Abstract 1680: Effect of folate and γ-glutamyl hydrolase modulation on in vitro chemosensitivity of colon and breast cancer cells to antifolates and 5-fluorouracil

Abstract

Abstract 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. In the present study, we investigated whether exogenous folate concentrations further influence the effects of GGH modulation on the chemosensitivity to antifolates and 5FU. 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 pemetrexed (MTA, positive control), trimetrexate (TMTX, negative control), MTX and 5FU under 2.3 μM folic acid (FA) and 100 nM and 50 nM of 5-methyltetrahydrofolate (5MTHF) was determined. Results: In HCT116 cells, GGH overexpression increased chemosensitivity to MTA and 5FU alone at all concentrations (p<0.05). At 2.3 uM FA, GGH overexpression decreased chemosensitivity to MTX and 5FU+leucovorin (LV) and enhanced TMTX chemosensitivity while it increased chemosensitivity to MTX and 5FU+LV and decreased TMTX chemosensitivity at 50 nM 5MTHF (p<0.05). At all concentrations, GGH inhibition decreased chemosensitivity to MTX and 5FU alone whereas it enhanced 5FU+LV chemosensitivity (p<0.05). GGH inhibition decreased chemosensitivity to MTA at 2.3 uM FA and 100 nM 5MTHF while MTA chemosensitivity increased at 50 nM 5MTHF (p<0.05). In MDA-MB-435 cells, GGH overexpression decreased chemosensitivity to all drugs at all concentration, and to 5FU alone at 50nM 5MTHF (p<0.05). GGH inhibition decreased chemosensitivity to MTX and TMTX at all concentrations while it increased chemosensitivity to MTA at 100 nM and 50 nM 5MTHF, and to 5FU+LV at 2.3 uM FA and 100 nM 5MTHF, respectively (p<0.05). Conclusions: As proof of principle, we provide functional evidence that GGH modulation affects the chemosensitivity of colon and breast cancer cells to antifolates and 5FU. These GGH modulation-induced changes in chemosensitivity are highly complex and 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 antifolates and 5FU. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1680. doi:10.1158/1538-7445.AM2011-1680