Caja Laia, Sancho Patricia, Bertran Esther, Iglesias-Serret Daniel, Gil Joan, Fabregat Isabel
Laboratori de Oncologia Molecular and Departament de Ciències Fisiològiques II, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain.
Cancer Res. 2009 Oct 1;69(19):7595-602. doi: 10.1158/0008-5472.CAN-09-1482. Epub 2009 Sep 22.
Transforming growth factor-beta (TGF-beta) induces apoptosis in hepatocytes, being considered a liver tumor suppressor. However, many human hepatocellular carcinoma (HCC) cells escape from its proapoptotic effects, gaining response to this cytokine in terms of malignancy. We have recently reported that the apoptosis induced by TGF-beta in hepatocytes requires up-regulation of the NADPH oxidase NOX4, which mediates reactive oxygen species (ROS) production. TGF-beta-induced NOX4 expression is inhibited by antiapoptotic signals, such as the phosphatydilinositol-3-phosphate kinase or the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. The aim of the present work was to analyze whether resistance to TGF-beta-induced apoptosis in HCC cells is related to the impairment of NOX4 up-regulation due to overactivation of survival signals. Results indicate that inhibition of the MAPK/ERK kinase (MEK)/ERK pathway in HepG2 cells, which are refractory to the proapoptotic effects of TGF-beta, sensitizes them to cell death through a mitochondrial-dependent mechanism, coincident with increased levels of BIM and BMF, decreased levels of BCL-XL and MCL1, and BAX/BAK activation. Regulation of BMF, BCL-XL, and MCL1 occurs at the mRNA level, whereas BIM regulation occurs post-transcriptionally. ROS production and glutathione depletion are only observed in cells treated with TGF-beta and PD98059, which correlates with NOX4 up-regulation. Targeting knockdown of NOX4 impairs ROS increase and all the mitochondrial-dependent apoptotic features by a mechanism that is upstream from the regulation of BIM, BMF, BCL-XL, and MCL1 levels. In conclusion, overactivation of the MEK/ERK pathway in liver tumor cells confers resistance to TGF-beta-induced cell death through impairing NOX4 up-regulation, which is required for an efficient mitochondrial-dependent apoptosis.
转化生长因子-β（TGF-β）可诱导肝细胞凋亡，被视为一种肝脏肿瘤抑制因子。然而，许多人类肝细胞癌（HCC）细胞可逃避其促凋亡作用，在恶性程度方面对这种细胞因子产生反应。我们最近报道，TGF-β在肝细胞中诱导的凋亡需要上调烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶NOX4，该酶介导活性氧（ROS）的产生。TGF-β诱导的NOX4表达受到抗凋亡信号的抑制，如磷脂酰肌醇-3-磷酸激酶或丝裂原活化蛋白激酶（MAPK）/细胞外信号调节激酶（ERK）途径。本研究的目的是分析HCC细胞对TGF-β诱导凋亡的抗性是否与生存信号过度激活导致的NOX4上调受损有关。结果表明，在对TGF-β的促凋亡作用具有抗性的HepG2细胞中，抑制MAPK/ERK激酶（MEK）/ERK途径可通过线粒体依赖性机制使它们对细胞死亡敏感，这与BIM和BMF水平升高、BCL-XL和MCL1水平降低以及BAX/BAK激活同时发生。BMF、BCL-XL和MCL1的调节发生在mRNA水平，而BIM的调节发生在转录后水平。仅在用TGF-β和PD98059处理的细胞中观察到ROS产生和谷胱甘肽耗竭，这与NOX4上调相关。靶向敲低NOX4通过一种在BIM、BMF、BCL-XL和MCL1水平调节上游的机制损害ROS增加和所有线粒体依赖性凋亡特征。总之，肝肿瘤细胞中MEK/ERK途径的过度激活通过损害NOX4上调赋予对TGF-β诱导细胞死亡的抗性，而NOX4上调是有效的线粒体依赖性凋亡所必需的。
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