基于MEK/ERK信号通路探讨阳和汤含药血清对乳腺癌4T1细胞的影响

Effect of Yanghetang-containing Serum on Breast Cancer 4T1 Cells Based on MEK/ERK Signaling Pathway

目的:通过阳和汤含药血清干预小鼠乳腺癌4T1细胞,探索阳和汤对乳腺癌4T1细胞及其内丝裂原活化蛋白激酶(MEK)/细胞外调节蛋白激酶(ERK)信号通路的影响。方法:制备阳和汤SD大鼠含药血清。将其随机分组为空白组、阳和汤低、中、高剂量组(5.8、11.6、23.2 g·kg^(-1)),空白组用生理盐水代替。各组按10%含药血清和90%RMPI 1640完全培养基均匀混合,干预细胞。采用细胞增殖与活性检测(CCK-8)法分别在24、48、72 h检测阳和汤含药血清对4T1细胞增殖的影响;采用流式细胞术检测4T1细胞的凋亡情况;采用划痕实验检测4T1细胞迁移情况;采用Transwell实验检测细胞侵袭能力;采用蛋白免疫印迹法(Western blot)检测MEK1/2、磷酸化(p)-MEK1/2、ERK1/2、p-ERK1/2、大鼠肉瘤病毒(RAS)蛋白的表达水平。结果:与空白组比较,阳和汤干预24、48、72 h,阳和汤含药血清低、中、高剂量组对4T1细胞增殖具有明显抑制作用(P<0.05,P<0.01);阳和汤干预48 h,阳和汤含药血清低、中、高剂量组细胞凋亡率显著升高(P<0.01);阳和汤含药血清低、中、高剂量组在24、48 h的细胞划痕愈合能力显著降低(P<0.01);阳和汤含药血清低、中、高剂量组细胞侵袭能力显著降低(P<0.01);阳和汤含药血清低、中、高剂量组细胞内MEK1/2、ERK1/2表达差异无统计学意义,p-MEK1/2、p-ERK1/2、RAS蛋白表达显著降低(P<0.01)。结论:阳和汤可抑制乳腺癌细胞4T1增殖、迁移、侵袭,促进其凋亡。其凋亡机制与MEK/ERK信号通路受抑制,p-MEK1/2、p-ERK1/2、RAS蛋白表达下调有关。

Objective:To investigate the effects of Yanghetang (YHT) on breast cancer 4T1 cells and their mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway.Method:The YHT-containing serum was prepared from SD rats.The rats were randomly assigned into a blank group (normal saline) and low-,medium-,and high-dose (5.8,11.6,23.2 g·kg^(-1),respectively) YHT groups.The serum containing 10%YHT in each group was mixed with 90%RMPI 1640 complete medium,and the mixture was used to interfere with the cells.Cell counting kit-8 (CCK-8) method was used to detect the proliferation of the 4T1 cells treated with YHT for 24,48,72 h.The apoptosis,migration,and invasion of 4T1cells were detected by flow cytometry,scratch test,and Transwell assay,respectively.Western blot was employed to determine the expression levels of MEK1/2,phosphorylation (p)-MEK1/2,ERK1/2,p-ERK1/2,and rat sarcoma virus (RAS) protein.Result:Compared with the blank group,the intervention with YHT-containing serum for 24,48,and 72 h had significant inhibitory effect on 4T1 cell proliferation (P<0.05,P<0.01).After intervention with YHT-containing serum for 48 h,the apoptosis rate of cells increased (P<0.01).Compared with the blank group,the intervention with YHT for 24 h and 48 h decreased the healing ability of cells in the scratch test (P<0.01).The invasive ability of cells treated with the low,medium,and high-dose YHT containing serum showed a decreasing trend (P<0.01).Compared with the blank group,YHT-containing serum did not change the expression of MEK1/2 and ERK1/2 while down-regulating the expression of p-MEK1/2,p-ERK1/2,and RAS protein (P<0.01).Conclusion:YHT can inhibit the proliferation,migration,and invasion and promote the apoptosis of breast cancer 4T1 cells.In may promote the apoptosis by inhibiting the MEK/ERK signaling pathway and down-regulating the expression of p-MEK1/2,p-ERK1/2,and RAS protein.