| Mechanistic target of rapamycin(MTOR)is a central regulator for cell proliferation,apoptosis and survival.MTOR inhibitors have immunosuppressive and anti-tumor effects,such as rapamycin.Everolimus,a rapamycin derivative,possesses the similar pharmaceutic function as rapamycin on inhibiting m TOR activity.However,whether everolimus has anti-tumor efficacy is unclear.In this study,we found that everolimus treatment led to the decreased phosphorylation level of MTOR.Moreover,everolimus induced the conversion of LC3-I to LC3-II,but not increased autophagy flux and the cleavage of caspase 3 and PARP were not found.We also found that everolimus treatment alone did not affect some crucial pathways associated cell death,but it dramatically enhanced cisplatin-induced cell death.However,everolimus treatment did not affect cisplatin-triggered apoptosis in COLO-16 cells,Interestingly,Everolimus inhibited the activation of checkpoint kinase 1(Chk1)in response to cisplatin treatment.Our study demonstrated that everolimus treatment enhances the cisplatin-induced cell death of human skin squamous carcinoma cell line COLO-16 through inhibiting activation of Chk1,and this effect is independent on apoptosis and autophagy.Our findings provided a molecular rationale for using everolimus in anti-tumor therapy[Objective] To evaluate the anti-tumor efficacy of Everolimus,and to investigate the mechanisms that Everolimus treatment enhances the cisplatin-induced cell death of human skin squamous carcinoma cell line COLO-16.[Method] COLO-16 cells were treated with different concentrations of Everolimus(50,100,200 nmol/L)for 12 and 24 hours.Then,cell appearance was observed by OLYMPUS FV1000 laser scanning confocal fluorescence microscope for acridine orange staining(AO),Lactate dehydrogenase(LDH)assay was performed to measure cell death,Western blot to measure the expression of microtubule—associated protein l light chain 3(LC3),the phosphorylation level of MTOR pathway,the phosphorylation level of Chk1 and the cleavage level of caspase3 and PARP,and Annexin V-EGFP Apoptosis detection to identified apoptotic cells.Western blot to measure the phosphorylation level of MTOR pathway,Akt,DNA damage pathway and Chk pathway when COLO-16 cells were treated with or without 50 nmol/L everolimus together with or without 25 μmol/L cisplatin.[Results] After treatment with different doses of Everolimus(50,100,200 nmol/L)for 12 and 24 hours,we found that Everolimus led to the decreased phosphorylation level of MTOR at ser2448 and ser2481 as well as Rictor at thr1135.However,we did not observed the significant change of the targeted pathways including ULK1 phosphorylation at ser757,p70 S6 phosphorylation at thr389.The conversions of LC3-I to LC3-II expressed at 12 hours were not significant in the treat groups(3.52±0.21、4.03±0.39、5.05±0.22,all P>0.05)and expressed significant higher than the control group(2.07±0.05,P<0.05)。 The conversions of LC3-I to LC3-II expressed at 24 hours were also no significant in the treat groups(3.38±0.26 、3,29±0.06、6.57±0.16,all P>0.05)and expressed significant higher than the control group((2.61±0.16,P<0.05)。After treatment with different doses of Everolimus(50,100,200 nmol/L)with E64 d and pepstatin for 12 hours,the ratios of LC3-Ⅱandβ-actin were not significant with the control groups which treated with only E64 d and pepstatin(1.26±0.40、1.16±0.34、1.21±0.39、1.19±0.27,all P>0.05),it demonstrated that autophagy flux was not increased.we validated that cisplatin treatment alone led to significant cell death of COLO-16 cells in time-dependent manner from 12 to 48 hours(percentage(%)of cell death 14.33±3.07 at 12 hour,18.20±1.46 at 24 hour respectively).Everolimus treatment did not change the protein level and phosphorylation of Akt.However,everolimus treatment alone induced a low level cell death from 12 to 24 hours(percentage(%)of cell death 0.82±0.47 at 12 hour,8.75±1.17 at 24 hour respectively).Interestingly,the cell death rate in cells treated cisplatin combined with everolimus(percentage(%)of cell death 28.27±2.12 at 12 hour,42.58±0.93 at 24 hour)was significantly higher than cells treated with cisplatin alone from 12 to 24 hours,suggesting that everolimus enhanced the cisplatin-induced anti-tumor effect.Cisplatin treatment for 24 hours activated the cleavage of apoptotic molecular marker caspase 3 and PARP.In addition,cells marked with annexin and PI were increased after cisplatin challenge.These findings indicated that cisplatin triggered apoptosis COLO-16 cells.Moreover,there were no significant difference of the cleavage level of caspase 3 and PARP and cells marked with annexin and PI between cells treated with cisplatin alone and cells treated with cisplatin and everolimus together for 24 hours(P>0.05).Furthermore,Compared with cells treated with cisplatin alone,the cleavage of caspase 3 and PARP,Annexin V marked cells and LC3-II formation were not changed in cells treated everolimus and cisplatin together.In cisplatin-treated COLO-16 cells,the phosphorylation of Rictor at thr1135 and Chk1 at ser345 was increased at 12 and 24 h after treatment,but the effect was not observed in everolimus-treated cells.Interestingly,the cisplatin-induced increase of Chk1 and Rictor phosphorylation was inhibited after COLO-16 cells were treated cisplatin in combination with everolimus.[Conclusion]1 Everolimus treatment enhanced the cisplatin-induced cell death in COLO-16 cells,and the effect was independent on apoptosis and autophagy.2 MTOR signaling is sensitive to everolimus in COLO-16 cells,but the targeted pathway of MTOR is not regulated simultaneously.Akt pathway is not sensitive to everolimus3 Inhibiting the activation of Chk1 may mediate the enhancing effect of everolimus to cisplatin-induced cell death,and everolimus could not aggravate the DNA damage induced by cisplatin. |
