| Objective:Through exploring the effects of autophagy inhibitor HCQ combined with chemotherapeutic drugs DOC on proliferation of castration-resistant prostate cancer22RV1 cell line in vitro and in vivo,and its mRNA expression changes of autophagy gene Bcelin-1,autophagy-specific substrate P62 gene,pro-apoptotic gene Bax and expression influence of autophagic protein Bcelin-1,autophagy specific marker LC3B protein,pro-apoptotic protein Bax,to further understand the biological mechanisms of CRPC patients'resistance to chemotherapeutic drugs,provide a research basis for the clinical treatment of CRPC through the regulation of autophagy level,and also provide a detection target for the resistance monitoring of CRPC patients.Methods:Castration-resistant prostate cancer 22RV1 cell line was cultured in vitro,nude mice transplanted tumor with 22RV1 cell line were established in vivo.Set the blank control group and DOC,HCQ+DOC experiment groups respectively.In the DOC and HCQ+DOC experimental groups in vitro,three subgroups with DOC concentrations of 10-6,10-7,and 10-88 mol/L were set.The HCQ+DOC experiment group received DOC after using the autophagy inhibitor HCQ for autophagy intervention.Detected the cell proliferation activity changes in vitro,observed the growth volume of transplanted tumors in vivo,and used qPCR to detect Beclin-1,P62,and Bax mRNA expression levels,used Western blot to detect Beclin-1.LC3B,Bax protein expression levels in 22RV1 cell lines and transplanted tumors.Results:1.Changes of cell proliferation in vitro:compared with the blank control group,the proliferation of cells in each experiment group showed a decrease,and the difference was significant?P<0.05?;in the DOC and HCQ+DOC experiment groups,the cells in each subgroup proliferation activity decreased with increasing DOC concentration,and the difference was significant?P<0.05?;at the same DOC concentration,the degree of proliferation of HCQ+DOC group cells was lower than that of DOC group,and the difference was significant?P<0.05?.The most significant difference was observed when the DOC concentration was 10-77 mol/L.The IC50 of the drug DOC in DOC+HCQ was also reduced to 10-7.536mol/L by the single action of10-6.120mol/L.2.Volume change of transplanted tumor in vivo:compared with the control group,the volume growth value of the transplanted tumors showed a downward trend with the increase in the number of weeks,and the difference was statistically significant?P<0.05?;among the DOC and HCQ+DOC experiments groups,The weekly volume growth of tumor in the HCQ+DOC group was lower than that in the DOC group,it was significantly lower in the fourth week,and the difference was significant?P<0.05?.3.mRNA detection results:?1?Cultured cell line mRNA detection in vitro:compared with the blank control group,the expression levels of Beclin-1,P62,and Bax genes in both experiment groups all increased;in comparison,the expression of Beclin-1,P62,and Bax genes in HCQ+DOC group was higher,and the difference was significant?P<0.05?.?2?Transplantation tumor mRNA detection in vivo:compared with the blank control group,the Beclin-1,Bax gene expression levels in DOC group and Beclin-1,P62,and Bax gene expression levels in HCQ+DOC group were all increased;the expression of Beclin-1,P62 and Bax genes in HCQ+DOC group was higher in compared with DOC group,and the difference was significant?P<0.05?.4.Protein detection results:?1?Cultured cell line protein detection in vitro:autophagic protein Beclin-1,autophagy specific marker LC3B protein,and pro-apoptotic protein Bax in DOC and HCQ+DOC experiment groups both showed higher expressions in compared with control group,and the expression of HCQ+DOC group was higher,the difference was significant?P<0.05?.?2?Transplantation tumor protein detection in vivo:compared with the blank control group,the autophagy specific markers LC3B protein and pro-apoptotic protein Bax of the experimental group DOC and HCQ+DOC were both up-regulated and the HCQ+DOC group expression was higher,the difference was significant?P<0.05?;the expression of autophagy protein Beclin-1 in HCQ+DOC group was significantly higher than that in blank conrol group and DOC group,and the difference was significant?P<0.05?.Conclusion:The results of this experiment showed that using the autophagy inhibitor HCQ can significantly inhibit the proliferation of prostate cancer cells,enhance the expressions of Beclin-1,P62,and Bax mRNA and beclin-1,LC3B,and Bax protein in vitro and vivo,which were involved in the down-regulation of autophagy expression.Therefore,we believe that autophagy inhibitor HCQ can increase the sensitivity of prostate cancer 22RV1 cell line to chemotherapeutic drug DOC. |
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