二氢杨梅素增强阿霉素对肝癌细胞的抑制作用

Enhancing doxorubicin-induced anti-tumor effects on human liver cancer cells by dihydromyricetin

目的研究二氢杨梅素(dihydromyricetin,DMY)对阿霉素(adriamycin,DOX)在肝癌化疗中的增效作用并研究其可能作用机制。方法不同浓度DMY作用肝癌细胞系MHCC97H后,CCK-8法筛选无毒剂量的DMY,用无毒剂量的DMY联合不同浓度DOX作用MHCC97H细胞,CCK-8法检测细胞活力;Hoschest染色法测定细胞凋亡情况;通过蛋白质印迹法测定PTEN、p-AKT、Bcl-2、Cyt-c、P-gp、裂解的caspase-3和裂解的caspase-9;合成特异性靶向PTEN基因的shRNA慢病毒表达载体,建立稳定沉默PTEN基因的MHCC97H细胞,检测沉默PTEN后是否影响DMY联合DOX对肝癌细胞凋亡的诱导作用;通过裸鼠皮下肿瘤细胞注射法建立MHCC97H裸鼠异种移植模型,观察DMY联合DOX给药对体内肝癌生长的影响,Tunel法测定体内肝癌细胞凋亡,Ki-67和PTEN在移植瘤组织中的表达通过免疫组织化学法测定。结果无毒剂量的DMY与DOX联合应用能显著提高DOX对体外MHCC97H细胞的生长抑制和凋亡诱导作用;DMY联合DOX可上调体外肝癌MHCC97H细胞中PTEN、Cyt-c、cleaved caspase-3和cleaved caspase-9含量,并下调p-AKT、Bcl-2和P-gp的表达;成功构建沉默PTEN基因的稳定细胞系,沉默PTEN基因可明显抑制DMY对DOX的辅助治疗效果,同时DMY联合DOX对MHCC97H细胞的凋亡诱导途径受到明显抑制。DMY联合DOX给药可抑制体内肝癌生长,有效诱导体内细胞凋亡,同时肿瘤组织中的Ki-67和PTEN表达受到DMY联合DOX的显著下调和上调作用。结论DMY可能通过PTEN/PI3K/AKT途径增强DOX对肝癌细胞的体外杀伤活性。

Objective To investigate the adjuvant effect of dihydromyricetin(DMY)on doxorubicin(DOX)-based chemotherapy on liver cancer and its mechanisms.Methods Human liver cancer MHCC97 H cells were exposed to increasing doses of DMY.Nontoxic dose of DMY was screened by CCK-8 assay.MHCC97 H cells were exposed to increasing doses of DOX and nontoxic dose of DMY.CCK-8 experiment was employed to analyze the viability of the MHCC97 H cells in vitro.Apoptotic MHCC97 H cells after Hosches 33342 staining were viewed by fluorescence microscopy.Western blot was employed to analyze the levels of PTEN,p-AKT,Bcl-2,Cyt-c,P-gp,cleaved caspase-3 and cleaved caspase-9 in the MHCC97 H cells.Human PTEN sequence was employed for the design of shRNA targeting PTEN.Then the MHCC97 H cells were infected using the lenti-virus packaging to observe whether silencing of PTEN affects the apoptosis induction effect of DMY and DOX.MHCC97 H cells were implanted subcutaneously into left flank of nude mice to testify the anti-cancer effect of DMY in vivo.The apoptosis of liver cancer in vivo was detected by Tunel test.Immunohistochemistry was employed to analyze the levels of Ki-67 and PTEN in the xenografts after DMY plus DOX treatment.Results Nontoxic dose of DMY significantly enhanced the growth inhibitory effect of DOX against liver cancer cells.Furthermore,the apoptotic cells induced by DMY and DOX were more than that of DOX alone.The levels of pro-apoptotic proteins(PTEN,Cyt-c,cleaved caspase-3 and cleaved caspase-9)were increased by the combination of DMY and DOX,while the protein levels of p-AKT,Bcl-2 and P-gp were markedly inhibited upon the same treatment.The down-regulation expression of PTEN significantly suppressed the adjuvant effect of DMY and DOX.Meanwhile,the regulatory effects of DMY and DOX on the apoptosis-related proteins were reversed by the knockdown of PTEN.In vivo experiment,the growth of subcutaneously implanted tumors were notably inhibited by DMY and DOX treatment.The protein expressions of Ki-67 and PTEN in tumor tissues were obviously decreased and increased,respectively,by the treatment of DMY and DOX.Furthermore,Co-treatment of DMY and DOX showed a significantly greater increase in tumor cell apoptosis in vivo.Conclusion DMY exerts a promotive effect on the cytotoxicity of DOX against human liver cancer cells through the PTEN/PI3 K/AKT pathway.