大黄酸调控Rac1/LIMK1/cofilin信号通路抑制卵巢癌细胞运动与侵袭

摘要

目的：探讨大黄酸对卵巢癌淋巴结高转移 SKOV3-PM4细胞运动和侵袭能力的影响,揭示大黄酸调控 Rac1/LIMK1/ cofilin信号通路与抑制卵巢癌细胞运动侵袭作用的机制。方法划痕实验观察大黄酸对卵巢癌细胞运动的影响,Matrigel-Transwell方法检测细胞侵袭能力,激光共聚焦扫描显微镜和扫描电镜观察大黄酸对卵巢癌细胞形态和细胞骨架超微结构的影响, Western blot分别检测 Rac1/LIMK1/cofilin通路上Rac1、LIMK1、PAK1、cofilin蛋白的表达。结果与空白对照组相比,大黄酸能抑制SKOV3-PM4细胞侵袭和迁移能力,浓度为8．8、17．60、26．40μmol · L-1的大黄酸处理24 h后,细胞体外迁移和侵袭能力均明显下降( P <0．05)；细胞出现伪足减少,细胞内微丝断裂、分布紊乱,质膜凹凸不平、细胞间的间隙变宽等超微结构改变； Western blot结果表明大黄酸能明显下调Rac1蛋白的表达水平,并呈浓度依赖性。卵巢癌细胞经大黄酸及Rac1抑制剂处理后,磷酸化LIMK1、cofilin、PAK1蛋白水平与空白对照组比较明显下降,且大黄酸联合Rac1抑制剂处理后的磷酸化蛋白下调更为明显( P<0．05)；而Rac1激活剂联合大黄酸组比大黄酸组磷酸化蛋白上调明显( P<0．05)。结论大黄酸为潜在的Rac1小分子抑制剂,其作用机制可能是调控 Rac1/LIMK1/ cofilin信号通路,抑制卵巢癌淋巴结转移细胞运动和侵袭的能力。%Aim To investigate the effect of Rhein on the movement and invasiveness of human ovarian carci-noma cells with directional high lymphatic metastasis SKOV3-PM4 cells and explore the role of Rac1/LIMK1/cofilin signaling pathway. Methods Migration assay and invasion assay were used to observe the effect of Rhein on the metastatic and invasive ability of SK-OV3-PM4 cells in vitro. The effect of Rhein on the morphology and cytoskeleton ultrastructure of ovarian cancer cells was observed by laser scanning confocal microscope and scanning electron microscope. The protein expression level of Rac1,LIMK1,PAK1 and co-filin were detected by Western blot, respectively. Re-sults Rhein inhibited the abilities of cell invasion and migration of SKOV3-PM4 cells,and the inhibitory rate increased along with the increase of the concentration and treatment duration. After treated with 8. 80 μmol· L-1 ,17. 60 μmol · L-1 , 26. 40 μmol · L-1 of Rhein for 24 h,the abilities of migration and invasion of SK-OV3-PM4 cells were inhibited ( P <0. 05 ); the mor-phology and cytoskeleton ultrastructure of SKOV3-PM4 cells were changed, cellular pseudopod reduced, cell microfilament fractured and its distribution disordered, plasma membrane was uneven and cell gap widened . After treatment of Rhein and Rac1 inhibitor , Rac1 protein expression and the expression of P-LIMK1 , P-PAK1 and P-cofilin notably decreased in a dose-de-pendent manner compared with the control group ( P<0. 05 ) . After Rhein and Rhein plus Rac1 inhibitor treatment ,P-LIMK1, P-cofilin, P-PAK1 protein levels of SKOV3-PM4 cells significantly decreased compared with the control group , and the group of Rac1 inhibi-tor plus Rhein treatment, the phosphorylated protein decreased more significantly ( P <0. 05 ) . After Rac1 activator plus Rhein treatment, phosphorylated protein expression of P-LIMK1 ,P-PAK1 and P-cofilin upregu-lated significantly ( P <0. 05 ) . Conclusions Rhein may be a potential inhibitor of Rac1 and can inhibit the migrating and invasive capabilities of directional high lymphatic metastasis SKOV3-PM4 cells through down-regulating the phosphorylation of Rac1/LIMK1 /cofilin pathway associated protein.