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Title: [Effects of the Rho-kinase inhibitor fasudil on the invasion, migration, and apoptosis of human prostate cancer PC3 and DU145 cells].
Author: Gao QQ, Chen H, Chen Y, Xu ZP, Zhu LL, Yu W, Han YF, Dai YT.
Journal: Zhonghua Nan Ke Xue; 2016 Jun; 22(6):483-490. PubMed ID: 28963834.
Abstract:
OBJECTIVE: To investigate the potential role of the RhoA/Rock signaling pathway in the formation of prostate cancer and the effects of the Rock inhibitor fasudil on the invasion, migration and apoptosis of human prostate cancer cells. METHODS: Human prostate cancer cell lines PC3 and DU145 were treated with fasudil at the concentrations of 5, 10, 20, 40, 80, and 160 μmol/L, respectively, and those as negative controls cultured in the Ham's-F12 medium, all for 24 hours. Then, MTT assay was used to measure the cell inhibition rate and half maximal inhibitory concentration (IC50) value of fasudil, with 1/4 of IC50 as the medication dose for further investigation. The expressions of RhoA, RockⅠ, and RockⅡ proteins in the PC3 and DU145 cells were detected by Western blot and immunohistochemistry, and the invasion, migration and apoptosis of the cells were determined using the Transwell chamber, scratch wound healing assay and flow cytometry. RESULTS: Fasudil inhibited the proliferation of the PC3 cells from (9.29±1.23)% at 5 μmol/L to (81.37±3.97)% at 160 μmol/L and that of DU145 from (7.59±1.54)% to (76.53±2.67)%, both in a dose-dependent manner (P<0.05 ). Significantly fewer PC3 and DU145 cells migrated into the lower compartment in the experimental group (39.2±8.4 and 34.2±6.7) than in the negative control (116.8±9.3 and 112.5±10.8) (P<0.05 ). The wound healing rates of the PC3 and DU145 cells were remarkably lower in the former (［37.26±1.17］% and ［32.38±2.73］%) than in the latter (［78.12±4.16］% and ［69.47±6.71］%) (P<0.05 ). Annexin V-FITC/PI double staining showed markedly increased apoptosis rates of PC3 and DU145 cells treated with fasudil (［31.88±2.49］% and ［28.65±2.99］%) as compared with the negative controls (［7.51±2.28］% and ［7.13±1.61］%) (P<0.05 ). The expressions of RockⅠ and RockⅡ were significantly reduced in the fasudil-treated cells in comparison with those of the control group (P<0.05 ) while that of RhoA showed no significant difference between the two groups (P>0.05 ). CONCLUSIONS: The RhoA/Rock signaling pathway may play an important role in the formation of prostate cancer. Fasudil can significantly inhibit the proliferation, migration, and invasion and promote the apoptosis of human prostate cancer PC3 and DU145 cells by reducing RhoA/Rho kinase activity. 目的: 探讨RhoA/Rock信号传导通路在前列腺癌形成过程中的可能作用；研究Rock蛋白抑制剂法舒地尔潜在抗肿瘤侵袭、迁移和促凋亡的作用。方法: 分别用5、10、20、40、80、160 μmol/L浓度的法舒地尔作用于前列腺癌PC3、DU145细胞24h，MTT法检测细胞增殖抑制率，并计算其IC50，取法舒地尔IC50的1/4作为给药剂量用于进一步研究。免疫细胞化学法检测法舒地尔对PC3、DU145细胞RhoA、RockⅠ、RockⅡ蛋白表达的影响；Western印迹检测法舒地尔对PC3、DU145细胞RhoA总蛋白、RhoA膜蛋白、RockⅠ、RockⅡ蛋白表达的影响；通过Transwell、划痕实验、流式细胞术评估细胞的侵袭、迁移、凋亡情况。结果：随着法舒地尔作用浓度的增加,对PC3、DU145的抑制率分别从（9.29±1.23）%、（7.59±1.54）%增加到（81.37±3.97）%、（76.53±2.67）%，呈一定剂量依赖性(P均<0.05)；细胞迁移实验中实验组穿入下室的PC3、DU145细胞为（39.2±8.4）个和（34.2±6.7）个，阴性对照组分别为（116.8±9.3）和（112.5±10.8）个，差异有统计学意义(P均<0.05)；划痕实验结果显示实验组PC3、DU145细胞24h划痕愈合率为（37.26±1.17）%和（32.38±2.73）%，阴性对照组愈合率为（78.12±4.16）%和（69.47±6.71）%(P均<0.05)；AnnexinV-FITC/PI双染色法的结果显示，法舒地尔作用PC3、DU145细胞后凋亡率分别为（31.88±2.49）%、（28.65±2.99）%,显著高于阴性对照组［（7.51±2.28）%、（7.13±1.61）%，P均<0.05］。免疫细胞化学检测显示，法舒地尔能明显降低RockⅠ和RockⅡ的表达(与阴性对照组比较P均<0.05)，而RhoA的表达与阴性对照组比较差异无统计学意义(P>0.05)；Western印迹也显示，法舒地尔也明显降低RhoA膜蛋白、RockⅠ、RockⅡ蛋白的表达(与阴性对照组比较P均<0.05)，而RhoA总蛋白表达与阴性对照组差异无统计学意义(P>0.05)。结论：RhoA/Rock信号传导途径可能参与了前列腺癌的形成；法舒地尔能够有效地抑制前列腺癌细胞的增殖、侵袭、迁移，并促进其凋亡，其机制可能与法舒地尔下调RhoA/Rock信号通路有关。.

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