Low dose Zebularine treatment enhances immunogenicity of tumor cells.

Strategy: We have investigated how alterations in gene expression induced by the demethylating drug Zebularine affect the immune response tumor cells elicit. The rational has been to treat syngeneic rat colon cancer cells with Zebularine at different concentrations and then use these cells to study gene expression of different genes involved in cancer immunogenicity. Gene expressions were monitored by semi-quantitative PCR and real-time PCR.

Results: Intriguingly there was a large increase in the production of indoleamine 2,3-dioxygenase (IDO) after treatment with 100 microM Zebularine as compared with untreated tumor cells, whereas treatment with 20 microM Zebularine caused a significant decrease of the IDO production. After immunization with syngeneic tumor cells, spleen cells were isolated and restimulated in vitro with irradiated tumor cells. Immune reactivity was measured by proliferation, and production of interferon gamma and interleukin10. The immunogenicity of tumor cells treated in vitro with a low dose of Zebularine increased, whereas it decreased after high dose exposure. The inhibition of immunogenicity by 100 microM Zebularine was shown to be counteracted by the IDO inhibitor 1-methyl-tryptophan (1 MT), confirming that this effect of Zebularine is mainly caused by IDO induction. Differences using Zebularine-treated or non-treated cells for in vitro restimulation were marginal.

Conclusion: Low dose treatment with Zebularine (20 microM) decreases the production of the immunosuppressive IDO from rat colon cancer cells and enhances their immunogenicity, whereas high dose Zebularine treatment (100 microM) enhances the IDO production from the cancer cells and suppresses their immunogenicity. This immunosuppression should be considered when cancer is treated with Zebularine or drugs acting in a similar way.