title: Enhancement effect of adenovirus-mediated antisense c-myc and caffeine on the cytotoxicity of cisplatin in osteosarcoma cell lines.
abstract: OBJECTIVE
  Studies on cancer biology have shown that overexpression of oncogenes (with or without functional loss of tumor suppressor genes), which is responsible for the progression of human malignancies via a multistep process, may be reduced by antisense technology. Caffeine enhances the effect of cisplatin (CDDP) chemotherapy on osteosarcoma cells. We constructed the recombinant adenovirus (Myc-AS) encoding the antisense c-myc fragment and investigated the synergic effect of caffeine and Myc-AS on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin.
  
  
  METHODS
  The recombinant adenovirus (Myc-AS) encoding the antisense c-myc fragment was constructed by cloning c-myc cDNA of about 750 bp in a reverse direction into adenovirus vector, then undergoing recombination, amplification and complementation in vivo. Myc-AS and caffeine were used either alone or in combination with CDDP to treat osteosarcoma MG-63 cells in vitro. Western blot, MTT, flow cytometry (FCM) and electron microscopy were used to evaluate the expression of c-myc protein, tumor cell proliferation in vitro and apoptosis and to perform cell cycle analysis.
  
  
  RESULTS
  Myc-AS encoding antisense c-myc fragment was obtained with a titer of 2 x 10(9) pfu/ml. Myc-AS downregulated the expression of c-myc protein after transfecting MG-63 cells for 48 h, induced tumor cell apoptosis and inhibited tumor cell proliferation in vitro. Myc-AS or caffeine can enhance the cytotoxic effects of 2.0 and 5.0 microg/ml CDDP on MG-63 cells. Moreover, the significantly enhancing effect of the Myc-AS-caffeine combination on CDDP chemotherapy of MG-63 cells was not restricted to apoptosis but also decreased tumor cell proliferation in vitro. Expression of the apoptosis-associated bcl-2 gene was downregulated and bax was upregulated, with no changes in E2F-1 expression. FCM analysis showed that CDDP treatment induced a block in S phase, and caffeine reversed this block and accelerated cell progression through the S phase.
  
  
  CONCLUSIONS
  Myc-AS can induce obvious G2/M phase arrest in transfected cells. Myc-AS combined with caffeine can enhance apoptosis induction and chemotherapeutic effects of CDDP on osteosarcoma MG-63 cells.
authors.affiliation: Department of Orthopedics, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hang Zhou, People's Republic of China. xiexkhz @ 163.com
authors.forename: Xian-kuan
authors.initials: XK
authors.lastname: Xie
authors.forename: Di-sheng
authors.initials: DS
authors.lastname: Yang
authors.forename: Zhao-ming
authors.initials: ZM
authors.lastname: Ye
authors.forename: Hui-min
authors.initials: HM
authors.lastname: Tao
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002110:Caffeine / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D016373:DNA, Antisense / Q000008:administration & dosage; D004357:Drug Synergism; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D008854:Microscopy, Electron; D012516:Osteosarcoma / Q000188:drug therapy / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000008:administration & dosage*
chemical_list: D000970:Antineoplastic Agents; D016373:DNA, Antisense; D016271:Proto-Oncogene Proteins c-myc; D002110:Caffeine; D002945:Cisplatin