatory strategy (Baran et al., 2018). IL-1β is one of the most potent proinflammatory cytokines, which affects a large number of cellular responses and mediates inflammatory processes at local and systemic levels (Dinarello, 2018; Borthwick, 2016). These proinflammatory mediators can induce cell and tissue damage and also activate macrophages in various inflammation-associated diseases (Yoon et al., 2009).