title: Novel synergistic antitumor effects of rapamycin with bortezomib on hepatocellular carcinoma cells and orthotopic tumor model.
abstract: BACKGROUND
  Despite recent advances in the treatment of hepatocellular carcinoma (HCC), the chemotherapy efficacy against HCC is still unsatisfactory. The mammalian target of rapamycin (mTOR) has been emerged as an important cancer therapeutic target. However, HCC cells often resistant to rapamycin because of the paradoxical activation of Akt by rapamycin. In this study, we investigated whether bortezomib could enhance the antitumor effects of rapamycin.
  
  
  METHODS
  The effects of rapamycin and bortezomib on HCC proliferation, apoptosis, migration, and invasiveness in vitro were assessed by CCK-8 analysis, flow cytometry, Hoechst 33342 staining and transwell assays, respectively. Total and phosphorylated protein levels of Akt were detected by Western blotting. The effects of rapamycin and/or bortezomib on the mRNA expression levels of p53, p27, p21 and Bcl-2 family in HCCLM3 cells were evaluated by RT-PCR. The roles of rapamycin and bortezomib on HCC growth and metastasis in xenograft models were evaluated by tumor volumes and fluorescent signals. The effects of rapamycin and bortezomib on cell proliferation and apoptosis in vivo were test by PCNA and TUNEL staining.
  
  
  RESULTS
  Bortezomib synergized with rapamycin to reduce cell growth, induce apoptosis, and inhibit cell mobility in vitro. Further mechanistic studies showed that bortezomib inhibited rapamycin-induced phosphorylated Akt, which in turn enhanced apoptosis of HCC cell lines. The alteration of the mRNA expression of cell cycle inhibitors p53, p27, p21 and apoptosis associated genes Bcl-2, Bax were also involved in the synergistic antitumor effects of rapamycin and bortezomib. P53 inhibitor PFT-α significantly attenuate the effect of rapamycin and bortezomib on cell apoptosis, which indicated that the pro-apoptotic effect of rapamycin and bortezomib may be p53-dependent. Treatment of HCCLM3-R bearing nude mice with rapamycin and bortezomib significantly enhanced tumor growth inhibition (72.4%), comparing with either rapamycin- (54.7%) or bortezomib-treated mice (22.4%). In addition, the lung metastasis was significantly suppressed in mice received the combination treatment (16.6%). The combination treatment of rapamycin and bortezomib significantly inhibited tumor cell proliferation and tumor angiogenesis in vivo.
  
  
  CONCLUSIONS
  The combination of rapamycin with bortezomib could be a novel and promising therapeutic approach to the treatment of HCC.
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai 200032, China.
authors.forename: Cun
authors.initials: C
authors.lastname: Wang
authors.forename: Dongmei
authors.initials: D
authors.lastname: Gao
authors.forename: Kun
authors.initials: K
authors.lastname: Guo
authors.forename: Xiaonan
authors.initials: X
authors.lastname: Kang
authors.forename: Kai
authors.initials: K
authors.lastname: Jiang
authors.forename: Chun
authors.initials: C
authors.lastname: Sun
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Lu
authors.initials: L
authors.lastname: Sun
authors.forename: Hong
authors.initials: H
authors.lastname: Shu
authors.forename: Guangzhi
authors.initials: G
authors.lastname: Jin
authors.forename: Haiyan
authors.initials: H
authors.lastname: Sun
authors.forename: Weizhong
authors.initials: W
authors.lastname: Wu
authors.forename: Yinkun
authors.initials: Y
authors.lastname: Liu
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001897:Boronic Acids / Q000008:administration & dosage / Q000494:pharmacology*; D000069286:Bortezomib; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004357:Drug Synergism; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D008175:Lung Neoplasms / Q000556:secondary; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009389:Neovascularization, Pathologic / Q000188:drug therapy; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D011719:Pyrazines / Q000008:administration & dosage / Q000494:pharmacology*; D020123:Sirolimus / Q000008:administration & dosage / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D047368:Tumor Burden / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D001897:Boronic Acids; D011719:Pyrazines; D016159:Tumor Suppressor Protein p53; D000069286:Bortezomib; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus