Combinatorial Antitumor Activity of Oxaliplatin with Epigenetic Modifying Agents, 5-Aza-CdR and FK228, in Human Gastric Cancer Cells

Abstract

Epigenetic silencing is considered to be a major mechanism for loss of activity in tumor suppressors. Reversal of epigenetic silencing by using inhibitors of DNA methyltransferase (DNMT) or histone deacetylases (HDACs) such as 5-Aza-CdR and FK228 has shown to enhance cytotoxic activities of several anticancer agents. This study aims to assess the combinatorial effects of gene-silencing reversal agents (5-Aza-CdR and FK228) and oxaliplatin in gastric cancer cells, i.e., Epstein-Barr virus (EBV)-negative SNU-638 and EBV-positive SNU-719 cells. The doublet combinatorial treatment of 5-Aza-CdR and FK228 exhibited synergistic effects in both cell lines, and this was further corroborated by Zta expression induction in SNU-719 cells. Three drug combinations as 5-Aza-CdR/FK228 followed by oxaliplatin, however, resulted in antagonistic effects in both cell lines. Simultaneous treatment with FK228 and oxaliplatin induced synergistic and additive effects in SNU-638 and SNU-719 cells, respectively. Three drug combinations as 5-Aza-CdR prior to FK228/oxaliplatin, however, again resulted in antagonistic effects in both cell lines. This work demonstrated that efficacy of doublet synergistic combination using DNMT or HDACs inhibitors can be compromised by adding the third drug in pre- or post-treatment approach in gastric cancer cells. This implies that the development of clinical trial protocols for triplet combinations using gene-silencing reversal agents should be carefully evaluated in light of their potential antagonistic effects.

Keywords: Antagonism; Combinatorial index; EBV; Synergism; Zta.

Fig. 1.

Effects of combined treatment with 5-Aza-CdR and FK228. SNU-638 (A) and SNU-719 cells (B) were treated with single agent of 5-Aza-CdR (0.1 μM) and FK228 (3 nM), or with doublet combination of 5-Aza-CdR/FK228 for 24 h. After removal of drug-containing media, cells were further incubated in drug-free media for an additional 72 h and were then subjected to MTS assay. Data are presented as the survival rate relative to the control, which was assigned a value of “100%”. Data are presented as the mean ± SD from three independent experiments. ^** indicates synergistic effects.

Fig. 2.

Zta induction after treatment with 5-Aza-CdR and/or FK228 in SNU-719 cells. Immunofluorescence was used to measure Zta. Representative fluorescence images are shown using anti-Zta primary antibody (1:40) after 24 h and 48 h of drug treatment. Bars, 100 μm. The data are expressed as relative fold change to control, 5-Aza-CdR single treatment. Data are presented as the mean ± SD from three independent experiments. ^** indicates synergistic effects.

Fig. 3.

Pre-treatment with 5-Aza-CdR/FK228 antagonized the anti-proliferative effect of oxaliplatin. Cells were pre-treated with 5-Aza-CdR (0.1 μM)/FK228 (1 nM or 3 nM) for 24 h, washed and then exposed to 1 μM of oxaliplatin for subsequent 72 h (A). Relative rate of proliferation was determined in SNU-638 (B) and SNU-719 (C) cells using the MTS assay. Data are presented as the mean ± SD from three independent experiments. All triplet combinations showed antagonism in both cell lines.

Fig. 4.

Combination indexes (CI_x) versus affected fraction (fa) determined for combination of FK228 and oxaliplatin. Cells were treated with FK228/oxaliplatin for 72 h at fixed equitoxic ratios. CI_x≤0.8, CI_x≥1.2, and 0.8<CI_x<1.2 indicate synergism, antagonism, and additivity, respectively (See methods for details).

Fig. 5.

Combination effects of 5-Aza-CdR pre-treatment combined with FK228/oxaliplatin treatment. Cells were treated with 0.25 μM of 5-Aza-CdR for 24 h, and then incubated for 72 h with FK228/oxaliplatin (A). SNU-638 (B) and SNU-719 (C) cells were exposed to drugs at the concentrations indicated. Relative rate of proliferation was determined using the MTS assay. Data are presented as the mean ± SD based on at least three replicate experiments. ^* indicates additive effects.