罗哌卡因调节JAK2/STAT3信号通路对前列腺癌细胞恶性生物学行为的影响

Ropivacaine inhibit the malignant biological behaviors of prostate cancer cells by inactivating the JAK2/STAT3 signaling pathway

目的探讨罗哌卡因调节Janus激酶2(JAK2)/信号转导和转录激活因子3(STAT3)信号通路对前列腺癌细胞恶性生物学行为的影响。方法前列腺癌细胞株PC⁃3分别以0、1、2.5、5、10、15 mmol/L的罗哌卡因处理后以CCK⁃8实验测定各组细胞存活率,并筛选罗哌卡因在细胞中的最佳作用浓度。将PC⁃3细胞随机分为对照组、罗哌卡因组、colivelin(JAK2/STAT3激活剂)组、罗哌卡因+colivelin组,采用罗哌卡因和colivelin分组处理后以CCK⁃8实验、流式细胞实验、细胞划痕实验、Transwell侵袭实验分别检测各组细胞增殖、凋亡、迁移与侵袭;以免疫印迹法检测各组细胞凋亡相关蛋白[cleaved多聚ADP核糖聚合酶(PARP)、Bax、Bcl⁃2]、上皮间质转化(EMT)相关蛋白(Vimentin、E⁃cadherin)表达。构建PC⁃3裸小鼠移植瘤模型以不同剂量罗哌卡因处理后检测其移植瘤生长情况,筛选罗哌卡因在裸小鼠中的最佳作用剂量。将PC⁃3裸小鼠移植瘤模型随机分为对照组、罗哌卡因组、colivelin组、罗哌卡因+colivelin组,采用罗哌卡因和colivelin分组处理后以免疫组化检测移植瘤中Ki67表达,评测各组移植瘤组织细胞增殖情况;以TUNEL染色检测各组移植瘤组织细胞凋亡情况。以免疫印迹法检测各组细胞与移植瘤组织JAK2/STAT3通路相关蛋白表达。结果与对照组相比,罗哌卡因组细胞凋亡率、Bax及cleaved PARP、E⁃cadherin蛋白表达、移植瘤组织细胞凋亡比例升高(P<0.05),细胞存活率、迁移率、侵袭数、Bcl⁃2与Vimentin蛋白表达、移植瘤组织细胞增殖比例、细胞及移植瘤组织p⁃JAK2/JAK2、p⁃STAT3/STAT3降低(P<0.05);colivelin组细胞凋亡率、Bax及cleaved PARP、E⁃cadherin蛋白表达、移植瘤组织细胞凋亡比例降低(P<0.05),细胞存活率、迁移率、侵袭数、Bcl⁃2与Vimentin蛋白表达、移植瘤组织细胞增殖比例、细胞及移植瘤组织p⁃JAK2/JAK2、p⁃STAT3/STAT3升高(P<0.05)。与罗哌卡因组相比,罗哌卡因+colivelin组细胞凋亡率、Bax及cleaved PARP、E⁃cadherin蛋白表达、移植瘤组织细胞凋亡比例降低(P<0.05),细胞存活率、迁移率、侵袭数、Bcl⁃2与Vimentin蛋白表达、移植瘤组织细胞增殖比例、细胞及移植瘤组织p⁃JAK2/JAK2、p⁃STAT3/STAT3升高(P<0.05)。结论罗哌卡因可抑制JAK2/STAT3信号激活,进而降低前列腺癌细胞增殖、迁移、侵袭活性并促进其凋亡,延缓其在裸小鼠体内生长,最终抑制其恶性生物学行为。

Objective To investigate the effects of ropivacaine on the malignant biological behaviors of prostate cancer cells and its potential mechanism.Methods Human prostate cancer cells(PC⁃3)were treated with different doses of ropivacaine(0,1,2.5,5,10,and 15 mmol/L)to determine the optimal concentration of ropivacaine by cell survival curve analysis.PC⁃3 cells were cultured and randomly divided into four groups including the control group,the ropivacaine group,the colivelin group,and the combined group of ropivacaine and colivelin(JAK2/STAT3 activator)group.After treated with ropivacaine and colivelin,cell proliferation,apoptosis,migration,and invasion in each group were detected by CCK⁃8 assay,flow cytometry assay,cell scratch assay,and Transwell invasion assay.The expressions of apoptosis related proteins such as PARP,Bax,Bcl⁃2,and the expressions of EMT related proteins such as Vimentin,E⁃cadherin in each group were analyzed by western blot.The subcutaneous tumor nude mica models from PC⁃3 cells were constructed and divided into the control group,the ropivacaine group,the colivelin group,and the combined group of ropivacaine and colivelin group.After treated with ropivacaine and colivelin,the growth of subcutaneous tumors in each group was observed and their tumor cell proliferation was evaluated by Ki67 expression.The apoptosis of tumor tissues in each group was measured using TUNEL staining.The expressions of the related proteins to JAK2/STAT3 pathway in each group were examined by western blot.Results Compared with those in the control group,the apoptosis rate of cells,the protein expressions of Bax,cleaved PARP,E⁃cadherin,and the cell apoptosis proportion in tumor tissue in the ropivacaine group were all significantly increased(P<0.05),but the survival rate of cells,the rate of cell migration,the number of invaded cells,the protein expressions of Bcl⁃2 and Vimentin,the proliferation ratio of cells in tumor tissues,the expression of p⁃JAK2/JAK2 and p⁃STAT3/STAT3 decreased(P<0.05).On the contrary,in the colivelin group,the apoptosis rate of cells,the protein expressions of Bax,cleaved PARP,E⁃cadherin,and the cell apoptosis proportion in tumor tissues were all obviuosly decreased(P<0.05),but the survival rate of cells,the rate of migration,the number of invaded cells,the protein expressions of Bcl⁃2 and Vimentin,the proliferation ratio of cells in tumor tissues,the expressions of p⁃JAK2/JAK2,p⁃STAT3/STAT3increased(P<0.05).Compared with those in the ropivacaine group,the cell apoptosis rate,the protein expressions of Bax,cleaved PARP,and E⁃cadherin,and the cell apoptosis proportion in tumor tissues in the ropivacaine+colivelin group were all prominently reduced(P<0.05),but the survival rate of cells,the migration rate of cells,the number of the invaded cells,the protein expressions of Bcl⁃2 and Vimentin,the proliferation ratio of cells in tumor tissue,the expressions of p⁃JAK2/JAK2,and p⁃STAT3/STAT3 increased(P<0.05).Conclusion Ropivacaine obviously inhibits the activation of JAK2/STAT3 pathway to suppress the proliferation,migration,invasion activities of prostate cancer cells,promotes their apoptosis and delays their growth in nude mice,ultimately represses their malignant biological behavior.