Metformin Synergizes With Sorafenib To Inhibit Post-Operative Recurrence And Metastasis Of Hepatocellular Carcinoma In Orthotopic Mouse Models

Objectives:Surgical resection has been regarded as the main treatment for HCC. However,postoperative recurrence and metastasis are still the main obstacles for long-term survival of HCC. Sorafenib is recognized as a standard treatment for advanced hepatocellular carcinoma(HCC). However, many patients have to adopt dose reduction or terminate the use of sorafenib because of side effects. In addition, a large number of patients are resistant to sorafenib to some extent. Thus it is essential to investigate the underlying mechanisms for the resistance to sorafenib and seek potential strategy to enhance its efficacy. Metformin is a widely used drug for treatment of type 2 diabetes. Recently, some articles pointed out that metformin may be associated with reduced cancer incidence in diabetic patients.Our work aims to study whether metformin could sensitize sorafenib to minimize postoperative recurrence and lung metastasis of HCC in tumor-bearing mouse and explore its mechanism.Contents:To study the impact of metformin combined with sorafenib on cell proliferation,apoptosis in vitro and recurrence and metastasis in vivo after hepatectomy in orthotopic HCC mouse models. To explore the mechanism of synergistic effect of metformin on sorafenib.Methods:Cell viability assays were performed to study the influence of metformin and sorafenib on cell proliferation. Annexin V-FITC Apoptosis assays were used to detect the influence of metformin and sorafenib on cell apoptosis. The protein expression of HIF2?, TIP30, E-Cadherin, N-Cadherin and pAMPK was detected by the Western blot. The relationship between HIF-2? and TIP30 was studied using gene silencing approach and chromatin immunoprecipitation assay. To investigate the effect of metformin and sorafenib on postoperative recurrence and lung metastasis of HCC in tumor-bearing mice, the mice were orally treated either with metformin or sorafenib once daily for 37 days after a second operation to remove the lobe where the tumor was implanted. CD31, Ki67 and TUNEL was examined by immunohistochemistry.The protein expression of HIF2?, TIP30, E-Cadherin, N-Cadherin and pAMPK in hepatocellular carcinoma tissue was also detected by the Western blot.Results:1. Low-dose sorafenib inhibited the expression of TIP30 by increasing the expression of HIF-2? to promote tumor invasion and metastasis.2. Metformin synergized with sorafenib to reduce HIF-2? expression.3. Metformin in combination with sorafenib inhibited CD31 and Ki67 expression but promoted TUNEL expression.4. Metformin sensitized sorafenib to minimize postoperative recurrence and lung metastasis of HCC in tumor-bearing mouse.Conclusions:Metformin enhances sorafenib to inhibit HCC recurrence and metastasis after liver resection by regulating the expression of HIF-2? and TIP30.