A Combinatorial Therapy Targeting Glutaminolysis And Redox Homeostasis In Hepatocellular Carcinoma Cells

Background and ObjectiveCancer has become the most significant threat to human health. Both incidence and mortality of hepatocellular carcinomas (HCC) are particularly serious in China. Apart from the traditional surgery and chemotherapy, novel and effective therapeutics against HCC are urgently needed. Many studies have shown that reprogrammed metabolism and redox homeostasis are potential targets in cancer therapy. Our previous studies have shown that kidney-type of glutaminase (GLS1) is highly expressed in HCC, and GLS1 can be used for effective anticancer therapy. Previous studies have shown that dihydroartemisinin (DHA) increases intracellular ROS levels in cancer cells and expose cancer cells to oxidative damage. However, the heterogeneity of cancer cells, such as metabolic status, hypoxia and pH values, often leads to various responses to oxidative lesions. Given that cancer cells are more dependent on glutamine metabolism, and heterogeneous sensitivity to ROS-induced cell death, we postulate that a combinatorial therapeutic cohort targeting glutaminolysis and redox homeostasis may enhance antitumor efficacy. In this study, we firstly disrupted glutaminolysis by the compound 968, by which not only targeted reprogrammed metabolism of HCC, but also drove HCC to be more sensitive to ROS-induced cell death due to reduced ROS elimination. Then we treated HCC with DHA to trigger high level of ROS generation. Our results may have practical implications for development of effective treatment in HCC patients.Materials and Method1. Glutamine metabolism determination:1) HCC cells were treated with 968, and the activity of glutaminase was detected,2) HCC cells were treated with 968, and the levels of GSH were detected in cancer cells,3) HCC cells were treated with 968, and the hepatocellular carcinoma proliferation was detected.2. ROS levels detection:1) The levels of intracellular ROS in cancer cells treated with 968 or DHA were detected,2) The levels of intracellular ROS in cancer cells treated with 968 and DHA were detected by the flow cytometry.3. Confirmation of synergistic effect:1) The effect of 968 and DHA on HCC viability is detected by MTT assay, and then the CI index was calculated.2) The synergistic effect of 968 and DHA on cell viability was detected by cell counts method.3)4. Detection of the mechanism of synergistic antitumor effect:1) The synergistic effect of GLS-siRNA and DHA on cell viability was assayed by cell counts method.2) HCC cells were pretreated with NAC, and then treated with 968 and DHA, then the viability of HCC cells was detected by cell counts method.2) The expression of caspase-3 and PARP was detected by western blot.Results1. The compound 968 inhibited the activity of glutaminase and the synthesis of GSH in HCC cells, and also inhibited the proliferation of liver cancer cell.2. Dihydroartemisinin (DHA) increased the intracellular ROS generation and exerted antitumor effect.3. Small molecule compound968 combined with DHA enhanced generation of intracellular ROS and inhibited the proliferation of HCC cell.4. The compound 968 and DHA enhanced apoptosis in HCC.ConclusionsTargeting redox homeostasis by the combination of 968 and DHA is an effective way to treat HCC, which may drive cancer cells more voluntary to ROS-induced apoptosis. Our results provide a practical therapeutic cohort in treatment of HCC patients.