Cisplatin-induced DNA hypermethylation in a low-folate environment

Cisplatin is a chemotherapy agent commonly used to treat non-small cell lung cancer (NSCLC). Unfortunately, most NSCLCs will develop resistance to cisplatin. Deoxyribonucleic acid (DNA) hypermethylation is one possible mechanism partly responsible for the development of cisplatin resistance. We sought to determine whether acute reductions of folate in the cell culture medium and treatment with a demethylating agent, 5-aza-2′-deoxycytidine (DAC), could prevent and/or reverse the cisplatin resistance associated with DNA hypermethylation. Moreover, complementary deoxyribonucleic acid (cDNA) microarray technology was used to analyze gene expression alterations associated with the development of cisplatin resistance. Cell proliferation and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)-based cytotoxicity assays were performed to determine the effect of acute reductions of folate in the cell culture medium on the growth and viability of three NSCLC cell lines and were extended to include three ovarian cancer cell lines. DNA methylation was determined with an enzymatic assay by using 3H-labelled S-adenosylmethionine and SssI methylase. Gene expression alterations associated with the development of acquired cisplatin resistance were determined in two NSCLC cell lines during a 5-week time-course study. During this study, the cells were treated weekly with cisplatin to induce cisplatin resistance; cells were harvested weekly, and ribonucleic acid (RNA) was extracted for subsequent cDNA microarray analysis. The effect of acute reductions of folate with and without cisplatin treatment on gene expression was determined during a 1-week study. Treatment of cells with cisplatin resulted in significant DNA hypermethylation. Acute reductions in folate could not reverse the DNA hypermethylating effect of cisplatin. Acute reductions of folate resulted in enhanced intrinsic resistance to cisplatin. Treatment of cells with DAC resulted in a mild but statistically significant increase in cisplatin resistance. cDNA microarray analysis revealed the expression of novel genes previously related to cancer progression but never before related to cisplatin resistance. These studies are the first to demonstrate that acute reductions of folate in the cell culture medium are associated with enhanced intrinsic resistance to cisplatin in NSCLC and ovarian cancer cell lines.