摘要
目的 探讨芹菜素与曲妥珠单抗联合应用对人表皮生长因子受体2(HER2)阳性乳腺癌细胞系BT474及SK-BR-3的抗肿瘤作用及其机制.方法 采用流式细胞术检测曲妥珠单抗与人乳腺癌细胞的结合活性;细胞计数试剂盒(CCK-8)检测芹菜素、曲妥珠单抗单药及联合应用对BT474和SK-BR-3细胞的生长抑制活性;EdU染色法检测芹菜素、曲妥珠单抗单药及联合应用对BT474和SK-BR-3细胞DNA复制的影响;蛋白质印迹法检测芹菜素、曲妥珠单抗单药及联合应用对BT474和SK-BR-3细胞中表皮生长因子受体(EGFR)、HER2、HER3、Akt等蛋白及其各自磷酸化蛋白表达水平的影响.设置芹菜素组、曲妥珠单抗组、芹菜素+曲妥珠单抗联合用药组、对照组和空白对照组.结果 生长抑制实验结果显示,芹菜素抑制BT474和SK-BR-3细胞生长的半数抑制浓度值分别为25.70和29.34 μmol/L.EdU染色实验结果表明,不同浓度芹菜素(16、32、64 μmol/L)单药作用于SK-BR-3和BT474细胞后,与对照组比较差异均有统计学意义,F值分别为99.25和309.10,均P＜0.001.芹菜素(16、32、64 μmol/L)与曲妥珠单抗(16.67 nmol/L)联合处理SK-BR-3和BT474细胞后,与芹菜素组EdU阳性率比较,差异均有统计学意义,F值分别为145.60和110.10,均P＜0.001;芹菜素+曲妥珠单抗联合用药组与曲妥珠单抗组EdU阳性率比较差异有统计学意义,F值分别为48.38和66.46,均P＜0.001.蛋白质印迹结果显示,芹菜素组、曲妥珠单抗组和芹菜素+曲妥珠单抗联合用药组p-EGFR蛋白相对表达量分别为(81.75±1.08)％、(45.84±1.71)％和(17.06±1.06)％,F=2 093.00,P＜0.001;p-HER2 蛋白相对表达量分别为(55.09±6.23)％、(41.33±2.48)％和(8.87±0.20)％,F=604.60,P＜0.001;p-HER3 蛋白相对表达量分别为(67.78±1.94)％、(17.89±0.84)％和(12.06±0.81)％,F=2 749.00,P＜0.001;p-Akt 蛋白相对表达量分别为(46.85±13.96)％、(42.35±2.71)％和(23.41±0.24)％,F=85.73,P＜0.001.结论 芹菜素与曲妥珠单抗可协同抑制HER2阳性乳腺癌细胞增殖,其作用机制可能与抑制Akt信号通路有关.
Abstract
Objective To explore the anti-tumor effect and mechanism of apigenin combined with trastuzumab on human ep-idermal growth factor receptor 2(HER2)positive breast cancer cell lines BT474 and SK-BR-3.Methods The binding ac-tivity of trastuzumab to human breast cancer cells was detected by flow cytometry;Cell counting kit(CCK)-8 was used to detect the growth inhibitory activity of apigenin,trastuzumab monotherapy,and their combination on BT474 and SK-BR-3 cells;EdU staining method was used to detect the effects of apigenin,trastuzumab monotherapy,and their combination on DNA replication in BT474 and SK-BR-3 cells;Protein blotting was used to detect the effects of apigenin,trastuzumab monotherapy,and their combination on the expression levels of epidermal growth factor receptor(EGFR),HER2,HER3,Akt,and their respective phosphorylated proteins in BT474 and SK-BR-3 cells.The experiment was divided into apigenin group,trastuzumab group,apigenin+trastuzumab combination group,control group,and blank control group.Results The results of the growth inhibition experiment showed that the IC50 values of apigenin inhibiting the growth of BT474 and SK-BR-3 cells were 25.70 and 29.34 μmol/L,respectively.The EdU staining experiment results showed that after different concentrations of apigenin(16,32,64 μmol/L)were applied to SK-BR-3 and BT474 cells,and there were statistically significant differences compared to the control group,with F values of 99.25 and 309.10,both P＜0.001.After treating SK-BR-3 and BT474 cells with a combination of apigenin and trastuzumab,there was a statistically signifi-cant difference in the positive rate of EdU compared to the apigenin group,with F values of 145.60 and 110.10,respec-tively,all P＜0.001;There was a statistically significant difference in the positive rate of EdU compared to the trastuzum-ab group,with F values of 48.38 and 66.46,respectively,both P＜0.001.The protein blot results showed that the rela-tive expression levels of p-EGFR protein in the apigenin group,trastuzumab group,and apigenin+trastuzumab combina-tion group were(81.75±1.08)％,(45.84±1.71)％and(17.06±1.06)％,F=2.093.00,P＜0.001.The relative ex-pression levels of p-HER2 protein were(55.09±6.23)％,(41.33±2.48)％and(8.87±0.20)％,F=604.60,P＜0.001.The relative expression levels of p-HER3 protein were(67.78±1.94)％,(17.89±0.84)％and(12.06±0.81)％,F=2 749.00,P＜0.001.The relative expression levels of p-Akt protein were(46.85±13.96)％,(42.35±2.71)％and(23.41±0.24)％,F=85.73,P＜0.001.Conclusion Apigenin and trastuzumab can synergistically inhibit the proliferation of HER2 positive breast cancer cells,and the mechanism may be related to the inhibition of Akt signaling pathway.
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