Enhancement effect of adenovirus-mediated antisense c-myc and caffeine on the cytotoxicity of cisplatin in osteosarcoma cell lines.
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.
Xie
Xian-kuan
XK
Department of Orthopedics, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hang Zhou, People's Republic of China. xiexkhz @ 163.com
Yang
Di-sheng
DS
Ye
Zhao-ming
ZM
Tao
Hui-min
HM
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*
D000970:Antineoplastic Agents; D016373:DNA, Antisense; D016271:Proto-Oncogene Proteins c-myc; D002110:Caffeine; D002945:Cisplatin