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目的研究PI3k/Akt信号通路在肝癌细胞对索拉菲尼耐药中作用机制,评估PI3k抑制剂LY294002逆转肝癌细胞对索拉菲尼耐药作用。方法 Western blot检测不同措施处理后索拉菲尼耐药肝癌细胞PI3k/Akt信号通路蛋白、EMT标记蛋白及凋亡相关蛋白Bax表达。CCK-8实验及Transwell实验检测低浓度索拉菲尼联合LY294002作用后Huh7R细胞增殖活力及迁移、侵袭能力。结果与亲本Huh7细胞相比,Huh7R的PI3k和p-Akt表达量升高,PTEN表达量下降,同时上皮标记蛋白E-cadherin表达下降,间质标记蛋白N-cadherin、Vimenti、Snail、Slug表达增加。LY294002作用后,EMT标志相关蛋白Snail、Slug、Vimenti、N-cadherin的表达量呈时间依赖性和浓度依赖性下降。低浓度索拉菲尼联合LY294002使Huh7R细胞增殖活力明显下降,同时抑制索拉菲尼耐药肝癌细胞迁移、侵袭能力。结论长期索拉菲尼暴露诱导肝癌细胞PI3k/Akt信号通路活化及促进肝癌细胞上皮间质变;LY294002通过抑制PI3k/Akt信号通路活化从而逆转索拉菲尼耐药肝癌上皮间质变作用;LY294002联合低浓度索拉菲尼可以协同作用逆转肝癌对索拉菲尼耐药,抑制索拉菲尼耐药肝癌细胞的迁移、侵袭能力。
AIM To investigate the mechanism of PI3k / Akt signaling pathway in sorafenib-resistant hepatocellular carcinoma cells and to evaluate the reversal effect of PI3k inhibitor LY294002 on sorafenib-resistant hepatocellular carcinoma cells. Methods The expressions of PI3k / Akt signaling pathway protein, EMT marker protein and apoptosis related protein Bax were detected by Western blot in different groups of treated Sorafenib-resistant hepatoma cells. CCK-8 and Transwell assays were used to detect the proliferation, invasion and migration of Huh7R cells after treated with low concentrations of sorafenib and LY294002. Results Compared with the parental Huh7 cells, the expression of PI3k and p-Akt in Huh7R cells increased and the expression of PTEN decreased. Meanwhile, the expression of E-cadherin in epithelial cells decreased and the expressions of N-cadherin, Vimenti, Snail and Slug increased . After LY294002 treatment, the expression of EMT marker-related proteins Snail, Slug, Vimenti and N-cadherin decreased in a time-dependent manner and in a concentration-dependent manner. Low concentration of sorafenib combined with LY294002 significantly decreased the proliferation activity of Huh7R cells and inhibited the migration and invasion ability of sorafenib-resistant hepatocellular carcinoma cells. Conclusion Long-term sorafenib exposure induces activation of PI3k / Akt signaling pathway and promotes epithelial-to-mesenchymal transition of hepatoma cells. LY294002 reverses the effect of sorafenib-resistant hepatocarcinogenesis by inhibiting the PI3k / Akt signaling pathway. LY294002 Combined with low concentration of sorafenib can synergistic effect reversal of liver cancer resistance to sorafenib and inhibit the migration and invasion of sorafenib resistant liver cancer cells.