Recombinant antisense C-myc adenovirus increase in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin.
C-myc is an oncogene with the important role of cell proliferation controller. It has been found to be amplified and overexpressed in osteosarcoma. Moreover, it can promote cell transformation and induce metastatic features. Some studies showed that overexpression of c-myc could induce resistance in response to antineoplastic agents. Currently, we constructed the recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment and investigated its effect on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin(CDDP). The osteosarcoma MG-63 cells were transfected by the Ad-Asc-myc in vitro, and Western Blot, MTT assay, RT-PCR, flow cytometry (FCM), and transmission electron microscopy (TEM) were used to study expression of c-myc and caspase-3 protein, tumor cell proliferation in vitro, cell apoptotic morphology and cell cycle change. Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2.0 x 10(9) pfu/ml. Ad-Asc-myc downregulated the expression of c-myc protein after transfected MG-63 cells for 48 hours, combined with the treatment of 2.0, 5.0 microg/ml cisplatin for 2 hours can inhibited tumor cells proliferation in vitro by 33.4 and 54.2 percent, respectively, which had significant difference compared with control recombinant adenovirus (Ad-LacZ) groups (P < 0.05). RT-PCR revealed that Ad-Asc-myc downregulated expression of bcl-2 and upregulated expression of Bax, and no appreciable changes were observed in the expression of E2F-1. Detection of caspase-3 protein TEM, and FCM analysis showed that Ad-Asc-myc could induce apoptosis of transfected cells, which was enhanced by the treatment of cisplatin. Cell cycle analysis showed that obvious G(2)/M phase arrested in transfected cells. In conclusion, Ad-Asc-myc increased the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.
Xie
Xian-Kuan
XK
Department of Orthopaedics, The Second Hospital Affiliated Zhejiang University, College of Medicine, Hangzhou, People's Republic of China. xiexk2@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 / Q000502:physiology; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000096:biosynthesis; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D016373:DNA, Antisense / Q000494:pharmacology*; D004274:DNA, Recombinant / Q000494:pharmacology; D005434:Flow Cytometry; D015316:Genetic Therapy; D006801:Humans; D066298:In Vitro Techniques; D046529:Microscopy, Electron, Transmission; D012516:Osteosarcoma / Q000235:genetics / Q000628:therapy* / Q000648:ultrastructure; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D000970:Antineoplastic Agents; D016373:DNA, Antisense; D004274:DNA, Recombinant; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002945:Cisplatin