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Effects of short-term supplementation of folic acid and L-5-methyltetrahydrofolate on cell proliferation and the expression of folate transporters in human colorectal adenocarcinoma (Caco2) cells Hempstock, Wendy

Abstract

Folate plays a role in the synthesis and repair of DNA and the generation of methyl groups. Folic acid (FA) is a synthetic oxidized form of folate used in food fortification and supplements in Canada. Increased colon cancer incidence has been correlated with FA fortification in several countries. The effect of FA on the development of colon cancer is controversial as other research shows a lack of association between FA fortification and colon cancer incidence. I hypothesize that FA affects proliferation and folate transporter expression in colon cancer cells differently than L-5-methyltetrahydrofolate (5MTHF). In addition, the forms of folate, reduced versus oxidized, would differentially affect the activity of the Wnt signaling pathway. The overall objective of my research is to investigate the effect of FA and 5MTHF on cell proliferation, the expression of selected folate transporters, and the activity of the Wnt signalling pathway in human colorectal adenocarcinoma (Caco2) cells. Caco2 cells were cultured for 3 or 5 days in folate-free RPMI 1640 medium supplemented with 10% dialyzed FBS and treated with 0, 0.9, 2.3, or 3.4 µM FA or MTHF. Cell viability was assessed using WST-1 colourimetric assay. Cell proliferation was assessed by BrdU colourimetric assay and cell cycle analysis with BrdU incorporation was measured by flow cytometry. The abundance of reduced folate transporter (RFC), folate receptor-α (FRα), proton-coupled folate transporter (PCFT), breast cancer resistance protein (BCRP) was assessed by Western blotting. β-Catenin nuclear localization was assessed by measuring the fluorescence of Alexa Fluor 488® using confocal microscopy. FA treatment increased cell proliferation compared to treatment with MTHF at all concentrations after 3 days. After 5 days, there was no difference in cell viability or cell proliferation. Cell cycle analysis after 5 days of 3.4 µM FA and 5MTHF treatment showed spikes in the pre-G1 phase compared to the control. Neither folate transporter expression nor β-Catenin nuclear localization was affected by FA and 5MTHF treatment under the conditions tested. This lack of effect of FA and 5MTHF on cell proliferation and the expression of selected folate transporters was possibly due to relatively short treatment duration.

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Attribution-NonCommercial-NoDerivs 2.5 Canada

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