Cytochrome P450 1B1 Expression Research Articles

Abstract LB296: Human CYP1B1 enhances cancer progression through induction of Sp1 via DNMT-mediated epigenetic regulation of miR-375

Abstract Human cytochrome P450 1B1 (CYP1B1) is a major catalytic enzyme for metabolism of 17β-estradiol (E2) to catechol estrogens, mainly carcinogenic metabolite 4-hydroxyestradiol (4-OHE2). To identify the unknown molecular mechanism for cancer progression induced by CYP1B1, we studied the role of CYP1B1 in human tumor cells, MCF-7, MDA-MB-231, and HeLa cells. Suppression of CYP1B1 by shRNA significantly reduced cancer cell viability and invasion. Clinical data from cancer patients showed that high level of CYP1B1 expression resulted in relatively lower survival rate. Interestingly, overexpression of CYP1B1 markedly activated Wnt/β-catenin signaling and EMT process, and suppression of specificity protein 1 (Sp1) by siRNA, an well-known transcription factor, demolished the promoting effects of CYP1B1 on cancer progression. To investigate the correlation between CYP1B1 and Sp1, we analyzed the clinical data from breast and cervical cancer patients and the results showed the positive correlation between two genes. Furthermore, TMS (tetramethoxystilbene), a specific CYP1B1 inhibitor, diminished the induction of Sp1 by CYP1B1, and 4-OHE2 exceedingly promoted the expression level of Sp1. Surprisingly, miR-375, known as an oncogenic microRNA, substantially decreased by 4-OHE2 and CYP1B1 overexpression in CYP1B1-transgenic (TG) mice. Methylation specific assay revealed that CYP1B1 suppressed miR-375 expression through induction of hyper-methylation on pre-miR-375. In addition, epigenetic regulation of miR-375 by CYP1B1 was executed via induction of DNA methyltransferases (DNMTs), DNMT1, 3a, and 3b. Enhanced expression level of Sp1 by 4-OHE2 was significantly deteriorated by 5-aza-dC, a DNMT inhibitor. Taken together, our data suggest that CYP1B1 enhances cancer progression through induction of cancer cell proliferation and invasion via DNMT-mediated epigenetic regulation of miR-375 and further promotion of Sp1 expression. Citation Format: Young-Jin Chun, Yeo-Jung Kwon, Tae-Uk Kwon. Human CYP1B1 enhances cancer progression through induction of Sp1 via DNMT-mediated epigenetic regulation of miR-375 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB296.

Activation of the Wnt/β-catenin/CYP1B1 pathway alleviates oxidative stress and protects the blood-brain barrier under cerebral ischemia/reperfusion conditions.

Abstract JOURNAL/nrgr/04.03/01300535-202407000-00033/figure1/v/2023-11-20T171125Z/r/image-tiff Accumulating evidence suggests that oxidative stress and the Wnt/β-catenin pathway participate in stroke-induced disruption of the blood-brain barrier. However, the potential links between them following ischemic stroke remain largely unknown. The present study found that cerebral ischemia leads to oxidative stress and repression of the Wnt/β-catenin pathway. Meanwhile, Wnt/β-catenin pathway activation by the pharmacological inhibitor, TWS119, relieved oxidative stress, increased the levels of cytochrome P450 1B1 (CYP1B1) and tight junction-associated proteins (zonula occludens-1 [ZO-1], occludin and claudin-5), as well as brain microvascular density in cerebral ischemia rats. Moreover, rat brain microvascular endothelial cells that underwent oxygen glucose deprivation/reoxygenation displayed intense oxidative stress, suppression of the Wnt/β-catenin pathway, aggravated cell apoptosis, downregulated CYP1B1 and tight junction protein levels, and inhibited cell proliferation and migration. Overexpression of β-catenin or knockdown of β-catenin and CYP1B1 genes in rat brain microvascular endothelial cells at least partly ameliorated or exacerbated these effects, respectively. In addition, small interfering RNA-mediated β-catenin silencing decreased CYP1B1 expression, whereas CYP1B1 knockdown did not change the levels of glycogen synthase kinase 3β, Wnt-3a, and β-catenin proteins in rat brain microvascular endothelial cells after oxygen glucose deprivation/reoxygenation. Thus, the data suggest that CYP1B1 can be regulated by Wnt/β-catenin signaling, and activation of the Wnt/β-catenin/CYP1B1 pathway contributes to alleviation of oxidative stress, increased tight junction levels, and protection of the blood-brain barrier against ischemia/hypoxia-induced injury.

Acetylcholinesterase activity, histopathological changes, lipid peroxidation and stress-related genes expression in Nile tilapia (Oreochromis niloticus) exposed to waterborne methidathion

The safety of aquatic system has been threatened due to extensive and inappropriate application of organophosphorus pesticide methidathion (MD). Hence, a 12-day stress trial was carried out to explore the impacts of waterborne MD on acetylcholinesterase (AChE) activity, histopathology, antioxidant status and stress-related genes expression in Nile tilapia (Oreochromis niloticus). Fish (initial mean weight 60 ± 2 g) were placed in 18 tanks (volume 460 L, 30 fish/tank), with triplicate treatment groups being exposed to diverse concentrations of MD (0, 0.8, 1.67, 5.34, 7.49 and 15.83 µg/L). The results showed that AChE activities in serum and liver were inhibited by MD. After the 12-day stress trial, malonaldehyde (MDA) content in serum and liver was higher than that of 0 µg/L group when MD concentration exceeded 0.8 and 5.34 µg/L, respectively. Catalase (CAT) and total superoxide dismutase (T-SOD) activity in serum and liver revealed irregular variations. In addition, MD (＞0.8 µg/L) caused lesions in gills and liver tissues. MD also down-regulated expression of cytochrome P450 1B1, heat shock protein 70 and cytochrome c oxidase subunit 7B, which contradicted the trend of phospholipase A2 and cytochrome P450 1 A (except in the 15.83 µg/L group). In a word, waterborne MD suppressed AChE activities, induced oxidative stress and harmed the liver and gills. Additionally, it may negatively influence resistance to stress, oxidative phosphorylation, detoxification and anti-inflammatory response in Nile tilapia.

CYP1B1 inhibits ferroptosis and induces anti-PD-1 resistance by degrading ACSL4 in colorectal cancer

Immune checkpoint blockade (ICB) is a promising treatment strategy for colorectal cancer (CRC) patients. However, most CRC patients do not response well to ICB therapy. Increasing evidence indicates that ferroptosis plays a critical role in immunotherapy. ICB efficacy may be enhanced by inducing tumor ferroptosis. Cytochrome P450 1B1 (CYP1B1) is a metabolic enzyme that participates in arachidonic acid metabolism. However, the role of CYP1B1 in ferroptosis remains unclear. In this study, we demonstrated that CYP1B1 derived 20-HETE activated the protein kinase C pathway to increase FBXO10 expression, which in turn promoted the ubiquitination and degradation of acyl-CoA synthetase long-chain family member 4 (ACSL4), ultimately inducing tumor cells resistance to ferroptosis. Furthermore, inhibiting CYP1B1 sensitized tumor cells to anti-PD-1 antibody in a mouce model. In addition, CYP1B1 expression was negatively correlated with ACSL4 expression, and high expression indicates poor prognosis in CRC. Taken together, our work identified CYP1B1 as a potential biomarker for enhancing anti-PD-1 therapy in CRC.

Functional variant rs10175368 which affects the expression of CYP1B1 plays a protective role against breast cancer in a Chinese Han population.

Cytochrome P450 1B1 ( CYP1B1 ) genetic variants are relevant in the pathogenesis of breast cancer. Exploring the relationships between CYP1B1 functional variants and breast cancer could improve our understanding of breast cancer molecular pathophysiology. This is a two-stage hospital-based case-control study of a Chinese Han population. Genotyping was performed to identify candidate gene variants. 3DSNP, ANNOVAR, and RegulomeDB were used to determine functional single nucleotide polymorphisms (SNPs). The relationship between candidate variants and breast cancer risk was evaluated through unconditional logistic regression analysis. The PancanQTL platform was used to perform cis and trans expression quantitative trait loci (eQTL) analysis of positive SNPs. The GSCA platform was then used to compare the gene expression levels of potential target genes between breast cancer tissue and normal tissue adjacent to the cancer. rs10175368-T acted as a protective factor against breast cancer based on an additive model [odds ratio (OR) = 0.722, 95% confidence interval (CI) = 0.613-0.850; P < 0.001], and was identified as a protective factor in the postmenopausal population (OR = 0.601; 95% CI, 0.474-0.764; P < 0.001). eQTL analysis and analysis of differential expression in carcinoma and paracancerous tissues revealed that the expression level of CYP1B1 - AS1 was associated with rs10175368 and that CYP1B1-AS1 had significantly higher expression levels in breast cancer tissues than in paracancerous tissues. We show, for the first time in a Chinese Han population, that the functional variant rs10175368 plays a protective role against breast cancer, especially in the postmenopausal population.

Cytochrome P450 1B1 Expression Regulates Intracellular Iron Levels and Oxidative Stress in the Retinal Endothelium.

Cytochrome P450 (CYP) 1B1 is a heme-containing monooxygenase found mainly in extrahepatic tissues, including the retina. CYP1B1 substrates include exogenous aromatic hydrocarbons, such as dioxins, and endogenous bioactive compounds, including 17β-estradiol (E2) and arachidonic acid. The endogenous compounds and their metabolites are mediators of various cellular and physiological processes, suggesting that CYP1B1 activity is likely important in maintaining proper cellular and tissue functions. We previously demonstrated that lack of CYP1B1 expression and activity are associated with increased levels of reactive oxygen species and oxidative stress in the retinal vasculature and vascular cells, including retinal endothelial cells (ECs). However, the detailed mechanism(s) of how CYP1B1 activity modulates redox homeostasis remained unknown. We hypothesized that CYP1B1 metabolism of E2 affects bone morphogenic protein 6 (BMP6)-hepcidin-mediated iron homeostasis and lipid peroxidation impacting cellular redox state. Here, we demonstrate retinal EC prepared from Cyp1b1-deficient (Cyp1b1-/-) mice exhibits increased estrogen receptor-α (ERα) activity and expresses higher levels of BMP6. BMP6 is an inducer of the iron-regulatory hormone hepcidin in the endothelium. Increased hepcidin expression in Cyp1b1-/- retinal EC resulted in decreased levels of the iron exporter protein ferroportin and, as a result, increased intracellular iron accumulation. Removal of excess iron or antagonism of ERα in Cyp1b1-/- retinal EC was sufficient to mitigate increased lipid peroxidation and reduce oxidative stress. Suppression of lipid peroxidation and antagonism of ERα also restored ischemia-mediated retinal neovascularization in Cyp1b1-/- mice. Thus, CYP1B1 expression in retinal EC is important in the regulation of intracellular iron levels, with a significant impact on ocular redox homeostasis and oxidative stress through modulation of the ERα/BMP6/hepcidin axis.

An integrated targeted metabolomics and network pharmacology approach to exploring the mechanism of ellagic acid against sleep deprivation-induced memory impairment and anxiety.

As a neuroprotective agent, ellagic acid (EA) is extremely beneficial. Our previous study found that EA can alleviate sleep deprivation (SD)-induced abnormal behaviors, although the mechanisms underlying this protective effect have not yet been fully elucidated. An integrated network pharmacology and targeted metabolomics approach was utilized in this study to investigate the mechanism of EA against SD-induced memory impairment and anxiety. Behavioral tests were conducted on mice after 72 h of SD. Hematoxylin and eosin staining and nissl staining were then carried out. Integration of network pharmacology and targeted metabolomics was performed. Eventually, the putative targets were further verified using molecular docking analyses and immunoblotting assays. The present study findings confirmed that EA ameliorated the behavioral abnormalities induced by SD and prevented histopathological and morphological damage to hippocampal neurons. Through multivariate analysis, clear clustering was obtained among different groups, and potential biomarkers were identified. Four key targets, catechol-O-methyltransferase (COMT), cytochrome P450 1B1 (CYP1B1), glutathione S-transferase A2 (GSTA2), and glutathione S-transferase P1 (GSTP1), as well as the related potential metabolites and metabolic pathways, were determined by further integrated analysis. Meanwhile, in-silico studies revealed that EA is well located inside the binding site of CYP1B1 and COMT. The experimental results further demonstrated that EA significantly reduced the increased expression of CYP1B1 and COMT caused by SD. The findings of this study extended our understanding of the underlying mechanisms by which EA treats SD-induced memory impairment and anxiety, and suggested a novel approach to address the increased health risks associated with sleep loss.

Comparative Inhibitory Effects of Natural Biflavones from Ginkgo against Human CYP1B1 in Recombinant Enzymes and MCF-7 Cells.

Human cytochrome P450 1B1 (CYP1B1) is an extrahepatic enzyme overexpressed in many tumors and associated with angiogenesis. Ginkgetin, isoginkgetin, sciadopitysin, and amentoflavone, the primary biflavones found in Ginkgo biloba, have excellent anti-inflammatory and anti-tumor effects. However, the effect of biflavones on CYP1B1 activities remains unknown. In this study, 7-ethoxyresorufin O-deethylation (EROD) was used to characterize the activities of CYP1 families. The impacts of four ginkgo biflavones on CYP1B1 activity and the cellular protein expression of CYP1B1 were systematically investigated. The results showed that amentoflavone with six hydroxyl substituents exhibited the most potent selective inhibitory effect on CYP1B1 activity with IC50 of 0.054 µM in four biflavones. Sciadopitysin, with three hydroxyl and three methoxy substituents, had the weakest inhibitory activity against CYP1B1. Ginkgetin and isoginkgetin, both with four hydroxyl and two methoxy substituents, showed similar inhibitory intensity towards CYP1B1 with IC50 values of 0.289 and 0.211 µM, respectively. Kinetic analysis showed that ginkgetin and amentoflavone inhibited CYP1B1 in a non-competitive mode, whereas sciadopitysin and isoginkgetin induced competitive or mixed types of inhibition. Notably, four ginkgo biflavones were also confirmed to suppress the protein expressions of CYP1B1 and AhR in MCF-7. Furthermore, molecular docking studies indicated more hydrogen bonds formed between amentoflavone and CYP1B1, which might explain the strongest inhibitory action towards CYP1B1. In summary, these findings suggested that biflavones remarkably inhibited both the activity and protein expression of CYP1B1 and the inhibitory activities enhanced with the increasing hydroxyl substitution, providing new insights into the anti-tumor potentials of biflavones.

CYP1B1 Augments the Mesenchymal, Claudin-Low, and Chemoresistant Phenotypes of Triple-Negative Breast Cancer Cells.

Cytochrome P4501B1 (CYP1B1) is elevated in breast cancer. Studies indicate a relationship between CYP1B1 and aggressive cancer phenotypes. Here, we report on in vitro studies in triple-negative breast cancer cell lines, where knockdown (KD) of CYP1B1 was used to determine the influence of its expression on invasive cell phenotypes. CYP1B1 KD in MDA-MB-231 cells resulted in the loss of mesenchymal morphology, altered expression of epithelial–mesenchymal genes, and increased claudin (CLDN) RNA and protein. CYP1B1 KD cells had increased cell-to-cell contact and paracellular barrier function, a reduced rate of cell proliferation, abrogation of migratory and invasive activity, and diminished spheroid formation. Analysis of clinical breast cancer tumor samples revealed an association between tumors exhibiting higher CYP1B1 RNA levels and diminished overall and disease-free survival. Tumor expression of CYP1B1 was inversely associated with CLDN7 expression, and CYP1B1HI/CLDN7LOW identified patients with lower median survival. Cells with CYP1B1 KD had an enhanced chemosensitivity to paclitaxel, 5-fluorouracil, and cisplatin. Our findings that CYP1B1 KD can increase chemosensitivity points to therapeutic targeting of this enzyme. CYP1B1 inhibitors in combination with chemotherapeutic drugs may provide a novel targeted and effective approach to adjuvant or neoadjuvant therapy against certain forms of highly metastatic breast cancer.

Hippocampal Expression of Cytochrome P450 1B1 in Penetrating Traumatic Brain Injury.

Hippocampal dysfunction contributes to multiple traumatic brain injury sequala. Female rodents’ outcome is superior to male which has been ascribed the neuroprotective sex hormones 17β-estradiol and progesterone. Cytochrome P450 1B1 (CYP1B1) is an oxidative enzyme influencing the neuroinflammatory response by creating inflammatory mediators and metabolizing neuroprotective 17β-estradiol and progesterone. In this study, we aimed to describe hippocampal CYP1B1 mRNA expression, protein presence of CYP1B1 and its key redox partner Cytochrome P450 reductase (CPR) in both sexes, as well as the effect of penetrating traumatic brain injury (pTBI). A total 64 adult Sprague Dawley rats divided by sex received pTBI or sham-surgery and were assigned survival times of 1-, 3-, 5- or 7 days. CYP1B1 mRNA was quantified using in-situ hybridization and immunohistochemistry performed to verify protein colocalization. CYP1B1 mRNA expression was present in all subregions but greatest in CA2 irrespective of sex, survival time or intervention. At 3-, 5- and 7 days post-injury, expression in CA2 was reduced in male rats subjected to pTBI compared to sham-surgery. Females subjected to pTBI instead exhibited increased expression in all CA subregions 3 days post-injury, the only time point expression in CA2 was greater in females than in males. Immunohistochemical analysis confirmed neuronal CYP1B1 protein in all hippocampal subregions, while CPR was limited to CA1 and CA2. CYP1B1 mRNA is constitutively expressed in both sexes. In response to pTBI, females displayed a more urgent but brief regulatory response than males. This indicates there may be sex-dependent differences in CYP1B1 activity, possibly influencing inflammation and neuroprotection in pTBI.

Constitutive expression of cytochrome P450 1B1 endows testicular Leydig cells with susceptibility to 7,12-dimethylbenzanthracene-induced cell death.

Testicular Leydig cells produce testosterone through the participation of steroidogenic proteins. The CYP1B1 enzyme has been shown to catalyze 7,12-dimethylbenzanthracene (DMBA), a representative polycyclic aromatic hydrocarbon. We hypothesized that exposure to DMBA causes Leydig cell cytotoxicity through activation of CYP1B1. Leydig cells were exposed to various concentrations of DMBA for the induction of CYP1B1 expression and activity. The status of CYP1B1 function was monitored by evaluation of cytotoxicity-mediated cell death. Our data show that exposure to DMBA causes cytotoxicity in Leydig cells by CYP1B1 activation. DMBA evoked a significant increase in the generation of reactive oxygen species (ROS) by which the depolarization of mitochondrial membrane potential (MMP) is initiated and caspase-3 activation is augmented. The knockdown of CYP1B1 expression resulted in the suppression of DMBA-induced apoptosis via reduced p53 activation and caspase-3 activation, suggesting that a final metabolite of DMBA (i.e., DMBA-DE) bioactivated by CYP1B1 induces p53 activation by binding to DNA and subsequently causing apoptosis via caspase-3 activation. This finding provides evidence for constitutive expression of CYP1B1 in Leydig cells, which is a trait that only requires an initiating signal for its activity. Further research on CYP1B1 activation-provoked steroid metabolism in Leydig cells may provide decisive clues for elucidating its innate function.

Cytochrome P450 1B1 Overexpression in Cervical Cancers: Cross-sectional Study.

BackgroundCurrent standard treatments for patients with recurrent cervical cancer are not very effective and are associated with severe toxicity. Recently, the rational approach for the discovery of new therapies for cervical cancer is based on the alterations in the molecular biology of cancer cells. One of the emerging molecular changes in cancer cells is the aberrant expression of cytochrome P450 1B1 (CYP1B1). This unique enzyme has been reported to be selectively overexpressed in several cancers.ObjectiveThe aim of this study was to examine CYP1B1 expression in cervical cancers and to assess the enzyme’s relationship with several clinicopathological features.MethodsImmunohistochemistry was performed to examine CYP1B1 expression in 100 patient samples with cervical cancer and 10 patient samples with normal healthy cervical tissues.ResultsCYP1B1 was expressed in the majority of the cervical cancer samples (91/100, 91.0%) but not in normal healthy cervical samples. The difference in the expression of CYP1B1 between healthy and tumorous cervical tissues was significant (P=.01). Moreover, the frequency of CYP1B1 expression was found to be significantly higher in patients with advanced grades of the disease (P=.03) and in patients having metastasis to the lymph nodes (P=.01). Surprisingly, there was a significantly higher expression of CYP1B1 in patients with a high prevalence of human papilloma virus 16/18 (P=.04).ConclusionsThe differential profile of CYP1B1 expression between cervical cancer tissues and normal cervical tissues suggests that CYP1B1 may be used as a target for future therapeutic exploitations.

Possible Role of Cytochrome P450 1B1 in the Mechanism of Gemcitabine Resistance in Pancreatic Cancer.

Patient-derived xenograft models reportedly represent original tumor morphology and gene mutation profiles. In addition, patient-derived xenografts are expected to recapitulate the parental tumor drug responses. In this study, we analyzed the pathways involved in gemcitabine resistance using patient-derived xenograft models of pancreatic cancer. The patient-derived xenograft models were established using samples from patients with pancreatic cancer. The models were treated with gemcitabine to better understand the mechanism of resistance to this anti-cancer drug. We performed comparative gene analysis through the next-generation sequencing of tumor tissues from gemcitabine-treated or non-treated patient-derived xenograft mice and gene set enrichment analysis to analyze mRNA profiling data. Pathway analysis of gemcitabine-treated patient-derived xenografts disclosed the upregulation of multiple gene sets and identified several specific gene pathways that could potentially be related to gemcitabine resistance in pancreatic cancer. Further, we conducted an in vitro analysis to validate these results. The mRNA expression of cytochrome P450 1B1 and cytochrome P450 2A6 was upregulated in a concentration-dependent manner following gemcitabine treatment. Moreover, the sensitivity to gemcitabine increased, and viable cells were decreased by the cytochrome P450 1B1 inhibitor, indicating that the cytochrome P450 1B1 pathway may be related to gemcitabine resistance in pancreatic cancer.

Upregulation of Aryl Hydrocarbon Receptor in Granulosa Cells by Endoplasmic Reticulum Stress Contributes to the PCOS Pathophysiology

Studies have demonstrated that endocrine disrupting chemicals (EDC) are involved in the pathophysiology of PCOS, and aryl hydrocarbon receptor (AHR) mediates the cellular effect of EDC by inducing xenobiotic metabolizing enzymes including cytochrome P450 1B1 (CYP1B1). However, recent studies suggest the novel role of AHR in various diseases, including obesity and cancer progression, independent from the EDC metabolism. We previously demonstrated that endoplasmic reticulum (ER) stress, a newly recognized local factor, contributes to PCOS pathology by affecting diverse functions of granulosa cells. We hypothesized that ER stress induces the expression of AHR and activates its downstream signaling in granulosa cells, irrespective of the presence of EDCs, thereby promoting PCOS pathogenesis. At first, we determined the upregulation of AHR, AHR nuclear translocator (ARNT), and AHR target gene cytochrome P450 1B1 (CYP1B1) in the granulosa cells of PCOS patients and model mice by immunohistochemical staining and qPCR. We examined CYP1B1 as a representative AHR target gene. Treatment of cultured human granulosa-lutein cells (GLCs) with tunicamycin (ER stress inducer) upregulated the expression of AHR, ARNT and CYP1B1. Knockdown of AHR decreased the tunicamycin-induced expression and activity of CYP1B1, suggesting the intermediary role of AHR in upregulation of AHR activity by ER stress. To confirm the role of AHR in vivo, we administered the AHR antagonist CH223191 to PCOS model mice. The administration of the antagonist restored estrous cycling and decreased the number of atretic antral follicles, concomitant with downregulation of AHR and CYP1B1 in granulosa cells. Taken together, this study indicates that AHR and downstream signaling are activated by ER stress in GLCs of PCOS. Moreover, downregulation of local AHR expression and activation restores a normal reproductive phenotype in a PCOS mouse model. Our findings demonstrate that AHR activated by ER stress in the follicular microenvironment contributes to PCOS pathology, and that AHR represents a novel therapeutic target for PCOS.

Identification of miRNAs that regulate human CYP2B6 expression

Human hepatic cytochrome P450 2B6 (CYP2B6) expressed is responsible for the metabolism of many drugs, such as cyclophosphamide, ifosfamid, and efavirenz. In the present study, the correlation between CYP2B6 mRNA and protein levels in human liver samples was found to be moderate, indicating a contribution of posttranscriptional regulation of CYP2B6. Thus, we examined the role of microRNAs (miRNAs) in the regulation of CYP2B6. We established two kinds of HEK293 cell lines stably expressing CYP2B6, including or excluding the full-length 3′-untranslated region (3′-UTR) (HEK/2B6+UTR and HEK/2B6 cells, respectively). We tested 14 miRNAs that were predicted to bind to the 3′-UTR of CYP2B6 and found that the overexpression of miR-145, miR-194, miR-222, and miR-378 decreased the CYP2B6 protein level and activity in HEK/2B6+UTR but not in HEK/2B6 cells. These results suggested that miR-145, miR-194, miR-222, and miR-378 negatively regulate CYP2B6 expression by binding to the 3′-UTR. A negative correlation was not observed between the translational efficiency of CYP2B6 and the expression level of miR-145, miR-194, miR-222, or miR-378. This is due to the contribution of multiple miRNAs to CYP2B6 regulation. In conclusion, this study demonstrated that human CYP2B6 is posttranscriptionally regulated by miR-145, miR-194, miR-222, and miR-378.

Induction of aryl hydrocarbon receptor in granulosa cells by endoplasmic reticulum stress contributes to pathology of polycystic ovary syndrome.

Recent studies have uncovered the critical role of aryl hydrocarbon receptor (AHR) in various diseases, including obesity and cancer progression, independent of its previously identified role as a receptor for endocrine-disrupting chemicals (EDCs). We previously showed that endoplasmic reticulum (ER) stress, a newly recognized local factor in the follicular microenvironment, is activated in granulosa cells from patients with polycystic ovary syndrome (PCOS) and a mouse model of the disease. By affecting diverse functions of granulosa cells, ER stress contributes to PCOS pathology. We hypothesized that expression of AHR and activation of its downstream signaling were upregulated by ER stress in granulosa cells, irrespective of the presence of EDCs, thereby promoting PCOS pathogenesis. In this study, we found that AHR, AHR nuclear translocator (ARNT), and AHR target gene cytochrome P450 1B1 (CYP1B1) were upregulated in the granulosa cells of PCOS patients and model mice. We examined CYP1B1 as a representative AHR target gene. AHR and ARNT were upregulated by ER stress in human granulosa-lutein cells (GLCs), resulting in an increase in the expression and activity of CYP1B1. Administration of the AHR antagonist CH223191 to PCOS mice restored estrous cycling and decreased the number of atretic antral follicles, concomitant with downregulation of AHR and CYP1B1 in granulosa cells. Taken together, our findings indicate that AHR activated by ER stress in the follicular microenvironment contributes to PCOS pathology, and that AHR represents a novel therapeutic target for PCOS.

Compound danshen dripping pills affect the pharmacokinetics of azisartan by regulating the expression of cytochrome P450 2B1, 2C6, and 2C11 in rats

Combination therapies of compound danshen dripping pill (CDDP) and Azilsartan (AZ) represent a promising treatment option in clinical practice in China, but there are no reports on drug-drug interactions between CDDP and AZ. This study investigated the effects of CDDP on the pharmacokinetics of AZ and clarified its potential mechanism. The pharmacokinetic profiles of oral administration of AZ (2 mg/kg) in Sprague-Dawley rats, with or without pre-treatment of CDDP (81, 405, 810 mg/kg/d for 7 d) were investigated using UPLC-MS/MS. The main pharmacokinetic parameters were calculated and compared. The MS analysis was performed in positive ionization mode. The purpose of chromatographic separation of AZ and the internal standard (IS, Valsartan) was finished on a Waters XBridge BEH C18 column (2.1 × 100 mm, 2.5 μm). The mobile phase was acetonitrile and 0.1 % formic acid-water with gradient elution at a flow rate of 0.4 mL/min. The mRNA and protein levels of CYP2B1, CYP2C6, and CYP2C11 in the rat liver were detected by qRT-PCR and western blot, respectively. The results indicated that low, medium and high doses of CDDP significantly increased the Cmax (6.47 ± 2.28, 6.51 ± 1.99, 7.04 ± 1.31 vs. 3.30 ± 1.87) of AZ, compared with that in the AZ single-drug group (p<0.05). The AUC0-t of AZ (47.77 ± 23.41, 50.69 ± 25.46, 54.50 ± 11.57 vs. 26.85 ± 16.79) tended to increase in combination with CDDP. The gene and protein expression levels of CYP2B1, CYP2C6, and CYP2C11 were significantly reduced in the rat liver by CDDP. CDDP may diminish the AZ metabolism in vivo by suppressing the expression of the CYP2B1, CYP2C6, and CYP2C11 enzymes. This observation suggested the occurrence of potential interactions between CDDP and AZ when clinically administered as combination therapy, which may require adjustment of the clinical dose of AZ.

Abstract 4668: Human CYP1B1 prevents TRAIL-DR4-mediated cancer cell apoptosis through downregulation of miR-375

Abstract Human cytochrome P450 1B1 (CYP1B1) is an important enzyme that catalyzes metabolism of 17β-estradiol (E2) to catechol estrogens, including carcinogenic metabolite 4-hydroxyestradiol (4-OHE2). CYP1B1 may play a crucial role in estrogen-mediated carcinogenesis since CYP1B1 shows tumor-specific expression pattern in various hormone-responsive tissues. However, the detailed mechanism of CYP1B1-dependent carcinogenesis remains still unclear. Here, we investigated the association between TRAIL, DR4, and CYP1B1 expression in human tumor cells, including MCF-7, MDA-MB-231, and HeLa cells. CYP1B1 can inhibit the expression and release of TRAIL simultaneously with the expression of the death receptor DR4/5, known as TRAIL receptors, to prevent apoptosis. Furthermore, we found that CYP1B1 represses the transcription of DR4 by inducing DNA methylation in the promoter region of DR4. In addition, we revealed that CYP1B1 regulates TRAIL and DR4 through activation of Sp1 transcription factor. CYP1B1 and its enzymatic product, 4-OHE2, promote Sp1 expression levels in both mRNA and protein levels. Interestingly, CYP1B1 elevated Sp1 through downregulation of miR-375, which is a candidate tumor suppressive microRNA and transcriptional inhibitor of Sp1. Taken together, our data suggest that CYP1B1 inhibits TRAIL-DR4-mediated cancer cell apoptosis through induction of Sp1 through repression of miR-375, which promotes cancer cell survival. Citation Format: Yeo-Jung Kwon, Dong-Jin Ye, Huoung-Seok Baek, Sangyun Shin, Tae-Uk Kwon, Eunah Cho, Hong-Gyu Ahn, Youngjin Chun. Human CYP1B1 prevents TRAIL-DR4-mediated cancer cell apoptosis through downregulation of miR-375 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4668.

Cytochrome P4501B1 in bone marrow is co-expressed with key markers of mesenchymal stem cells. BMS2 cell line models PAH disruption of bone marrow niche development functions

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants that are metabolized to carcinogenic dihydrodiol epoxides (PAHDE) by cytochrome P450 1B1 (CYP1B1). This metabolism occurs in bone marrow (BM) mesenchymal stem cells (MSC), which sustain hematopoietic stem and progenitor cells (HSPC). In BM, CYP1B1-mediated metabolism of 7, 12-dimethylbenz[a]anthracene (DMBA) suppresses HSPC colony formation within 6 h, whereas benzo(a)pyrene (BP) generates protective cytokines. MSC, enriched from adherent BM cells, yielded the bone marrow stromal, BMS2, cell line. These cells express elevated basal CYP1B1 that scarcely responds to Ah receptor (AhR) inducers. BMS2 cells exhibit extensive transcriptome overlap with leptin receptor positive mesenchymal stem cells (Lepr+ MSC) that control the hematopoietic niche. The overlap includes CYP1B1 and the expression of HSPC regulatory factors (Ebf3, Cxcl12, Kitl, Csf1 and Gas6). MSC are large, adherent fibroblasts that sequester small HSPC and macrophage in the BM niche (Graphic abstract). High basal CYP1B1 expression in BMS2 cells derives from interactions between the Ah-receptor enhancer and proximal promoter SP1 complexes, boosted by autocrine signaling. PAH effects on BMS2 cells model Lepr+MSC niche activity. CYP1B1 metabolizes DMBA to PAHDE, producing p53-mediated mRNA increases, long after the in vivo HSPC suppression. Faster, direct p53 effects, favored by stem cells, remain possible PAHDE targets. However, HSPC regulatory factors remained unresponsive. BP is less toxic in BMS2 cells, but, in BM, CYP1A1 metabolism stimulates macrophage cytokines (Il1b > Tnfa> Ifng) within 6 h. Although absent from BMS2 and Lepr+MSC, their receptors are highly expressed. The impact of this cytokine signaling in MSC remains to be determined.

Resveratrol attenuates angiotensin II-induced cellular hypertrophy through the inhibition of CYP1B1 and the cardiotoxic mid-chain HETE metabolites.

Several reports demonstrated the direct contribution of cytochrome P450 1B1 (CYP1B1) enzyme and its associated cardiotoxic mid-chain, hydroxyeicosatetraenoic acid (HETEs) metabolites in the development of cardiac hypertrophy. Resveratrol is commercially available polyphenol that exerts beneficial effects in wide array of cardiovascular diseases including cardiac hypertrophy, myocardial infarction and heart failure. Nevertheless, the underlying mechanisms responsible for these effects are not fully elucidated. Since resveratrol is a well-known CYP1B1 inhibitor, the purpose of this study is to test whether resveratrol attenuates angiotensin II (Ang II)-induced cellular hypertrophy through inhibition of CYP1B1/mid-chain HETEs mechanism. RL-14 and H9c2 cells were treated with vehicle or 10 μM Ang II in the absence and presence of 2, 10 or 50 μM resveratrol for 24 h. Thereafter, the level of mid-chain HETEs was determined using liquid chromatography–mass spectrometry (LC/MS). Hypertrophic markers and CYP1B1 gene expression and protein levels were measured using real-time PCR and Western blot analysis, respectively. Our results demonstrated that resveratrol, at concentrations of 10 and 50 μM, was able to attenuate Ang-II-induced cellular hypertrophy as evidenced by substantial inhibition of hypertrophic markers, β-myosin heavy chain (MHC)/α-MHC and atrial natriuretic peptide. Ang II significantly induced the protein expression of CYP1B1 and increased the metabolite formation rate of its associated mid-chain HETEs. Interestingly, the protective effect of resveratrol was associated with a significant decrease of CYP1B1 protein expression and mid-chain HETEs. Our results provided the first evidence that resveratrol protects against Ang II-induced cellular hypertrophy, at least in part, through CYP1B1/mid-chain HETEs-dependent mechanism.