罗格列酮对乳腺癌4T1细胞迁移侵袭和p38 MAPK信号通路的影响

Effects of rosiglitazone on migration and invasion of breast cancer 4T1 cells and p38 MAPK signaling pathway

目的探讨罗格列酮(RGZ)对高转移性乳腺癌4T1细胞迁移侵袭和p38丝裂原活化蛋白激酶(p38 MAPK)信号通路的影响。方法4T1细胞经不同浓度RGZ(0、0.1、1、10μmol/L)处理后,采用活细胞计数试剂盒CCK-8检测增殖能力,流式细胞术检测凋亡情况,划痕实验和Transwell小室检测迁移和侵袭情况,Western blotting检测基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)、磷酸化细胞外信号调节激酶(p-ERK)、磷酸化c-Jun氨基末端激酶(p-JNK)和磷酸化p38 MAPK(p-p38 MAPK)的蛋白水平。结果4T1细胞经RGZ处理24 h后,CCK-8检测显示,与0μmol/L比较,10μmol/L浓度的增殖能力降低(P<0.05),故后续实验RGZ浓度采用10μmol/L。流式细胞术检测显示,与对照组比较,RGZ组的早期凋亡率和总凋亡率轻微升高,但差异均无统计学意义(P>0.05)。划痕实验和Transwell小室实验显示,与对照组比较,RGZ组细胞划痕愈合率和穿膜细胞数均减少,差异有统计学意义(P<0.05);Western blotting显示,与对照组比较,RGZ组MMP-2、MMP-9和p-p38的蛋白水平均降低(P<0.05),但两组p-ERK和p-JNK蛋白水平的差异无统计学意义(P>0.05)。与RGZ组相比,p38 MAPK抑制剂SB203580联合RGZ组的细胞划痕愈合率增加且增殖活性增强并伴有MMP-2和MMP-9水平升高,差异有统计学意义(P<0.05)。结论RGZ可以抑制乳腺癌4T1细胞的增殖和迁移侵袭能力,降低细胞内MMP-2和MMP-9的蛋白水平,其作用可能与RGZ促进p38的磷酸化有关。

Objective To investigate the effects of rosiglitazone(RGZ)on migration and invasion of highly metastatic breast cancer 4 T1 cells and p38 mitogen-activated protein kinase(p38 MAPK)signaling pathway.Methods 4 T1 cells were treated with different concentrations of RGZ(0,0.1,1,10μmol/L).Proliferative ability was detected by CCK-8 and apoptosis was detected by flow cytometry.Migration and invasion of 4 T1 cells were detected by scratch test and Transwell chamber.Western blotting was used to measure the protein levels of matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9),phosphorylated extracellular signal-regulated kinase(p-ERK),phosphorylated c-Jun amino-terminal kinase(p-JNK)and phosphorylated p38 MAPK(p-p38 MAPK).Results After 24 h of RGZ treatment,CCK-8 assay showed that the proliferative ability of 4 T1 cells at 10μmol/L was lower than that at 0μmol/L(P<0.05),so the concentration of RGZ was 10μmol/L in subsequent experiments.Flow cytometry showed that compared with the Control group,the early apoptotic rate and total apoptotic rate in RGZ group increased slightly,but there was no significant difference(P>0.05).Scratch test and Transwell chamber test showed that compared with the Control group,the healing rate and the number of penetrating cells in RGZ group decreased significantly(P<0.05).Western blotting showed that the levels of MMP-2,MMP-9 and p-p38 in RGZ group were lower than those in Control group(P<0.05).There was no significant difference in p-ERK and p-JNK protein levels(P>0.05).Compared with RGZ group,the healing rate of cell scratch wounds and proliferative activity in SB203580(p38 MAPK inhibitor)plus RGZ group increased,and the levels of MMP-2 and MMP-9 elevated simultaneously(P<0.05).Conclusion RGZ can inhibit the proliferation,migration and invasion of 4 T1 cells and decrease the levels of MMP-2 and MMP-9 in breast cancer,possibly by the up-regulation of p-p38 induced by RGZ.