Effects of 7-ketocholesterol on tamoxifen efficacy in breast carcinoma cell line models in vitro.

Oxysterols play significant roles in many physiological and pathological processes including cancer. They modulate some of the cancer hallmarks pathways, influence the efficacy of anti-cancer drugs, and associate with patient survival. In this study, we aimed to analyze the role of 7-ketocholesterol (7-KC) in breast carcinoma cells and its potential modulation of the tamoxifen effect. 7-KC effects were studied in two estrogen receptor (ER)-positive (MCF-7 and T47D) and one ER-negative (BT-20) breast cancer cell lines. First, we tested the viability of cells in the presence of 7-KC. Next, we co-incubated cells with tamoxifen and sublethal concentrations of 7-KC. We also tested changes in caspase 3/7 activity, deregulation of the cell cycle, and changes in expression of selected genes/proteins in the presence of tamoxifen, 7-KC, or their combination. Finally, we analyzed the effect of 7-KC on cellular migration and invasion. We found that the presence of 7-KC slightly decreases the efficacy of tamoxifen in MCF-7 cells, while an increased effect of tamoxifen and higher caspase 3/7 activity was observed in the BT-20 cell line. In the T47D cell line, we did not find any modulation of tamoxifen efficacy by the presence of 7-KC. Expression analysis showed the deregulation in CYP1A1 and CYP1B1 with the opposite trend in MCF-7 and BT-20 cells. Moreover, 7-KC increased cellular migration and invasion potential regardless of the ER status. This study shows that 7-KC can modulate tamoxifen efficacy as well as cellular migration and invasion, making 7-KC a promising candidate for future studies.

Plasma oxysterol levels in luminal subtype breast cancer patients are associated with clinical data.

Oxygenated metabolites of cholesterol (oxysterols) have been previously demonstrated to contribute to progression of various cancers and to modulate resistance to breast cancer endocrine therapy in vitro. We measured prognostic roles of circulating levels of seven major oxysterols in the progression of luminal subtype breast carcinoma. Liquid chromatography coupled with tandem mass spectrometry was used for determination of levels of non-esterified 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, 7-ketocholesterol, cholesterol-5α,6α-epoxide, cholesterol-5ß,6ß-epoxide, and cholestane-3ß,5α,6ß-triol in plasma samples collected from patients (n = 58) before surgical removal of tumors. Oxysterol levels were then associated with clinical data of patients. All oxysterols except cholesterol-5α,6α-epoxide were detected in patient plasma samples. Circulating levels of 7α-hydroxycholesterol and 27-hydroxycholesterol were significantly lower in patients with small tumors (pT1) and cholesterol-5ß,6ß-epoxide and cholestane-3ß,5α,6ß-triol were lower in patients with stage IA disease compared to larger tumors or more advanced stages. Patients with higher than median cholestane-3ß,5α,6ß-triol levels had significantly worse disease-free survival than patients with lower levels (p = 0.037 for all patients and p = 0.015 for subgroup treated only with tamoxifen). In conclusion, this study shows, for the first time, that circulating levels of oxysterols, especially cholestane-3ß,5α,6ß-triol, may have prognostic roles in patients with luminal subtype breast cancer.

Roles of Cytochrome P450 in Metabolism of Ethanol and Carcinogens.

Cytochrome P450 (P450) enzymes are involved in the metabolism of carcinogens, as well as drugs, steroids, vitamins, and other classes of chemicals. P450s also oxidize ethanol, in particular P450 2E1. P450 2E1 oxidizes ethanol to acetaldehyde and then to acetic acid, roles also played by alcohol and aldehyde dehydrogenases. The role of P450 2E1 in cancer is complex in that P450 2E1 is also induced by ethanol, P450 2E1 is involved in the bioactivation and detoxication of a number of chemical carcinogens, and ethanol is an inhibitor of P450 2E1. Contrary to some literature, P450 2E1 expression and induction itself does not cause global oxidative stress in vivo, as demonstrated in studies using isoniazid treatment and gene deletion studies with rats and mice. However, a major fraction of P450 2E1 is localized in liver mitochondria instead of the endoplasmic reticulum, and studies with site-directed rat P450 2E1 mutants and natural human P450 2E1 N-terminal variants have shown that P450 2E1 localized in mitochondria is catalytically active and more proficient in producing reactive oxygen species and damage. The role of the mitochondrial oxidative stress in ethanol toxicity is still under investigation, as is the mechanism of altered electron transport to P450s that localize inside mitochondria instead of their typical endoplasmic reticulum environment.

The relationships between cytochromes P450 and H2O2: Production, reaction, and inhibition.

In this review we address the relationship between cytochromes P450 (P450) and H2O2. This association can affect biology in three distinct ways. First, P450s produce H2O2 as a byproduct either during catalysis or when no substrate is present. This reaction, known as uncoupling, releases reactive oxygen species that may have implications in disease. Second, H2O2 is used as an oxygen-donating co-substrate in peroxygenase and peroxidase reactions catalyzed by P450s. This activity has proven to be important mainly in reactions involving prokaryotic P450s, and investigators have harnessed this reaction with the aim of adaptation for industrial use. Third, H2O2-dependent inhibition of human P450s has been studied in our laboratory, demonstrating heme destruction and also the inactivating oxidation of the heme-thiolate ligand to a sulfenic acid (-SOH). This reversible oxidative modification of P450s may have implications in the prevention of uncoupling and may give new insights into the oxidative regulation of these enzymes. Research has elucidated many of the chemical mechanisms involved in the relationship between P450 and H2O2, but the application to biology is difficult to evaluate. Further studies are needed reveal both the harmful and protective natures of reactive oxygen species in an organismal context.

Human cytochrome P450 enzymes 5-51 as targets of drugs and natural and environmental compounds: mechanisms, induction, and inhibition - toxic effects and benefits.

Cytochrome P450 (P450, CYP) enzymes have long been of interest due to their roles in the metabolism of drugs, pesticides, pro-carcinogens, and other xenobiotic chemicals. They have also been of interest due to their very critical roles in the biosynthesis and metabolism of steroids, vitamins, and certain eicosanoids. This review covers the 22 (of the total of 57) human P450s in Families 5-51 and their substrate selectivity. Furthermore, included is information and references regarding inducibility, inhibition, and (in some cases) stimulation by chemicals. We update and discuss important aspects of each of these 22 P450s and questions that remain open.

Pre-Steady-State Kinetic Analysis of Single-Nucleotide Incorporation by DNA Polymerases.

Pre-steady-state kinetic analysis is a powerful and widely used method to obtain multiple kinetic parameters. This protocol provides a step-by-step procedure for pre-steady-state kinetic analysis of single-nucleotide incorporation by a DNA polymerase. It describes the experimental details of DNA substrate annealing, reaction mixture preparation, handling of the RQF-3 rapid quench-flow instrument, denaturing polyacrylamide DNA gel preparation, electrophoresis, quantitation, and data analysis. The core and unique part of this protocol is the rationale for preparation of the reaction mixture (the ratio of the polymerase to the DNA substrate) and methods for conducting pre-steady-state assays on an RQF-3 rapid quench-flow instrument, as well as data interpretation after analysis. In addition, the methods for the DNA substrate annealing and DNA polyacrylamide gel preparation, electrophoresis, quantitation and analysis are suitable for use in other studies. © 2016 by John Wiley & Sons, Inc.

Aryl hydrocarbon receptor response to indigoids in vitro and in vivo.

Indigo and indirubin have been reported to be present at low levels in human urine. The possibility that indigoids are physiological ligands of the aryl hydrocarbon receptor (AhR) has been suggested by initial studies in yeast, where indirubin was found to be 50 times more potent than 2,3,7,8-tetrachlorodibenzo[p]dioxin (TCDD), and indigo was found to be equipotent. To demonstrate that these indigoids are bona fide agonists in mammalian systems, we employed a number of in vitro and in vivo measures of AhR agonist potency. In a hepatoma cell reporter system, indigo yielded an EC50 of approximately 5x10(-6)M (indirubin 3' -oxime EC50 approximately 5x10(-7)M, indirubin EC50 approximately 1x10(-7)M). A comparison of these EC50 values with that of 2,3,7,8-tetrachlorodibenzofuran (TCDBF) ( approximately 3x10(-9)M) indicated that these compounds are less potent than classic halogenated-dibenzofurans or -dibenzo-p-dioxins. Competitive binding assays for AhR occupancy showed similar IC50 values for indirubin and TCDBF ( approximately 2x10(-9) and 5x10(-9)M), with the IC50 values of indigo and indirubin 3' -oxime being approximately 10-fold higher. When rats were treated with these indigoids in the range of 1.5-50mg/kg, induction of hepatic cytochrome P450 1A1 was detected. Differences in the rank-order of potency observed in vivo and in vitro could, in part, be explained by metabolism. Although their biological potencies are not as high as has been previously suggested, collectively the results show that these indole-derived pigments are agonists of AhR in vivo. The in vivo results suggest that solubility, distribution, and metabolism influence the response to the compounds.

Diterpene quinone tanshinone IIA selectively inhibits mouse and human cytochrome p4501A2.

1. Tanshinone IIA is the main active diterpene quinone in the herbal medicine Salvia miltiorrhiza. In untreated mouse liver microsomes, tanshinone IIA selectively inhibited 7-ethoxyresorufin O-deethylation (EROD) and 7-methoxyresorufin O-demethylation (MROD) activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, N-nitrosodimethylamine and nifedipine. Tanshinone IIA was a competitive inhibitor of MROD activity with a K(i) of 7.2 +/- 0.7 nM. 2. In 3-methylcholanthrene-treated mouse liver microsomes, tanshinone IIA and two minor tanshinones, tanshinone I and cryptotanshinone, inhibited liver microsomal MROD activity without affecting EROD and benzo(a)pyrene hydroxylation activities at the concentrations up to 1 microM. Tanshinone IIA induced a type I binding spectrum with a spectral dissociation constant K(s) of 2.3 +/-0.8 microM without cooperativity. 3. In human liver microsomes, tanshinone IIA decreased EROD and MROD activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, chlorzoxazone and nifedipine. 4. In Escherichia coli membranes expressing bicistronic human CYP1A enzymes, tanshinone IIA inhibited EROD activity of CYP1A1 with an IC(50) 48 times higher than that for CYP1A2. Tanshinone I and cryptotanshinone had the same IC(50) ratio (1A1/1A2) of 4. 5. The results indicate that tanshinone represents a new group of CYP1A inhibitors, and tanshinone IIA had the highest selectivity in inhibition of CYP1A2.

Cytochrome P450 1B1: a target for inhibition in anticarcinogenesis strategies.

Cytochrome P450 (P450) 1B1 is expressed in a number of human tissues in which cancers occur (e.g. prostate, ovary, uterus, mammary gland). P450 1B1 activates many environmental mutagens and also catalyzes the 4-hydroxylation of estrogens, considered to be an important step in hormonal carcinogenesis. We have examined the activities of several of the major allelic variants of human P450 1B1 in these reactions. Another interest has been the development of chemical inhibitors of P450 1B1. 2-Ethynylpyrene and alpha-naphthoflavone preferentially inhibit human P450 1B1 compared to P450 1A1, which may be present in the same tissue sites. The natural product resveratrol is also an inhibitor of P450 1B1. Further studies with rhapontigenin and synthetic stilbenes led to the discovery of 2,4,3',5'-tetramethoxystilbene, a selective inhibitor of P450 1B1 relative to other P450s. Inhibition is competitive, with a K(i) value of 3nM, and the inhibitor is resistant to metabolism. In addition to blocking 17beta-estradiol 4-hydroxylation, this stilbene also inhibited the activation of heterocyclic amines to mutagens. 2,4,3',5'-Tetramethoxystilbene also suppressed expression of P450 1B1 and growth of human mammary tumor cells. 3,3',4',5,5'-Pentamethoxystilbene was a selective inhibitor of P450 1A1, showing mixed inhibition, and also suppressed P450 1A1 expression in HepG2 cells. Substituted stilbenes may be useful in preventing cancer caused by estrogens and xenobiotics.