Role of hMLH1 promoter hypermethylation in drug resistance to 5‐fluorouracil in colorectal cancer cell lines

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Abstract
Loss of DNA mismatch repair (MMR) occurs in 10–15% of sporadic colorectal cancer, is usually caused by hMLH1 hypermethylation, and has been shown to confer resistance to various chemotherapeutic reagents, including 5‐fluorouracil (5‐FU). We tested the hypothesis that demethylation of the hMLH1 promoter in hypermethylated colorectal cancer cells would restore MMR proficiency and drug sensitivity to 5‐FU. We used the MMR‐deficient cell lines SW48, HCT116, HCT116+chr2 and the ‐proficient cell line HCT116+chr3. After treatment with the demethylating agent 5‐Aza‐2′‐deoxycytidine (5 aza‐dC), hMLH1 mRNA and protein expression were determined by RT‐PCR and immunoblots. The methylation status for hMLH1 was investigated by methylation‐specific PCR. Cells were subsequently treated with 5‐FU and the growth characteristics ascertained by clonogenic assays. hMLH1 hypermethylation was reverted in SW48 cells 24 hr after treatment with 5 aza‐dC and was accompanied by hMLH1 mRNA and protein reexpression. While 5 aza‐dC alone did not affect the growth of SW48 cells, all other cell lines responded with a pronounced growth inhibition. 5‐FU treatment strongly reduced the colony formation of HCT116+chr3 cells. These effects were significantly less in the MMR‐deficient cells. Combined treatment of SW48 cells resulted in a similar growth pattern as seen in 5‐FU only treated HCT116+chr3 cells. We demonstrate that in vitro resistance to 5‐FU can be overcome by reexpression of hMLH1 protein through 5 aza‐dC‐induced demethylation in hypermethylated cell lines. Induction of the expression of methylated tumor suppressor or MMR genes could have a significant impact on the development of future chemotherapy strategies.