Discovery of a small-molecule protein kinase Cd-selective activator with promising application in colon cancer therapy

Bessa e al.Cell Dea h and Disease (2018) 9:23 DOI 10.1038/s41419-017-0154-9 Cell Dea h & Disease ARTICLE Open Access Disco e y o a small-molecule p o ein kinase Cδ-selec i e ac i a o wi h p omising applica ion in colon cance he apy Cláudia Bessa 1 , Joana Soa es 1 , Liliana Raimundo 1 ,JoanaB.Lou ei o 1 , Célia Gomes 2 , Flá io Reis 2 ,MiguelL.Soa es 3 , Daniel San os 4 , Che na Du eja 5 ,SaumyaR.Chaudhu i 5 , Cyn hia Lopez-Habe 6 ,Ma celoG.Kazanie z 6 , Jo ge Gonçal es 7 , Ma ia F. Simões 8,9 , Pa ícia Rijo 8,9 and Lucília Sa ai a 1 Abs ac P o ein kinase C (PKC) isozymes play majo oles in human diseases, including cance . Ye , he poo unde s anding o isozymes-specific unc ions and he limi ed a ailabili y o selec i e pha macological modula o s o PKC isozymes ha e limi ed he clinical ansla ion o PKC- a ge ing agen s. He e, we epo he fi s small-molecule PKCδ-selec i e ac i a o , he 7α-ace oxy-6β-benzoyloxy-12-O-benzoyl oyleanone (Roy-Bz), which binds o he PKCδ-C1-domain. Roy- Bz po en ly inhibi ed he p oli e a ion o colon cance cells by inducing a PKCδ-dependen mi ochond ial apop o ic pa hway in ol ing caspase-3 ac i a ion. In HCT116 colon cance cells, Roy-Bz specifically igge ed he ansloca ion o PKCδbu no o he pho bol es e esponsi e PKCs. Roy-Bz caused a ma ked inhibi ion in mig a ion o HCT116 cells in a PKCδ-dependen manne . Addi ionally, he impai men o colonosphe e g ow h and o ma ion, associa ed wi h deple ion o s emness ma ke s, indica e ha Roy-Bz also a ge s d ug- esis an cance s em cells, p e en ing umo dissemina ion and ecu ence. No ably, in xenog a mouse models, Roy-Bz showed a PKCδ-dependen an i umo e ec , h ough an i-p oli e a i e, p o-apop o ic, and an i-angiogenic ac i i ies. Besides, Roy-Bz was non-geno oxic, and in i o i had no appa en oxic side e ec s. Collec i ely, ou findings e eal a no el p omising an icance d ug candida e. Mos impo an ly, Roy-Bz opens he way o a new e a on PKC biology and pha macology, con ibu ing o he po en ial edefini ion o he s uc u al equi emen s o isozyme-selec i e agen s, and o he e-es ablishmen o PKC isozymes as easible he apeu ic a ge s in human diseases. In oduc ion P o ein kinase C (PKC) is a amily o se ine- h eonine kinases g ouped based on hei dis inc egula ion in o “con en ional”(cPKCs α,βΙ,βΙΙ,γ), “no el”(nPKCs δ,ε,η, θ), and “a ypical”(aPKCs ι/λ,ζ). PKC isozymes sha e a conse ed N- e minal egula o y egion comp ising he C1- domain and C2-domain, and a C- e minal ca aly ic egion esponsible o ATP binding and phospho ans e ase ac i i y. The C1-domain is he pho bol es e and dia- cylglyce ol (DAG) binding si e in cPKCs and nPKCs, whe e i is duplica ed in andem (C1a and C1b). The C2-domain in cPKCs binds calcium, whe eas in nPKCs i is p ima ily a calcium-un esponsi e phospho- y osine binding mo i . On he o he hand, aPKCs bind nei he pho bol es e /DAG no calcium 1–3 . PKC isozymes a e in ol ed in he ac i a ion o © The Au ho (s) 2018 Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o heC ea i eCommons license, and indica e i changes we e made. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons license, unless indica ed o he wise in a c edi line o he ma e ial. I ma e ial is no included in he a icle’s C ea i e Commons license and you in ended use is no pe mi ed by s a u o y egula ion o exceeds he pe mi ed use, you will need o ob ain pe mission di ec ly om he copy igh holde . To iew a copy o his license, isi h p://c ea i ecommons.o g/licenses/by/4.0/. Co espondence: Pa ícia Rijo (p1609@uluso ona.p ) o Lucília Sa ai a ([email p o ec ed]) 1 UCIBIO/REQUIMTE, Labo a ó io de Mic obiologia, Depa amen o de Ciências Biológicas, Faculdade de Fa mácia, Uni e sidade do Po o, Po o, Po ugal 2 Labo a o y o Pha macology and Expe imen al The apeu ics, Ins i u e o Biomedical Imaging and Li e Sciences (IBILI), Facul y o Medicine, & CNC.IBILI Resea ch Conso ium, Uni e si y o Coimb a, Coimb a, Po ugal Full lis o au ho in o ma ion is a ailable a he end o he a icle Edi ed by M. Daugaa d O ficial jou nal o he Cell Dea h Di e en ia ion Associa ion 1234567890 1234567890 mul iple signaling pa hways, and hence hey a e ecognized he apeu ic a ge s o se e al human diseases. Thei wide- anging e ec s in c ucial p ocesses o umo igenesis and me as a ic dissemina ion jus i y he e o s o de elop PKC- a ge ed d ugs o cance ea men . Ye , he pha macolo- gical modula ion o PKCs in an icance he apy has p o ed gene ally ine ec i e in clinical ials. This can be explained by he complex biological unc ions egula ed by PKC iso- zymes, bo h edundan and opposi e, and hei significan exp ession he e ogenei y in di e en cance ypes. Un o - una ely, he e is a e y limi ed a ailabili y o isozyme- selec i e PKC modula o s, which ep esen a majo limi a ion o achie ing he apeu ic success. As such, he gene a ion o isozyme-selec i e PKC modula o s has been o high p io i y. Howe e , his has been a majo challenge om a pha macological s andpoin , as PKCs a e highly ela ed among hem, as well as o o he s uc u ally ela ed kinases 1–3 . Whe eas PKCs ha e been gene ally iewed as oncogenic kinases, se e al labo a o ies ha e es ablished ha PKC ac i i y is o en los in cance due o loss-o - unc ion mu a ions, hus suppo ing a po en ial unc ion o PKC isozymes as umo supp esso s a he han umo p o- mo e s 3 . Mo eo e , specific PKC isozymes induce an i- p oli e a i e o p o-apop o ic e ec s in cance cells upon ac i a ion 4–7 . This led esea che s o belie e ha cance he apies should be ocused on es o ing a he han inhi- bi ing hei ac i i y, and may jus i y he ine ec i eness o mo e han h ee decades o clinical ials using non-selec i e PKC inhibi o s. The e o e, he selec i e PKC ac i a ion may be a aluable he apeu ic s a egy in cance ea men 3–7 . Fo long, PKC signaling has been closely associa ed wi h in es inal ca cinogenesis 5,8,9 , one o he mos p e alen cance s and a leading cause o cance mo ali y wo ld- wide 10 . Pa icula ly, clinical da a ha e e ealed educed p o ein le els o PKCδin colon cance issues compa ed o no mal issues 11 . In ac , se e al s udies co obo a ed he no ion ha PKCδbeha es as umo supp esso in colon cance 11–15 . The e o e, besides hei po en ial applica ion in o he cance ypes, i is highly concei able ha PKCδ-selec i e ac i a o s may be he apeu ically beneficial o pa ien s wi h colon cance . He e, we iden- ified he compound 7α-ace oxy-6β-benzoyloxy-12-O- benzoyl oyleanone (Roy-Bz) as a PKCδ-selec i e ac i a o wi h p omising a ge ed an icance ac i i y in colon can- ce . Impo an ly, Roy-Bz may also ep esen a aluable he apeu ic al e na i e in o he human pa hologies in ol ing an impai men o PKCδ-signaling pa hway. Resul s Roy-Bz is a selec i e ac i a o o PKCδ ha binds o he C1-domain In o de o iden i y no el PKC ac i a o agen s, se e al na u al di e penoids isola ed om plan species belonging o he Lamiaceae amily and semi-syn he ic de i a i es we e es ed using a p e iously de eloped yeas PKC sc eening assay 16 . In his assay, PKC ac i a o s induce a significan g ow h inhibi ion in mammalian PKC- exp essing yeas , which is p opo ional o he deg ee o PKC ac i a ion, ha ing no e ec on con ol yeas (emp y ec o ) 16 . Using his app oach, i was obse ed ha whe eas 0.1–30 μM pho bol 12-my is a e 13-ace a e (PMA; an es ablished ac i a o o cPKCs and nPKCs) inhibi ed he g ow h o yeas -exp essing cPKCs (αand βI) and nPKCs (δand ε), 0.1–30 μM o Roy-Bz (Fig. 1a) only inhibi ed he g ow h o PKCδ-exp essing yeas wi hou significan e ec s on yeas exp essing o he PKCs (Fig. 1b). In e es ingly, in PKCδ-exp essing yeas , while PMA- induced g ow h inhibi ion was associa ed wi h G2/M- phase cell cycle a es , Roy-Bz-induced g ow h inhibi ion was media ed by apop o ic cell dea h, as demons a ed by he inc ease in DNA agmen a ion wi h p ese a ion o plasma memb ane in eg i y (Fig. 1d, e). Addi ionally, Roy- Bz-induced g ow h inhibi ion was comple ely abolished in yeas exp essing a PKCδin which he C1-domain has been dele ed (ΔC1-PKCδ; Fig. 1c) (Fig. 1b). In ac , a simila esul was ob ained wi h PMA, which is known o bind PKCs a he C1-domain (Fig. 1b). These esul s indica e ha Roy-Bz is a PKCδ-selec i e ac i a o and, like PMA, i should ha e he C1-domain as he p edic ed binding si e. To confi m he PKC isozyme selec i i y obse ed in he yeas assay, we nex pe o med an in i o kinase assay using ecombinan human cPKCs (a mix o PKCα,β,andγ), nPKCδ,nPKCε,andaPKCζ. All ecombinan PKCs we e e ec i ely ac i a ed a 10 −4 µM PMA/a achidonic acid (ARA) (posi i e con ols: PMA o cPKCs and nPKCs; ARA o aPKCζ)(Fig.1 ). Rema kably, 10 −7 –10 −3 μMRoy-Bz only inc eased he ac i i y o PKCδ(Fig. 1g). Roy-Bz ac i- a ed PKCδwi h an EC 50 (concen a ion equi ed o induce 50% e ec ) alue o 58.8 ±3.6 nM (n=5), simila o ha o PMA (61.9 ±4.3 nM, n=5). Thus, he in i o kinase assays, in acco dance wi h he yeas assays, e ealed a ema kable selec i i y o Roy-Bz o PKCδ. Besides, hese esul s show ha Roy-Bz is as e ec i e as PMA o ac i a e PKCδ. Nex , he po en ial binding mode o Roy-Bz o PKCδ was explo ed by molecula docking s udies (Fig. 1h, i). Like 13-ace ylpho bol (PRB; posi i e con ol), which es ablishes di ec s ong hyd ogen bond in e ac ions wi h Gly253 and Th 242 (hyd ogen bond dono s), and Leu251 (hyd ogen bond accep o ) in he C1-domain o PKCδ, Roy-Bz also in e ac ed wi h Gly253 and Th 242, bu exchanged Leu251 wi h Gln257, all ac ing as hyd ogen dono s o ca bonyl oxygen unc ional g oups. I is in e - es ing o no e ha like in he case o PRB, hese in e ac- ions also s abilized he molecule om bo h sides o he cle . The es ima ed au odock ee ene gy o binding o PRB was −7.54 kcal/mol and o Roy-Bz was −7.87 kcal/ mol, and a clus e ing analysis o he molecula docking Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 2 o 16 O ficial jou nal o he Cell Dea h Di e en ia ion Associa ion Fig. 1 (See legend on nex page.) Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 3 o 16 O ficial jou nal o he Cell Dea h Di e en ia ion Associa ion esul s showed o he h ee poses simila o he op- anked ( he mos popula ed one; in a o al o 10 poses and 6 di e en clus e s). These p edic ed binding models sup- po he Roy-Bz binding o he PKCδ-C1-domain. The ansloca ion o PKC om he cy osol o mem- b anes, ei he plasma memb ane o in e nal memb anes, is a well-known hallma k o PKC ac i a ion 1,17 . As such, we e alua ed he impac o Roy-Bz on he ansloca ion o (see figu e on p e ious page) Fig. 1 Roy-Bz selec i ely ac i a es PKCδ h ough binding o he C1-domain. a Chemical s uc u e o Roy-Bz. bDose– esponse cu es o he inhibi o y e ec o PMA and Roy-Bz on he g ow h o yeas exp essing mammalian PKC isozymes and con ol yeas (emp y ec o ), o 42 h ea men ; g ow h o yeas incuba ed wi h ehicle was se as 100%. cSchema ic ep esen a ion o dele ion o C1-domain a amino acids 231–283 in he PRKCD gene. d,eE ec o 10 μMPMAandRoy-Bzon(d) cell cycle p og ession and (e) DNA agmen a ion (TUNEL+)/loss o plasma memb ane in eg i y (PI+) o PKCδ- exp essing yeas (and con ol yeas in (e)), o 42 h ea men . ,gIn i o kinase assay wi h ecombinan PKCs; inc ease o PKC ac i i y by 10 −4 μMPMA/ ARA and 10 −4 μMRoy-Bz;gdose– esponsecu es o heinc easeo PKCac i i y by Roy-Bz; kinase ac i i y o endogenous PKC ac i a o phospha idylse ine was se as 1; wo-way analysis o a iance (ANOVA) (p< 0.01). h,iMolecula docking s udies o Roy-Bz in e ac ion wi h he C1-domain o PKCδ.hTop anked docking pose o Roy-Bz wi h Gly253, Th 242, and Gln257 highligh ed h ough a CPK ep esen a ion. The hyd ogen bonds be ween Roy-Bz and hese esidues a e also displayed h ough a do ed blue line. The in e ac ion be ween he alpha-hyd ogen o T p252 and he ca bonyl (CH•••O) is also ep esen ed and he esidue is highligh ed in lico ice. All esidues wi hin 4.5 Å o Roy-Bz a e ep esen ed and he molecula su ace o he pocke is displayed h ough a mesh ha changes om g een in lipophilic egions o pink (hyd ophilic). iScheme o PRB and Roy-Bz in e ac ions. The pocke p oximi y con ou is ep esen ed by g ay do ed lines, he ligand exposu e is depic ed in iole and he ecep o exposu e in blue. Di ec in e ac ions a e ep esen ed by a do ed o g een line along wi h he in e ac ion ene gy (kcal/mol) es ima ed by MOE. In b,d– , da a a e mean ± SEM o fi e independen expe imen s; alues significan ly di e en om con ol yeas b,eo ehicle d, :*p < 0.05, **p < 0.01, unpai ed S uden ’s - es Fig. 2 Roy-Bz selec i ely ansloca es PKCδ o he pe inuclea egion in human umo cells. HCT116 cells ans ec ed wi h pEGFP-N1-PKCα, pEGFP-N1-PKCδ,o pEGFP-N1-PKCεwe e ea ed wi h PMA, Roy-Bz, o ehicle o 1 h; PKC localiza ion was de e mined by con ocal mic oscopy. aImages a e ep esen a i e o h ee independen expe imen s; scale ba = 10 µm and magnifica ion = ×100. bQuan ifica ion o cells wi h PKC ansloca ion; da a a e mean ± SEM o h ee independen expe imen s; alues significan ly di e en a e indica ed (***p< 0.001), unpai ed S uden ’s - es Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 4 o 16 O ficial jou nal o he Cell Dea h Di e en ia ion Associa ion Fig. 3 (See legend on nex page.) Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 5 o 16 O ficial jou nal o he Cell Dea h Di e en ia ion Associa ion PKCα, PKCδ, and PKCεin human HCT116 colon cance cells using GFP- used PKCs (Fig. 2a, b). As p e iously epo ed in LNCaP p os a e cance cells 18 , also in HCT116 cells he ea men wi h PMA igge ed he subcellula edis ibu ion o he h ee PKC isozymes. The e was a clea ansloca ion o he plasma memb ane o PKCα, PKCδand PKCε, and as epo ed in o he models 18 , PKCδalso ansloca ed o a pe inuclea com- pa men . When we assessed he abili y o Roy-Bz o ansloca e PKCs, he e we e ob ious di e ences wi h ega d o PMA. Indeed, Roy-Bz was unable o ansloca e PKCαo PKCε, while i selec i ely ansloca ed PKCδ o he pe inuclea egion (Fig. 2a, b). A dose-dependen analysis o PKCδ ansloca ion in HCT116 cells e ealed an EC 50 alue o 0.19 ±0.07 μM(n=3). Al oge he , hese esul s s ongly indica e ha Roy-Bz is a selec i e PKCδac i a o , ac ing a he C1-domain. Roy-Bz inhibi s he p oli e a ion o colon cance cells The e ec o Roy-Bz on p oli e a ion o human colo - ec al cance cells was e alua ed using a sul o hodamine B (SRB) assay (Fig. 3a, b). T ea men o HCT116, HT-29, and SW-837 cells (wi h simila PKCδexp ession le els; Sup- plemen a y Fig. S1) wi h Roy-Bz o 48 h esul ed in a dose-dependen inhibi ion o cell g ow h (IC 50 alues o 0.58 ±0.05 μM o HCT116, 1.50 ±0.06 μM o HT-29, and 1.08 ±0.03 μM o SW-837; n=5). The p onounced inhibi o y e ec o Roy-Bz on cell p oli e a ion/ iabili y o colo ec al cance cells was u he demons a ed by assessing he colony- o ming abili y (Fig. 3c). In ac , e en in he mos esis an SW-837 cells, 0.3 μM Roy-Bz educed he colony- o ming abili y by ~75% ela i e o ehicle. In e es ingly, while in HT-29 and SW-837 cells he Roy- Bz g ow h inhibi o y e ec was associa ed wi h G2/M- phase cell cycle a es (Fig. 3d; pa icula ly p onounced in HT-29) and apop osis (Fig. 3e), in HCT116 cells he Roy- Bz-induced g ow h inhibi ion was only media ed by apop osis. In ac , Roy-Bz had no e ec on cell cycle p og ession (Fig. 3d; simila esul s we e ob ained o 8, 16, and 48 h ea men , da a no shown) and ma kedly inc eased Annexin V-posi i e cells (Fig. 3e). Con e sely, in HCT116 cells, PMA, wi h an IC 50 alue abou 14- old highe han ha o Roy-Bz (7.83 ±1.67 μM, n=5; Fig. 3a), inhibi ed cell g ow h h ough induc ion o S-phase cell cycle a es and apop osis (Supplemen a y Fig. S2). In HCT116 cells, he induc ion o apop osis by Roy-Bz was u he ein o ced by he occu ence o caspase-3 and PARP clea age, an inc ease in p o-apop o ic p53 and Bax le els, and a educ ion in he le els o an i-apop o ic p o eins Bcl-2 and su i in (Fig. 3 , g). The in ol emen o he mi ochond ial pa hway in Roy-Bz-induced apop osis was also e idenced by he inc ease o mi ochond ial eac i e oxygen species (ROS) gene a ion and Δψ m dis- sipa ion (Fig. 3h), as well as he elease o cy och ome c (cy c) o cy osol (Fig. 3i). In an a emp o explo e he an i umo ac i i y o Roy- Bz in a sys em ha mo e closely esembles he in i o ea u es o he umo mic oen i onmen and is highly en iched in a small popula ion o cance s em cells (CSCs), a colonosphe e cul u e model was gene a ed om HCT116 cells. In ac , he sphe oid- o ma ion (colono- sphe e) assay is ecognized as a aluable ool o assess- men and expansion o s em cells in colon cance 19 .In addi ion, acco ding o se e al s udies, colon CSCs can mainly be iden ified by he exp ession o cell bioma ke s, such as CD44 and ALDH 20–23 . Ac ually, a posi i e co - ela ion be ween he CD44 dec ease and he he apeu ic esponse o pa ien s wi h colo ec al cance has been ecen ly iden ified 22,23 . To es he e ec o Roy-Bz in colonosphe e g ow h, 3-day-old sphe oids we e ea ed wi h Roy-Bz o up o 96 h (Fig. 3j). A significan educ- ion in colonosphe e diame e by Roy-Bz ela i e o ehicle was obse ed bo h a 72 h (a 3 and 3.5 μM) and 96 h (a 2–3.5 μM). Acco dingly, 3.5 μM Roy-Bz ma kedly educed he exp ession le els o he s emness ma ke s CD44 and ALDH2 a e 96 h ea men (Fig. 3k). O no e, a p onounced educ ion o ma ix me allop o einase 9 (MMP-9) and inc ease o E-cadhe in, p ominen playe s (see figu e on p e ious page) Fig. 3 Roy-Bz inhibi s he g ow h o colon cance cells. a,bDose– esponse cu es o he g ow h o (a) HCT116, (b) HT-29 and SW-837 cells ea ed wi h 0.11–10 μM Roy-Bz o PMA o 48 h; da a a e mean ± SEM o fi e independen expe imen s; g ow h ob ained wi h ehicle was se as 100%. cColony o ma ion assay o HCT116, HT-29, and SW-837 cells ea ed wi h Roy-Bz; images co espond o a ep esen a i e expe imen o ou ; g aphs ep esen mean ± SEM o ou independen expe imen s. dCell cycle a es and eAnnexin V-posi i e cells we e de e mined o 24 h ea men wi h 1 μM Roy-Bz; da a a e mean ± SEM o ou independen expe imen s. ,gWes e n blo analysis o ( ) caspase-3 and (g) PARP clea age, Bax, p53, Bcl-2, and su i in exp ession le els, o 24 h ea men wi h Roy-Bz in HCT116 cells. hMi ochond ial ROS gene a ion ( o 24 h) and Δψ m dissipa ion ( o 16 h) igge ed by 0.5 μM Roy-Bz ea men in HCT116 cells; da a a e mean ± SEM o ou independen expe imen s. iMi ochond ial cy c elease in HCT116 cells o 24 h ea men wi h Roy-Bz. jE alua ion o 3-day-old HCT116 sphe oids diame e o 72 and 96 h ea men wi h Roy- Bz o ehicle. kCD44, ALDH2, MMP-9, and E-cadhe in exp ession le els o 96 h ea men wi h 3.5 μM Roy-Bz. lE alua ion o sphe oids diame e o med a e 48 h ea men o seeded HCT116 cells wi h Roy-Bz o ehicle. In jand l, b igh field imaging o sphe oids (scale ba = 50 μm, magnifica ion = ×100); g aphs ep esen mean ± SEM o ou independen expe imen s. In ,g,iand kimmunoblo s ep esen one o h ee independen expe imen s; loading con ol o cy osolic (GAPDH) and mi ochond ial (COX IV) ac ions. In c,d,e,h,jand l, alues significan ly di e en om ehicle (*p < 0.05, **p < 0.01, ***p < 0.001), unpai ed S uden ’s - es Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 6 o 16 O ficial jou nal o he Cell Dea h Di e en ia ion Associa ion in ex acellula ma ix homeos asis and me as asis 24 , we e also obse ed unde hese ea men condi ions (Fig. 3k). The e ec i eness o Roy-Bz in colonosphe e o ma ion was also checked by e alua ing he colonosphe e diame e a e 48 h ea men wi h Roy-Bz added a he seeding ime o HCT116 cell suspension (Fig. 3l). The esul s Fig. 4 (See legend on nex page.) Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 7 o 16 O ficial jou nal o he Cell Dea h Di e en ia ion Associa ion showed a no able dose-dependen educ ion in colono- sphe e o ma ion abili y by Roy-Bz, wi h an abolishmen o colonosphe e o ma ion a 1 μM Roy-Bz. Al oge he , hese esul s e idence a po en inhibi o y ac i i y o Roy-Bz in he g ow h o colon cance cells, including in CSCs, sugges ing a po en ial e ec o he d ug agains cance chemo esis ance, dissemina ion, and ecu ence. Roy-Bz p o-apop o ic and an i-mig a o y ac i i y in HCT116 cance cells is media ed by PKCδ-selec i e ac i a ion To e alua e he in ol emen o PKCδin Roy-Bz- induced apop osis, a PKCδs able knockdown model was gene a ed upon ans ec ion o he me as a ic colon cance HCT116 cells wi h a PKCδRNAi plasmid. As con ol, we used HCT116 cells ans ec ed wi h non- a ge con ol RNAi. PKCδsilencing was confi med by Wes e n blo (Supplemen a y Fig. S3). The impac o 0.1–6μM Roy-Bz on he iabili y o PKCδ-deple ed and con ol HCT116 cells was he ea e e alua ed by a ypan blue assay. As shown in Fig. 4a, silencing PKCδdec eased Roy-Bz-induced cell dea h compa ed o con ol HCT116 cells. Acco dingly, he deple ion o PKCδin HCT116 cells abolished Roy-Bz-induced Annexin V-posi i e cells (Fig. 4b), PARP clea age, and inc ease o Bax and p53 exp ession le els (Fig. 4c). The impac o Roy-Bz on MMP-9 and E-cadhe in exp ession le els in colonosphe e cul u es led us o u he in es iga e he e ec o Roy-Bz on he mig a ion o HCT116 cells and i s dependence on PKCδ.Usinga wound healing assay, we ound ha Roy-Bz caused a p onounced inhibi ion o mig a ion. No ably, he inhibi o y e ec o Roy-Bz was no obse ed in HCT116 cells subjec o PKCδ-knockdown. I is impo an o no e ha a he concen a ion o Roy-Bz used in hese assays (0.25 μM), he e we e no significan e ec s on cell iabili y (Fig. 4d, e). These esul s we e also ecapi ula ed using a chemo axis cell mig a ion assay, in which he an i-mig a o y e ec o Roy-Bz obse ed in con ol HCT116 cells was significan ly p e en ed by RNAi silencing o PKCδexp es- sion (Fig. 4 ). I has been p e iously es ablished in ce ain cellula models ha PKCδunde goes a p o eoly ic clea age by caspases ha gene a es a ca aly ically ac i e agmen (PKCδ-CF) 6,7 . In e es ingly, we ound ha in HCT116 cells, Roy-Bz caused a ime-dependen inc ease in PKCδ- CF le els, wi hou a isible in e e ence in o al PKCδ exp ession le els (Fig. 4g). Since i was p e iously epo ed ha ac i a ion o PKCδ is a equi ed ac o o his one H3 phospho yla ion on Se -10 (pSe -10 his one H3), which is a c ucial e en in apop osis 25 , we nex assessed he e ec o Roy-Bz on pSe -10 his one H3 le els in HCT116 cells. To his end, HCT116 cells we e synch onized in G1-phase by hyd o- xyu ea, in o de o exclude mi o ic pSe -10 his one H3, ollowed by ea men wi h 1 μM Roy-Bz o up o 8 h. We obse ed ha 1 μM Roy-Bz caused a p onounced inc ease o pSe -10 his one H3 le els ha was no iceable a 8 h. This e ec was abolished by PKCδ-knockdown (Fig. 4h, i), again ein o cing he concep ha Roy-Bz is a po en induce o apop o ic cance cell dea h ia ac i a ion o PKCδ. Al oge he , hese esul s showed ha Roy-Bz is a po en p o-apop o ic and an i-mig a o y agen in human colon cance cells h ough PKCδ-selec i e ac i a ion. Roy-Bz is non-geno oxic in human cance and no mal cells and has in i o PKCδ-dependen an i umo ac i i y wi h no appa en oxic side e ec s The geno oxici y o Roy-Bz in human cance cells was e alua ed by checking come -posi i e cells and his one H2AX phospho yla ion on Se ine-139 (γH2AX) as ma - ke s o DNA damage (single/double-s and b eaks). Ou esul s showed ha , con e sely o 25 μM e oposide (ETOP; posi i e con ol), 48 h ea men wi h Roy-Bz ailed o induce DNA damage in HCT116 cells, as demons a ed by he negligible numbe o cells wi h mo e han 5% o DNA in ail (Fig. 5a, b), by he low ail momen (Fig. 5a, c), as well as by he absence o inc eased γH2AX (see figu e on p e ious page) Fig. 4 P o-apop o ic and an i-mig a o y ac i i y o Roy-Bz is media ed by selec i e ac i a ion o PKCδin HCT116 cance cells. a Cell iabili y dose– esponse cu es we e de e mined by ypan blue assay o con ol (Mock) and PKCδ-knockdown (PKCδRNAi) HCT116 cells ea ed wi h 0.1–6 μM Roy-Bz o 24 h; cell iabili y o ehicle was se as 100%. bAnnexin V-posi i e cells we e de e mined in con ol and PKCδ-knockdown HCT116 cells a e 24 h ea men wi h 1 μM Roy-Bz o ehicle. cWes e n blo analysis o PARP clea age, p53 and Bax exp ession le els o 16 h ea men wi h 1 μM Roy-Bz o ehicle in con ol and PKCδ-knockdown HCT116 cells; GAPDH (loading con ol). d,eWound healing assay o con ol and PKCδ- knockdown HCT116 confluen cells ea ed wi h 0.25 µM Roy-Bz o ehicle, a di e en ime-poin s; in d, scale ba = 50 µm and magnifica ion = ×100; in e, quan ifica ion o wound closu e in fi e andomly selec ed mic oscopic fields. Chemo axis mig a ion assay o con ol and PKCδ-knockdown HCT116 cells o 24 h ea men wi h 0.25 µM Roy-Bz; mig a o y cells we e quan ified by fluo escence in ensi y, which was se as 100% o un ea ed cells. gWes e n blo analysis o ull-leng h PKCδ(PKCδ-FL) and clea ed PKCδ agmen (PKCδ-CF) in HCT116 cells ea ed wi h 1 μM Roy-Bz o up o 24 h. h,iLe els o his one H3 phospho yla ion on Se -10 (pSe -10) in con ol and PKCδ-knockdown HCT116 cells ea ed wi h 1 μM Roy-Bz o up o 8 h; his one H3 (loading con ol); in i, quan ifica ion o pSe -10 le els no malized o his one H3, alues o asynch onized cells we e se as 1. In c,g, and h, immunoblo s ep esen one o h ee independen expe imen s; in a,b,e, , and ida a a e mean ± SEM o h ee independen expe imen s; alues significan ly di e en a e indica ed (*p< 0.05, **p < 0.01, ***p< 0.001), unpai ed S uden ’s - es Bessa e al.Cell Dea h and Disease (2018) 9:23 Page 8 o 16 O ficial jou nal o he Cell Dea h Di e en ia ion Associa ion Fig. 5 (See legend on nex page.) 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