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Abstract：
BACKGROUND: Increased level of serum cholic acid (CA) is often accompanied with decreased CYP2E1 expression in hepatocellular carcinoma (HCC) patients. However, the roles of CA and CYP2E1 in hepatocarcinogenesis have not been elucidated. This study aimed to investigate the roles and the underlying mechanisms of CYP2E1 and CA in HCC cell growth.
METHODS: The proteomic analysis of liver tumors from DEN-induced male SD rats with CA administration was used to reveal the changes of protein expression in the CA treated group. The growth of CA-treated HCC cells was examined by colony formation assays. Autophagic flux was assessed with immunofluorescence and confocal microscopy. Western blot analysis was used to examine the expression of CYP2E1, mTOR, AKT, p62, and LC3II/I. A xenograft tumor model in nude mice was used to examine the role of CYP2E1 in CA-induced hepatocellular carcinogenesis. The samples from HCC patients were used to evaluate the clinical value of CYP2E1 expression.
RESULTS: CA treatment significantly increased the growth of HCC cells and promoted xenograft tumors accompanied by a decrease of CYP2E1 expression. Further studies revealed that both in vitro and in vivo, upregulated CYP2E1 expression inhibited the growth of HCC cells, blocked autophagic flux, decreased AKT phosphorylation, and increased mTOR phosphorylation. CYP2E1 was involved in CA-activated autophagy through the AKT/mTOR signaling. Finally, decreased CYP2E1 expression was observed in the tumor tissues of HCC patients and its expression level in tumors was negatively correlated with the serum level of total bile acids (TBA) and gamma-glutamyltransferase (GGT).
CONCLUSIONS: CYP2E1 downregulation contributes to CA-induced HCC development presumably through autophagy regulation. Thus, CYP2E1 may serve as a potential target for HCC drug development.
METHODS: The proteomic analysis of liver tumors from DEN-induced male SD rats with CA administration was used to reveal the changes of protein expression in the CA treated group. The growth of CA-treated HCC cells was examined by colony formation assays. Autophagic flux was assessed with immunofluorescence and confocal microscopy. Western blot analysis was used to examine the expression of CYP2E1, mTOR, AKT, p62, and LC3II/I. A xenograft tumor model in nude mice was used to examine the role of CYP2E1 in CA-induced hepatocellular carcinogenesis. The samples from HCC patients were used to evaluate the clinical value of CYP2E1 expression.
RESULTS: CA treatment significantly increased the growth of HCC cells and promoted xenograft tumors accompanied by a decrease of CYP2E1 expression. Further studies revealed that both in vitro and in vivo, upregulated CYP2E1 expression inhibited the growth of HCC cells, blocked autophagic flux, decreased AKT phosphorylation, and increased mTOR phosphorylation. CYP2E1 was involved in CA-activated autophagy through the AKT/mTOR signaling. Finally, decreased CYP2E1 expression was observed in the tumor tissues of HCC patients and its expression level in tumors was negatively correlated with the serum level of total bile acids (TBA) and gamma-glutamyltransferase (GGT).
CONCLUSIONS: CYP2E1 downregulation contributes to CA-induced HCC development presumably through autophagy regulation. Thus, CYP2E1 may serve as a potential target for HCC drug development.
摘要：
背景：在肝细胞癌（HCC）患者中，血清胆酸（CA）水平升高通常伴随CYP2E1表达降低。然而,CA和CYP2E1在肝癌发生中的作用尚未阐明。本研究旨在探讨CYP2E1和CA在肝癌细胞生长中的作用及其机制。
方法：对DEN诱导的雄性SD大鼠给予CA的肝肿瘤进行蛋白质组学分析，以揭示CA治疗组蛋白质表达的变化。通过集落形成测定法检查CA处理的HCC细胞的生长。用免疫荧光和共聚焦显微镜评估自噬通量。Westernblot分析CYP2E1、mTOR、AKT,p62,andLC3II/I.使用裸鼠中的异种移植肿瘤模型来检查CYP2E1在CA诱导的肝细胞癌变中的作用。来自HCC患者的样本用于评估CYP2E1表达的临床价值。
结果：CA治疗可显着增加HCC细胞的生长，并促进异种移植肿瘤，同时CYP2E1表达降低。进一步的研究表明，在体外和体内，CYP2E1表达上调抑制肝癌细胞的生长，阻断自噬通量，AKT磷酸化减少，和mTOR磷酸化增加。CYP2E1通过AKT/mTOR信号参与CA激活的自噬。最后,在HCC患者的肿瘤组织中观察到CYP2E1表达降低，其在肿瘤中的表达水平与血清总胆汁酸（TBA）和γ-谷氨酰转移酶（GGT）水平呈负相关。
结论：CYP2E1下调可能通过自噬调节促进CA诱导的HCC发展。因此，CYP2E1可能成为HCC药物开发的潜在靶点。
方法：对DEN诱导的雄性SD大鼠给予CA的肝肿瘤进行蛋白质组学分析，以揭示CA治疗组蛋白质表达的变化。通过集落形成测定法检查CA处理的HCC细胞的生长。用免疫荧光和共聚焦显微镜评估自噬通量。Westernblot分析CYP2E1、mTOR、AKT,p62,andLC3II/I.使用裸鼠中的异种移植肿瘤模型来检查CYP2E1在CA诱导的肝细胞癌变中的作用。来自HCC患者的样本用于评估CYP2E1表达的临床价值。
结果：CA治疗可显着增加HCC细胞的生长，并促进异种移植肿瘤，同时CYP2E1表达降低。进一步的研究表明，在体外和体内，CYP2E1表达上调抑制肝癌细胞的生长，阻断自噬通量，AKT磷酸化减少，和mTOR磷酸化增加。CYP2E1通过AKT/mTOR信号参与CA激活的自噬。最后,在HCC患者的肿瘤组织中观察到CYP2E1表达降低，其在肿瘤中的表达水平与血清总胆汁酸（TBA）和γ-谷氨酰转移酶（GGT）水平呈负相关。
结论：CYP2E1下调可能通过自噬调节促进CA诱导的HCC发展。因此，CYP2E1可能成为HCC药物开发的潜在靶点。
