Cytochrome P450 1B1 Inhibitor Research Articles

Resveratrol Protects Against Angiotensin II‐Induced Cellular Hypertrophy through Inhibition of CYP1B1/Mid‐Chain Hydroxyeicosatetraenoic Acid Mechanism

ObjectiveCytochrome P450 1B1 (CYP1B1) and its associated cardiotoxic mid‐chain hydroxyeicosatetraenoic acid (HETEs) metabolites have been reported to directly contribute to the development of cardiac hypertrophy. Resveratrol (RESV) is naturally occurring and commercially available polyphenol that possess beneficial effects in wide array of cardiovascular diseases. Several reports have shown the capacity of RESV to treat cardiac hypertrophy, myocardial infarction and heart failure. However, the underlying mechanisms, responsible for RESV beneficial effects, are not fully elucidated. Since RESV is a well‐known CYP1B1 inhibitor, the purpose of this study is to investigate whether RESV protects against angiotensin II (Ang II)‐induced cellular hypertrophy through inhibition of CYP1B1/mid‐chain HETEs mechanism.MethodsHuman ventricular cardiomyocytes RL‐14 and rat cardiomyoblast H9c2 cells were treated with vehicle or 10 μM Ang II in the absence and presence of 2, 10 or 50 μM RESV for 24 h. Thereafter, the level of mid‐chain HETEs was determined using liquid chromatography–mass spectrometry (LC/MS). Hypertrophic markers and CYP gene expression and protein levels were measured using real‐time PCR and Western blot analysis, respectively.ResultsOur results demonstrated that RESV, at concentrations of 10 and 50 μM, was able to protect against Ang‐II‐induced cellular hypertrophy as evidenced by a substantial inhibition of hypertrophic markers, β‐myosin heavy chain (MHC)/α‐MHC and atrial natriuretic peptide (ANP). Ang II significantly induced the protein expression of CYP1B1 and increased the metabolite formation rate of its associated mid‐chain HETEs namely 5‐, 8‐, 9‐, 12‐ and 15‐HETE in both cell lines. Interestingly, the protective effect of RESV, at concentrations 10 and 50 μM, was associated with a significant decrease of CYP1B1 protein expression and mid‐chain HETEs to nearly control levels.ConclusionOur results provided the first evidence that RESV protects against Ang II‐induced cellular hypertrophy, at least in part, through CYP1B1/mid‐chain HETEs‐dependent mechanism.Support or Funding InformationThis work was supported by a grant from the Canadian Institutes of Health Research [Grant 106665] to A.O.S.E. S.M.S. is the recipient of Antoine Noujaim Graduate Scholarship in Pharmaceutical Sciences.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of 19-(S/R)Hydroxyeicosatetraenoic Acid as the First Endogenous Noncompetitive Inhibitor of Cytochrome P450 1B1 with Enantioselective Activity.

The overexpression of cytochrome P450 1B1 (CYP1B1) is a common characteristic of several diseases and conditions, such as inflammation, cancer, and cardiac hypertrophy. CYP1B1 is believed to contribute to pathogenesis of these diseases by mediating the formation of toxic compounds, either from exogenous or endogenous origin. We recently reported that an arachidonic acid metabolite, 19(S/R-)hydroxyeicosatetraenoic (HETE) acid, protects from cardiac hypertrophy by inhibiting the formation of toxic compounds, midchain HETEs, known to be formed by CYP1B1. This raised the question whether 19(S/R)-HETE can directly inhibit CYP1B1. In the current study, we report that 19(S/R)-HETE enantioselectively inhibits human recombinant CYP1B1 activity measured by 7-ethoxyresorufin O-deethylation assay. 19(S)-HETE is more potent than the R enantiomer (K i = 37.3 and 89.1 nM, respectively). Noncompetitive inhibition was identified as the mechanism of CYP1B1 inhibition, which underlines the potentially important physiologic role of 19(S/R)-HETE as an endogenous CYP1B1 inhibitor; to our knowledge, 19(S/R)-HETE is the first inhibitor of its kind to be reported.

Resveratrol improves cardiac function and exercise performance in MI-induced heart failure through the inhibition of cardiotoxic HETE metabolites

Numerous epidemiological studies have demonstrated that approximately 40% of myocardial infarctions (MI) are associated with heart failure (HF). Resveratrol, a naturally occurring polyphenol, has been shown to be beneficial in the treatment of MI-induced HF in rodent models. However, the mechanism responsible for the effects of resveratrol are poorly understood. Interestingly, resveratrol is known to inhibit cytochrome P450 1B1 (CYP1B1) which is involved in the formation of cardiotoxic hydroxyeicosatetraenoic acid (HETE) metabolites. Therefore, we investigated whether resveratrol could improve MI-induced cardiac remodeling and HF in rats through the inhibition of CYP1B1 and its metabolites. To do this, rats were subjected to either sham surgery or a surgery to ligate the left anterior descending artery to induce a MI and subsequent HF. Three weeks post-surgery, rats with established HF were treated with control diet or administered a diet containing low dose of resveratrol. Our results showed that low dose resveratrol treatment significantly improves % ejection fraction in MI rats and reduces MI-induced left ventricular and atrial remodeling. Furthermore, non-cardiac symptoms of HF such as reduced physical activity improved with low dose resveratrol treatment. Mechanistically, low dose resveratrol treatment of rats with established HF restored levels of fatty acid oxidation and significantly improved cardiac energy metabolism as well as significantly inhibited CYP1B1 and cardiotoxic HETE metabolites induced in MI rats. Overall, the present work provides evidence that low dose resveratrol reduces the severity of MI-induced HF, at least in part, through the inhibition of CYP1B1 and cardiotoxic HETE metabolites.

2-Methoxyestradiol protects against pressure overload-induced left ventricular hypertrophy

Numerous experimental studies have supported the evidence that 2-methoxyestradiol (2 ME) is a biologically active metabolite that mediates multiple effects on the cardiovascular system, largely independent of the estrogen receptor. 2 ME is a major cytochrome P450 1B1 (CYP1B1) metabolite and has been reported to have vasoprotective and anti-inflammatory actions. However, whether 2 ME would prevent cardiac hypertrophy induced by abdominal aortic constriction (AAC) has not been investigated yet. Therefore, the overall objectives of the present study were to elucidate the potential antihypertrophic effect of 2 ME and explore the mechanism(s) involved. Our results showed that 2 ME significantly inhibited AAC-induced left ventricular hypertrophy using echocardiography. The antihypertrophic effect of 2 ME was associated with a significant inhibition of CYP1B1 and mid-chain hydroxyeicosatetraenoic acids. Based on proteomics data, the protective effect of 2 ME is linked to the induction of antioxidant and anti-inflammatory proteins in addition to the modulation of proteins involved in myocardial energy metabolism. In vitro, 2 ME has shown a direct antihypertrophic effect through mitogen-activated protein kinases- and nuclear factor-κB-dependent mechanisms. The present work shows a strong evidence that 2 ME protects against left ventricular hypertrophy. Our data suggest the potential of repurposing 2 ME as a selective CYP1B1 inhibitor for the treatment of heart failure.

The role of cytochrome P450 1B1 and its associated mid-chain hydroxyeicosatetraenoic acid metabolites in the development of cardiac hypertrophy induced by isoproterenol.

Numerous experimental studies have demonstrated the role of cytochrome P450 1B1 (CYP1B1) and its associated mid-chain hydroxyeicosatetraenoic acids (mid-chain HETEs) metabolite in the pathogenesis of cardiac hypertrophy. However, the ability of isoproterenol (ISO) to induce cardiac hypertrophy through mid-chain HETEs has not been investigated yet. Therefore, we hypothesized that ISO induces cardiac hypertrophy through the induction of CYP1B1 and its associated mid-chain HETE metabolites. To test our hypothesis, Sprague-Dawley rats were treated with ISO (5mg/kg i.p.) for 12 and 72h whereas, human ventricular cardiomyocytes RL-14 cells were exposed to 100μM ISO in the presence and absence of 0.5μM tetramethoxystilbene (TMS) a selective CYP1B1 inhibitor, or 25 nM CYP1B1-siRNA. Moreover, RL-14 cells were transiently transfected with the CRISPR-CYP1B1 plasmid. Thereafter, real-time PCR, western blot analysis, and liquid chromatography-electrospray ionization mass spectroscopy were used to determine the level of gene expression, protein expression, and mid-chain HETEs, respectively. Our results showed that ISO induced CYP1B1 protein expression and the level of cardiac mid-chain HETEs in vivo at pre-hypertrophic and hypertrophic stage. In vitro, inhibition of CYP1B1 using TMS or CYP1B1-siRNA significantly attenuates ISO-induced hypertrophy. Furthermore, overexpression of CYP1B1 significantly induced cellular hypertrophy and mid-chain HETEs metabolite. Mechanistically, the protective effect of TMS against cardiac hypertrophy was mediated through the modulation of superoxide anion, mitogen-activated protein kinases (MAPKs), and nuclear factor-κB (NF-κB). In conclusion, our study provides the first evidence that CYP1B1 and its associated mid-chain HETE metabolites are directly involved in the ISO-induced cardiac hypertrophy.

Development of an In Vitro Model to Screen CYP1B1-Targeted Anticancer Prodrugs

Cytochrome P450 1B1 (CYP1B1) is an anticancer therapeutic target due to its overexpression in a number of steroid hormone–related cancers. One anticancer drug discovery strategy is to develop prodrugs specifically activated by CYP1B1 in malignant tissues to cytotoxic metabolites. Here, we aimed to develop an in vitro screening model for CYP1B1-targeted anticancer prodrugs using the KLE human endometrial carcinoma cell line. KLE cells demonstrated superior stability of CYP1B1 expression relative to transiently transfected cells and did not express any appreciable amount of cognate CYP1A1 or CYP1A2, which would have compromised the specificity of the screening assay. The effect of two CYP1B1-targeted probe prodrugs on KLE cells was evaluated in the absence and presence of a CYP1B1 inhibitor to chemically “knock out” CYP1B1 activity (CYP1B1 inhibited). Both probe prodrugs were more toxic to KLE cells than to CYP1B1-inhibited KLE cells and significantly induced G0/G1 arrest and decreased the S phase in KLE cells. They also exhibited pro-apoptotic effects in KLE cells, which were attenuated in CYP1B1-inhibited KLE cells. In summary, a KLE cell–based model has been characterized to be suitable for identifying CYP1B1-targeted anticancer prodrugs and should be further developed and employed for screening chemical libraries.

Targeting cytochrome P450 (CYP) 1B1 with steroid derivatives

Inhibition of cytochrome P450 1B1 (CYP1B1) represents a promising therapeutic strategy, because it would enable action at three different levels: (1) by inhibiting the formation of mutagenic 4-hydroxy-estradiol, (2) by inhibiting the bioactivation of procarcinogens, and (3) by reducing drug-resistance. Surprisingly, few steroids were reported as inhibitors of CYP1B1. From a screening performed with 90 steroid derivatives, we identified thioestrone (B19) as an inhibitor (IC50=3.4μM) of CYP1B1. Molecular modeling studies showed that the 3-SH group of B19 is closer (3.36Å) to the iron atom of the heme system than the 3-OH group of enzyme substrates estrone and estradiol (4.26Å and 3.58Å, respectively). B19 also produced a better docking GOLD score that correlated with the inhibitory results obtained. The estrane derivative B19 represents an interesting lead compound that can be easily modified to extend the structure–activity relationship study and to provide a next generation of more powerful CYP1B1 inhibitors.

The role of cytochrome P450 1B1 and its associated mid‐chain hydroxyeicosatetraenoic acids metabolites in the development of cellular hypertrophy induced by isoproterenol

Heart failure is the leading cause of death, with a prevalence of over 23 million worldwide. The lifetime risk of developing heart failure is one in five. Numerous experimental studies have demonstrated the role cytochrome P450 1B1 (CYP1B1) and its associated mid‐chain hydroxyeicosatetraenoic acids (mid‐chain HETEs) metabolites in the pathogenesis of heart failure and cardiac hypertrophy. However, the ability of isoproterenol (ISO) to induce cardiac hypertrophy through mid‐chain HETEs has not been investigated yet. Therefore, we hypothesized that ISO induced cardiac hypertrophy through the induction of CYP1B1 and its associated mid‐chain HETEs metabolites. To test our hypothesis, the human ventricular cardiomyocytes, RL‐14 cells, were treated with ISO in the presence and absence of tetramethoxystilbene (TMS), a selective CYP1B1 inhibitor. Thereafter, the cellular hypertrophy markers, cell volume and mid‐chain HETEs metabolites were determined using real‐time polymerase chain reaction, phase contrast imaging and liquid chromatography‐electron spray ionization‐mass spectrometry, respectively. Our results showed that ISO induced cellular hypertrophy in RL‐14 cells as evidenced by the significant induction of β‐myocin heavy chain/α‐myocin heavy chain (β‐MHC/α‐MHC) and cell volume. Interestingly, ISO‐induced cellular hypertrophy was associated with a proportional increase in the protein expression of CYP1B1 and the formation of mid‐chain HETEs metabolites. The direct evidence for the involvement of CYP1B1 in the ISO‐induced cellular hypertrophy was supported by the ability of TMS and CYP1B1 siRNA to significantly inhibit the ISO‐mediated the induction of β‐MHC/α‐MHC and the increase cell volume. Mechanistically, the protective effect of TMS against ISO‐induced cellular hypertrophy was mediated through the inhibition of NF‐κB signaling pathway. In conclusion, our study provides the first evidence that the inhibition of CYP1B1 and hence mid‐chain HETEs attenuate ISO‐induced cellular hypertrophy.Support or Funding InformationThis work was supported by a grant from the CIHR to A.O.S.E.

The serotonin transporter promotes a pathological estrogen metabolic pathway in pulmonary hypertension via cytochrome P450 1B1.

Pulmonary arterial hypertension (PAH) is a devastating vasculopathy that predominates in women and has been associated with dysregulated estrogen and serotonin signaling. Overexpression of the serotonin transporter (SERT(+)) in mice results in an estrogen-dependent development of pulmonary hypertension (PH). Estrogen metabolism by cytochrome P450 1B1 (CYP1B1) contributes to the pathogenesis of PAH, and serotonin can increase CYP1B1 expression in human pulmonary arterial smooth muscle cells (hPASMCs). We hypothesized that an increase in intracellular serotonin via increased SERT expression may dysregulate estrogen metabolism via CYP1B1 to facilitate PAH. Consistent with this hypothesis, we found elevated lung CYP1B1 protein expression in female SERT(+) mice accompanied by PH, which was attenuated by the CYP1B1 inhibitor 2,3',4,5'-tetramethoxystilbene (TMS). Lungs from female SERT(+) mice demonstrated an increase in oxidative stress that was marked by the expression of 8-hydroxyguanosine; however, this was unaffected by CYP1B1 inhibition. SERT expression was increased in monocrotaline-induced PH in female rats; however, TMS did not reverse PH in monocrotaline-treated rats but prolonged survival. Stimulation of hPASMCs with the CYP1B1 metabolite 16α-hydroxyestrone increased cellular proliferation, which was attenuated by an inhibitor (MPP) of estrogen receptor alpha (ERα) and a specific ERα antibody. Thus, increased intracellular serotonin caused by increased SERT expression may contribute to PAH pathobiology by dysregulation of estrogen metabolic pathways via increased CYP1B1 activity. This promotes PASMC proliferation by the formation of pathogenic metabolites of estrogen that mediate their effects via ERα. Our studies indicate that targeting this pathway in PAH may provide a promising antiproliferative therapeutic strategy.

CYP1B1 inhibition attenuates doxorubicin-induced cardiotoxicity through a mid-chain HETEs-dependent mechanism

Doxorubicin (DOX) has been reported to be a very potent and effective anticancer agent. However, clinical treatment with DOX has been greatly limited due to its cardiotoxicity. Furthermore, several studies have suggested a role for cytochrome P450 1B1 (CYP1B1) and mid-chain hydroxyeicosatetraenoic acids (mid-chain HETEs) in DOX-induced cardiac toxicity. Therefore, we hypothesized that DOX induced cardiotoxicity is mediated through the induction of CYP1B1 and its associated mid-chain HETEs metabolite. To test our hypothesis, Sprague-Dawley rats and RL-14 cells were treated with DOX in the presence and absence of 2,3′,4,5′-tetramethoxystilbene (TMS), a selective CYP1B1 inhibitor. Thereafter, cardiotoxicity parameters were determined using echocardiography, histopathology, and gene expression. Further, the level of mid-chain HETEs was quantified using liquid chromatography-electron spray ionization-mass spectrometry. Our results showed that DOX induced cardiotoxicity in vivo and in vitro as evidenced by deleterious changes in echocardiography, histopathology, and hypertrophic markers. Importantly, the TMS significantly reversed these changes. Moreover, the DOX-induced cardiotoxicity was associated with a proportional increase in the formation of cardiac mid-chain HETEs both in vivo and in our cell culture model. Interestingly, the inhibition of cardiotoxicity by TMS was associated with a dramatic decrease in the formation of cardiac mid-chain HETEs suggesting a mid-chain HETEs-dependent mechanism. Mechanistically, the protective effect of TMS against DOX-induced cardiotoxicity was mediated through the inhibition of mitogen activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). In conclusion, our study provides the first evidence that the inhibition of CYP1B1 and mid-chain HETE formation attenuate DOX-induced cardiotoxicty.

Abstract 2754: Induction of Sp1 factor by CYP1B1 inhibits TRAIL-mediated apoptotic pathway

Abstract Human cytochrome P450 1B1 (CYP1B1) belongs to the CYP1 family and is known as a major enzyme for estradiol 4-hydroxylation. It has been reported that CYP1B1 expression is higher in the tumor tissues than the normal ones, especially in hormone-related cancers such as breast, ovarian and prostate. Previously, we identified that CYP1B1 induces cell proliferation by activation of Wnt/β-catenin signal pathway. To explore the role of CYP1B1 on cancer cell proliferation further, we investigated whether CYP1B1 blocks apoptotic pathways. Using Western blot and RT-PCR, the expression of TRAIL was examined in MCF-7 cells, and we found that CYP1B1 overexpression or treatment with a CYP1B1 inducer, 7, 12-dimethylbenz[α]anthracene (DMBA) caused suppression of TRAIL and its receptors (DR4 and DR5) expression. However, when cells were treated with CYP1B1 siRNA or a selective CYP1B1 inhibitor, tetramethoxystilbene (TMS), expression of TRAIL and its receptors were significantly increased. Sp1, a zinc finger-containing transcription factor, has been reported to suppress TRAIL gene expression by binding TRAIL promoter. Interestingly, CYP1B1 overexpression and DMBA induced Sp1 expression and TMS was able to prevent DMBA-induced Sp1 expression. Sp1 knockdown with siRNA suppressed c-FLIP, an inhibitor protein of TRAIL-mediated apoptosis while it enhanced TRAIL and DR4 expression. These results were reversed by Sp1 overexpession. Moreover, attenuation of Sp1 DNA binding activity using mithramycin A, a Sp1 binding inhibitor, resulted in recovery of TRAIL expression suppressed by DMBA. Taken together, these data suggest that CYP1B1 prevents apoptosis via TRAIL suppression and this is mediated by Sp1 induction. Citation Format: Yeo-Jung Kwon, Nahee Park, Sangyun Shin, Dong-Jin Ye, Mihye Hong, Young-Jin Chun. Induction of Sp1 factor by CYP1B1 inhibits TRAIL-mediated apoptotic pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2754. doi:10.1158/1538-7445.AM2014-2754

Lipidomics Reveals a Link between CYP1B1 and SCD1 in Promoting Obesity

Cytochrome P450 1B1 (CYP1B1) is involved in the metabolism of xenobiotic compounds and endogenous metabolites. Disruption of Cyp1b1 in mice results in suppression of high-fat diet (HFD)-induced obesity and an extensive change in hepatic energy regulation despite minimal constitutive expression of CYP1B1 in hepatocytes. Lack of CYP1B1 is correlated with altered lipid metabolism, especially lysophosphatidylcholines, contributing to protection against obesity. Ultraperformance liquid chromatography coupled to electrospray ionization quadrupole mass spectrometry (UPLC-ESI-QTOFMS)-based metabolomics revealed lysophosphatidylcholine 18:0 (LPC 18:0) as a biomarker positively related to HFD-induced obesity. The increased serum LPC 18:0 in wild-type mice is reduced in Cyp1b1-null mice on a HFD, which is reversed in CYP1B1-humanized mice. CYP1B1-humanized mice show higher diet-induced obesity compared with Cyp1b1-null mice, suggesting that human CYP1B1 shows a similar response to HFD as mouse Cyp1b1. In addition, hepatic stearoyl-CoA desaturase 1 (SCD1) expression was decreased in Cyp1b1-null mice, and the attenuated diet-induced obesity and lower serum LPC 18:0 in the Cyp1b1-null mice is elevated after SCD1 overexpression, suggesting that SCD1 is correlated with CYP1B1-induced obesity. These studies establish a biochemical link between cytochromes P450, lipids, and metabolic disorders and suggest that inhibition of CYP1B1 could be target for antiobesity drugs.

Abstract 2580: Pharmacologic inhibition of cytochrome P450 1B1 attenuates the motility and proliferation of cultured human dysplastic oral leukoplakia cells.

Abstract Oral leukoplakia is a precancerous lesion that may progress to squamous cell carcinoma of the head and neck (HNSCC). The long latency period to invasive cancer provides an opportunity for intervention at the precancerous stage. The inability of surgical resection to prevent the recurrence of oral leukoplakia speaks clearly to the need to develop an efficacious regimen for the chemoprevention of this disease. Tobacco smoke constituents and ethanol, the major risk factors for oral leukoplakia and HNSCC, are metabolized by cytochrome P450 1B1 (CYP1B1) to form potentially carcinogenic species. Our previous data indicate that knockdown of CYP1B1 in human oral leukoplakia cells (MSK-Leuk1) by shRNA decreases cell motility and proliferation, relative to control cells overexpressing empty vector. Homoeriodictyol (HED), a flavonoid found in citrus fruits, is a selective inhibitor of CYP1B1 (IC50 0.24 μM). As a next step, we hypothesized that pharmacologic inhibition of CYP1B1 by HED would attenuate cell motility and proliferation in a manner similar to genetic knockdown. Because HED is commonly found in our diet, its toxicity is expected to be low, making it a promising agent for preventive or therapeutic intervention. The ability of HED to inhibit CYP1B1 activity in MSK-Leuk1 cells was established by incubating cells with either vehicle (0.01% ethanol) or HED (5-10 μM) for 72 hrs and quantifying 7-ethoxyresorufin o-deethylation (EROD), a measure of the activity of CYP1B1 and other enzymes of the CYP1 subfamily. HED decreased EROD activity in a dose-dependent manner, with 40% inhibition observed at 10 μM, as compared to vehicle-treated controls. Western blot analyses revealed that CYP1B1 levels were unaltered following HED treatment. The effect of CYP1B1 inhibition on cell motility was examined by exposing MSK-Leuk1 cells to HED (0-10 μM) for 96 hrs followed by a wound-healing assay, in which the ability of cell monolayers to close a gap was monitored for 23 hrs. HED inhibited cell motility in a concentration dependent manner, with maximum inhibition of gap closure reached at 10 μM (6.7 fold relative to vehicle). To assess the effect of CYP1B1 inhibition on cell growth, MSK-Leuk1 cells were exposed to HED (0-10 μM) for 5 days and proliferation was analyzed (Fluorescent DNA Quantitation kit). A direct association was observed between HED dose and inhibition of proliferation, with a 33% decrease observed at 10 μM. In summary, inhibition of CYP1B1 activity by HED resulted in attenuation of the motility and proliferation of cultured MSK-Leuk1 cells. These data confirm that CYP1B1 is a promising molecular target for intervention in HNSCC and provide strong support for the further development of CYP1B1 inhibitors as efficacious agents for the prevention of HNSCC in patients with oral leukoplakia. (Supported by NCI fellowship 1 F32 CA144199-01). Citation Format: Ekaterina G. Shatalova, Margie L. Clapper. Pharmacologic inhibition of cytochrome P450 1B1 attenuates the motility and proliferation of cultured human dysplastic oral leukoplakia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2580. doi:10.1158/1538-7445.AM2013-2580

2,4,3′,5′‐Tetramethoxystilbene Reduces Blood Pressure and Associated Cardiac Fibrosis Via Inhibition of Cytochrome P450 1B1 and Decreased Oxidative Stress in SHR

Previously, we reported that inhibition of cytochrome P450 (CYP) 1B1 activity with 2,4,3′,5′‐tetramethoxystilbene (TMS) reversed angiotensin II‐ and deoxycoticosterone acetate salt‐induced hypertension and associated pathophysiology in rats. This study was conducted to investigate the effect of TMS on blood pressure (BP), and associated cardiac fibrosis and oxidative stress in male, normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). TMS (600 μg/kg, i.p. daily for 6 weeks) beginning at 8 weeks of age, did not alter systolic BP in WKY, but decreased it in SHR (207 ± 7 mmHg vehicle, vs. 129 ± 2 mmHg TMS, P < 0.05). Cardiac CYP1B1 activity that was increased in SHR was reduced by TMS treatment. Cardiac fibrosis, as indicated by increased collagen deposition and myofibroblast accumulation, was observed in SHR, which was prevented by TMS. SHR had increased cardiac levels of reactive oxygen species, increased NADPH oxidase activity, and increased plasma levels of H2O2, NOx, and TBARS that were prevented by TMS, suggesting that in addition to inhibiting CYP1B1 activity and limiting cardiac damage, TMS regulates cardiac oxidative stress in SHR. These data suggest that CYP1B1 contributes to increased BP, cardiac damage, and associated oxidative stress in SHR. Moreover, CYP1B1 could serve as a novel target for the development of drugs such as TMS to treat cardiac disease associated with hypertension.

Dexfenfluramine and the oestrogen-metabolizing enzyme CYP1B1 in the development of pulmonary arterial hypertension

AimsPulmonary arterial hypertension (PAH) occurs more frequently in women than men. Oestrogen and the oestrogen-metabolising enzyme cytochrome P450 1B1 (CYP1B1) play a role in the development of PAH. Anorectic drugs such as dexfenfluramine (Dfen) have been associated with the development of PAH. Dfen mediates PAH via a serotonergic mechanism and we have shown serotonin to up-regulate expression of CYP1B1 in human pulmonary artery smooth muscle cells (PASMCs). Thus here we assess the role of CYP1B1 in the development of Dfen-induced PAH.Methods and resultsDfen (5 mg kg−1 day−1 PO for 28 days) increased right ventricular pressure and pulmonary vascular remodelling in female mice only. Mice dosed with Dfen showed increased whole lung expression of CYP1B1 and Dfen-induced PAH was ablated in CYP1B1−/− mice. In line with this, Dfen up-regulated expression of CYP1B1 in PASMCs from PAH patients (PAH-PASMCs) and Dfen-mediated proliferation of PAH-PASMCs was ablated by pharmacological inhibition of CYP1B1. Dfen increased expression of tryptophan hydroxylase 1 (Tph1; the rate-limiting enzyme in the synthesis of serotonin) in PAH-PASMCs and both Dfen-induced proliferation and Dfen-induced up-regulation of CYP1B1 were ablated by inhibition of Tph1. 17β-Oestradiol increased expression of both Tph1 and CYP1B1 in PAH-PASMCs, and Dfen and 17β-oestradiol had synergistic effects on proliferation of PAH-PASMCs. Finally, ovariectomy protected against Dfen-induced PAH in female mice.ConclusionCYP1B1 is critical in the development of Dfen-induced PAH in mice in vivo and proliferation of PAH-PASMCs in vitro. CYP1B1 may provide a novel therapeutic target for PAH.

Cytochrome P450 1B1 Gene Disruption Minimizes Deoxycorticosterone Acetate-Salt–Induced Hypertension and Associated Cardiac Dysfunction and Renal Damage in Mice

Previously, we showed that the cytochrome P450 1B1 inhibitor 2,3′,4,5′-tetramethoxystilbene reversed deoxycorticosterone acetate (DOCA)-salt–induced hypertension and minimized endothelial and renal dysfunction in the rat. This study was conducted to test the hypothesis that cytochrome P450 1B1 contributes to cardiac dysfunction, and renal damage and inflammation associated with DOCA-salt–induced hypertension, via increased production of reactive oxygen species and modulation of neurohumoral factors and signaling molecules. DOCA-salt increased systolic blood pressure, cardiac and renal cytochrome P450 1B1 activity, and plasma levels of catecholamines, vasopressin, and endothelin-1 in wild-type ( Cyp1b1 +/+ ) mice that were minimized in Cyp1b1 −/− mice. Cardiac function, assessed by echocardiography, showed that DOCA-salt increased the thickness of the left ventricular posterior and anterior walls during diastole, the left ventricular internal diameter, and end-diastolic and end-systolic volume in Cyp1b1 +/+ but not in Cyp1b1 −/− mice; stroke volume was not altered in either genotype. DOCA-salt increased renal vascular resistance and caused vascular hypertrophy and renal fibrosis, increased renal infiltration of macrophages and T lymphocytes, caused proteinuria, increased cardiac and renal nicotinamide adenine dinucleotide phosphate-oxidase activity, caused production of reactive oxygen species, and increased activities of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and cellular-Src; these were all reduced in DOCA-salt–treated Cyp1b1 −/− mice. Renal and cardiac levels of eicosanoids were not altered in either genotype of mice. These data suggest that, in DOCA-salt hypertension in mice, cytochrome P450 1B1 plays a pivotal role in cardiovascular dysfunction, renal damage, and inflammation, and increased levels of catecholamines, vasopressin, and endothelin-1, consequent to generation of reactive oxygen species and activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and cellular-Src independent of eicosanoids.

Activity of the Estrogen-Metabolizing Enzyme Cytochrome P450 1B1 Influences the Development of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a hyperproliferative vascular disorder observed predominantly in women. Estrogen is a potent mitogen in human pulmonary artery smooth muscle cells and contributes to PAH in vivo; however, the mechanisms attributed to this causation remain obscure. Curiously, heightened expression of the estrogen-metabolizing enzyme cytochrome P450 1B1 (CYP1B1) is reported in idiopathic PAH and murine models of PAH. Here, we investigated the putative pathogenic role of CYP1B1 in PAH. Quantitative reverse transcription-polymerase chain reaction, immunoblotting, and in situ analysis revealed that pulmonary CYP1B1 is increased in hypoxic PAH, hypoxic+SU5416 PAH, and human PAH and is highly expressed within the pulmonary vascular wall. PAH was assessed in mice via measurement of right ventricular hypertrophy, pulmonary vascular remodeling, and right ventricular systolic pressure. Hypoxic PAH was attenuated in CYP1B1(-/-) mice, and the potent CYP1B1 inhibitor 2,3',4,5'-tetramethoxystilbene (TMS; 3 mg · kg(-1) · d(-1) IP) significantly attenuated hypoxic PAH and hypoxic+SU5416 PAH in vivo. TMS also abolished estrogen-induced proliferation in human pulmonary artery smooth muscle cells and PAH-pulmonary artery smooth muscle cells. The estrogen metabolite 16α-hydroxyestrone provoked human pulmonary artery smooth muscle cell proliferation, and this mitogenic effect was greatly pronounced in PAH-pulmonary artery smooth muscle cells. ELISA analysis revealed that 16α-hydroxyestrone concentration was elevated in PAH, consistent with CYP1B1 overexpression and activity. Finally, administration of the CYP1B1 metabolite 16α-hydroxyestrone (1.5 mg · kg(-1) · d(-1) IP) caused the development of PAH in mice. Increased CYP1B1-mediated estrogen metabolism promotes the development of PAH, likely via the formation of mitogens, including 16α-hydroxyestrone. Collectively, this study reveals a possible novel therapeutic target in clinical PAH.

Involvement of cytochrome P-450 1B1 in renal dysfunction, injury, and inflammation associated with angiotensin II-induced hypertension in rats

We investigated the contribution of cytochrome P-450 1B1 (CYP1B1) to renal dysfunction and organ damage associated with ANG II-induced hypertension in rats. ANG II (300 ng·kg(-1)·min(-1)) or vehicle were infused for 2 wk, with daily injections of a selective CYP1B1 inhibitor, 2,4,3',5'-tetramethoxystilbene (TMS; 300 μg/kg ip), or its vehicle. ANG II increased blood pressure and renal CYP1B1 activity that were prevented by TMS. ANG II also increased water intake and urine output, decreased glomerular filtration rate, increased urinary Na(+) and K(+) excretion, and caused proteinuria, all of which were prevented by TMS. ANG II infusion caused hypertrophy, endothelial dysfunction, and increased reactivity of renal and interlobar arteries to vasoconstrictor agents and renal vascular resistance and interstitial fibrosis as indicated by accumulation of α-smooth muscle actin, fibronectin, and collagen, and inflammation as indicated by increased infiltration of CD-3(+) cells; these effects were inhibited by TMS. ANG II infusion also increased production of reactive oxygen species (ROS) and activities of NADPH oxidase, ERK1/2, p38 MAPK, and c-Src that were prevented by TMS. TMS alone had no effect on any of the above parameters. These data suggest that CYP1B1 contributes to the renal pathophysiological changes associated with ANG II-induced hypertension, most likely via increased ROS production and activation of ERK1/2, p38 MAPK, and c-Src and that CYP1B1 could serve as a novel target for treating renal disease associated with hypertension.

Trans-Stilbenoids: potent and selective inhibitors for human cytochrome P450 1B1

On the basis of our previous insights into the structural requirements of stilbenoids for the inhibition of cytochrome P450 1B1 (CYP1B1), a series of 2,4-dimethoxy group-containing stilbenes was prepared and evaluated for their inhibitory effects on the activity of CYP1s with the ultimate goal of identifying a potent and selective CYP1B1 inhibitor. Among the thirteen derivatives prepared, five compounds exhibited similar or greater potency compared to the previous lead compound, 2,4,3′,5′-tetramethoxystilbene (2,4,3′,5′-TMS), in inhibiting CYP1B1. In particular, 2,2′,3′,4,6′-pentamethoxystilbene was found to be a more selective and more potent CYP1B1 inhibitor than 2,4,3′,5′-TMS. 2,4,2′,6′-TMS showed remarkably potent inhibitory activity against CYP1B1 (IC50 = 1.77 ± 0.14 nM) and also had a very high selectivity toward CYP1 isoenzymes. Molecular modeling was performed to determine the key molecular interactions with the CYP1B1 and CYP1A2 structures. On the basis of these structural and biological studies, the design of more potent and more selective drug-like derivatives can be envisaged.

Tetra-methoxystilbene modulates ductal growth of the developing murine mammary gland

Extensive data suggest that estradiol contributes to the development of breast cancer by acting as a mitogen and exerting direct genotoxic effects after enzymatic conversion to 4-hydroxyestradiol (4-OHE2) via cytochrome P450 1B1 (CYP1B1). The mammary gland, ovary, and uterus all express CYP1B1. Overexpression of this enzyme has been associated with an increased risk of breast cancer and blockade might reduce this carcinogenic effect. For this reason, we conducted systematic in vitro and in vivo studies of a CYP1B1 inhibitor, TMS (2,3',4,5'-tetramethoxystilbene). We found that TMS blocked the enzymatic conversion of radiolabeled estradiol to both 2-hydroxyestradiol (2-OHE2) and 4-OHE2, but did not inhibit Cyp1b1 message formation. In vivo studies using mass spectrometry showed that TMS inhibited formation of 2-OHE2 and 4-OHE2 and the resulting estrogen-DNA adducts. To examine its biologic actions in vivo, we investigated whether TMS could block the hyperplastic changes that occur in the developing breast of aromatase-transfected mice. We found that TMS induced a significant reduction of ductal structures in mice less than 6 months in age. In older mice, no reduction in breast morphology occurred. These latter studies uncovered unexpected estrogen agonistic actions of TMS at high doses, including a paradoxical stimulation of breast ductal structures and the endometrium. These studies suggest that the enzyme inhibitory properties of TMS, as well as the effects on developing breast, could implicate a role for TMS in breast cancer prevention, but only in low doses and on developing breast.