title: Metformin sensitizes sorafenib to inhibit postoperative recurrence and metastasis of hepatocellular carcinoma in orthotopic mouse models.
abstract: BACKGROUND
  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. Thus, it is essential to investigate the underlying mechanisms of the resistance to sorafenib and seek potential strategy to enhance its efficacy.
  
  
  METHODS
  The protein expression of hypoxia-inducible factors (HIF)-2α, 30-kDa HIV Tat-interacting protein (TIP30), E-cadherin, N-cadherin, and pAMPK was detected by Western blot. 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 relationship between HIF-2α 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 a day for continuous 37 days after the operation to remove the lobe where the tumor was implanted. CD31, Ki67, and TUNEL were examined by immunohistochemistry.
  
  
  RESULTS
  Our study demonstrated that metformin synergized with sorafenib reduced HIF-2α expression as examined by Western blot. Gene silencing approach indicated TIP30 was upregulated after knocking-down of HIF-2α and chromatin immunoprecipitation assay revealed that HIF-2α could bind to TIP30 promoter under hypoxic condition. Cell Counting Kit-8 (CCK8) cell viability assay and Annexin V-FITC apoptosis assay showed that metformin in combination with sorafenib suppressed cell proliferation and promoted cell apoptosis. Besides, combined therapy suppressed epithelial-mesenchymal transition (EMT) process both in vitro and in vivo. Moreover, metformin in combination with sorafenib significantly minimized postoperative recurrence and lung metastasis of HCC in orthotopic mouse model. Combined therapy inhibited CD31 and Ki67 expression but promoted TUNEL expression.
  
  
  CONCLUSIONS
  Metformin may potentially enhance the effect of sorafenib to inhibit HCC recurrence and metastasis after liver resection by regulating the expression of HIF-2α and TIP30.
authors.affiliation: Department of Hepatobiliary Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, 24 Bin Shui Road, Hexi District, Tianjin, 300060, People's Republic of China.
authors.forename: Abin
authors.initials: A
authors.lastname: You
authors.affiliation: Liver Cancer Institute and Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China.|Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, 180 Fenglin Road, Shanghai, 200032, China.
authors.forename: Manqing
authors.initials: M
authors.lastname: Cao
authors.affiliation: Department of Hepatobiliary Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, 24 Bin Shui Road, Hexi District, Tianjin, 300060, People's Republic of China.
authors.forename: Zhigui
authors.initials: Z
authors.lastname: Guo
authors.affiliation: Research Center of Basic Medical Science, Tianjin Medical University, Qixiangtai Road, Heping District, Tianjin, 300070, China.
authors.forename: Bingfeng
authors.initials: B
authors.lastname: Zuo
authors.affiliation: Academy of Medical Image, Tianjin Medical University, Tianjin, 300070, People's Republic of China.
authors.forename: Junrong
authors.initials: J
authors.lastname: Gao
authors.affiliation: Department of Hepatobiliary Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, 24 Bin Shui Road, Hexi District, Tianjin, 300060, People's Republic of China.
authors.forename: Hongyuan
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Department of Hepatobiliary Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, 24 Bin Shui Road, Hexi District, Tianjin, 300060, People's Republic of China.
authors.forename: Huikai
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Hepatobiliary Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, 24 Bin Shui Road, Hexi District, Tianjin, 300060, People's Republic of China.
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Cui
authors.affiliation: Department of Hepatobiliary Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, 24 Bin Shui Road, Hexi District, Tianjin, 300060, People's Republic of China.
authors.forename: Feng
authors.initials: F
authors.lastname: Fang
authors.affiliation: Department of Hepatobiliary Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, 24 Bin Shui Road, Hexi District, Tianjin, 300060, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Hepatobiliary Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, 24 Bin Shui Road, Hexi District, Tianjin, 300060, People's Republic of China.
authors.forename: Tianqiang
authors.initials: T
authors.lastname: Song
authors.affiliation: Department of Hepatobiliary Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, 24 Bin Shui Road, Hexi District, Tianjin, 300060, People's Republic of China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Hepatobiliary Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, 24 Bin Shui Road, Hexi District, Tianjin, 300060, People's Republic of China.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Research Center of Basic Medical Science, Tianjin Medical University, Qixiangtai Road, Heping District, Tianjin, 300070, China. haifangyin@tmu.edu.cn.
authors.forename: Haifang
authors.initials: H
authors.lastname: Yin
authors.affiliation: Liver Cancer Institute and Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China. sun.huichuan@zs-hospital.sh.cn.|Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, 180 Fenglin Road, Shanghai, 200032, China. sun.huichuan@zs-hospital.sh.cn.
authors.forename: Huichuan
authors.initials: H
authors.lastname: Sun
authors.affiliation: Department of Hepatobiliary Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key laboratory of Cancer Prevention and Therapy, 24 Bin Shui Road, Hexi District, Tianjin, 300060, People's Republic of China. zhangti@tjmuch.com.
authors.forename: Ti
authors.initials: T
authors.lastname: Zhang
mesh_terms: D000123:Acetyltransferases / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000601:surgery; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004357:Drug Synergism; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D006801:Humans; D007004:Hypoglycemic Agents / Q000008:administration & dosage / Q000494:pharmacology; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000601:surgery; D008297:Male; D008687:Metformin / Q000008:administration & dosage / Q000494:pharmacology*; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009362:Neoplasm Metastasis; D009364:Neoplasm Recurrence, Local; D009536:Niacinamide / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000008:administration & dosage / Q000494:pharmacology*; D000077157:Sorafenib; D014157:Transcription Factors / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D000123:Acetyltransferases; D000970:Antineoplastic Agents; D051792:Basic Helix-Loop-Helix Proteins; D007004:Hypoglycemic Agents; D008687:Metformin; D009536:Niacinamide; D010671:Phenylurea Compounds; D000077157:Sorafenib; D014157:Transcription Factors; D000099162:Endothelial PAS Domain-Containing Protein 1; C111246:HTATIP2 protein, human