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.

The Molecular Pathogenesis of Osteosarcoma: A Review

“…Downregulation of Myc enhanced the therapeutic activity of methotrexate against osteosarcoma cells [50]. Adenovirus-mediated transfection with the antisense Myc fragment led to cell-cycle arrest and enhanced apoptosis in the MG-63 osteosarcoma cell line [51]. Using a conditional transgenic mouse model, Arvanitis et al [52] showed that Myc inactivation caused proliferative arrest and promoted differentiation in osteosarcoma.…”

Suppression of metastasis by mirtazapine via restoration of the Lin-7C/β-catenin pathway in human cancer cells

“…Although IPA did not identify any reagents affecting Lin-7C expression, five different ligands specific for HTR2C, which is an upstream molecule of Lin-7C, were nominated. It is noteworthy that while caffeine was thought to be a promising reagent for enhancing cisplatin in human osteosarcoma cells15 and effective for treating patients with lung metastases16, and we thought that caffeine might be a strong candidate as an antimetastatic reagent with Lin-7C up-regulation, we did not observe a significant effect of caffeine or other reagents examined on Lin-7C expression in our metastatic cell lines and the non-metastatic cells. Despite evidence for an association between caffeine and apoptosis induction through the AKT/mTOR/S6K, NF-κB, and MAPK pathways17, the reason why the effect of caffeine depends on cellular types is unclear.…”

Inhibition of malignant phenotypes of human osteosarcoma cells by a gene silencer, a pyrrole–imidazole polyamide, which targets an E‐box motif

“…It has been also shown that c‐ MYC is amplified in several osteosarcoma‐derived cell lines, which are sometimes resistant to anti‐cancer drugs such as doxorubicin and methotrexate [10]. Notably, knockdown of c ‐MYC resulted in a marked enhancement of sensitivity to cisplatin in osteosarcoma MG63 cells [11]. Since these findings indicate that the amplification of c‐ MYC plays a pivotal role in the regulation of osteosarcoma development and acquisition of chemo‐resistance, molecule(s) targeting c‐ MYC and/or E‐box might become a novel and attractive chemotherapeutic drug.…”