Abstract 1607: Effect of folate and GGH modulation on chemosensitivity of colon and breast cancer cells to 5-fluorouracil and methotrexate

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: γ-Glutamyl hydrolase (GGH) removes terminal glutamates of polyglutamylated folates and antifolates, thereby facilitating hydrolysis and efflux of folates and antifolates from the cell. Therefore, GGH plays an important role in regulating intracellular folates and antifolates for optimal folate-dependent one-carbon transfer reactions and antifolate-induced cytotoxic effects, respectively. We have previously reported that GGH modulation significantly affects chemosensitivity of colon and breast cancer cells to 5-fluorouracil (5FU) and methotrexate (MTX) by changing intracellular retention of a folate cofactor (5,10-methylenetetrahydrofolate) necessary for the cytotoxic effects of 5FU and by changing intracellular retention of MTX, respectively. We investigated whether the GGH modulation-induced changes in chemosensitivity might be counterbalanced by GGH modulation-induced changes in polyglutamylation of other intracellular folate cofactors and total intracellular folate pools. Methods: Human HCT116 colon and MDA-MB-435 breast cancer cells were stably transfected with the sense GGH cDNA or GGH-targeted siRNA, respectively, to generate an in vitro model of GGH overexpression and inhibition. In vitro chemosensitivity to 5FU and MTX under 2.3 μM folic acid (FA) and 50 nM and 100 nM of 5-methyltetrahydrofolate (5MTHF) was determined. Results: GGH overexpression was associated with lower total and long-chain polyglutamylated intracellular folate concentrations, thymidylate synthase (TS) catalytic enzyme activity, and dihydrofolate reductase (DHFR) protein expression than controls, while GGH inhibition showed higher total and long-chain polyglutamylated intracellular folate concentrations, TS activity, and DHFR protein expression than controls (P<0.05). In HCT116 cells, GGH overexpression decreased chemosensitivity to 5FU and MTX at 2.3 uM FA while it enhanced chemosensitivity to 5FU and MTX at 50 nM 5MTHF (P<0.05). At all concentrations, GGH inhibition increased chemosensitivity to 5FU while it decreased chemosensitivity to MTX (P<0.05). In MDA-MB-435 cells, GGH overexpression decreased chemosensitivity to 5FU and MTX at all concentration (P<0.05). GGH inhibition increased chemosensitivity to 5FU at 2.3 uM FA and 100 nM 5MTHF whereas it decreased chemosensitivity to MTX at all concentrations (P<0.05). Conclusions: As proof of principle, we provide functional evidence that GGH overexpression and inhibition can modulate chemosensitivity of colon and breast cancer cells to 5FU and MTX. These GGH modulation-induced changes in chemosensitivity are further influenced by different folate forms and concentrations. Our data suggest that both GGH modulation and folate status affect chemosensitivity of colon and breast cancer cells to 5FU and MTX. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1607.