It has been hypothesized that the ubiquitously expressed Phase II drug metabolizing enzyme human arylamine N-acetyltransferase 1 (NAT1) has a role in cell biology other than the metabolism of xenobiotics. The identification of p-aminobenzoylglutamate (pABG) as an endogenous substrate for NAT1 led to speculation that the enzyme might be involved in folate regulation. NAT1 is overexpressed in human luminal breast cancers and is a proposed biomarker for estrogen receptor positive breast cancers in women as well as luminal M2 breast cancers in men. Moreover, NAT1 has been shown to support human cancer cell growth, survival and invasion both in vitro and in vivo. Overall, the aim of this work was to identify possible endogenous roles for NAT1 that might explain its wide tissue expression.
Initially, mouse Nat2 (homologue to human NAT1) was considered, and the levels of pABG and 5-methyltetrahydrofolate, the predominant circulating folate, were measured in wild-type and Nat2-deleted (Nat2-/-) mice. Both metabolites were unaltered in Nat2-/- mice suggesting the enzyme did not regulate either of these folate products in vivo under normal conditions. Therefore, the effect of human NAT1 on pABG and 5-methyltetrahydrofolate in the human colon adenocarcinoma cell line, HT-29, was investigated to determine if NAT1 regulated folate in cancer cells. As with the mouse study, pABG and 5-methyltetrahydrofolate were unaltered when NAT1 was knocked down in these cells. Overall, these results suggests that NAT1 is not essential for the regulation of 5-methyltetrahydrofolate nor is the acetylation of pABG physiologically relevant in these models.
Metabolomic analysis following human NAT1 inhibition was also carried out. Firstly, changes in the S-adenosylmethionine (SAM) cycle was also investigated because 5-methyltetrahydrofolate provides the methyl group to the SAM cycle necessary for methylation reactions of DNA, proteins and lipids. Secondly, differences in amino acids present in cell culture medium were investigated following human NAT1 knocked down. SAM cycle homeostasis was maintained in HT-29 cells independent of human NAT1 activity. Furthermore, there were very few changes in SAM cycle components in Nat2-/- tissues compared to wild-type. This suggests that NAT1 was not involved in regulating methylation reactions. However, changes in alanine and homocysteine concentrations in cell culture medium between control and human NAT1 knock-down cells were identified.
Homocysteine levels in the medium initially increased with time for both control and knock-down cells. However, by day 3 in culture, levels began to decrease in the knocked-down cells whereas they continued to increase in the controls. This correlated with the depletion of methionine from the medium suggesting the knocked down cells required homocysteine whereas the control cells did not. The methionine salvage pathway is a multi-enzyme multi-reaction process that recycles methylthioadenosine (MTA), a by-product of polyamine synthesis, back to methionine. This pathway is important for supplying methionine when other sources are exhausted.
Cancer cells were grown in medium deficient of methionine but containing MTA as an assay for a functional methionine salvage pathway. In HT-29 cells, human NAT1 knockdown resulted in an inability to convert MTA to methionine. This was due to a loss in the enzyme methylthioribose isomerase 1 (MRI1), which is involved in the salvage pathway. To confirm these results, NAT1 and MRI1 were individually and collectively knocked down in HeLa cells. When either enzyme was knocked down, growth in MTA was sustained, albeit at a lower rate than in complete medium. By contrast, simultaneous knock down of both enzymes completely inhibited growth in medium deficient of methionine but containing MTA. Furthermore, ectopically expressed human NAT1 in HT-29 cells supported the methionine salvage pathway when MRI1 was knocked down. LC-MS/MS was used to characterise the isomerase reaction and there is evidence to suggest that human NAT1 is capable of performing this reaction. However, under the in vitro conditions used, this reaction was very inefficient and at times remained undetected. Further work is required to characterise the role of human NAT1 as a methylthioribose isomerase. Overall, this shows that human NAT1 has a novel role in the methionine salvage pathway and suggests a redundancy between NAT1 and MRI1.