Elsevier

Urology

Volume 71, Issue 6, June 2008, Pages 1220-1225
Urology

Basic and Translational Science
Demethylating Agent 5-Aza-2′-Deoxycytidine Enhances Susceptibility of Bladder Transitional Cell Carcinoma to Cisplatin

https://doi.org/10.1016/j.urology.2007.11.029Get rights and content

Objectives

We investigated the effect of 5-aza-2′-deoxycytidine (DAC), a DNA methyltransferase inhibitor, on transitional cell carcinoma (TCC) cell lines, and examined the synergistic suppression of TCC growth by DAC and five chemotherapeutic agents.

Methods

We measured the cytotoxicity of DAC and chemotherapeutic agents against five TCC cell lines using the WST-1 assay, and analyzed the synergy of DAC and these agents by isobolographic analysis. The effects of each single agent or the combined treatment on apoptosis induction and cell cycle arrest were analyzed by flow cytometric analysis. We also investigated caspase activity and PCNA expression to clarify the mechanism of the synergistic actions of DAC and chemotherapeutic agents against TCC.

Results

We demonstrated that DAC could significantly increase the susceptibility of TCC cells to cisplatin (CDDP). Synergistic growth suppression by DAC and CDDP was confirmed in all TCC cell lines tested, but not by DAC combined with other chemotherapeutic agents. DAC inhibited proliferation via inducing G2/M cell cycle arrest, whereas CDDP inhibited proliferation via inducing both apoptosis and G2/M arrest. DAC enhanced the CDDP-induced upregulation of caspase activity and antiproliferative effect, resulting in an increase of cells in subG1 and G2/M phases. In addition, the synergy of DAC and CDDP was independent of p53 status in TCC.

Conclusions

The synergy of DAC and CDDP against TCC suggested that combination chemotherapy with these two agents might be a new strategy to improve the clinical response rate of this malignancy, regardless of p53 mutation.

Section snippets

Cell Lines and Agents

We used bladder transitional cell carcinoma cell lines: RT112 (wild-type p53), 253J (wild-type p53), TCCSUP (mutant p53) and T24 (p53 −/−). All cell lines were cultured in complete medium and maintained as monolayers and incubated in a humidified atmosphere containing 5% CO2 at 37°C.

We purchased DAC, paclitaxel (PTX), VBL, and 5-fluorouracil (5-FU) from Sigma Aldrich Japan (Tokyo, Japan); CDDP and ADM were purchased from Nippon Kayaku (Tokyo, Japan) and Kyowa Hakko Kogyo (Tokyo, Japan),

Synergistic Growth Suppression by DAC and CDDP

5-Aza-2′-deoxycytidine (Fig. 1A) and five chemotherapeutic agents (data not shown) each caused dosage-dependent cell growth suppression of TCC cells. DAC could increase the susceptibility of TCC cells to CDDP (Fig. 1B and C, results for RT112 and TCCSUP), and combined treatment with DAC and CDDP caused synergistic growth suppression in all TCC cell lines examined in a p53-independent manner, as shown by isobolographic analysis (Fig. 1D). However, synergy of DAC and PTX, 5-FU, VBL, and ADM was

Comment

Promising new antitumor agents usually appear as our understanding of oncogenesis advances. Although decitabine (Dacogen), a pharmacological form of DAC, has only been approved for use against hematopoietic malignancies at present, it also showed clinical efficacy in the treatment of metastatic lung carcinoma.5 Since 2004, studies concerning combination chemotherapy of DAC and chemotherapeutic agents have been performed, and the results suggested that DAC increased the cytotoxicity of

Conclusions

We observed synergistic growth suppression by DAC and CDDP in TCC cell lines, and the synergy of the two agents was independent of p53 status. This suggests that DAC might enhance the efficacy of CDDP against TCC, and combination chemotherapy with DAC and CDDP might become a new clinical therapeutic strategy to overcome TCC.

References (21)

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This research was supported by a grant from Ministry of Education, Culture, Sports, Science and Technology, Japan.

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