Enhancement Effect of Adenovirus-Mediated Antisense c-myc and Caffeine on the Cytotoxicity of Cisplatin in Osteosarcoma Cell Lines

Abstract

Aims: 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 × 10⁹ 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 μg/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.