| BACKGROUDEsophageal cancer is a malignant lesion caused by abnormal hyperplasia of esophageal squamous epithelium and glandular epithelium.It is one of the most common malignant tumors.Besides,there are significant regional differences in the incidence of esophageal cancer.Chemotherapy is the most important treatment for patients with advanced esophageal cancer,but the current chemotherapy drugs usually cause severe problems leading to treatment failure.In order to cope with this unfavorable situation,the development of new chemotherapeutic drugs and the study of the molecular mechanism underlying this cancer are particularly important.Bcl-2 family protein regulate apoptosis by interfering with mitochondrial membrane integrity.The high expression of Bcl-2 and Bcl-xl protein is closely related to esophagus cancer occurrence and development.The Bcl-2 gene amplification leads to abnormal proliferation of esophageal carcinoma cells.The abnormal expression of Bcl-2 and Bcl-xl protein results in tolerance of tumors to chemotherapy.Furthermore,the expression level of Bcl-2 is an important index to distinguish the differentiation of esophageal cancer cells,and it may also influence the prognosis of esophageal carcinoma.Therefore,Bcl-2 family protein are attractive targets for the treatment of esophageal cancer.AT-101[R-(-)-enantiomer of gossypol]is an inhibitor of Bcl-2 screened through the Bcl-2 site.Since it was originally used as an effective oral anti fertility drug,we can learn from its natural product safety data.AT-101 can inhibit Bcl-2,Bcl-xl and Mcl-1 simultaneously.AT-101 can inhibit the proliferation of malignant tumors and induce apoptosis in vivo and in vitro.Although AT-101 can inhibit the proliferation of tumor cells,and the treatment of solid tumors has entered the second clinical phase,there is no clear mechanism between AT-101 and esophageal cancer cells.At present,there are a lot of drugs that can change the cell cycle to play the anti-tumor effect.Therefore,we investigated whether AT-101 can inhibit the vaibility of human esophageal cancer cells and further explored the mechanism underlying its inhibition of cell viability in this study.Moreover,we explored the mechanism whereby AT-101 induced cell cycle arrest and apoptosis.We hope that this study may provide an experimental basis for clinical application of AT-101 for esophageal cancer.OBJECTIVEWe investigated the effect of AT-101 on the cell viability of human esophageal cancer cells,and further explored the mechanism whereby AT-101 induced cell cycle arrest and apoptosis.METHODS AND RESULTSMTT assay was used to investigate the effect of AT-101 on the viability of human esophageal cancer cell lines,EC109 and CaES-17.AT-101 could significantly inhibit the viability of human esophageal cancer cells.Flow cytometry was utilized to determine cell cycle distribution as well as apoptosis.In this regard,we found that AT-101 could induce G1/G0 phase arrest and apoptosis.AT-101 increased the mRNA levels of p53 and p21wafl/Cipl measured by fluorescence quantitative PCR.Furthermore,our data showed that AT-101 elevated the expression of p-p53(Ser 15),p53 and p21wafl/Cipl,but it did not change the expression of p27,CDK2/4/6 and Rb protein by western blot analysis.At the same time,the expression of cyclin D1,p-Rb(Ser 780)and E2F-1 protein were decreased by AT-101.In further study,we found that AT-101 significantly suppressed the expression of ?-catenin protein.Interfering with the expression of ?-catenin protein by small molecule compounds,we found that the ?-catenin activator WAY-262611 was able to reverse the G1/G0 cell cycle arrest induced by AT-101 and attenuated the inhibitory effect of AT-101 on the expression of P-catenin and cyclin D1 proteins.In contrast,?-catenin inhibitor XAV-939 enhanced AT-101-induced G1/G0 cell cycle arrest.Moreover,XAV-939 enhanced the inhibitory effect of AT-101 on cyclin D1 protein expression.In addition,western blot analysis showed that AT-101 could inhibit the expression of anti-apoptotic protein Bcl-2 and Mcl-1,activating cytochrome C/caspase-9/caspase-3/PARP proapoptotic signaling pathway in esophageal cancer cells.CONCLUSION1.AT-101 significantly inhibits the viability of human esophageal cancer cell lines EC 109 and CaES-17,with IC50 value of 2-3 ?M.2.AT-101 induces G1/G0 cell cycle arrest and apoptosis in EC 109 and CaES-17 cells.3.AT-101 induces G1/G0 cell cycle arrest in human esophageal cancer cells by inhibiting ?-catenin/cyclin D1 pathway.4.AT-101 promotes the expression of p53/p21wafl/Cipl mRNAs and protein whereas it suppresses p-Rb and E2F-1 protein levels.5.AT-101 inhibits the expression of anti-apoptotic protein Bcl-2 and Mcl-1,activating cytochrome C/caspase-9/caspase-3/PARP proapoptotic signaling pathway... |
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