光甘草定抑制结直肠癌细胞迁移和诱导凋亡及其分子机制研究
发布时间：2018-10-23 15:04
【摘要】：目的探究光甘草定(glabridin)对人结直肠癌细胞RKO和HCT116细胞的迁移和凋亡的影响,并探讨其可能的分子机制。方法不同浓度的光甘草定、MLCK抑制剂ML-7、ROCK抑制剂H-1152以及p38通路抑制剂SB203580、JNK抑制剂SP600125和PKC激活剂PMA处理RKO和(或)HCT116细胞;采用MTT比色法检测光甘草定对RKO和HCT116细胞活力的影响,平板克隆实验检测光甘草定对结直肠癌细胞克隆形成能力的影响,通过Hoechst 33258染色检测光甘草定对RKO细胞凋亡的影响;通过细胞划痕和Transwell实验观察光甘草定对RKO和HCT116细胞迁移和侵袭能力的影响;免疫荧光检测光甘草定处理结直肠癌细胞后E-cadherin、N-cadherin以及HCT116细胞内紧密连接蛋白Occludin和ZO-1的变化。Western blot检测RKO细胞中凋亡相关蛋白(BCL-2、Bax、Caspase家族),HCT116细胞中紧密连接蛋白ZO-1、occludin,RKO和HCT116细胞中E-cadherin、N-cadherin以及迁移相关蛋白(Rho A、ROCK、MLCK、MLCP、p-MLC)以及MAPK信号转导通路相关蛋白的表达情况,并加入抑制剂和激活剂分析其可能的机制。结果光甘草定作用于RKO和HCT116细胞后,对细胞的增殖、克隆形成能力和迁移、侵袭有明显抑制作用。光甘草定诱导RKO细胞凋亡,并下调抗凋亡蛋白Bcl-2、Procaspase 9表达水平和上调Bax、Caspase 3蛋白表达,而对Caspase 8蛋白表达影响不明显。光甘草定处理后,RKO和HCT116细胞迁移相关蛋白MYPT1磷酸化水平及MLC磷酸化水平下调,RKO细胞内Rho A、ROCK1、ROCK2表达水平均明显下调,而HCT116细胞内Rho A、ROCK1、ROCK2蛋白无明显变化;为进一步的验证,我们采用MLCK的特异性抑制剂ML-7或ROCK的特异性抑制剂H-1152处理RKO和(或)HCT116细胞,观察到二者对RKO和(或)HCT116细胞的迁移存在抑制作用且能下调MLCK、ROCK1或p-MYPT1蛋白的表达,提示光甘草定可能通过抑制MLCK和ROCK活性而抑制RKO和(或)HCT116细胞的迁移能力。光甘草定在不同时间点处理结直肠癌细胞后,两株细胞中ERK无明显变化,RKO细胞内JNK磷酸化水平先上升后下降,p38通路上调,而HCT116细胞中JNK通路下调后恢复,p38通路上调。加入JNK和p38/MAPK通路抑制剂后能下调迁移相关蛋白MYPT1和MLC的磷酸化水平,表明光甘草定可能部分通过JNK信号通路,下调MLC磷酸化水平,从而导致细胞运动能力降低而抑制迁移。光甘草定能抑制RKO和HCT116细胞上皮间质转化过程,上调上皮细胞标志物E-cadherin,下调间叶细胞标志物N-cadherin;并且上调HCT116细胞膜上紧密连接蛋白Occludin和ZO-1的蛋白水平。结论光甘草定抑制结直肠癌RKO和HCT116细胞的增殖与迁移,并诱导RKO细胞凋亡;其抑制迁移的机制可能是通过JNK信号通路下调MLC磷酸化水平,增加E-cadherin表达,降低N-cadherin表达,逆转上皮间质转化,并上调紧密连接蛋白ZO-1和Occludin的表达抑制结直肠癌细胞的迁移。
[Abstract]:Objective to investigate the effects of (glabridin) on the migration and apoptosis of RKO and HCT116 cells, and to explore its possible molecular mechanism. Methods RKO and / or HCT116 cells were treated with different concentrations of Glycyrrhizin, MLCK inhibitor ML-7,ROCK inhibitor H-1152, p38 pathway inhibitor SB203580,JNK inhibitor SP600125 and PKC activator PMA. Plate cloning assay was used to detect the effect of Gangliandrine on the clone forming ability of colorectal cancer cells, and the effect of GG on the apoptosis of RKO cells was detected by Hoechst 33258 staining. The effects of Glycyrrhizin on the migration and invasion of RKO and HCT116 cells were observed by cell scratch and Transwell assay. Changes of E-cadherin and Occludin and ZO-1 in HCT116 cells after treatment of Colorectal Cancer cells with Light-Glycyrrhizin by immunofluorescence Detection of apoptosis-related proteins (BCL-2,Bax,Caspase family) in RKO cells and ZO-1,occludin,RKO and HCT116 cells in HCT116 cells by. Western blot The expression of E-cadherin, Rho, MLCP p-MLC and MAPK signal transduction pathway related proteins were observed. The possible mechanisms were analyzed by adding inhibitors and activators. Results after treated with RKO and HCT116 cells, Guanglianding inhibited cell proliferation, clone formation, migration and invasion. Glycyrrhizin induced apoptosis of RKO cells, down-regulated the expression of anti-apoptotic protein Bcl-2,Procaspase 9 and up-regulated the expression of Bax,Caspase 3 protein, but had no significant effect on the expression of Caspase 8 protein. The level of MYPT1 phosphorylation and MLC phosphorylation of migration related protein in RKO and HCT116 cells were down-regulated, and the expression level of Rho AroCK1 and ROCK2 in RKO cells were significantly down-regulated, while Rho AroCK1 and ROCK2 protein in HCT116 cells had no significant changes after treatment with Ligandrine. We treated RKO and / or HCT116 cells with ML-7, a specific inhibitor of MLCK, or H-1152, a specific inhibitor of ROCK, and observed that both of them could inhibit the migration of RKO and / or HCT116 cells and down-regulate the expression of MLCK,ROCK1 or p-MYPT1 protein. These results suggest that Licorrhizine inhibits the migration of RKO and / or HCT116 cells by inhibiting the activities of MLCK and ROCK. There was no significant change of ERK in the two cell lines after treated with licorice at different time points. The level of JNK phosphorylation in RKO cells increased at first, then decreased, the p38 pathway increased, while the JNK pathway in HCT116 cells was down-regulated, and the p38 pathway was up-regulated. The addition of JNK and p38/MAPK pathway inhibitors could down-regulate the phosphorylation levels of MYPT1 and MLC, suggesting that Glycyrrhizin might down-regulate the phosphorylation level of MLC through JNK signaling pathway, resulting in the decrease of cell motility and inhibition of migration. Glycyrrhizin inhibited the process of epithelial interstitial transformation of RKO and HCT116 cells, up-regulated E-cadherin, down-regulated N-cadherin, and up-regulated the protein levels of Occludin and ZO-1 on HCT116 cell membrane. Conclusion Gliangandrine inhibits the proliferation and migration of RKO and HCT116 cells and induces apoptosis of RKO cells in colorectal cancer, and its mechanism may be to down-regulate the level of MLC phosphorylation, increase E-cadherin expression and decrease N-cadherin expression through JNK signaling pathway. Reverse epithelial interstitial transformation and up-regulate the expression of ZO-1 and Occludin to inhibit the migration of colorectal cancer cells.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
本文编号：2289599
[Abstract]:Objective to investigate the effects of (glabridin) on the migration and apoptosis of RKO and HCT116 cells, and to explore its possible molecular mechanism. Methods RKO and / or HCT116 cells were treated with different concentrations of Glycyrrhizin, MLCK inhibitor ML-7,ROCK inhibitor H-1152, p38 pathway inhibitor SB203580,JNK inhibitor SP600125 and PKC activator PMA. Plate cloning assay was used to detect the effect of Gangliandrine on the clone forming ability of colorectal cancer cells, and the effect of GG on the apoptosis of RKO cells was detected by Hoechst 33258 staining. The effects of Glycyrrhizin on the migration and invasion of RKO and HCT116 cells were observed by cell scratch and Transwell assay. Changes of E-cadherin and Occludin and ZO-1 in HCT116 cells after treatment of Colorectal Cancer cells with Light-Glycyrrhizin by immunofluorescence Detection of apoptosis-related proteins (BCL-2,Bax,Caspase family) in RKO cells and ZO-1,occludin,RKO and HCT116 cells in HCT116 cells by. Western blot The expression of E-cadherin, Rho, MLCP p-MLC and MAPK signal transduction pathway related proteins were observed. The possible mechanisms were analyzed by adding inhibitors and activators. Results after treated with RKO and HCT116 cells, Guanglianding inhibited cell proliferation, clone formation, migration and invasion. Glycyrrhizin induced apoptosis of RKO cells, down-regulated the expression of anti-apoptotic protein Bcl-2,Procaspase 9 and up-regulated the expression of Bax,Caspase 3 protein, but had no significant effect on the expression of Caspase 8 protein. The level of MYPT1 phosphorylation and MLC phosphorylation of migration related protein in RKO and HCT116 cells were down-regulated, and the expression level of Rho AroCK1 and ROCK2 in RKO cells were significantly down-regulated, while Rho AroCK1 and ROCK2 protein in HCT116 cells had no significant changes after treatment with Ligandrine. We treated RKO and / or HCT116 cells with ML-7, a specific inhibitor of MLCK, or H-1152, a specific inhibitor of ROCK, and observed that both of them could inhibit the migration of RKO and / or HCT116 cells and down-regulate the expression of MLCK,ROCK1 or p-MYPT1 protein. These results suggest that Licorrhizine inhibits the migration of RKO and / or HCT116 cells by inhibiting the activities of MLCK and ROCK. There was no significant change of ERK in the two cell lines after treated with licorice at different time points. The level of JNK phosphorylation in RKO cells increased at first, then decreased, the p38 pathway increased, while the JNK pathway in HCT116 cells was down-regulated, and the p38 pathway was up-regulated. The addition of JNK and p38/MAPK pathway inhibitors could down-regulate the phosphorylation levels of MYPT1 and MLC, suggesting that Glycyrrhizin might down-regulate the phosphorylation level of MLC through JNK signaling pathway, resulting in the decrease of cell motility and inhibition of migration. Glycyrrhizin inhibited the process of epithelial interstitial transformation of RKO and HCT116 cells, up-regulated E-cadherin, down-regulated N-cadherin, and up-regulated the protein levels of Occludin and ZO-1 on HCT116 cell membrane. Conclusion Gliangandrine inhibits the proliferation and migration of RKO and HCT116 cells and induces apoptosis of RKO cells in colorectal cancer, and its mechanism may be to down-regulate the level of MLC phosphorylation, increase E-cadherin expression and decrease N-cadherin expression through JNK signaling pathway. Reverse epithelial interstitial transformation and up-regulate the expression of ZO-1 and Occludin to inhibit the migration of colorectal cancer cells.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
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