The effect of folic acid and methionine deficiency and excess on DNA damage and cancer growth in HT29 colon cancer cells and the Apc Min mouse model

Abstract

Folate and methionine are critical for one-carbon metabolism, impacting DNA synthesis, repair, and methylation processes, as well as polyamine synthesis. These micronutrients have been implicated in colorectal cancer risk. The aim of this thesis was to examine in greater detail the role of folate and methionine in colon cancer initiation and progression by assessing DNA stability and tumour incidence. Studies were performed in vitro (using human colorectal adenocarcinoma HT29 cell line) and in vivo (using ApcMin/+ [Min/+ superscript] mouse model). The in vitro studies examining the effects of various folic acid and methionine concentrations within the physiological range on cell proliferation and genomic instability of HT29 cells, showed that restriction of folic acid or methionine inhibited cellular proliferation, while supra-physiological folate induced apoptosis. HT29 cells may be resistant to genome instability induced by folic acid or methionine deficiency under the experimental conditions reported for this study because no significant increases in micronuclei, nuclear buds or nucleoplasmic bridges were observed in the Cytokinesisblock micronucleus cytome (CBMN-Cyt) assay. The investigation on the effect of folic acid and methionine depletion on telomere length and DNA methylation in HT29 cells demonstrated that folate and methionine depletion may increase both telomere length and DNA methylation in HT29 cells. The length of telomere was positively correlated with DNA methylation. In the in vivo studies using the ApcMin/+ [Min/+ superscript] mouse model, the effect of supplementing a western-style diet with dietary folic acid and/or methionine on intestinal tumour development was assessed. A total of 113 mice were randomised to receive one of the four diet treatments; New Western Diet (NWD) as control diet, NWD with additional folic acid, NWD with additional methionine, and NWD with additional folic acid and methionine, administered at age of 3 until 13 weeks, with wild type (WT) mice used as controls. Supplementation of folic acid and methionine separately, resulted in marginally lower tumour numbers, when compared to the control diet. However, supplementation with both folic acid and methionine together appeared to annul the marginal protective effect of supplementing individually. The investigation on the effect of supplementing a western-style diet with dietary folic acid and/or methionine on genomic stability (measured via micronucleated erythrocyte assay on blood sample; telomere length and DNA methylation on the colon tissue) showed insufficient evidence that additional folic acid and/or methionine promotes DNA stability or instability in ApcMin/+ [Min/+ superscript] or WT mice. Dietary supplementation with folic acid and/or methionine at the levels and duration used in this study did not substantially promote or protect against DNA damage in WT or intestinal cancer-prone ApcMin/+ [Min/+ superscript] mouse model fed a western-style diet although a marginal effect on tumour number was evident. In conclusion, the results of this thesis support a role of methionine and folate in affecting intestinal cell proliferation and possibly tumour number. However, the impact of supplementation with folate and methionine on genome stability was marginal.