甘氨鹅脱氧胆酸钠通过MAPK/ERK1/2介导Bcl-2在Ser70位点的磷酸化引起人肝细胞癌的抗药

B细胞淋巴瘤-2（Bcl-2）是一种重要的抗凋亡蛋白质，在多种人类肿瘤中普遍过表达。甘氨鹅脱氧胆酸钠（GCDA）与消化道肿瘤的发生发展密切相关，并能介导肝癌细胞对化疗药物的抵抗。本文旨在探讨在GCDA介导的人肝细胞癌（HCC）耐药性中Bcl-2的作用及其机制。本研究以肝癌细胞系为研究对象，Western印迹结果显示，Bcl-2在多种肝癌细胞系中均有表达。设计靶向Bcl-2的siRNA沉默HCC细胞系内源性Bcl-2的表达，发现Bcl-2沉默之后促进了化疗药物5-FU介导的HCC细胞凋亡。机制上，GCDA可介导Bcl-2在Ser70位点的磷酸化，而Ser70位点的磷酸化能够被PD98059（MAPK/ERK1/2抑制剂）所抑制。构建huBcl2-WT和huBcl2-S70A真核表达载体，脂质体转染HCC细胞系。用Annexin V-FITC/PI流式细胞术检测凋亡细胞。结果显示，huBcl2-WT过表达能抑制5 FU介导的凋亡，S70位点失活突变成A后，Bcl-2的过表达不能抑制5-FU介导的凋亡。本研究提示，GCDA通过MAPK/ERK1/2通路介导的Bcl-2 Ser70位点的磷酸化，在肝癌细胞的存活和抗药中发挥重要作用。抑制Bcl-2能够促进化疗药物5-FU介导的HCC细胞凋亡，该结果为治疗GCDA介导的耐药性肝癌提供新的思路。

Glycochenodeoxycholate Induces Drug Resistance of Human Hepatocellular Carcinoma by Phosphorylation of Bcl-2 at Ser70 via MAPK/ERK1/2

B-cell lymphoma-2 (Bcl-2) is an important anti-apoptotic protein and is overexpressed in many human tumors. Glycochenodeoxycholate (GCDA) is closely related to the occurrence and development of digestive tract tumors and can mediate the resistance of hepatoma cells to chemotherapeutic drugs. The aim of this study is to investigate the effect and mechanism of Bcl-2 in GCDA-mediated drug resistance of human hepatocellular carcinoma (HCC). In this study, Western blot results showed that Bcl-2 was expressed in several HCC cell lines. siRNA targeting Bcl-2 was designed to silence endogenous Bcl-2 expression. Silence of Bcl-2 promoted apoptosis of HCC cells mediated by 5-FU. GCDA can induce the phosphorylation of Bcl-2 at Ser70, which may be inhibited by PD98059 (MAPK/ERK1/2 inhibitor). HuBcl2-WT and huBcl2-S70A eukaryotic expression vectors were constructed and transfected into HCC cell lines. Apoptotic cells were detected by Annexin V-FITC/PI flow cytometry. Overexpression of huBcl2-WT could inhibit 5-FU-mediated apoptosis. When S70 of Bcl-2 was mutated to A, Bcl-2 inhibition of 5-FU-mediated apoptosis was abolished. This study suggests that phosphorylation of Bcl-2 at S70 by GCDA was through MAPK/ERK1/2 pathway, which may play an important role in the survival and drug resistance of HCC cells. Inhibition of Bcl-2 promotes 5-FU-mediated apoptosis in HCC cells, which can be used in the treatment of GCDA-mediated drug-resistance in hepatocellular carcinoma.