Pleiotropic effects of the sirtuin inhibitor sirtinol involves concentration‐dependent modulation of multiple nuclear receptor‐mediated pathways in androgen‐responsive prostate cancer cell LNCaP

Sirtinol is a purported specific inhibitor of the nicotinamide adenine dinucleotide (NAD)‐dependent type III histone deacetylase (also known as sirtuin). Sirtinol has been used extensively to identify chemopreventive/chemotherapeutic agents that modulate the sirtuins. However, the molecular effect of sirtinol other than serving as sirtuin inhibitor in cells is less clear. The present study addressed this deficiency in the literature. Based on structural similarity with plant‐derived cancer preventive/therapeutic compounds such as 3', 3'‐diindolylmethane, resveratrol, and genistein, we hypothesized that sirtinol may act on pathways similar to that affected by these compounds in the human prostate cancer cell LNCaP. We found that treatment of LNCaP cells with sirtinol led to concentration‐dependent effects on multiple pathways. Sirtinol inhibited LNCaP cell cycle and growth that was correlated with up‐regulation of cyclin‐dependent kinase inhibitor 1A mRNA and protein levels. This effect of sirtinol may due in part to modulation of androgen, estrogen, and insulin‐like growth factor‐1 mediated pathways as sirtinol treatment led to inhibition of mRNA and protein expression of marker genes involved in these pathways. We also found sirtinol activates aryl hydrocarbon‐dependent pathways in LNCaP cells. The effects of sirtinol were observed at 25 µM, a concentration lower than Ki (38 µM) for sirtuin activity. Based on these results we reasoned that sirtinol exerts pleiotropic effects in cells and that biological effects of sirtinol may not be due solely to inhibition of sirtuin. © 2012 Wiley Periodicals, Inc.     

A comprehensive review of cytochrome P450 2E1 for xenobiotic metabolism

Abstract

Cytochrome P450 2E1 (CYP2E1) plays a vital role in drug-induced hepatotoxicity and cancers (e.g. lung and bladder cancer), since it is responsible for metabolizing a number of medications and environmental toxins to reactive intermediate metabolites. CYP2E1 was recently found to be the highest expressed CYP enzyme in human livers using a proteomics approach, and CYP2E1-related toxicity is strongly associated with its protein level that shows significant inter-individual variability related to ethnicity, age, and sex. Furthermore, the expression of CYP2E1 demonstrates regulation by extensive genetic polymorphism, endogenous hormones, cytokines, xenobiotics, and varying pathological states. Over the past decade, the knowledge of pharmacology, toxicology, and biology about CYP2E1 has grown remarkably, but the research progress has yet to be summarized. This study presents a timely systematic review on CYP2E1’s xenobiotic metabolism, genetic polymorphism, and inhibitors, with the focus on their clinical relevance for the efficacy and toxicity of various CYP2E1 substrates. Moreover, several knowledge gaps have been identified towards fully understanding the potential interactions among different CYP2E1 substrates in clinical settings. Through in-depth analyses of these knowns and unknowns, we expect this review will aid in future drug development and improve management of CYP2E1 related clinical toxicity.     

Evaluation of Genetic Polymorphisms of N-acetyltransferase 2 and Relation with Chronic Myeloid Leukemia

Objectives: The N-Acetyltransferase 2 (NAT2) gene encodes a key enzyme involved in xenobiotic metabolism, which contributes to the detoxification of numerous cancer therapy-induced products. However, the NAT2 genotype/phenotype is not fully understood and few studies have reported its relationship with CML. The aim of this study was to determine whether its polymorphisms (C481T, G590A, 803A>G and 857G>A) have a role in chronic myeloid leukemia susceptibility (CML) in Sudanese population. Methods: We performed a case- control study. DNA from 200 CML patients and 100 controls was analyzed for the NAT2 polymorphisms using PCR-RFLP assay. Results: The study showed NAT2 polymorphisms 803AG are associated with CML protection by a factor of 2.3, (OR = 0.044, 95% CI: 0.020-0.095, p = 0. 000). The study indicated that the heterozygous (GA) and mutant (AA) variants of the G857A genotype also offer protection, (OR = 0.002, 95% CI: 0.002-0.019, p = 0. 000) and (OR = 0.018, 95% CI: 0.002-0.133, p = 0. 000), respectively. Conclusion: There was no significant difference in CML diagnosis among Sudanese cases with the 481C→T and 590G→A polymorphisms. But patients with the compound NAT2 genotypes 481CT/803 AG, 590AG/ 803AG, 590AG/ 803GG, 590AA/ 803AG and 590GG/ 803AG were found to have a reduced risk. The current study demonstrates that polymorphisms of NAT2 A803G and G857A might also act as protective factors against developing the disease.     

Cytochrome P450 1A1, 2E1 and GSTM1 Gene Polymorphisms and Susceptibility to Colorectal Cancer in the Saudi Population

Background: The Saudi population has experienced a sharp increase in colorectal and gastric cancer incidenceswithin the last few years. The relationship between gene polymorphisms of xenobiotic metabolizing enzymesand colorectal cancer (CRC) incidence has not previously investigated among the Saudi population. The aimof the present study was to investigate contributions of CYP1A1, CYP2E1, and GSTM1 gene polymorphisms.Materials and Methods: Blood samples were collected from CRC patients and healthy controls and genotypeswere determined by polymerase chain reaction restriction fragment length polymorphism and sequencing.Results and Conclusions: CYP2E1*6 was not significantly associated with CRC development (odd ratio=1.29;confidence interval 0.68-2.45). A remarkable and statistically significant association was observed amongpatients with CYP1Awt/*2A (odd ratio=3.65; 95% confidence interval 1.39-9.57). The GSTM1*0/*0 genotypewas found in 2% of CRC patients under investigation. The levels of CYP1A1, CYP2E1 and GSTM1 mRNAgene expression were found to be 4, 4.2 and 4.8 fold, respectively, by quantitative real time PCR. The results ofthe present case-control study show that the studied Saudi population resembles Caucasians with respect to theconsidered polymorphisms. Investigation of genetic risk factors and susceptibility gene polymorphisms in ourSaudi population should be helpful for better understanding of CRC etiology.     

Aryl hydrocarbon receptor biology and xenobiotic responses in hematopoietic progenitor cells

Studying the biological functions of the aryl hydrocarbon receptor (AhR) other than its function in xenobiotic drug metabolism may answer the questions as to why AhR orthologues have long been conserved phylogenically widely in the animal kingdom, and why homologues have diverged from nonvertebrate species such as nematodes and drosophila to all the vertebrate species. In this review, we focused on the mechanism of longevity possibly derived from evolution of AhRs and compared the functional difference of hematopoietic progenitors between wild-type (AhR^+/+) mice and AhR-deficiencies (AhR^+/−, AhR^−/−). Particular advantages found in wild-type mice compared with AhR-deficiencies were as follows: first, higher antioxidative function in the hematopoietic microenvironment with low oxidative tension seemed to have developed with the evolution of AhR; second, primitive hematopoietic progenitor-cell-specific deceleration and dormancy of cell-cycle regulation may have developed also with AhR evolution, which keeps hematopoietic progenitor cell compartment dormant without extinction by continuous differentiation; third, the consequent evolution of genomic stabilization with a longer lifespan in wild-type mice developed with the evolution of AhR. Experimentally, mice showed a significant extension of lifespan in a gene-dosage-dependent manner with a delayed onset of leukemogenicity. Another possible additional advantage observed in wild-type mice, the mechanism of which is not yet clarified, is an improved efficiency of fertilization in wild-type mice as compared with AhR-deficiencies, which seems to have developed with the evolution of AhR. Four advantages altogether, including the anti-aging feature mentioned above may have induced the AhR molecule to diverge various of species in the animal kingdom.     

Intestinal cell-specific vitamin D receptor (VDR)-mediated transcriptional regulation of CYP3A4 gene

CYP3A4 is the most important drug-metabolizing enzyme that is involved in biotransformation of more than 50% of drugs. Pregnane X receptor (PXR) dominantly controls CYP3A4 inducibility in the liver, whereas vitamin D receptor (VDR) transactivates CYP3A4 in the intestine by secondary bile acids. Four major functional PXR-binding response elements of CYP3A4 have been discovered and their cooperation was found to be crucial for maximal up-regulation of the gene in hepatocytes. VDR and PXR recognize similar response element motifs and share DR3(XREM) and proximal ER6 (prER6) response elements of the CYP3A4 gene.In this work, we tested whether the recently discovered PXR response elements DR4(eNR3A4) in the XREM module and the distal ER6 element in the CLEM4 module (CLEM4-ER6) bind VDR/RXRα heterodimer, whether the elements are involved in the intestinal transactivation, and whether their cooperation with other elements is essential for maximal intestinal expression of CYP3A4.Employing a series of gene reporter plasmids with various combinations of response element mutations transiently transfected into four intestinal cell lines, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP), we found that the CLEM4-ER6 motif interacts with VDR/RXRα heterodimer and partially cooperates with DR3(XREM) and prER6 in both basal and VDR-mediated inducible CYP3A4 regulation in intestinal cells. In contrast, eNR3A4 is involved only in the basal transactivation in intestinal cells and in the PXR-mediated rifampicin-induced transactivation of CYP3A4 in LS174T intestinal cells.We thus describe a specific ligand-induced VDR-mediated transactivation of the CYP3A4 gene in intestinal cells that differs from PXR-mediated CYP3A4 regulation in hepatocytes.     

Comparison of rat liver parenchymal and nonparenchymal cells in the activation of promutagens.

While the liver consists of both parenchymal cells (PC) and nonparenchymal cells (NPC), virtually all studies on promutagen activation have been performed using PC. To evaluate the comparative roles of PC and NPC in promutagen activation, we cocultivated a cell line generally considered to have an insignificant level of xenobiotic metabolism, Chinese hamster ovary (CHO) cells, with either PC, NPC, or a combination of both. The mixed culture was treated with two promutagens: dimethylnitrosamine (DMN) and 3-methylcholanthrene (3-MC). The induction of 6-thioguanine resistant mutants was evaluated using the well-established CHO/hypoxanthine-guanine phosphoribosyl transferase (HGPRT) assay. Activation of promutagens, as indicated by an increase in mutant frequency in CHO cells, was observed only when the PC were present with the CHO cells during the treatment period. No activation was observed with NPC. Coculturing of PC and NPC yielded essentially the same results as PC alone. P-450 mixed function monooxygenase activity measured by the 7-ethoxycoumarin-O-deethylase assay further substantiates that PC had a significantly higher xenobiotic metabolism activity than NPC. Our study therefore indicates that PC, not NPC, are the major cell population in the liver responsible for the activation of promutagens.