The Effects of ɤ-Glutamyl Hydrolase Modulation on Cancer Treatment and DNA Methylation in Human Cancer Cells

Folate and antifolates are retained in cells by polyglutamylation mediated by folylpolyglutamate synthase (FPGS), and are exported from cells after hydrolysis to monoglutamates by γ-glutamyl hydrolase (GGH). Polyglutamylated (anti)folates are retained in cells longer and are better substrates than their monoglutamate counterparts for intracellular folate-dependent enzymes. GGH modulation may therefore affect chemosensitivity of cancer cells to antifolates and 5-fluorouracil by altering polyglutamylation of antifolates and a specific target intracellular folate cofactor for 5-fluorouracil (5,10-methylenetetrahydrofolate), respectively. We generated an in vitro model of GGH modulation in HCT116 and MDA-MB-435 cells with predictable functional consequences and investigated chemosensitivity to 5-fluorouracil and antifolates. Overall, GGH overexpression decreased chemosensitivity to 5-fluorouracil+ leucovorin and methotrexate, while GGH inhibition increased chemosensitivity to 5-fluorouracil +leucovorin. However, these effects appeared to depend not only on the GGH modulation-induced changes in polyglutamylation of 5,10-methylenetetrahydrofolate and antifolates but also on intracellular folate levels as well as adaptive and compensatory changes in other enzymes involved in intracellular folate and antifolate accumulation and metabolism in response to GGH modulation. Polyglutamylation is also important in DNA methylation as polyglutamylated folates are better substrates for methylenetetrahydrofolate reductase and methionine synthase involved in the generation of S-adenosylmethionine, which is a substrate for DNA methylation. We hypothesized that GGH and FPGS modulation may affect DNA methylation at global and gene-specific levels with consequent functional ramifications. GGH and FPGS modulation demonstrated the inverse relationship between GGH activity and global DNA methylation as well as was associated with differential gene expression and altered CpG promoter DNA methylation involved in important biological pathways. Some of the observed altered gene expression appear to be regulated by DNA methylation. Whether or not GGH modulation may be an important determinant of chemosensitivity to 5-fluorouracil- and antifolate-based chemotherapy, and the potential roles of GGH and FPGS modulation in DNA methylation need further exploration.Ph