The multikinase inhibitor Sorafenib targets mitochondria and synergizes with glycolysis blockade for cancer cell killing.

Objective: identify novel and relevant aspects of Sorafenib action on liver cancer cells. We found that in rat hepatocholangiocarcinoma (LCSC-2) cells, exposure to the MEK/multikinase inhibitor sorafenib did not inhibit ERK phosphorylation nor induced appreciable cell death in the low micromolar range; instead, the drug elicited a raise of intracellular reactive oxygen species (ROS) accompanied by a severe decrease of oxygen consumption and intracellular ATP levels, all changes consistent with mitochondrial damage. Moreover, Sorafenib induced depolarization of isolated rat liver mitochondria, indicating a possible direct effect on the organelle. Microarray analysis of gene expression in sorafenib-trated cells revealed a metabolic reprogramming toward aerobic glycolysis, that likely accounts for resitance to drug toxicity in this cell line. Importantly, cytotoxicity was strongly potentiated by glucose withdrawal from the culture medium or by the glycolytic inhibitor 2-deoxy-glucose, a finding also confirmed in the highly malignant melanoma cell line B16F10. Mechanistic studies revealed that ROS are pivotal to cell killing by the Sorafenib + 2DG combination, and that a low content of intracellular oxidants is associated with resistance to the drug; instead, Thr172phosphorylation/activation of the AMP-activated protein kinase (AMPK), induced by Sorafenib, may exert protective effects, since cytotoxicity was enhanced by an AMPK specific inhibitor and prevented by the AMPK activator Metformin. Overall, this study identifies novel and relevant aspects of Sorafenib action on liver cancer cells, including mitochondrial damage, induction of ROS and a metabolic cell reprogramming towards “glucose addiction”, potentially exploitable in therapy.研究目标：明确索拉非尼（Sorafenib）对肝癌细胞作用的新型相关机制。本研究发现，在大鼠肝胆管癌（LCSC-2）细胞中，暴露于MEK/多靶点激酶抑制剂索拉非尼后，ERK磷酸化未受到抑制，低微摩尔浓度范围内亦未诱导明显的细胞死亡；相反，该药物可引发细胞内活性氧（ROS）水平升高，同时伴随耗氧量显著降低与细胞内ATP水平大幅下降，上述变化均与线粒体损伤相符。此外，索拉非尼可诱导分离的大鼠肝脏线粒体发生去极化，提示其可能对该细胞器存在直接作用。对索拉非尼处理后的细胞进行基因芯片分析显示，细胞发生了向有氧糖酵解方向的代谢重编程，这或许解释了该细胞系对药物毒性产生耐药性的原因。值得注意的是，从培养基中撤除葡萄糖或使用糖酵解抑制剂2-脱氧葡萄糖（2-DG）均可显著增强细胞毒性，这一现象在高转移性黑色素瘤细胞系B16F10中也得到了验证。机制研究表明，活性氧（ROS）是索拉非尼联合2-DG杀伤细胞的关键介导因子，而细胞内低水平氧化应激与药物耐药性相关；反之，索拉非尼诱导的腺苷酸活化蛋白激酶（AMPK）Thr172位点磷酸化/激活可能发挥保护作用，因为使用AMPK特异性抑制剂可增强细胞毒性，而AMPK激活剂二甲双胍（Metformin）则可抑制该毒性。综上，本研究明确了索拉非尼对肝癌细胞作用的全新相关机制，包括线粒体损伤、活性氧（ROS）诱导以及细胞向“葡萄糖成瘾”的代谢重编程，这些发现有望为肿瘤治疗提供新的潜在应用方向。