三叶青总黄酮增强三阴性乳腺癌细胞对EGFR-TKI敏感性的机制研究

Effect and mechanism radix tetrastigmae enhances EGFR-TKI sensitivity in breast cancer cells

目的探讨三叶青总黄酮(RTF)对三阴性乳腺癌MDA-MB-231细胞表皮生长因子受体酪氨酸激酶抑制药(EGFR-TKI)药物吉非替尼(GEF)敏感性的影响及其机制。方法以不同浓度的RTF或RTF联合不同浓度的GEF处理MDA-MB-231细胞后,采用噻唑蓝(MTT)法检测细胞活力;将细胞分为对照组(培养基处理)、RTF(25μg·mL^(-1) RTF处理)组、GEF(10μg·mL^(-1) GEF处理)组、GEF+RTF组(10μg·mL^(-1) GEF和25μg·mL^(-1) RTF共同处理)和GEF+RTF+RAPA组(10μg·mL^(-1) GEF、25μg·mL^(-1) RTF和10 nmol·L^(-1) RAPA共同处理)。流式细胞仪检测细胞周期与凋亡;蛋白质印迹法检测凋亡相关蛋白B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、剪切的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)和自噬相关蛋白微管相关蛋白1轻链3(LC3)、P62和Beclin-1的表达。结果对照组、RTF组、GEF组、GEF+RTF组和GEF+RTF+RAPA组处于S期的细胞比例分别为(43.89±3.20)%,(36.36±3.73)%,(30.28±2.87)%,(18.12±1.54)%和(23.82±2.20)%;凋亡细胞的比例分别为(3.60±0.68)%,(12.46±2.80)%,(22.77±3.10)%,(36.19±3.87)%和(27.04±2.84)%;上述5组Cleaved Caspase-3蛋白表达水平分别为0.12±0.03,0.36±0.03,0.48±0.03,0.74±0.06和0.56±0.03;Bcl-2蛋白表达水平分别为0.84±0.06,0.71±0.06,0.57±0.04,0.36±0.03和0.48±0.04;Bax蛋白表达水平分别为0.15±0.03,0.20±0.02,0.30±0.03,0.45±0.03和0.38±0.03;LC3Ⅱ/Ⅰ蛋白表达水平分别为0.98±0.05,0.20±0.02,4.36±0.91,1.10±0.07和2.75±0.58;P62蛋白表达水平分别为0.27±0.03,0.53±0.04,0.12±0.03,0.38±0.03和0.18±0.02;Beclin-1蛋白表达水平分别为0.11±0.02,0.06±0.01,0.40±0.03,0.22±0.03和0.33±0.03。RTF组和GEF组与对照组比较,各项指标差异均具有统计学意义(均P<0.05);GEF+RTF+RAPA组与GEF+RTF组比较,各项指标差异均具有统计学意义(均P<0.05)。结论RTF可通过抑制自噬增强MDA-MB-231细胞对EGFR-TKI药物敏感性的敏感性。

Objective To investigate the effect of total flavonoids from radix tetrastigmae(RTF)on sensitivity of triple negative breast cancer MDA-MB-231 cell to epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI)gefitinib(GEF)and its mechanism.Methods MDA-MB-231 cells were treated with different concentrations of RTF or RTF combined with different concentrations of GEF,and the cell viability was detected by MTT assay;the cells were divided into control group(medium treatment),RTF(treated with 25μg·mL^(-1) RTF)group,GEF(treated with 10μg·mL^(-1) GEF)group,GEF+RTF group(treated with 10μg·mL^(-1) GEF and 25μg·mL^(-1) RTF)and GEF+RTF+RAPA(treated with 10μg·mL^(-1) GEF,25μg·mL^(-1) RTF and 10 nmol·L^(-1) RAPA)group.The cell cycle and apoptosis were detected by flow cytometry,and the expressions of apoptosis related proteins B cell lymphoma 2 family protein(Bcl-2),Bcl-2 associated X(Bax),Cleaved Caspase-3,autophagy related proteins microtubule-associated protein 1 light 3(LC3),p62 and beclin-1 were detected by Western blot.Results The proportions of cells in S phase of the control group,RTF group,GEF group,GEF+RTF group and GEF+RTF+RAPA group were(43.89±3.20)%,(36.36±3.73)%,(30.28±2.87)%,(18.12±1.54)%and(23.82±2.20)%;the proportions of apoptotic cells were(3.60±0.68)%,(12.46±2.80)%,(22.77±3.10)%,(36.19±3.87)%and(27.04±2.84)%;the protein levels of Cleaved Caspase-3 were 0.12±0.03,0.36±0.03,0.48±0.03,0.74±0.06 and 0.56±0.03;the protein levels of Bcl-2 were 0.84±0.06,0.71±0.06,0.57±0.04,0.36±0.03 and 0.48±0.04;the protein levels of Bax were 0.15±0.03,0.20±0.02,0.30±0.03,0.45±0.03 and 0.38±0.03;the protein levels of LC3Ⅱ/Ⅰprotein were 0.98±0.05,0.20±0.02,4.36±0.91,1.10±0.07 and 2.75±0.58;the protein levels of P62 were 0.27±0.03,0.53±0.04,0.12±0.03,0.38±0.03 and 0.18±0.02;Beclin-1 protein levels were 0.11±0.02,0.06±0.01,0.40±0.03,0.22±0.03 and 0.33±0.03,respectively.Compared with the control group,the differences in each index between the RTF group and the GEF group were statistically significant(all P<0.05).Compared with the GEF+RTF+RAPA group,the differences in each index were statistically significant(all P<0.05).Conclusion RTF can enhance the sensitivity of MDA-MB-231 cells to EGFR-TKI by inhibiting autophagy.