Hepatocellular carcinoma (HCC) is a principal type of liver cancer with high incidence and mortality rates. Regorafenib is a novel oral multikinase inhibitor for second-line therapy for advanced HCC. However, resistance to regorafenib is gradually becoming a dilemma for HCC and the mechanism remains unclear. In this study, we aimed to reveal the metabolic profiles of regorafenib-resistant cells and the key role and mechanism of the most relevant metabolic pathway in regorafenib resistance. Metabolomics was performed to detect the metabolic alteration between drug-sensitive and regorafenib-resistant cells. Colony formation assay, CCK-8 assay and flow cytometry were applied to observe cell colony formation, cell proliferation and apoptosis, respectively. The protein and mRNA levels were detected by western blot and RT-qPCR. Cell lines of Glucose-6-phosphate dehydrogenase(G6PD) knockdown in regorafenib-resistant cells or G6PD overexpression in HCC cell lines were stably established by lentivirus infection technique. G6PD activity, NADPH level, NADPH/NADP+ ratio, the ratio of ROS positive cells, GSH level, and GSH/GSSG ratio were detected to evaluate the anti-oxidative stress ability of cells. Phosphorylation levels of NADK were evaluated by immunoprecipitation. Metabonomics analysis revealed that pentose phosphate pathway (PPP) was the most relevant metabolic pathway in regorafenib resistance in HCC. Compared with drug-sensitive cells, G6PD enzyme activity, NADPH level and NADPH/NADP+ ratio were increased in regorafenib-resistant cells, but the ratio of ROS positive cells and the apoptosis rate under the conditions of oxidative stress were decreased. Furthermore, G6PD suppression using shRNA or an inhibitor, sensitized regorafenib-resistant cells to regorafenib. In contrast, G6PD overexpression blunted the effects of regorafenib to drug-sensitive cells. Mechanistically, G6PD, the rate-limiting enzyme of PPP, regulated the PI3K/AKT activation. Furthermore, PI3K/AKT inhibition decreased G6PD protein expression, G6PD enzymatic activity and the capacity of PPP to anti-oxidative stress possibly by inhibited the expression and phosphorylation of NADK. Taken together, a feedback loop of PPP and PI3K/AKT signal pathway drives regorafenib-resistance in HCC and targeting the feedback loop could be a promising approach to overcome drug resistance.

中文翻译：

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PPP 和 PI3K/AKT 信号通路的反馈回路驱动 HCC 中的瑞戈非尼耐药

肝细胞癌（HCC）是肝癌的主要类型，发病率和死亡率较高。瑞戈非尼是一种新型口服多激酶抑制剂，用于晚期 HCC 的二线治疗。然而，瑞戈非尼耐药逐渐成为HCC的困境，且其机制尚不清楚。在本研究中，我们旨在揭示瑞戈非尼耐药细胞的代谢特征，以及最相关的代谢途径在瑞戈非尼耐药中的关键作用和机制。进行代谢组学来检测药物敏感细胞和瑞戈非尼耐药细胞之间的代谢变化。集落形成实验、CCK-8实验和流式细胞术分别观察细胞集落形成、细胞增殖和凋亡。通过蛋白质印迹和RT-qPCR检测蛋白质和mRNA水平。通过慢病毒感染技术稳定建立了瑞戈非尼耐药细胞中葡萄糖-6-磷酸脱氢酶（G6PD）敲低或HCC细胞系中G6PD过表达的细胞系。检测G6PD活性、NADPH水平、NADPH/NADP+比值、ROS阳性细胞比例、GSH水平、GSH/GSSG比值来评价细胞的抗氧化应激能力。通过免疫沉淀评估 NADK 的磷酸化水平。代谢组学分析表明，磷酸戊糖途径（PPP）是肝癌瑞戈非尼耐药中最相关的代谢途径。与药物敏感细胞相比，瑞戈非尼耐药细胞中G6PD酶活性、NADPH水平和NADPH/NADP+比值升高，但氧化应激条件下ROS阳性细胞比例和凋亡率降低。此外，使用 shRNA 或抑制剂抑制 G6PD 可使瑞戈非尼耐药细胞对瑞戈非尼敏感。相比之下，G6PD 过度表达减弱了瑞戈非尼对药物敏感细胞的作用。从机制上讲，G6PD（PPP 的限速酶）调节 PI3K/AKT 的激活。此外，PI3K/AKT 抑制可能通过抑制 NADK 的表达和磷酸化来降低 G6PD 蛋白表达、G6PD 酶活性和 PPP 抗氧化应激的能力。综上所述，PPP 和 PI3K/AKT 信号通路的反馈环路驱动 HCC 中的瑞格非尼耐药，而靶向反馈环路可能是克服耐药性的有前途的方法。