Investigation Of The Effect And Mechanism Of Romidepsin In Human Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC),which is one of the most aggressive malignant tumors,especially,it is the second major reason of cancer related death in China.Surgical resection and liver transplantation are widely used to control its pathology,however,the prognosis is very poor in HCC patients,which might be due to their development of resistance to chemotherapy.Hence,novel therapeutic agents are required for the treatment immediately.Histone deacetylase inhibitor(HDACi)possesses potent and significant inhibitory effects on the growth of human solid tumor implant in mice.HDACis exert their antitumor activities through the induction of differentiation,cell cycle arrest and apoptotic cell death in a variety of cancer cells.Several HDACis have been well studied previously,including(FK228,Depsipeptide).Romidepsin is one of the most successful and widely used and an approved HDACi in clinical applications against refractory cutaneous and peripheral T cell lymphoma.However,its role in the treatment of solid tumors,including HCC remains still unclear and is under investigation.In the present study,we demonstrate that Romidepsin inhibited the growth of HCC cells by inducing cell cycle arrest and apoptosis in vivo and in vitro.This study is expected to reveal the mechanism of Romidepsin drug resistance to HCC.Our study will help to illustrate the mechanism of Romidepsin drug resistance and may provide a new therapeutic target for HCC.Objectives:The purpose of this study is to demonstrate that Romidepsin inhibited the growth of HCC cells in vivo and in vitro.Next,this study is expected to reveal the mechanism of Romidepsin drug resistance to HCC.Methods:1 The effects of Romidepsin on proliferation of HCC cells were investigated in four HCC cell lines using CCK-8 assay,colony formation assays and EdU,respectively.2 To verify the effects on cell cycle distribution by Romidepsin,the cells were examined by using the Cell Cycle Detection Kit.Furthermore,to confirm the G2/M phase arrest in HCC cells by Romidepsin induction of,p21,cyclinB1,p-cdc2,cdc2,p-cdc25C and p27expressions were monitored using Western blot analysis.3 The number of apoptotic cells were examined by fluorescein isothiocyanate(FITC)Annexin V and propidium iodide(PI).With regard to apoptosis signaling,the protein expression of c-caspase-3,c-caspase-9,and c-PARP was detected by Western blot.4 To verify the mechanism of Romidepsin on the effects of MAP kinase activation,Western blot analysis was used to evaluate the activation of Erk,JNK and p38 MAP kinase in HCC cells.5 Subsequently,Huh7 cells were subcutaneously inoculated into the nude mice,which were employed to further probe the tumor-suppressive effect of Romidepsin in vivo.6 To evaluate the effect of Romidepsin on autophagy,we examined the occurrence of autophagy in Romidepsin-treated HCC cells by Western blot analysis,Autophagy Assay Kit and immunofluorescence staining,respectively.7 The cells were treated with Romidepsin combined with 3MA or HCQ,the dateshow inhibition of autophagy potentiates the anti-tumor effect of Romidepsin in vitro and in vivo.To exclude the potential off-target pharmacological effects of drugs inhibiting autophagy,we treated cells with small interfering RNAs against ATG5 or ATG7,two essential components for the formation of autophagosome.Results:1 Romidepsin substantially inhibited HCC cell growth in a time-and dose-dependent manner by using CCK-8 assay.Cell proliferation and colony formation with 5-ethynyl-20-deoxyuridine(EdU)results showed that Romidepsin inhibited the proliferation of HCC cells and colony formation.2 Romidepsin treatment led to a time-and dose-dependent induction of cell cycle arrest in the G2/M phase.Furthermore,to confirm the G2/M phase arrest in HCC cells by Romidepsin induction of,p21,cyclinB1 p-cdc2,cdc2,p-cdc25C and p27 expressions were monitored using Western blot analysis in Huh7 and HCC-LM3 cells.3 The flow cytometric analysis indicated a significant increase in the number of apoptotic cells after Romidepsin treatment.Romidepsin treatment resulted in a marked dose-and time-dependent increase in apoptosis.With regard to apoptosis signaling,the protein expression of c-caspase-3,c-caspase-9,and c-PARP was detected by Western blot.4 Romidepsin treatment resulted in the increased concentrations of Erk and JNK phosphorylations in a dose-dependent manner,but the phosphorylation of p38 was not affected.The results suggested that G2/M phase arrest and the apoptosis of HCC cells induced by Romidepsin were mediated by the activation of Erk pathways and the JNK/caspase-3 pathways.5 The tumor size of the negative control group was significantly larger than that of the Romidepsin group.The tumor mass of Romidepsin(1 mg/kg)treated mice was significantly less than that of the control group.The immunohistochemistry staining of xenograft tissues revealed a higher expression of p-cdc25C,ki67,c-caspase-3 and c-PARP,and a lower expression of Ki-67 in Romidepsin treated tumors.Also the potential toxicity effects of Romidepsin on normal tissues showed no significant loss in body weight when treated with Romidepsin(1 mg/kg)and DMSO.Furthermore,H&E staining of the organs also suggested non-toxicity results.6 In Huh7 and HCC-LM3 cells,Romidepsin treatment led to upregulation of LC3,ATG5,ATG7 and Beclin-1,but downregulation of p62 in a dose dependent manner,indicating that autophagy was activated by Romidepsin.The results indicate that the accumulation of Autophagy BlueTM-labeled vesicles correlates with the induction of autophagy by Romidepsin,and is abrogated by proven inhibitors of the autophagic pathway such as 3MA.The number of autophagosomes and autophagolysosomes increased significantly in cells treated with Romidepsin compared with untreated control cells by immunofluorescence.Furthermore,more autophagic vesicles containing engulfed organelles were detected by transmission electron microscopy in Romidepsin-treated cells.7 We hypothesized that inhibiting autophagy could sensitize HCC cells to the Romidepsin treatment.Indeed,3MA and HCQ significantly augmented growth inhibition induced by Romidepsin in vitro and in vivo.ShRNA against ATG5 or ATG7 could efficiently knockdown its target gene,and blocked Romidepsin-induced autophagy in Huh7 cells.The cytotoxicity induced by Romidepsin was enhanced in the ATG5 or ATG7 knockdown cells,the results were confirmed by flow cytometric analysis.Conclusion:1 In conclusion,our results revealed that Romidepsin inhibits HCC cells in the G2/M phase arrest and apoptosis,possibly by mechanisms including cell cycle arrest which targets Erk/cdc25C/cdc2/cyclinBl pathway,and apoptosis that targets JNK/c-jnk/caspase-3 pathway.2 Inhibition of autophagy by pharmacological or genetic approaches sensitized HCC cells to Romidepsin in vitro and in vivo,at least partially,through promoting Romidepsin-induced apoptosis.Autophagy inhibition represents a promising approach to surmount intrinsic or acquired resistance to Romidepsin treatment.Thus,Romidepsin combined with autophagy inhibitors could be translated into clinical practice in the future.