Modulation of CAT-2B-Mediated l-Arginine Uptake and Nitric Oxide Biosynthesis in HCT116 Cell Line Through Biological Activity of 40-Geranyloxyferulic Acid Extract from Quinoa Seeds

International Journal of Molecular Sciences Article Modulation of CAT-2B-Mediated l-Arginine Uptake and Nitric Oxide Biosynthesis in HCT116 Cell Line Through Biological Activity of 40-Geranyloxyferulic Acid Extract from Quinoa Seeds Sara Franceschelli 1,* , Daniela Maria Pia Gatta 2, Mirko Pesce 1, Alessio Ferrone 2, JoséLuis Quiles 3, Salvatore Genovese 4, Francesco Epifano 4, Serena Fiorito 4, Vito Alessandro Taddeo 4, Antonia Patruno 2, Alfredo Grilli 1, Mario Felaco 2 and Lorenza Speranza 2,* 1 Department of Psychological, Health and Territorial Sciences, University G. D’Annunzio, 66100 Chieti, Italy 2Department of Medicine and Science of Aging, University G. D’Annunzio, 66100 Chieti, Italy 3Department of Physiology, Institute of Nutrition and Food Technology JoséMataix, Biomedical Research Centre, University of Granada, 18071 Granada, Spain 4Department of Pharmacy, University Gabriele D’Annunzio of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti Scalo (CH), Italy *Correspondence: [email protected] (S.F.); [email protected] (L.S.); Tel.: +39871-3554552 (S.F.); +39871-3554550 (L.S.); Fax: +39871-3554551 (S.F. & L.S.) Received: 3 May 2019; Accepted: 30 June 2019; Published: 2 July 2019   Abstract: Chenopodium quinoa Wild is a “pseudocereal” grain which attracts a lot of attention in the scientific community as it has a positive effect on health. Here, we investigate the presence of biologically active O-prenylated phenylpropanoids in the ethanol extract of commercially available quinoa seeds. We claim that 4 0 -Geranyloxyferulic acid (GOFA) was the only phytochemical product found that belongs to quinoa’s group secondary metabolites. We studied the changes in the oxidative and inflammatory status of the cellular environment in HCT 116 cell line processed with quinoa extract and its component GOFA; the implementation was done through the analysis of the antioxidant enzymes (SOD and CAT), the pro-inflammatory components (iNOS, IL-6 and TNFα ), and the products of intermediary metabolism (ONOO − , O 2− ). Moreover, the l-arginine uptake was proposed as a target of the tested compounds. We demonstrated that the GOFA, through a decrease of the CAT-2B expression, leads to a reduction of the l-arginine uptake, downregulating the harmful iNOS and restoring the altered redox state. These results propose a new molecular target involved in the reduction of the critical inflammatory process responsible for the cancer progression. Keywords: nitric oxide; l-arginine; oxidative stress; CAT-2B; inflammation; IL-6; TNF-α 1. Introduction The control of colorectal cancer (CRC) can be implemented by the potential of alternative therapies. In the past few years, plant extracts, which have anti-inflammatory, antioxidant, anti-proliferative, and anti-angiogenic properties, and are thus able to block, reverse, or prevent tumor growth, have been used as anticancer therapy [ 1 ]. The potential of nutraceutical compounds in reducing cancer progression has been largely explored. In fact, modern scientific researches have evidenced that a vast variety of medicinal aspects of plant extracts can be referred to the components, which can have a successful anticancer effect [2]. Chronic inflammation, accompanied by an imbalance of the cellular redox state, has been implicated as a potential biological mechanism in the progression of colorectal cancer in humans. Int. J. Mol. Sci. 2019,20, 3262; doi:10.3390/ijms20133262 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019,20, 3262 2 of 18 This condition leads to the release of inflammatory cytokines and chemokines which promote proliferation, angiogenesis, invasion, and metastasis, and facilitate tumor growth [ 3 ]. Moreover, inflammation is related to the formation of reactive oxygen and nitrogen species (ROS/RNS) which have been suggested as a mechanism underlying the pathophysiology of CRC [ 4 ]. In fact, the phlogistic mediators NO, and the pro-inflammatory cytokine TNF-alpha, can be considered as key mediators of inflammatory processes which activate angiogenic processes and determine the synthesis of different chemokines in the colonic mucosa of CRC patients [5,6]. Arginine is implicated in several biosynthetic pathways which significantly influence cancer cell proliferation and tumor biology. Arginine traverses the cell membrane via the specific CAT-family proteins (cell cationic amino acid transporters) [ 7 ]. CAT-2B is usually induced under inflammatory conditions in a variety of cells. The upregulated CAT-2B is the main arginine transporter for activated macrophages, which import large amounts of extracellular arginine for iNOS-derived NO synthesis. It would be interesting to study any possible involvement of this protein in the mechanism of cell exasperation induced by oxidative stress, which if prolonged might lead to inflammation and ultimately cancer [8,9]. A fundamental mechanism in changing gene expression, reducing oxidative damage, and decreasing inflammation is represented by the use of natural compounds [ 10 , 11 ]. Over the past two decades, an increased number of researches have been focused on non-essential components such as phytochemicals of quinoa due to their potential capacity in reducing the inflammatory process beyond nutritional functions provided by its components [12]. Chenopodium quinoa Wild has attracted interest in the scientific community due to its nutritional features. It is a “pseudocereal” grain, a gluten-free food, rich in macronutrients such as carbohydrates, fats, and proteins of high nutritional quality for the correct balance of the essential amino acids, and micronutrients such as vitamins and minerals [ 13 , 14 ]. We have demonstrated that Amaranthus retroflexus L. belongs to the Amaranthaceae family and contains the oxyprenylated phenylpropanoids, a rare class of biologically active natural products as 7-isopentenyloxycoumarin, auraptene, umbelliprenin, and 4 0 -geranyloxyferulic acid (GOFA) [ 15 ]. As C. quinoa belongs to the same taxonomic family we also investigated the presence of oxyprenylated phenylpropanoids in the ethanol extract of C. quinoa seeds using commercially available roasted seeds (which is world-wide the most popular edible form of this plant). Recently, we have reported that the dietary administration of an inclusion complex of GOFA with β -cyclodextrin or its co-drug, with a known NOS inhibitor (L-NAME), effectively suppresses colitis-associated colorectal carcinogenesis in the AOM/DSS mouse model reducing the levels of inflammation mediators and inhibiting cell proliferation by inducing apoptosis [ 16 , 17 ]. However, the underlying mechanisms are still unclear. In this study we investigated the possible effects C. quinoa components might have in the modulation of oxidative stress and phlogosis in the human colon cancer cell line HCT 116 focusing on l-arginine-related signaling which can lead to cancer progression. 2. Results The presence of oxyprenylated secondary metabolites was assessed using maceration for 96 h with EtOH. Using the gradient program already employed in the case of A. retroflexus [ 18 ], only GOFA was detected. The main retention times and other relevant parameters recorded for GOFA are summarized in Table 1. Conditions set for column purge and re-equilibration ensured stability for column pressure and chromatogram background. The dead retention time, calculated with uracil, was 1.83 min. LOD was assessed to be 0.1 µ g/mL. Calibration curves were linear over the range tested for pure analyte. The determination coefficient (r 2 ) was ≥ 0.9991. Bias values ranged from –5% to 5.8%. The back-calculated concentration value, obtained from the calibration curves, allowed calculating 0.3 µ L/mL as the validated LOQ. The weighting factor consisted of 1/x 2 values. The mean imprecision values (CVs) of the retention time were 0.5% with no statistical differences compared with the inaccuracy for the Int. J. Mol. Sci. 2019,20, 3262 3 of 18 normalized retentions time of the standard. Short-term stability of the analyte in vegetable matrix was studied under two experimental conditions: storage in an autosampler (20 ◦ C) for 15 h and after three freeze–thaw cycles. A representative HPLC chromatogram of quinoa roasted seeds ethanolic extract is presented in Figure 1A. Table 1. Mean linear calibration curve parameters obtained by weighted linear least-squares regression analysis of three independent twelve non-zero concentration point. Entry Linearity Range (µg/mL) Calibration Curve Weighting Factor Determination Coefficient (r2)Rt (min) * GOFA 1–100 Y =24142x +3054 1/x20.9993 41.77 * Rt =Retention time. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 21 Figure 1. (A) HPLC chromatogram of quinoa roasted seeds ethanolic extract. Peak with Rt of 26.2 min identifies 4’-geranyloxyferulic acid (GOFA). (B) Chemical structure of GOFA. (C) The effects of the quinoa extract (10–500 μg/mL) and GOFA (1–100 μM) on HCT116 cell viability, measured by the MTT assay (24 h). Data are expressed as the mean ± SEM of at least three independent experiments, performed in triplicate. 2.1. Quantification of GOFA in Roasted Quinoa Seeds Extracts The recorded content of GOFA in quinoa seeds ethanol extract was 2.01 ± 0.08 mg/g of dry plant. Analytical data for this isolated phytochemical were in full agreement with those previously reported for the same compound [15]. Although we used several other solvents to accomplish the extraction process, the best experimental conditions leading to the highest yields in GOFA consisted of performing maceration with only EtOH. In all other cases, yields were by far lower. The ethanolic extract of quinoa roasted seeds was thus selected to carry out biological assays, and as a comparison with the activity displayed with pure GOFA. Figure 1. ( A ) HPLC chromatogram of quinoa roasted seeds ethanolic extract. Peak with Rt of 26.2 min identifies 4 0 -geranyloxyferulic acid (GOFA). ( B ) Chemical structure of GOFA. ( C ) The effects of the quinoa extract (10–500 µ g/mL) and GOFA (1–100 µ M) on HCT116 cell viability, measured by the MTT assay (24 h). Data are expressed as the mean ± SEM of at least three independent experiments, performed in triplicate. Int. J. Mol. Sci. 2019,20, 3262 4 of 18 2.1. Quantification of GOFA in Roasted Quinoa Seeds Extracts The recorded content of GOFA in quinoa seeds ethanol extract was 2.01 ± 0.08 mg/g of dry plant. Analytical data for this isolated phytochemical were in full agreement with those previously reported for the same compound [ 15 ]. Although we used several other solvents to accomplish the extraction process, the best experimental conditions leading to the highest yields in GOFA consisted of performing maceration with only EtOH. In all other cases, yields were by far lower. The ethanolic extract of quinoa roasted seeds was thus selected to carry out biological assays, and as a comparison with the activity displayed with pure GOFA. 2.2. Quinoa and GOFA Effects on Cell Viability First, HCT116 cell viability after treatment with various concentrations of the quinoa extract (10, 25, 50, 100, 250 and 500 µ g/mL) and GOFA (1, 10 and 100 µ M) was performed. As shown in Figure 1C, quinoa extracts did not show cytotoxic effects in any of the different concentrations applied. Similarly, the results of MTT test performed with the GOFA showed that the tested concentrations were not toxic to the cells (1, 10 and 100 µM). 2.3. Quinoa and GOFA Effects on Redox Balance Inflammation leads to the production of reactive oxygen species (ROS), resulting in oxidative stress, activation of inflammatory response, and cellular damage [ 19 , 20 ]. The measure of ROS intracellular production represents a very useful parameter to quantify the ability of quinoa extract or GOFA in reducing oxidative stress. Therefore, the cytoprotective effects of quinoa extract and GOFA were also confirmed by NBT assay, in which the level of superoxide anion was measured. Quinoa extract and GOFA display an antioxidant effect on ROS production in a dose-dependent manner (Figure 2A). In HCT 116 cells, a significant reduction for ROS was obtained with a quinoa extract concentration of 100 µ g/mL (p<0.05). Instead, GOFA treatment showed a reduction of the ROS amount compared to the untreated cells, which became significant at 10 µ M (p<0.05). These preliminary data allowed obtaining a first observation of the concentration of quinoa extracts as well as GOFA, and these will be processed for further analysis. The doses of quinoa extract and GOFA used for all further analysis was 100 µg/mL and 10 µM respectively. The ability of GOFA to attenuate the oxidative stress caused by inflammation in cancer cells was confirmed by the analysis to detect the antioxidant enzymes activity of superoxide dismutase (SOD) and catalase which is responsible for scavenging metabolites generated by free radicals, in cell homogenates. The untreated cells exhibited a reduction in the activity of antioxidant enzymes ( p<0.05 , Figure 2B,C). Cells treatment with quinoa and GOFA promotes significant increase in the activity of these enzymes, 24 h post stimulation. However, the activity level of SOD in cells treated with quinoa and GOFA was significantly increased compared to the untreated cells (p<0.05; Figure 2B). Regarding catalase, the treated cells showed a weak activity compared to the untreated cells (p<0.01). Quinoa extract/GOFA-treatment resets the enzyme function which regained its normal metabolic and detoxifying function (p<0.05; Figure 2C). Thus, quinoa and GOFA both act in restoring the cellular turnover of antioxidant enzymes of the HTC 116. Int. J. Mol. Sci. 2019,20, 3262 5 of 18 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 21 Figure 2. The effect of quinoa extract and GOFA on reactive oxygen species (ROS) production and anti-oxidative enzyme activity in HCT116 cells. (A) ROS production was measured by NBT assay. (B) Superoxide dismutase (SOD) and (C) catalase activity were analyzed. The results are representative of three different assays. Data are expressed as the mean ± SD. *p < 0.05 vs. untreated cells. Figure 2. The effect of quinoa extract and GOFA on reactive oxygen species (ROS) production and anti-oxidative enzyme activity in HCT116 cells. ( A ) ROS production was measured by NBT assay. ( B ) Superoxide dismutase (SOD) and ( C ) catalase activity were analyzed. The results are representative of three different assays. Data are expressed as the mean ±SD. * p<0.05 vs. untreated cells. 2.4. Quinoa and GOFA Effects on iNOS/NO/3-Nitrotyrosine The expression of the inducible isoform of NOS and/or its catalytic activity increases in oxidative stress condition, as well as in inflammation. Thus, compounds that can selectively inhibit irregular expression of iNOS might be potential antioxidant and anti-inflammatory agents [ 21 ]. Our experimental Int. J. Mol. Sci. 2019,20, 3262 6 of 18 study evidenced an elevated mRNA and protein expression of iNOS in HTC 116 cells (Figure 3A,B). In this context, it is interesting to note that real-time data and immunoblotting, point out that quinoa extract and GOFA induced a significant down regulation of iNOS. Moreover, NO production was also significantly decreased in quinoa and GOFA treated cells (Figure 3C). Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 21 The ability of GOFA to attenuate the oxidative stress caused by inflammation in cancer cells was confirmed by the analysis to detect the antioxidant enzymes activity of superoxide dismutase (SOD) and catalase which is responsible for scavenging metabolites generated by free radicals, in cell homogenates. The untreated cells exhibited a reduction in the activity of antioxidant enzymes (p < 0.05, Figure 2B,C). Cells treatment with quinoa and GOFA promotes significant increase in the activity of these enzymes, 24 h post stimulation. However, the activity level of SOD in cells treated with quinoa and GOFA was significantly increased compared to the untreated cells (p < 0 .05; Figure 2b). Regarding catalase, the treated cells showed a weak activity compared to the untreated cells (p < 0.01). Quinoa extract/GOFA-treatment resets the enzyme function which regained its normal metabolic and detoxifying function (p < 0.05; Figure 2c). Thus, quinoa and GOFA both act in restoring the cellular turnover of antioxidant enzymes of the HTC 116. 2.4. Quinoa and GOFA Effects on iNOS/NO/3-Nitrotyrosine The expression of the inducible isoform of NOS and/or its catalytic activity increases in oxidative stress condition, as well as in inflammation. Thus, compounds that can selectively inhibit irregular expression of iNOS might be potential antioxidant and anti-inflammatory agents [21]. Our experimental study evidenced an elevated mRNA and protein expression of iNOS in HTC 116 cells (Figure 3 A,B). In this context, it is interesting to note that real-time data and immunoblotting, point out that quinoa extract and GOFA induced a significant down regulation of iNOS. Moreover, NO production was also significantly decreased in quinoa and GOFA treated cells (Figure 3C). Figure 3. The effects of quinoa and GOFA on iNOS/NO/3-NitroTyrosine signaling in HCT-116 cells. The effects of quinoa extract and GOFA on: (A) iNOS mRNA levels, (B) iNOS protein expression. (C) NO production. (D) 3-nitrotyrosine protein expression. Each bar represents mean ± SD (n = 3, *p < 0.01 vs. untreated cells). Figure 3. The effects of quinoa and GOFA on iNOS/NO/3-NitroTyrosine signaling in HCT-116 cells. The effects of quinoa extract and GOFA on: ( A ) iNOS mRNA levels, ( B ) iNOS protein expression. ( C ) NO production. ( D ) 3-nitrotyrosine protein expression. Each bar represents mean ± SD (n=3, *p<0.01 vs. untreated cells). Generally, NO and O 2•− , are observed at very low levels in the cellular microenvironment, when produced by constitutive enzymes. In altered redox state condition, if NO is produced by inducible isoform of nitric oxide synthase, it becomes highly reactive and might combine with O 2•− forming peroxynitrite (ONOO − ), which is a strong oxidant and a nitrating agent. High levels of peroxynitrite induce nitrosative stress, which is due to the nitration of many amino acids, like tyrosine and tryptophan [ 21 ]. The level of nitrosylated proteins indirectly represents peroxynitrite levels in vitro . As expected, the 3-NT formation increased remarkably in the HCT116 cells as revealed by immunoblotting analysis of cell homogenates, which were attenuated by quinoa and GOFA pretreatment (Figure 3D). 2.5. Effects of GOFA on the Expression of Cationic Amino Acid Transporters in HCT-116 Cells The formation of NO depends not only on the activity of iNOS, but also on the availability of its substrate arginine (Arg). As stated above, arginine is taken up into cells by cationic amino acid transporter (CAT) systems in the plasma membrane. To test this, we first evaluated the mRNA expression of the l-Arg transporter, CAT-2B, in HCT 116 cells with the presence or absence of quinoa extract or GOFA. As reported in Figure 4A,B, the addition of quinoa extract or GOFA decreased CAT-2B expression suggesting that l-Arg uptake via CAT-2B was modified in quinoa extract/GOFA-treated cells. Subsequently, to evaluate the functional activity of CAT-2B, we directly measured l-Arg uptake Int. J. Mol. Sci. 2019,20, 3262 7 of 18 through radioactivity incorporation in HTC-116 cells (Figure 4C). l-Arg uptake was significantly greater in untreated cells. In cells treated with quinoa extract or GOFA, the concentration of intracellular l-Arg was decreased comparing to untreated cells. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 9 of 21 Figure 4. The effect of quinoa extract and GOFA on L-arginine transport in HCT116 cells. (A) mRNA expression of the CAT-2B. (B) Representative Western blot and densitometric analysis of CAT-2B Figure 4. The effect of quinoa extract and GOFA on l-arginine transport in HCT116 cells. ( A ) mRNA expression of the CAT-2B. ( B ) Representative Western blot and densitometric analysis of CAT-2B expression. ( C ) ( 3 H) l-arginine uptake. Data are expressed as the mean ± SD. * p<0.05 vs. untreated cells. Int. J. Mol. Sci. 2019,20, 3262 8 of 18 2.6. Quinoa Extract and GOFA Effects on the Pro-Inflammatory Markers Given that ROS overproduction and antioxidant enzyme activities reduction play an important role in the inflammatory response in cancer cells, we measured the levels of cytokines as TNFα and IL-6 within HCT 116 cells using ELISA test (Figure 5). Cells were treated with QUINOA 100 µ g/mL and GOFA 10 µ M for a period of 24 h. We observed that TNFα and IL-6 increased in untreated cells. The Quinoa extract and GOFA attenuated the level of pro-inflammatory cytokines studied (TNF-αand IL-6). Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 10 of 21 expression. (C) (3H) L-arginine uptake. Data are expressed as the mean ± SD. *p < 0.05 vs. untreated cells. 2.6. Quinoa Extract and GOFA Effects on the Pro-Inflammatory Markers Given that ROS overproduction and antioxidant enzyme activities reduction play an important role in the inflammatory response in cancer cells, we measured the levels of cytokines as TNF-α and IL-6 within HCT 116 cells using ELISA test (Figure 5). Cells were treated with QUINOA 100 μg/mL and GOFA 10 μM for a period of 24 h. We observed that TNF-α and IL-6 increased in untreated cells. The Quinoa extract and GOFA attenuated the level of pro-inflammatory cytokines studied (TNF-α and IL-6). Figure 5. The effect of quinoa extract and GOFA on TNF-α and IL-6 production in HCT-116 cells. TNF-α (A) and IL-6 (B) levels were measured by ELISA. Values are means standard deviations; n = 3, * p < 0.05 vs. untreated cells. 2.7. Effect of CAT-2B Silencing on the L-Arg/iNOS Pathways Levels To assess whether the GOFA regulates iNOS by interfering with the intracellular concentration of L-arginine in HCT116 cells, the transporter CAT-2B was silenced with a siRNA specific for the coding sequence. Cells were transfected with scrambled or CAT-2B siRNA. Data presented in Figure 6A show the expression of CAT-2B transporter, evident in untreated cells, in comparison to cells transfected with CAT-2B siRNA in which it was abolished. The observed changes of gene expression were associated with changes in protein levels (Figure 6B). Furthermore, the cellular uptake of arginine decreased in cells silenced for CAT-2B compared to untreated ones (Figure 6C). Figure 5. The effect of quinoa extract and GOFA on TNFα and IL-6 production in HCT-116 cells. TNFα ( A ) and IL-6 ( B ) levels were measured by ELISA. Values are means standard deviations; n=3, * p<0.05 vs. untreated cells. 2.7. Effect of CAT-2B Silencing on the l-Arg/iNOS Pathways Levels To assess whether the GOFA regulates iNOS by interfering with the intracellular concentration of l-arginine in HCT116 cells, the transporter CAT-2B was silenced with a siRNA specific for the coding sequence. Cells were transfected with scrambled or CAT-2B siRNA. Data presented in Figure 6A show the expression of CAT-2B transporter, evident in untreated cells, in comparison to cells transfected with CAT-2B siRNA in which it was abolished. The observed changes of gene expression were associated with changes in protein levels (Figure 6B). Furthermore, the cellular uptake of arginine decreased in cells silenced for CAT-2B compared to untreated ones (Figure 6C). In order to verify whether GOFA acts as CAT-2B repressor in eliciting l-Arg uptake in HCT116 cells, we performed iNOS activity assay experiments (Figure 7A). First, the GOFA induced NO to decrease and it also decreased its selectivity towards the inducible isoform; this was confirmed by comparing it to the selective iNOS inhibitor, the W1400. When the cells were treated with a specific siCAT-2B we noted a reduction of iNOS activity compared to untreated cells, confirming that iNOS activation was dependent on CAT-2B inducted l-arginine uptake. In cells with poor expression of CAT-2B, treated with GOFA, we did not observe a significant change in iNOS activity compared to the cells treated only with GOFA. This data was confirmed by co-incubation, in silenced cells, with both the W1400 and GOFA. The data reported in Figure 7A suggest that GOFA effects are mediated by its ability to downregulate the expression of inducible CAT-2B transporter. To further confirm our hypothesis, cells were supplied with l-arginine 100 µ M, a concentration that can induce iNOS activity in untreated cells. The dose-response investigation showed that the levels of NO were significantly elevated. It is important to note that, in HTC116 cells with high enough levels of l-arginine and elevate expression of CAT-2B, the treatment with W1400 significantly reduced iNOS activity. We did not observe any variations in GOFA treated cells under the same experimental conditions. Int. J. Mol. Sci. 2019,20, 3262 9 of 18 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 11 of 21 Figure 6. The effect of quinoa extract and GOFA on CAT2B expression and L-arginine uptake in HTC116 transfected with CAT2 siRNA. (A) mRNA expression of the CAT2B. (B) Representative Figure 6. The effect of quinoa extract and GOFA on CAT2B expression and l-arginine uptake in HTC116 transfected with CAT2 siRNA. ( A ) mRNA expression of the CAT2B. ( B ) Representative Western blot and densitometric analysis of CAT 2B expression. ( C ) ( 3 H) l-Arg uptake by HCT116 cells. Data are expressed as the mean ± SD. * p<0.01 vs. untreated cells; # p<0.01 vs. relative control cells not treated with GOFA. Int. J. Mol. Sci. 2019,20, 3262 16 of 18 4.7. siRNA Transfection HCT116 cells (1 × 10 6 ) were seeded in a six-well plate. Fresh siRNA transfection reagent was prepared diluting first Lipofectamine RNAiMAX in Opti-MEM for 5 min and then mixed with an equal volume of Opti-MEM containing the siRNA (final concentration 25 nM). After 20 min of incubation, 200 µ L of the resulting RNAiMAX/siRNA was added directly onto the cells and cultured for 6 h. Then, the supernatant was removed and cells were further cultured with fresh complete growth medium for 12–1 h before any treatment with 1400W (50 µ M), l-arginine (150 µ M) and GOFA. qRT-PCR was used to confirm siRNA-mediated downregulation of the target gene. Control cells were without siRNA. Cell death was detected using MTT, showing no reduction of cell viability. siRNA for CAT-2 (sc-77441) was purchased from Santa Cruz Biotechnology. OptiMEM and Lipofectamine RNAiMax reagents were purchased from Invitrogen Life Technologies, USA. 4.8. Cytokine’s Levels Measurement Briefly, HCT-116 cells were pretreated with GOFA (10 µ M) and Quinoa (100 mg/mL) for 24 h. The supernatants were collected and assayed using the Searchligth Elisa kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Rockford, IL, USA). 4.9. Statistical Analysis All results were expressed as mean ± SD or SEM from three independent experiments. For statistical analyses, quantitative data were analyzed by Student t test for unpaired data between treated to non-treated cells. A probability of null hypothesis of <5% (p<0.05) was considered as statistically significant. Author Contributions: Conceptualization, L.S.; Investigation, D.M.P.G., A.F. and A.P.; Methodology, S.F., S.G., F.E., S.F., V.A.T. and A.P.; Project administration, A.G. and M.F.; Software, M.P.; Supervision, L.S.; Validation, J.L.Q., S.G., F.E., S.F. and V.A.T.; Visualization, J.L.Q., A.G. and M.F.; Writing—original draft, L.S.; Writing—review & editing, S.F. Funding: This research received no external funding. Acknowledgments: The Italian Ministry for University and Research is acknowledged for financial support. Conflicts of Interest: The authors declare no conflict of interest. References 1. Wang, H.; Khor, T.O.; Shu, L.; Su, Z.Y.; Fuentes, F.; Lee, J.H.; Kong, A.N. Plants vs. cancer: A review on natural phytochemicals in preventing and treating cancers and their druggability. Anticancer Agents Med. Chem. 2012,12, 1281–1305. [CrossRef] [PubMed] 2. Nobili, S.; Lippi, D.; Witort, E.; Donnini, M.; Bausi, L.; Mini, E.; Capaccioli, S. 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