Effects Of Autophagy Gene Beclin-1 In Adrenocortical Carcinoma And Experimental Study Of Chemosensitivity

Objectives 1.To detect the expression of autophagy gene Beclin-1 and to explore its clinical significance in adrenocortical carcinoma(ACC);2.To investigate the regulatory effects of autophagy gene Beclin-1 of proliferation in vitro,autophagy,apoptosis,invasion and cell cycle of cell biology on ACC SW13 cells,and to explore its mechanism;3.To investigate the role of the autophagy in the regulation of chemosensitivity to cisplatin in ACC SW13 cells,and to explore the feasibility of induce of down-regulation of autophagy gene Beclin-1 in treating ACC.Methods 1.The expression of Beclin-1 was detected with Immunohistochemistry and Western Blot analysis in specimens of ACC and normal adrenal tissue,and correlations of expression of Beclin-1 to clinicopathologic factors of ACC were analyzed statistically;2.The target gene Beclin-1 was designed to construct the eukaryotic expression vector p LVX-IRES-Puro/Beclin-1,and Beclin-1-specific small hairpin RNA(sh RNA)was designed to construct sh RNA expression vector p SUPER/Beclin-1;3.The vectors were transfected into SW13 cells via lipofectamine.After the vectors were transfected into SW13 cells via lipofectamine,the expression levels of Beclin-1,hvps34,p110? and p-PKB m RNA and protein were detected by RT-PCR and Western Blot analysis,Monodansylcadaverine(MDC)staining for qualitative autophagy detection by fluorescence microscopy,cell viability assay by the MTT method,Annexin V-FITC/PI double staining for apoptosis evaluation by flow cytometry and cell invasive determined by Transwell;4.After SW13 cells were treated with cisplatin and/or chloroquine,the expression levels of autophagy related proteins(Beclin-1,LC3 B,and p62)and apoptosis related proteins(Bax and Bcl-2)were detected by Western Blot analysis,cell viability assay by the MTT method,Monodansylcadaverine(MDC)staining for qualitative autophagy detection by fluorescence microscopy and Annexin V-FITC/PI double staining for apoptosis evaluation by flow cytometry;5.The nude mice with subcutaneous ACC SW13 cell xenografts were treated with cisplatin and/or chloroquine by intraperitoneal injection.The growth of nude mice was observed and the weight and volume of their tumors were measured.Results 1.The positive expression of Beclin-1 in ACC tissues was significantly lower than that in normal adrenal tissues(P < 0.05),and negative expression of Beclin-1 was significantly correlated with advanced stage,regional lymph node metastasis,increasing T stage,and poor differentiation(P = 0.032,0.007,0.019,and 0.018,respectively);2.It was confirmed that the eukaryotic expression vector p LVX-IRES-Puro/Beclin-1 and sh RNA expression vectors p SUPER/Beclin-1 were constructed successfully by DNA sequencing;3.The eukaryotic expression vector p LVX-IRES-Puro/Beclin-1 significantly improved the expression of of Beclin-1 and hvps34(m RNA and protein),and decreased the expression of p110? and p-PKB in SW13 cell(P < 0.05),cell proliferation activity was decreased,autophagy and apoptotic cells were significantly increased and the ability of cell invasion was decreased;4.The sh RNA expression vectors p SUPER/Beclin-1 significantly decreased the expression of of Beclin-1 and hvps34(m RNA and protein),and improved the expression of p110? and p-PKB in SW13 cell(P < 0.05),the ability of cell invasion was increased,autophagy cells were increased,but there was no significant effect on the cell proliferation activity and the number of apoptotic cells;5.After SW13 cells were treated with cisplatin,autophagy and apoptotic cells were increased;when treated with chloroquine,the expression of Beclin-1 protein was down-regulated,which increased the sensitibity to cisplatin of SW13 cell;6.The nude mice with subcutaneous ACC SW13 cell xenografts were constructed successfully,and cisplatin can inhibit the growth of tumor to some extent,but the concomitant therapy approach(cisplatin and chloroquine)was more effective in reducing the tumor weight and size compared with cisplatin monotherapy(both P < 0.01),and can prolong the survival of nude mice bearing xenografted tumors.Conclusions 1.Beclin-1 expression was significantly down-regulated in ACC tissues,which may be correlated with the occurrence and development of ACC;Beclin-1 overexpression can effectively inhibit the cell proliferation and cell invasion,and induce autophagy and apoptosis in SW13 cell;2.Application of chloroquine which inhibited beclin-1 expression may contribute to the sensibility to cisplatin of SW13 cell,and concomitant therapy with cisplatin and chloroquine can increase the efficacy of cisplatin monotherapy in treating ACC.