title: Cyproheptadine, an antihistaminic drug, inhibits proliferation of hepatocellular carcinoma cells by blocking cell cycle progression through the activation of P38 MAP kinase.
abstract: BACKGROUND
  Hepatocellular carcinoma (HCC) is a major cause of cancer deaths worldwide. However, current chemotherapeutic drugs for HCC are either poorly effective or expensive, and treatment with these drugs has not led to satisfactory outcomes. In a 2012 case report, we described our breakthrough finding in two advanced HCC patients, of whom one achieved complete remission of liver tumors and the other a normalized α-fetoprotein level, along with complete remission of their lung metastases, after the concomitant use of thalidomide and cyproheptadine. We assumed the key factor in our effective therapy to be cyproheptadine. In this study, we investigated the antiproliferative effects and molecular mechanisms of cyproheptadine.
  
  
  METHODS
  The effect of cyproheptadine on cell proliferation was examined in human HCC cell lines HepG2 and Huh-7. Cell viability was assayed with Cell Counting Kit-8; cell cycle distribution was analyzed by flow cytometry. Mechanisms underlying cyproheptadine-induced cell cycle arrest were probed by western blot analysis.
  
  
  RESULTS
  Cyproheptadine had a potent inhibitory effect on the proliferation of HepG2 and Huh-7 cells but minimal toxicity in normal hepatocytes. Cyproheptadine induced cell cycle arrest in HepG2 cells in the G1 phase and in Huh-7 cells at the G1/S transition. The cyproheptadine-induced G1 arrest in HepG2 cells was associated with an increased expression of HBP1 and p16, whereas the G1/S arrest in Huh-7 cells was associated with an increase in p21 and p27 expression and a dramatic decrease in the phosphorylation of the retinoblastoma protein. Additionally, cyproheptadine elevated the percentage of Huh-7 cells in the sub-G1 population, increased annexin V staining for cell death, and raised the levels of PARP and its cleaved form, indicating induction of apoptosis. Finally, cyproheptadine-mediated cell cycle arrest was dependent upon the activation of p38 MAP kinase in HepG2 cells and the activation of both p38 MAP kinase and CHK2 in Huh-7 cells.
  
  
  CONCLUSIONS
  Our results demonstrate that a non-classical p38 MAP kinase function, regulation of cell cycle checkpoints, is one of the underlying mechanisms promoted by cyproheptadine to suppress the proliferation of HCC cells. These results provide evidence for the drug's potential as a treatment option for liver cancer.
authors.affiliation: Department of Internal Medicine, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chia-Yi, Taiwan. fengyumin2@gmail.com.
authors.forename: Yu-Min
authors.initials: YM
authors.lastname: Feng
authors.affiliation: Department of Biological Science, National Sun Yat-sen University, Kaohsiung, Taiwan. feng1584@gmail.com.
authors.forename: Chin-Wen
authors.initials: CW
authors.lastname: Feng
authors.affiliation: Department of Medical Research, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chia-Yi, Taiwan. cych06246@gmail.com.
authors.forename: Syue-Yi
authors.initials: SY
authors.lastname: Chen
authors.affiliation: Department of Medical Research, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chia-Yi, Taiwan. hsiaoyen.hsieh@gmail.com.
authors.forename: Hsiao-Yen
authors.initials: HY
authors.lastname: Hsieh
authors.affiliation: Department of Medical Research, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chia-Yi, Taiwan. b0912070057@gmail.com.
authors.forename: Yu-Hsin
authors.initials: YH
authors.lastname: Chen
authors.affiliation: Department of Medical Research, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chia-Yi, Taiwan. cych06390@gmail.com.
authors.forename: Cheng-Da
authors.initials: CD
authors.lastname: Hsu
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000201:enzymology*; D002453:Cell Cycle / Q000187:drug effects* / Q000502:physiology; D059447:Cell Cycle Checkpoints / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D003533:Cyproheptadine / Q000494:pharmacology* / Q000627:therapeutic use; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D056945:Hep G2 Cells; D006634:Histamine H1 Antagonists / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000201:enzymology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: D006634:Histamine H1 Antagonists; D003533:Cyproheptadine; D048051:p38 Mitogen-Activated Protein Kinases