ApoG2诱导乳腺癌细胞MDA-MB231自噬的最低浓度探讨

Rational use of ApoG2 through autophagy inhibition exploiting and apoptosis inducing in breast cancer MDA- MB231 cells

目的:探究不同浓度棉酚衍生物Apo G2对乳腺癌细胞株MDA-MB231诱导自噬的最低浓度,寻找抗肿瘤效应的最适合浓度。方法:CCK8法检测细胞的增殖活性;透射电镜下观察细胞超微结构;流式细胞仪检测细胞凋亡及周期阻滞情况;细胞免疫荧光法检测各组细胞LC3-II荧光强度;RT-PCR及Western blot法检测细胞自噬蛋白LC3-I及LC3-II表达强度。结果:CCK8法示Apo G2对乳腺癌MDA-MB231细胞呈浓度、时间依赖性抑制增殖且各组间差异存在统计学意义;透射电镜下发现Apo G2可诱导凋亡及自噬;流式细胞仪检测发现Apo G2诱导凋亡呈浓度依赖性,使细胞周期阻滞于S期且10μmol/L浓度组最明显;细胞免疫荧光法发现10μmol/L组细胞LC3-II荧光最弱;RT-PCR及Western blot法示细胞自噬蛋白LC3-I表达各组间无明显差异,LC3-II在10μmol/L浓度组表达量最弱,80μmol/L浓度组表达量最强。结论:10μmol/L为Apo G2诱导乳腺癌细胞MDA-MB231自噬最低浓度,也是抑制自噬,促进凋亡的抗肿瘤最适浓度。

Objective:To investigate the rational dose of Apogosspolone by inhibiting antophagy in anticancer ther-apy.Methods:Qualitative verification of apoptosis and autophagy was conducted with transmission electron micro-scope.Semi -quantitative determination of autophapy was performed by immunofluorescence staining,flow cytometry, and quantitative determination of autophagy by CCK8,real -time polymerase chain reaction,Western blotting.Re-sults:CCK8 showed ApoG2 was at inhibition proliferation in concentration and time dependence for MDA -MB231 of brest cancer.It was found that ApoG2 can induce apoptosis and autophagy under the transmission electron micro-scope.ApoG2 induced apoptosis with concentration dependence and arrested in S stage for cell cycle,with obvious-ness in 10μmol/L group.Immunofluorescence staining showed the weakest cell fluorescence in 10μmol/L group.Cell autophagy protein LC3 -I expression was no significant difference among groups,with the weakest expression of LC3-II in 10μmol/L group and the strongest in 80μmol/L group by RT -PCR and Western blot.Conclusion:We rec-ommend 10μmol/L as the rational use of apogosspolone to abrogate autophagy in breast cancer cell MDA -MB231.