The role of methylation leading to capecitabine resistant malignant mesothelioma
17105 Background: Malignant mesothelioma is a deadly malignancy whose global incidence continues to be on the rise. Established therapies have been less than optimal. The current therapeutic standard is intravenous pemetrexed, an antifolate medication. Yet, another folate antimetabolite, capecitabine, is significantly less effective than pemetrexed. The enzymes thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and thymidylate phosphatase (TP) are critical to the efficacy of antifolates. Specifically, for capecitabine to be converted into a potent cytotoxic agent, the enzyme TP must be present and active. In one of four mesothelioma cell lines examined, the gene that encodes for TP, extracellular growth factor-1 (ECGF-1), is methylated. Methylation of this gene and the subsequent downregualtion of the TP enzyme confer a diminished cytotoxic effect by a capecitabine prodrug, dioxyfluridine (DFUR). Methods: Cells were cultured treated with and without 1uM decitabine (DAC) under identical conditions. DNA, RNA, and protein lysates were collected after 72 hours. Bisulfite-treated DNA was examined by MS-PCR for evidence of methylation of TS, DPD, and TP. RNA was collected and cDNA was synthesized. Real time PCR was utilized to detect the relative difference in RNA quantity. Western blots were done to evaluate the differences in protein expression between DAC treated and untreated cells. MTT assay was performed with DAC pretreated and untreated cell lines subsequently treated with DFUR and 5-fluorouracil. Results: One of the four mesothelioma cell lines showed consistent evidence of TP methylation by MS-PCR. The addition of 1uM DAC to the cell lines conferred a six-fold difference in expression of the methylated gene by both real time PCR as well as by Western blot. The prodrug DFUR subsequently shows increased cytotoxicity in the methylated cell line by MTT assay when pretreated with DAC compared when not exposed to the DAC. Conclusion: By demethylating the ECGF-1 gene with DAC, the now upregulated TP enzyme has an increased ability to convert DFUR to 5-FU; thus enhancing the cytotoxicity of a drug thought to be ineffective in malignant mesothelioma. No significant financial relationships to disclose.