Antitumoral activity of 1,2-diaminocyclohexane derivatives in breast, colon and skin human cancer cells

31/1/2017 An i- umo al Ac i i y o 1, 2‐Diaminocyclohexane De i a i es in B eas , Colon and Skin Human Cance Cells h p://powe xedi o .ap a aco p.com/ sg/Po al/Edi o /Edi o .aspx?asse id=MagMGzL2u%2 s%3d& oleid=TCw3dUK 1OA%3d&edi s a us=1LHEL 8bezw%3d 1/14 DOI: 10.4155/ mc-2016-0212 [Q ue y-Q 1: Ka is hma Bhan( CE) o All(AU)] I is ou hous e s y le o i alic iz e genes o di e en ia e hem om p o eins . Can y ou pleas e c hec k and c on i m ha any genes in y ou a ic le a e i alic iz ed c o ec ly ? Mo ales, Ramí ez, Mo a a-Ta i a e al.[Q ue y- Q 2: Ka is hma Bhan( CE) o All( AU) ] I is ou hous e s y le o lea e all p o eins , genes and g ow h ac o s in hei abb e ia ed o m only . Pleas e c ould y ou ens u e none o hes e ha e been de ined ( inc luding he igu es and ables o a ic les )? An i umo al ac i i y o 1,2‐diaminocyclohexane de i a i es in b eas , colon & skin human cance cells S h o C o m m u n i c a i o n An i umo al ac i i y o 1,2‐diaminocyclohexane de i a i es in b eas , colon and skin human cance cells[Q ue y-Q 3: Ka is hma Bhan( CE) o All(AU)] Pleas e c hec k all au ho names , in pa ic ula y ou c o-au ho ’s names , and a ilia ion de ails a e p es en ed c o ec ly (inc luding in he unning heade a he op o he page) . We will no c hange hes e onc e publis hed in mos c as es . Fá ima Mo ales1, Albe o Ramí ez2,3, Cyn hia Mo a a-Ta i a2,4, Saúl A Na a o2,4, Juan A Ma chal2,4, Joaquín M Campos‡,1,4 & Ana Conejo-Ga cía*,‡,1,4 1Depa amen o de Química Fa macéu ica y O gánica, Facul ad de Fa macia, G anada, Spain [Q ue y- Q 4: Ka is hma Bhan( CE) o All( AU) ] Pleas e p o ide he pos al c odes o a ilia ions 1,2 and 3. 2Ins i u o de Biopa ología y Medicina Regene a i a (IBIMER), Depa amen o de Ana omía y Emb iología Humana, Facul ad de Medicina, G anada, Spain 3Depa amen o de Ciencias de la Salud, Facul ad de Ciencias Expe imen ales y de la Salud, Jaén, Spain 4Ins i u o Biosani a io de G anada (ibs.GRANADA), Hospi ales Uni e si a ios de G anada-Uni e sidad de G anada, 18071, G anada, Spain *Au ho o co espondence: aconejo@ug .es [Q ue y- Q 5: Ka is hma Bhan( CE) o All(AU) ] Pleas e p o ide he elephone and ax numbe o he au ho o co espondence. ‡Au ho s con ibu ed equally Abs ac Aim: Cance is among he leading causes o dea h wo ldwide. Medical in e es has ocused on mac ocyclic polyamines because o hei p ope ies as an i umo agen s. Resul s/Me hodology: We ha e designed and syn hesized a se ies o 1,2‐ diaminocyclohexane de i a i es wi h no able in i o an ip oli e a i e ac i i ies agains he MCF‐7, HCT‐116 and A375 cance cell lines. Cell cycle and apop osis analyses we e also ca ied ou . Ou esul s show ha all he compounds a e po en cy o oxic agen s, especially agains he A375 cell line. Conclusion: The selec i e ac i i y o he mac ocyclic de i a i e agains A375, ia apop osis, supposes a g ea ad an age o u u e he apeu ic use. This exempli ies he po en ial o 1,2‐diaminocyclohexane de i a i es o quali y as lead s uc u es o u u e an icance d ug de elopmen due o hei easy syn heses and no ewo hy bioac i i y. G aphical abs ac 31/1/2017 An i- umo al Ac i i y o 1, 2‐Diaminocyclohexane De i a i es in B eas , Colon and Skin Human Cance Cells h p://powe xedi o .ap a aco p.com/ sg/Po al/Edi o /Edi o .aspx?asse id=MagMGzL2u%2 s%3d& oleid=TCw3dUK 1OA%3d&edi s a us=1LHEL 8bezw%3d 2/14 Keywo ds: 1, 2‐diaminocyclohexanes • an i umo • apop osis • benzenesul onamides • b eas cance • caspases • colon cance • mac ocycles • melanoma Fi s d a submi ed: 11 No embe 2016; Accep ed o publica ion: 4 Janua y 2017; Published online: TBC Cance is among he leading causes o dea h wo ldwide, being esponsible o mo e dea hs han all co ona y hea disease o s oke, accoun ing o 8.2 million dea hs in 2012. I is expec ed ha annual cance cases will ise om 14million in 2012 o 22 wi hin he nex wo decades. Thus, cance con inues o be a majo heal h p oblem in de eloping, as well as unde eloped coun ies [1,2]. The MCF‐7, HCT‐116 and A375 cell lines a e ep esen a i e o b eas , colon and melanoma umo s, espec i ely. B eas cance is he main cance in women bo h in he de eloped and he de eloping coun ies [3], wi h an es ima ed 1.7million new cases and 521,900dea hs in 2012, ep esen ing 25% o all cance diagnoses and 15% o all cance dea hs among emales. MCF‐7 is a luminal epi helial-like cell line ha exp esses low le els o he enzyme phosphodies e ase ype 5 and i s pheno ype is conse ed in hei de i ed umo s [4,5]. Colo ec al cance is a leading cause o cance dea h in de eloped coun ies, being he hi d mos commonly diagnosed cance in men and he second in women, wi h an es ima ed 1.4million cases and 693,900dea hs in 2012 [6]. Colon cance is associa ed wi h a high- equency a ia ion o mic osa elli e epea s as he HCT‐116 cell line shows [7]. A cance -speci ic hype me hyla ion e en o he small nucleola RNAs SNORD123, U70C and ACA59B has ecen ly been ound in his cell line [8]. The incidence o bo h nonmelanoma and melanoma skin cance s has been inc easing o e he pas decades [9], malignan melanoma, being he one ha accoun s o app oxima ely 75% o all dea hs om skin cance [10]. A375 is a human malignan melanoma cell line de i ed om skin biopsies ha is highly umo igenic and me as a ic in animal models [11,12]. Medical in e es has ocused on mac ocyclic polyamines in he las decades because o hei chemical and biological p ope ies as an i umo agen s. Thei an ip oli e a i e ac i i y is due o hei a ini y o he DNA [13]. 1,2‐diamines possess a wide ange o bioac i i ies, such as he an icance and an i ube culosis ones [14]. The amine g oups a e use ul o modula ing he solubili y o he d ug, as well as o dona ing o accep ing hyd ogen bonds o and om a biological ecep o [15]. Diamines (-)- ans-, (+)- ans- and meso‐1 (Figu e 1) showed an ip oli e a i e ac i i y agains he MCF‐7 cell line [16]. They induce g ow h inhibi ion wi h an IC50 o 0.4, 0.6 and 1.8µM, espec i ely. The mos ac i e compound (-)- ans‐1 shows g ea e cy o oxic ac i i y owa d he MCF‐7 cells han he no mal MCF‐10A cells. Real- ime RT-PCR analysis 31/1/2017 An i- umo al Ac i i y o 1, 2‐Diaminocyclohexane De i a i es in B eas , Colon and Skin Human Cance Cells h p://powe xedi o .ap a aco p.com/ sg/Po al/Edi o /Edi o .aspx?asse id=MagMGzL2u%2 s%3d& oleid=TCw3dUK 1OA%3d&edi s a us=1LHEL 8bezw%3d 3/14 demons a ed ha (-)- ans‐1 is an ex emely e icien egula o o he an iapop o ic genes Bcl-xl, Bcl‐2 and cyclin D1. La e s udies o he molecule con i med ha i also egula es p53 [16,17]. Figu e 1. Diamines wi h an ip oli e a i e ac i i y agains he MCF‐7 cell line. Da a aken om [12]. [Q ue y - Q 6: Ka is hma Bhan( CE) o All( AU)] Pleas e c hec k and c on i m ha any igu es / ables ha appea in he a ic le ha e been ed awn c o ec ly , and i no , pleas e des c ibe any c o ec ions ha need o be made. Mac ocyclic compounds ep esen a s uc u al class wi h an excep ional po en ial o biological ac i i y and speci ici y [18]. Nowadays, mac ocycles a e p omising chemo ypes, wi h mo e han 100 ma ke ed mac ocycle d ugs ha included an i umo d ugs [19]. Benzenesul onamides also show an icance ac i i y h ough a a ie y o mechanisms such as cell- cycle pe u ba ion on he G1 phase, he dis up ion o mic o ubule assembly o angiogenesis inhibi ion [20]. We ha e p e iously designed di e en molecules ha a ge cance (Figu e 2). Compound 2 is one o he mos po en human choline kinase inhibi o epo ed o da e, showing ac i i y in he low mic omola ange (IC50=0.3µM) [21], wi h simila alues o o he s ecen ly published [22-24]. Mac ocycle ans-bis(5‐FU O, N-ace al) (3) p esen s an an ip oli e a i e ac i i y o 5.5µM agains he MCF‐7 cell line and p o okes a G0/G1 cell cycle a es in he MCF‐7 b eas cance cells [25]. The benzenesul onamide-con aining compound, named bozepinib (4), is a po en an i umo agen wi h an IC50 agains he MCF‐7 cell line o 0.355µM [26] ha induces apop osis in b eas and colon cance cells. The double- s anded RNA-dependen p o ein kinase PKR is a a ge o bozepinib (4). Mo eo e , he combina ion wi h IFN-α po en ia es he apop osis induced by bozepinib (4) and also enhances au ophagy and senescence, bo h p ocesses o g ea impo ance in umo cells ha show esis ance o con en ional chemo he apy [27]. Bozepinib (4) also shows in i o an i umo and an ime as a ic e icacy in xeno ansplan ed nude mice wi hou p esen ing subacu e oxici y and a selec i e ac i i y agains cance s em cells [28]. Figu e 2. An icance agen s p e iously epo ed by us. In his pape we p esen he syn hesis and biological e alua ion o a se ies o compounds (Figu e 3) designed o s udy he in luence o h ee s uc u al modi ica ions in he 1,2‐diaminocyclohexane de i a i es (1): (A) ansi ion o he phenol 31/1/2017 An i- umo al Ac i i y o 1, 2‐Diaminocyclohexane De i a i es in B eas , Colon and Skin Human Cance Cells h p://powe xedi o .ap a aco p.com/ sg/Po al/Edi o /Edi o .aspx?asse id=MagMGzL2u%2 s%3d& oleid=TCw3dUK 1OA%3d&edi s a us=1LHEL 8bezw%3d 4/14 g oup o me a (m-) and pa a (p-) posi ions (5 and 6, Figu e 3), (B) in oduc ion o wo p-ni obenzenesul onyl g oups (7– 9, Figu e 3) and (C) i s con e sion in o a 14‐membe ed mac ocycle wi h an e hylene b idge ha links he wo phenolic g oups o 1 (10, Figu e 3). [Q ue y -Q 7: Leigh Nugen (PE) o All(AU) ] Wha a e A, B and C e e ing o in he p ec eding s en enc e? Figu e 3. Molecules syn hesized and analyzed as an i umo agen s. Ma e ials & me hods Chemis y Mel ing poin s we e aken in open capilla ies on an elec o he mal mel ing poin appa a us and a e unco ec ed. Analy ical hin laye ch oma og aphy was pe o med using Me ck Kieselgel 60 F254 aluminum shee s, he spo s being de eloped wi h UV ligh (λ=254nm). All e apo a ion was ca ied ou in acuo wi h a Büchi o a y e apo a o and he p essu e con olled by a Vacuub and CVCII appa a us. Fo lash ch oma og aphy, Me ck silica gel 60 wi h a pa icle size o 0.040– 0.063mm (230–400 mesh ASTM) was used. [Q ue y -Q 8: Ka is hma Bhan( CE) o All( AU)] Pleas e de ine he ollowing e m: ‘AST M.’ Pu i y o all compounds was de e mined by NMR s udies, mass spec oscopy and elemen al analysis. [Q ue y -Q 9: Ka is hma Bhan(CE) o All(AU)] Pleas e de ine he ollowing e m: ‘NMR.’ Elemen al analyses we e pe o med on he The mo Scien i ic Flash 2000 analyze and he measu ed alues indica ed by he symbols o he elemen s o unc ions we e wi hin±0.4% o he heo e ical alues. Nuclea magne ic esonance spec a ha e been ca ied ou a he Cen o de Ins umen ación Cien í ica (CIC)/Uni e sidad de G anada and eco ded on a 300MHz 1H and 75MHz 13CNMR Va ian Ino a-TM spec ome e s a ambien empe a u e. Chemical shi s (δ) a e quo ed in pa s pe million (ppm) and a e e e enced o he esidual sol en peak. Signals a e designa ed as ollows: s, single ; bs, b oad single ; d, double ; dd, double double ; ddd, double double double ; , iple ; m, mul iple . High- esolu ion nano-assis ed lase deso p ion/ioniza ion o elec osp ay ioniza ion mass spec a we e ca ied ou on a B uke Au o lex o a Wa e s LCT 31/1/2017 An i- umo al Ac i i y o 1, 2‐Diaminocyclohexane De i a i es in B eas , Colon and Skin Human Cance Cells h p://powe xedi o .ap a aco p.com/ sg/Po al/Edi o /Edi o .aspx?asse id=MagMGzL2u%2 s%3d& oleid=TCw3dUK 1OA%3d&edi s a us=1LHEL 8bezw%3d 5/14 P emie Mass Spec ome e , espec i ely. Anhyd ous CH2Cl2 was pu chased om he VWR In e na ional Eu olab. Anhyd ous condi ions we e pe o med unde a gon. All eagen s we e pu chased om Ald ich. Compounds 1, 5–10 ha e been named acco ding o IUPAC ecommenda ions o phane s uc u es [29,30]. In e media e 11 was syn hesized as p e iously desc ibed [31]. Ta ge compounds meso‐1,5–9, (±)- ans‐1,5–9 and meso‐10 we e syn hesized acco ding o he p e iously epo ed p o ocols [16,32,33] wi h he modi ica ions desc ibed in he Supplemen a y In o ma ion. Biology Cell cul u e MCF‐7 (ECACC: 86012803) and HCT‐116 (ECACC: 91091005) p o ided by he Cell Bank o he Uni e si y o G anada and A375 cells, a gi om Bosse ho (Ins i u e o Pa hology, Regensbu g Uni e si y, Ge many), we e g own a 37°C in an a mosphe e con aining 5% CO2, wi h Dulbecco’s modi ied Eagle Medium (Gibco, NY, USA) supplemen ed wi h 10% hea -inac i a ed e al bo ine se um (Gibco), 2% L-glu amine, 2.7% sodium bica bona e, 1% Hepes bu e , 40 mg/l gen amicin and 500mg/l ampicillin [34-36]. D ug ea men Compounds we e dissol ed in DMSO and s o ed a -20°C. Fo each expe imen , he s ock solu ions we e u he dilu ed in medium o ob ain he desi ed concen a ions. The inal sol en concen a ion in cell cul u e was≤0.1% / o DMSO, a concen a ion wi hou any e ec on cell eplica ion. Pa allel cul u es o MCF‐7, HCT‐116 and A375 cells in medium wi h DMSO we e used as con ols. Biological assays The e ec o he compounds on cell iabili y was assessed using he sul o hodamine-B (SRB) colo ime ic assay. The assay p ocedu e is explained in he supplemen a y in o ma ion ile, as well as he cell-cycle dis ibu ion analysis, he apop osis de ec ion by s aining wi h annexin V-FITC and p opidium iodide and he caspase assay. S a is ical analyses All he quan i a i e da a in he p esen s udy a e epo ed as means±s anda d de i a ion om a leas h ee independen expe imen s. Two-way ANOVA was used o g ouped analysis o di e ences ollowed by Bon e oni pos - es s. Resul s & discussion Chemis y meso- and (±)- ans‐1,5,6 we e syn hesized by educ i e amina ion [16]. The yield o meso‐1 was imp o ed o 100% by inc easing he eac ion ime o he Schi -base condensa ion om 2 o 48h. Sul onyla ion o diamines meso- and (±)- ans‐1,5,6 wi h p-ni obenzenesul onyl chlo ide ga e meso- and (±)- ans‐7,8,9 espec i ely (Figu e 4). [Q ue y - Q 10: Leigh Nugen ( PE) o All( AU)] Pleas e igno e he s pac e below F igu e 5; his will be ec i ied in he inal lay ou . PXE is no ep es en a i e o he inal lay ou , pleas e only us e i o e iew he c on en . 31/1/2017 An i- umo al Ac i i y o 1, 2‐Diaminocyclohexane De i a i es in B eas , Colon and Skin Human Cance Cells h p://powe xedi o .ap a aco p.com/ sg/Po al/Edi o /Edi o .aspx?asse id=MagMGzL2u%2 s%3d& oleid=TCw3dUK 1OA%3d&edi s a us=1LHEL 8bezw%3d 6/14 Figu e 4. Reagen s and condi ions. (A) E 3N/E OH, , 48 h; NaBH4, , 4h [100% o meso‐1, 41% o (±)- ans‐1, 75% o meso‐5, 74% o (±)- ans‐5, 51% o meso‐6, 57% o (±)- ans‐6]; (B) p-NO2-Ph-SO2Cl, anhyd ous CH2Cl2, , 4.5h [63% o meso‐7, 26% o (±)- ans‐7, 24% o meso‐8, 24% o (±)- ans‐8, 16% o meso‐9, 9% o (±)- ans‐9]. The i s s ep in he syn hesis o meso‐10 (Figu e 5) was he eac ion o 1,2‐dib omoe hane wi h salicylaldehyde [31]. The educ i e amina ion o 11 yield meso‐10 wi hou u he pu i ica ion [32,33]. Figu e 5. Reagen s and condi ions. (A) NaOH 2%/E OH, e lux, 72 h (27%); (B) meso‐1,2‐diaminocyclohexane, CH3OH, e lux, 5h; NaBH4, 50°C, 3.5h (38%). Biology Compounds meso‐1, 5–9, (±)- ans‐1, 5–9 and meso‐10 we e assayed o hei in i o an ip oli e a i e ac i i y agains he MCF‐7, HCT‐116 and A375 cell lines. The an i umo d ug oxalipla in, ha con ains a diamine in i s s uc u e, was used as a con ol in he assays and hei IC50 alues a e concu en wi h he ones in li e a u e [37]. The esul s a e 31/1/2017 An i- umo al Ac i i y o 1, 2‐Diaminocyclohexane De i a i es in B eas , Colon and Skin Human Cance Cells h p://powe xedi o .ap a aco p.com/ sg/Po al/Edi o /Edi o .aspx?asse id=MagMGzL2u%2 s%3d& oleid=TCw3dUK 1OA%3d&edi s a us=1LHEL 8bezw%3d 7/14 summa ized in Table 1. All he compounds a e po en an ip oli e a i e agen s wi h he highes po ency agains he A375 melanoma cance cell line (submic omola alues o IC50 o compounds 1, 7–10). meso- and (±)- ans‐1 a e he bes an ip oli e a i e agen s in he h ee cell lines s udied (MCF‐7, HCT‐116 and A375 (IC50=58nM o meso‐1 s 2.5µM o oxalipla in in MCF‐7 cells, IC50 =66 nM o (±)- ans‐1 s 3.8µM o oxalipla in in HCT‐116 cells). [Q ue y -Q 11: Leigh Nugen ( PE) o All( AU) ] Whe e do he b ac k e s be o e MCF - 7 and IC50 end in he p ec eding s en enc e? We should highligh ha he al e a ion o he phenol g oup om o ho (o-) o pa a (p-) posi ion dec eases he an ip oli e a i e ac i i y o compounds 5 (m-) and 6 (p-) subs an ially ( om IC50=0.06–1.2µM o 1 o IC50=10.7–54.6µM o 5 and 6). Howe e , he e is no a di e ence be ween he molecules wi h he p-ni obenzenesul onyl g oup (7–9), whe e he an ip oli e a i e ac i i y is simila be ween o-, m- and p- de i a i es (IC50 = 0.94–6.3µM). The bes alues o an ip oli e a i e ac i i y o he p- ni obenzenesul onyl de i a i es a e meso‐9 agains HCT‐116 cell line (IC50=1.1µM) and (±)- ans‐9 agains A375 cell line (IC50=94nM). meso‐10 shows simila alues o an ip oli e a i e ac i i y as oxalipla in agains he MCF‐7 cell line (IC50=2.9, 2.5µM, espec i ely), being a be e agen han oxalipla in agains HCT‐116 and A375 cell lines (IC50=1.5, 1.1µM agains 3.8, 3.4µM, espec i ely). As a esul , we should men ion ha he modi ica ion o he phenol g oups in compounds 5 and 6, he in oduc ion o he p-ni obenzenesul onyl g oup in o meso- and (±)- ans‐1 o gi e meso- and (±)- ans‐7–9 as well as he mac ocycliza ion o p oduce meso‐10, do no imp o e he an ip oli e a i e ac i i y in ela ion o hose o meso- and (±)- ans‐1. Table 1. An ip oli e a i e ac i i y o meso- and (±)- ans‐1,5–9, meso‐10 and oxalipla in agains he MCF‐7, HCT‐116 and A375 cell lines. Compound MCF‐7 IC50µM HCT‐116 IC50µM A375 IC50µM meso‐10.058±0.014 0.236±0.060 0.099±0.018 (±)- ans‐11.240±0.018 0.066±0.017 0.205±0.015 meso‐542.30±0.012 12.79±1.08 40.30±1.01 (±)- ans‐548,40±0.76 14.84±0.12 35.65±0.92 meso‐654.56±1.16 31.30±0.92 38.70±1.32 (±)- ans‐640.90±0.78 10.65±0.82 32.18±0.69 meso‐77.010±0.044 5.070±0.032 3.413±0.016 (±)- ans‐72.630±0.044 3.680±0.045 2.398±0.020 meso‐86.320±0.011 5.040±0.005 2.040±0.001 (±)- ans‐86.100±0.047 4.390±0.010 2.030±0.001 meso‐94.130±0.014 1.090±0.007 4.390±0.019 (±)- ans‐94.080±0.024 4.330±0.002 0.940±0.003 meso‐10 2.900±0.018 1.460±0.001 1.106±0.064 Oxalipla in 2.500±0.003 3.770±0.001 3.350±0.002 All expe imen s we e conduc ed in iplica e and ga e simila esul s. Da a a e he mean±SD o h ee independen de e mina ions. µM: Mic omola . The cell cycle is he p ocess by which cells duplica e hemsel es, g ow and p epa e o di ide again. I egula es cell di ision in mul icellula o ganisms and i is essen ial o he eplacemen o damaged cells. Regula ion o cell p oli e a ion 31/1/2017 An i- umo al Ac i i y o 1, 2‐Diaminocyclohexane De i a i es in B eas , Colon and Skin Human Cance Cells h p://powe xedi o .ap a aco p.com/ sg/Po al/Edi o /Edi o .aspx?asse id=MagMGzL2u%2 s%3d& oleid=TCw3dUK 1OA%3d&edi s a us=1LHEL 8bezw%3d 8/14 is one o he s a egies o he ea men o cance . As a consequence, in o de o e alua e whe he he an ip oli e a i e e ec o hese molecules in ol es changes in cell-cycle dis ibu ion, A375 cells we e ea ed wi h he compounds ha p esen be e an ip oli e a i e ac i i y: 1, 7–10 and hen analyzed by low cy ome y (see Table 2). We ound a cell- cycle a es in he G0/G1 phase induced by meso- and (±)- ans‐7 and 9 and meso‐10. meso‐1 and meso and (±)- ans‐8 did no modi y he cell cycle p o ile and (±)- ans‐1 p o oked a G2/M and S-phase cell-cycle a es a he expense o G0/G1 phase. The an ip oli e a i e p ope ies o se e al an i umo agen s a e media ed by he modula ion o cell-cycle checkpoin . Palbociclib is an inhibi o o CDK4 and CDK6 ha p o okes a p onounced G0/G1 a es , as molecules 7,9 and 10. I is a highly selec i e and o ally ac i e d ug, blocking e inoblas oma phospho yla ion in a low nanomola ange [38]. I s an i umo ac i i y is associa ed wi h educed Rb phospho yla ion and wi h a dec eased exp ession o Ki‐67 [39], a cell p oli e a ion ma ke ha is associa ed wi h ibosomal RNA ansc ip ion. I dec eased caspase 3/7 ac i a ion and CDKN2A and inc eased le els o RB1 and cyclin D1 [40]. An example o a d ug ha causes cell-cycle G2/M a es , as (±)- ans‐1, is panobinos a [41-43]. I is demons a ed ha i dec eases in i o umo igenesis o iple-nega i e b eas cance cells. Panobinos a is a po en inhibi o wi h ac i i y agains Class I, II and IV HDAC enzymes, wi h hype ace yla ion o his ones H3 (Lys9) and H4 (Lys8) [41]. Table 2. Cell cycle analysis in he A375 melanoma cell line upon ea men wi h meso- and (±)- ans‐ 1,7–9, meso‐10 compounds and oxalipla in (a 3×IC50 doses, 48h). Compound Cell cycle G0/G1SG2/M Con ol 38.97±0.22 44.42±0.66 16.61±0.45 meso‐139.04±1.63 42.24±2.98 18.73±1.35 (±)- ans‐119.97±0.13 54.78±1.95 25.26±1.83 meso‐758.01±0.36 30.14±0.24 11.85±0.11 (±)- ans‐762.28±0.14 28.54±1.33 9.19±1.20 meso‐838.95±2.69 46.90±2.62 14.15±1.70 (±)- ans‐836.68±1.91 50.40±0.14 12.93±0.92 meso‐955.63±1.98 36.56±1.20 7.79±0.28 (±)- ans‐942.35±0.99 44.16±0.64 13.49±0.57 meso‐10 66.97±3.59 15.22±1.05 17.82±4.63 Oxalipla in 53.15±0.71 30.22±0.85 16.62±0.42 De e mined by low cy ome y [44]. All expe imen s we e conduc ed in iplica e and ga e simila esul s. Da a a e he mean±SD o h ee independen de e mina ions. Fu he s udies we e pe o med o de e mina e i he obse ed g ow h inhibi ion was due o apop osis. Apop osis is a gene ic o de ly p ocess in which cells lead o hei own dea h in esponse o di e en s imuli and i is an essen ial p ocess o main ain homeos asis in mul icellula o ganisms. The e o e, apop osis ac i a ion is a undamen al s a egy in he imp o emen o cance he apy. Apop osis was de e mined using an Annexin V-based assay in A375 cells ea ed wi h compounds: 1, 7–10 (see Supplemen a y Table 1). A ligh apop o ic esponse was obse ed by compound (±)- ans‐7 (28.3%) and no signi ican esponse by molecules 1-, meso‐7 and 9 (6.35–14.91%). Howe e , we can emphasize ha †‡ † ‡ 31/1/2017 An i- umo al Ac i i y o 1, 2‐Diaminocyclohexane De i a i es in B eas , Colon and Skin Human Cance Cells h p://powe xedi o .ap a aco p.com/ sg/Po al/Edi o /Edi o .aspx?asse id=MagMGzL2u%2 s%3d& oleid=TCw3dUK 1OA%3d&edi s a us=1LHEL 8bezw%3d 9/14 he mac ocycle meso‐10, which is he compound ha induces mo e G0/G1 phase accumula ion, is also he d ug ha p o okes ex emely high le els o apop osis a 48h in he A375 cance cells. This compound inc eases he pe cen age o apop o ic cells (ea ly and la e apop osis) om 1.05% in DMSO- ea ed cells o 95.0%. Highly apop o ic d ugs ha e demons a ed e icien an i umo ac i i ies in pa ien s. The po en apop o ic p ope ies o he mac ocycle meso‐10 in melanoma cells sugges he po en ial clinical in e es o his d ug and encou age u u e deep s udies and in i o expe imen s. Finally, s udies o apop osis we e pe o med by he ac i a ion o caspases o de e mine which me abolic pa hway ac i a ed hese d ugs. Compound meso‐10 (Figu e 6) ac i a es he canonical in insic caspase‐8/caspase‐3 apop o ic pa hway on he MCF‐7 cell line, bu also his compound induces sligh ly caspase‐2 ac i a ion (Figu e 6A). Howe e , his compound does no signi ican ly ac i a e signi ican ly any caspase in HCT‐116 and A‐375 cell lines (Figu es 4B &C). Di e en cell dea h modali ies exposed o he same molecule in di e se cell lines ha e been p e iously desc ibed [45]. Indi ubin, a CDK inhibi o , induces caspase-independen cell dea h in human neu oblas oma [46], while o he indi ubin de i a i es induce cell dea h h ough he in insic caspase‐9 pa hway and/o ac i a ion o caspase‐8 in b eas cance cells [47].