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Human UDP-Glucuronosyltransferases: Effects of altered expression in breast and pancreatic cancer cell lines.

ABSTRACT: Increased aerobic glycolysis and de novo lipid biosynthesis are common characteristics of invasive cancers. UDP-glucuronosyltransferases (UGTs) are phase II drug metabolizing enzymes that in normal cells possess the ability to glucuronidate these lipids and speed their excretion; however, de-regulation of these enzymes in cancer cells can lead to an accumulation of bioactive lipids, which further fuels cancer progression. We hypothesize that UGT2B isoform expression is down-regulated in cancer cells and that exogenous re-introduction of these enzymes will reduce lipid content, change the cellular phenotype, and inhibit cancer cell proliferation. In this study, steady-state mRNA levels of UGT isoforms from the 2B family were measured using qPCR in 4 breast cancer and 5 pancreatic cancer cell lines. Expression plasmids for UGT2B isoforms known to glucuronidate cellular lipids, UGT2B4, 2B7, and 2B15 were transfected into MCF-7 and Panc-1 cells, and the cytotoxic effects of these enzymes were analyzed using trypan blue exclusion, annexin V/PI staining, TUNEL assays, and caspase-3 immunohistochemistry. There was a significant decrease in cell proliferation and a significant increase in the number of dead cells after transfection with each of the 3 UGT isoforms in both cell lines. Cellular lipids were also found to be significantly decreased after transfection. The results presented here support our hypothesis and emphasize the important role UGTs can play in cellular proliferation and lipid homeostasis. Evaluating the effect of UGT expression on the lipid levels in cancer cell lines can be relevant to understanding the complex regulation of cancer cells, identifying the roles of UGTs as "lipid-controllers" in cellular homeostasis, and illustrating their suitability as targets for future clinical therapy development.

SUBMITTER: Dates CR

PROVIDER: S-EPMC4622877 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Human UDP-Glucuronosyltransferases: Effects of altered expression in breast and pancreatic cancer cell lines.

Dates Centdrika R CR Fahmi Tariq T Pyrek Sebastian J SJ Yao-Borengasser Aiwei A Borowa-Mazgaj Barbara B Bratton Stacie M SM Kadlubar Susan A SA Mackenzie Peter I PI Haun Randy S RS Radominska-Pandya Anna A

Cancer biology & therapy 20150101 5

Increased aerobic glycolysis and de novo lipid biosynthesis are common characteristics of invasive cancers. UDP-glucuronosyltransferases (UGTs) are phase II drug metabolizing enzymes that in normal cells possess the ability to glucuronidate these lipids and speed their excretion; however, de-regulation of these enzymes in cancer cells can lead to an accumulation of bioactive lipids, which further fuels cancer progression. We hypothesize that UGT2B isoform expression is down-regulated in cancer c ...[more]

PMID: 25996841

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Project description:2-Amino-9H-pyrido[2,3-b]indole (A?C) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogenic heterocyclic aromatic amines (HAA) that arise during the burning of tobacco and cooking of meats. UDP-glucuronosyltransferases (UGT) detoxicate many procarcinogens and their metabolites. The genotoxic N-hydroxylated metabolite of A?C, 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-A?C), undergoes glucuronidation to form the isomeric glucuronide (Gluc) conjugates N(2)-(?-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (A?C-HON(2)-Gluc) and O-(?-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (A?C-HN(2)-O-Gluc). A?C-HON(2)-Gluc is a stable metabolite but A?C-HN(2)-O-Gluc is a biologically reactive intermediate, which covalently adducts to DNA at levels that are 20-fold higher than HONH-A?C. We measured the rates of formation of A?C-HON(2)-Gluc and A?C-HN(2)-O-Gluc in human organs: highest activity occurred with liver and kidney microsomes, and lesser activity was found with colon and rectum microsomes. A?C-HN(2)-O-Gluc formation was largely diminished in liver and kidney microsomes, by niflumic acid, a selective inhibitor UGT1A9. In contrast, A?C-HON(2)-Gluc formation was less affected and other UGT contribute to N(2)-glucuronidation of HONH-A?C. UGT were reported to catalyze the formation of isomeric Gluc conjugates at the N(2) and N3 atoms of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), the genotoxic metabolite of PhIP. However, we found that the N3-Gluc of HONH-PhIP also covalently bound to DNA at higher levels than HONH-PhIP. The product ion spectra of this Gluc conjugate acquired by ion trap mass spectrometry revealed that the Gluc moiety was linked to the oxygen atom of HONH-PhIP and not the N3 imidazole atom of the oxime tautomer of HONH-PhIP as was originally proposed. UGT1A9, an abundant UGT isoform expressed in human liver and kidney, preferentially forms the O-linked Gluc conjugates of HONH-A?C and HONH-PhIP as opposed to their detoxicated N(2)-Gluc isomers. The regioselective O-glucuronidation of HONH-A?C and HONH-PhIP, by UGT1A9, is a mechanism of bioactivation of these ubiquitous HAAs.

Dataset Information

Human UDP-Glucuronosyltransferases: Effects of altered expression in breast and pancreatic cancer cell lines.

Publications

Human UDP-Glucuronosyltransferases: Effects of altered expression in breast and pancreatic cancer cell lines.

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