CYP1B1 affects the integrity of the blood-brain barrier and oxidative stress in the striatum: An investigation of manganese-induced neurotoxicity.

AIMS: Excessive influx of manganese (Mn) into the brain across the blood-brain barrier induces neurodegeneration. CYP1B1 is involved in the metabolism of arachidonic acid (AA) that affects vascular homeostasis. We aimed to investigate the effect of brain CYP1B1 on Mn-induced neurotoxicity. METHOD: Brain Mn concentrations and α-synuclein accumulation were measured in wild-type and CYP1B1 knockout mice treated with MnCl2 (30 mg/kg) and biotin (0.2 g/kg) for 21 continuous days. Tight junctions and oxidative stress were analyzed in hCMEC/D3 and SH-SY5Y cells after the treatment with MnCl2 (200 µM) and CYP1B1-derived AA metabolites (HETEs and EETs). RESULTS: Mn exposure inhibited brain CYP1B1, and CYP1B1 deficiency increased brain Mn concentrations and accelerated α-synuclein deposition in the striatum. CYP1B1 deficiency disrupted the integrity of the blood-brain barrier (BBB) and increased the ratio of 3, 4-dihydroxyphenylacetic acid (DOPAC) to dopamine in the striatum. HETEs attenuated Mn-induced inhibition of tight junctions by activating PPARγ in endothelial cells. Additionally, EETs attenuated Mn-induced up-regulation of the KLF/MAO-B axis and down-regulation of NRF2 in neuronal cells. Biotin up-regulated brain CYP1B1 and reduced Mn-induced neurotoxicity in mice. CONCLUSIONS: Brain CYP1B1 plays a critical role in both cerebrovascular and dopamine homeostasis, which might serve as a novel therapeutic target for the prevention of Mn-induced neurotoxicity.

Circ_0020887 Silencing Combats Hypoxic-Induced Cardiomyocyte Injury in an MiR-370-3p/CYP1B1-Dependent Manner.

Targeting circular RNA has been a novel approach to preventing and limiting acute myocardial infarction (AMI). Here, we planned to investigate the role and mechanism of circ_0020887 in AMI progression.Hypoxic injury in human cardiomyocytes (AC16) was measured using cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine assay, flow cytometry, and colorimetric assay kits. RNA and protein expressions were determined using real-time quantitative PCR and western blotting. Direct interplay between RNAs was determined using dual-luciferase reporter, RNA pull-down, and RIP assays.In the plasma and hypoxia-induced AC16 cells of patients with AMI, circ_0020887 and miR-370-3p were upregulated and downregulated, respectively, concomitant with the upregulation of cytochrome P450 1B1 (CYP1B1). Circ_0020887 interference could inhibit hypoxia-induced AC16 cell apoptosis, oxidative stress, and inflammatory response. Circ_0020887 could sponge miR-370-3p, and miR-370-3p could target CYP1B1. The inhibition effect of circ_0020887 knockdown on hypoxia-induced AC16 cell injury could be reversed by the miR-370-3p inhibitor. Besides, CYP1B1 overexpression also overturned the suppressive effect of miR-370-3p on hypoxia-induced AC16 cell apoptosis, oxidative stress, and inflammatory response.In conclusion, circ_0020887 regulated the miR-370-3p/CYP1B1 axis to regulate hypoxia-induced cardiomyocyte injury, confirming that circ_0020887 might promote cardiomyocyte injury.

Single-nucleotide polymorphism in CYP1A1, CYP1B1, CYP2B6, CYP2C8, and CYP2C9 genes and their association with gastrointestinal cancer: A hospital-based case-control study.

BACKGROUND: Cytochrome P450 (CYP) comprises a group of phase-I metabolizing enzymes that are important in xenobiotics metabolism. Genetic polymorphism of CYPs has been comprehensively studied for their association with a range of diseases. In this study, we assessed single-nucleotide polymorphism (SNP) of CYP1A, CYP1B, CYP2B, and CYP2C and their role in gastrointestinal (GI) cancer susceptibility in the rural population of Maharashtra. MATERIALS AND METHODS: In this hospital-based case-control study, the association of polymorphism of CYP genes was studied by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The study subjects included 200 clinically confirmed GI cancer patients and equal number of healthy controls. Odds ratio (OR) with 95% confidence interval (CI) and P value were evaluated to find out the level of association, where P ≤ 0.005 was considered statistically significant. RESULTS: After the analysis of CYP1A1*2A (rs4646903), CYP1B1*3 (rs1059836), CYP2B6*5 (rs3211371), CYP2C8*2 (rs11572103), CYP2C9*2 (rs1799853), and CYP2C9*3 (rs1057910), we noticed that variant (T) allele of CYP2B6*5 possessed significantly elevated risk (OR = 4.43; 95% CI: 2.20-8.90; P < 0.0001) of GI cancer in studied population. The genotypic distribution of G/C heterozygote allele of CYP1B1*3 (OR = 0.19, 95% CI = 0.12-0.32; P < 0.0001) and homozygous variant C/C allele (OR = 0.24, 95% CI = 0.13-0.45; P < 0.0001) showed a negative association with the development of GI cancer. CONCLUSION: The findings from this study supported that polymorphism of CYP2B6*5gene may be involved in the development of GI cancer. However, other SNPs of CYP1A, CYP1B, and CYP2C genes did not signify the risk for GI cancer in the studied population of rural Maharashtra.

Design of Novel 2-Phenylquinazolin-4-amines as Selective CYP1B1 Inhibitors for Overcoming Paclitaxel Resistance in A549 Cells.

Cytochrome P450 1B1 (CYP1B1) contributes to the metabolic inactivation of chemotherapeutics when overexpressed in tumor cells. Selective inhibition of CYP1B1 holds promise for reversing drug resistance. In our pursuit of potent CYP1B1 inhibitors, we designed and synthesized a series of 2-phenylquinazolin-4-amines. A substantial proportion of these newly developed inhibitors demonstrated inhibitory activity against CYP1B1, accompanied by improved water solubility. Remarkably, compound 14b exhibited exceptional inhibitory efficacy and selectivity toward CYP1B1. Molecular docking studies suggested that the expansion of the π-system through aromatization, the introduction of an amine group, and iodine atom augmented the binding affinity. Furthermore, inhibitors 14a, 14b, and 14e demonstrated the ability to significantly reduce the resistance in A549 cells to paclitaxel, while also inhibiting the migration and invasion of these cells. Finally, radioiodine labeling experiments shed light on the metabolic pathway of compound 5l in mice, highlighting the potential of 125I-5l as a radioactive probe for future research endeavors.

CircPPAP2B controls metastasis of clear cell renal cell carcinoma via HNRNPC-dependent alternative splicing and targeting the miR-182-5p/CYP1B1 axis.

BACKGROUND: Renal cell carcinoma (RCC) is one of the most common malignant tumor worldwide. Metastasis is a leading case of cancer-related deaths of RCC. Circular RNAs (circRNAs), a class of noncoding RNAs, have emerged as important regulators in cancer metastasis. However, the functional effects and regulatory mechanisms of circRNAs on RCC metastasis remain largely unknown. METHODS: High-throughput RNA sequencing techniques were performed to analyze the expression profiles of circRNAs and mRNAs in highly and poorly invasive clear cell renal cell carcinoma (ccRCC) cell lines. Functional experiments were performed to unveil the regulatory role of circPPAP2B in the proliferation and metastatic capabilities of ccRCC cells. RNA pulldown, Mass spectrometry analysis, RNA methylation immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), co-immunoprecipitation (CoIP), next-generation RNA-sequencing and double luciferase experiments were employed to clarify the molecular mechanisms by which circPPAP2B promotes ccRCC metastasis. RESULTS: In this study, we describe a newly identified circular RNA called circPPAP2B, which is overexpressed in highly invasive ccRCC cells, as determined through advanced high-throughput RNA sequencing techniques. Furthermore, we observed elevated circPPAP2B in ccRCC tissues, particularly in metastatic ccRCC tissues, and found it to be associated with poor prognosis. Functional experiments unveiled that circPPAP2B actively stimulates the proliferation and metastatic capabilities of ccRCC cells. Mechanistically, circPPAP2B interacts with HNRNPC in a m6A-dependent manner to facilitate HNRNPC nuclear translocation. Subcellular relocalization was dependent upon nondegradable ubiquitination of HNRNPC and stabilization of an HNRNPC/Vimentin/Importin α7 ternary complex. Moreover, we found that circPPAP2B modulates the interaction between HNRNPC and splicing factors, PTBP1 and HNPNPK, and regulates pre-mRNA alternative splicing. Finally, our studies demonstrate that circPPAP2B functions as a miRNA sponge to directly bind to miR-182-5p and increase CYP1B1 expression in ccRCC. CONCLUSIONS: Collectively, our study provides comprehensive evidence that circPPAP2B promotes proliferation and metastasis of ccRCC via HNRNPC-dependent alternative splicing and miR-182-5p/CYP1B1 axis and highlights circPPAP2B as a potential therapeutic target for ccRCC intervention.

Regulation of Benzo[a]pyrene-Induced Hepatic Lipid Accumulation through CYP1B1-Induced mTOR-Mediated Lipophagy.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is caused by lipid accumulation within the liver. The pathogenesis underlying its development is poorly understood. Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon and a group 1 carcinogen. The aryl hydrocarbon receptor activation by B[a]P induces cytochrome P450 (CYP) enzymes, contributing to hepatic lipid accumulation. However, the molecular mechanism through which the B[a]P-mediated induction of CYP enzymes causes hepatic lipid accumulation is unknown. This research was conducted to elucidate the role of CYP1B1 in regulating B[a]P-induced lipid accumulation within hepatocytes. B[a]P increased hepatic lipid accumulation, which was mitigated by CYP1B1 knockdown. An increase in the mammalian target of rapamycin (mTOR) by B[a]P was specifically reduced by CYP1B1 knockdown. The reduction of mTOR increased the expression of autophagic flux-related genes and promoted phagolysosome formation. Both the expression and translocation of TFE3, a central regulator of lipophagy, were induced, along with the expression of lipophagy-related genes. Conversely, enhanced mTOR activity reduced TFE3 expression and translocation, which reduced the expression of lipophagy-related genes, diminished phagolysosome production, and increased lipid accumulation. Our results indicate that B[a]P-induced hepatic lipid accumulation is caused by CYP1B1-induced mTOR and the reduction of lipophagy, thereby introducing novel targets and mechanisms to provide insights for understanding B[a]P-induced MASLD.

Lower overall survival in male patients with advanced disease undergoing allogeneic hematopoietic stem cell transplantation is associated with CYP1B1 Leu432Val polymorphism.

Human cytochrome P450 1B1 (CYP1B1) is an extrahepatic key enzyme involved in estrogen metabolism, steroid synthesis, and pro-carcinogen activation. In a single-center retrospective study, 382 patients who underwent allogeneic hematopoetic stem cell transplantation and their donors were genotyped for CYP1B1 C432G polymorphism by reverse transcription polymerase chain reaction. One hundred and sixty-nine patients (44%) were homozygous wild-type (wt) gene CC, 157 (41%) heterozygous CG and 56 (15%) homozygous gene mutated GG. Of interest, mutated CYP1B1 was more common in male (62%) than in female patients (48%) P=0.006, unlike in donors. Five-year estimate for overall survival (OS) was 58±4% (CC) versus 48±3% (CG and GG), P=0.048. Surprisingly, this difference was only evident in males (P=0.024): OS 58±6% versus 42±4%, whereas it was virtually absent in females. Importantly, this difference was only evident in male patients with advanced disease (AD) (n=118, P=0.002): OS 44±8% (CC) versus 32±6% (CG) versus 6±6% (GG), whereas it was virtually absent in male patients with early disease. One-year non-relapse mortality in male patients with AD was 8±4% (CC) versus 21±5% (CG) versus 50±12% (GG), P=0.002. Three-year relapse rate in male patients with AD was 31±7% (wt) versus 42±6% (mut), P=0.04. Multivariate analysis for OS in male patients with AD revealed CYP1B1 polymorphism as the only prognostic factor: RR 1.78, P=0.001. In conclusion, these results suggest that male patients with AD and mutant CYP1B1 polymorphism have lower OS after allogeneic hematopoetic stem cell transplantation due to a higher non-relapse mortality and a higher relapse rate.

Gestational exposure to 1-NP induces ferroptosis in placental trophoblasts via CYP1B1/ERK signaling pathway leading to fetal growth restriction.

Fetal growth restriction (FGR) is a prevalent complication in obstetrics, yet its exact aetiology remains unknown. Numerous studies suggest that the degradation of the living environment is a significant risk factor for FGR. 1-Nitropyrene (1-NP) is a widespread environmental pollutant as a representative substance of nitro-polycyclic aromatic hydrocarbons. In this study, we revealed that 1-NP induced FGR in fetal mice by constructing 1-NP exposed pregnant mice models. Intriguingly, we found that placental trophoblasts of 1-NP exposed mice exhibited significant ferroptosis, which was similarly detected in placental trophoblasts from human FGR patients. In this regard, we established a 1-NP exposed cell model in vitro using two human trophoblast cell lines, HTR8/SVneo and JEG-3. We found that 1-NP not only impaired the proliferation, migration, invasion and angiogenesis of trophoblasts, but also induced severe cellular ferroptosis. Meanwhile, the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively rescued 1-NP-induced trophoblast biological function impairment. Mechanistically, we revealed that 1-NP regulated ferroptosis by activating the ERK signaling pathway. Moreover, we innovatively revealed that CYP1B1 was essential for the activation of ERK signaling pathway induced by 1-NP. Overall, our study innovatively identified ferroptosis as a significant contributor to 1-NP induced trophoblastic functional impairment leading to FGR and clarified the specific mechanism by which 1-NP induced ferroptosis via the CYP1B1/ERK signaling pathway. Our study provided novel insights into the aetiology of FGR and revealed new mechanisms of reproductive toxicity of environmental pollutants.

CYP1B1-AS1 Delays the Malignant Progression of Colorectal Cancer by Binding with NOP58.

BACKGROUND: Colorectal cancer (CRC) is a prevalent type of gastrointestinal cancer, and its poor prognosis is mainly attributed to the occurrence of invasion and metastasis. CYP1B1-AS1, as non-coding RNA, plays an important role in tumorigenesis and progression. However, the mechanism by which CYP1B1-AS1 acts in CRC is not yet understood. AIMS: The objective of this study was to investigate how CYP1B1-AS1 contributes to the development of CRC, and provide a base for CRC diagnosis and treatment. METHODS: RT-qPCR was used to detect the expression level of CYP1B1-AS1 in CRC and adjacent tissues. CCK-8, Edu, scratch healing, and transwell experiments were used to detect the changes of proliferation, migration, and invasion ability of CRC cells after overexpression or knockdown of CYP1B1-AS1 respectively. The RNA binding protein NOP58 combined with CYP1B1-AS1 was verified by RIP and RNA Pull-down experiments. Functional recovery experiments validated the interaction between CYP1B1-AS1 and NOP58 in CRC cells. The changes of EMT-related proteins were detected by Western blot, and the half-life of transcription factor SNAIL mRNA were detected by RT-qPCR after overexpression or knockdown of NOP58. RESULTS: CYP1B1-AS1 was found to be significantly downregulated in CRC compared to adjacent noncancerous tissues. Experiments conducted in vitro and in vivo confirmed that upregulation of CYP1B1-AS1 significantly inhibited the proliferation, migration, and invasion of CRC cells. In addition, CYP1B1-AS1 can directly bind to NOP58 and negatively regulate NOP58. The effect of overexpression CYP1B1-AS1 was reversed by NOP58 overexpression. NOP58 regulates the EMT process of CRC cells by affecting the stability of EMT-related transcription factor SNAIL mRNA, and then affects the progress of CRC. CONCLUSION: This research proves that CYP1B1-AS1 can inhibit the occurrence of EMT in CRC by binding with NOP58, thus delaying the progress of CRC. This finding indicates that CYP1B1-AS1 may be a novel biomarker to improve the diagnosis and treatment of CRC.

Variable Phenotype of Congenital Corneal Opacities in Biallelic CYP1B1 Pathogenic Variants.

PURPOSE: The aim of this study is to describe the variable phenotype of congenital corneal opacities occurring in patients with biallelic CYP1B1 pathogenic variants. METHODS: A retrospective chart review was conducted to identify patients with congenital corneal opacities and CYP1B1 pathogenic variants seen at UPMC Children's Hospital of Pittsburgh. Ophthalmic examination, high-frequency ultrasound, anterior segment optical coherence tomography, histopathologic images, and details of genetic testing were reviewed. RESULTS: Three children were identified. All presented with raised intraocular pressure. Two patients showed bilateral limbus-to-limbus avascular corneal opacification that did not resolve with intraocular pressure control; 1 showed unilateral avascular corneal opacity with a crescent of clear cornea, iridocorneal adhesions, iridolenticular adhesions, and classical features of congenital glaucoma in the fellow eye (enlarged corneal diameter, Haab striae, and clearing of the corneal clouding with appropriate intraocular pressure control). The first 2 patients were visually rehabilitated with penetrating keratoplasty. Histopathology revealed distinct features: a variably keratinized epithelium; a thick but discontinuous Bowman-like layer with areas of disruption and abnormal cellularity; Descemet membrane, when observed, showed reduced endothelial cells; and no pathological changes of Haab striae were identified. Two patients had compound heterozygous pathogenic variants in CYP1B1 causing premature stop codons, whereas 1 was homozygous for a pathogenic missense variant. CONCLUSIONS: Congenital corneal opacities seen in biallelic CYP1B1 pathogenic variants have a variable phenotype. One is that commonly termed as Peters anomaly type 1 (with iridocorneal adhesions, with or without iridolenticular adhesions) and the other is a limbus-to-limbus opacity, termed CYP1B1 cytopathy. Clinicians should be aware of this phenotypic variability.

Dihydrotanshinone I-Induced CYP1 Enzyme Inhibition and Alteration of Estradiol Metabolism.

Dihydrotanshinone I (DHTI) is a pharmacologically active component occurring in the roots of the herbal medicine Salvia miltiorrhiza Bunge. This study investigated DHTI-induced inhibition of CYP1A1, CYP1A2, and CYP1B1 with the aim to determine the potential effects of DHTI on the bioactivation of estradiol (E2), possibly related to preventive/therapeutic strategy for E2-associated breast cancer. Ethoxyresorufin as a specific substrate for CYP1s was incubated with human recombinant CYP1A1, CYP1A2, or CYP1B1 in the presence of DHTI at various concentrations. Enzymatic inhibition and kinetic behaviors were examined by monitoring the formation of the corresponding product. Molecular docking was further conducted to define the interactions between DHTI and the three CYP1s. The same method and procedure were employed to examine the DHTI-induced alteration of E2 metabolism. DHTI showed significant inhibition of ethoxyresorufin O-deethylation activity catalyzed by CYP1A1, CYP1A2 and CYP1B1 in a concentration-dependent manner (IC50 = 0.56, 0.44, and 0.11 µM, respectively). Kinetic analysis showed that DHTI acted as a competitive type of inhibitor of CYP1A1 and CYP1B1, whereas it noncompetitively inhibited CYP1A2. The observed enzyme inhibition was independent of NADPH and time. Molecular docking analysis revealed hydrogen bonding interactions between DHTI and Asp-326 of CYP1B1. Moreover, DHTI displayed preferential activity to inhibit 4-hydroxylation of E2 (a genotoxic pathway) mediated by CYP1B1. Exposure to DHTI could reduce the risk of genotoxicity induced by E2. SIGNIFICANCE STATEMENT: CYP1A1, CYP1A2, and CYP1B1 enzymes are involved in the conversion of estradiol (E2) into 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) through oxidation. 2-OHE2 is negatively correlated with breast cancer risk, and 4-OHE2 may be a significant initiator and promoter of breast cancer. The present study revealed that dihydrotanshinone I (DHTI) competitively inhibits CYP1A1/CYP1B1 and noncompetitively inhibits CYP1A2. DHTI exhibits a preference for inhibiting the genotoxicity associated with E2 4-hydroxylation pathway mediated by CYP1B1, potentially reducing the risk of 4-OHE2-induced genotoxicity.

AhR and CYP1B1 Control Oxygen Effects on Bone Marrow Progenitor Cells: The Enrichment of Multiple Olfactory Receptors as Potential Microbiome Sensors.

Polycyclic aromatic hydrocarbon (PAH) pollutants and microbiome products converge on the aryl hydrocarbon receptor (AhR) to redirect selective rapid adherence of isolated bone marrow (BM) cells. In young adult mice, Cyp1b1-deficiency and AhR activation by PAH, particularly when prolonged by Cyp1a1 deletion, produce matching gene stimulations in these BM cells. Vascular expression of Cyp1b1 lowers reactive oxygen species (ROS), suppressing NF-κB/RelA signaling. PAH and allelic selectivity support a non-canonical AhR participation, possibly through RelA. Genes stimulated by Cyp1b1 deficiency were further resolved according to the effects of Cyp1b1 and Cyp1a1 dual deletions (DKO). The adherent BM cells show a cluster of novel stimulations, including select developmental markers; multiple re-purposed olfactory receptors (OLFR); and α-Defensin, a microbial disruptor. Each one connects to an enhanced specific expression of the catalytic RNA Pol2 A subunit, among 12 different subunits. Mesenchymal progenitor BMS2 cells retain these features. Cyp1b1-deficiency removes lymphocytes from adherent assemblies as BM-derived mesenchymal stromal cells (BM-MSC) expand. Cyp1b1 effects were cell-type specific. In vivo, BM-MSC Cyp1b1 expression mediated PAH suppression of lymphocyte progenitors. In vitro, OP9-MSC sustained these progenitors, while Csf1 induced monocyte progenitor expansion to macrophages. Targeted Cyp1b1 deletion (Cdh5-Cre; Cyp1b1fl/fl) established endothelium control of ROS that directs AhR-mediated suppression of B cell progenitors. Monocyte Cyp1b1 deletion (Lyz2-Cre; Cyp1b1fl/fl) selectively attenuated M1 polarization of expanded macrophages, but did not enhance effects on basal M2 polarization. Thus, specific sources of Cyp1b1 link to AhR and to an OLFR network to provide BM inflammatory modulation via diverse microbiome products.

Design and Synthesis of Novel Near-Infrared Fluorescence Probes Based on an Open Conformation of a Cytochrome P450 1B1 Complex for Molecular Imaging of Colorectal Tumors.

Cytochrome P450 1B1 (CYP1B1) is induced during the early stage of cancer and is universally overexpressed in tumors. Thus, it was considered as a potential biomarker for the monitoring of cancer. In this study, we designed and synthesized CYP1B1-targeted near-infrared (NIR) fluorescence molecular probes based on the latest reported open conformation of the CYP1B1-α-naphthoflavone (ANF) complex. According to the architecture of the open channel, we introduced linkers and a Cy5.5 fragment at the 5' position of ANF derivatives with strong CYP1B1 inhibitory activity to obtain probes 19-21. Then, in vitro cell-based studies showed that the probes could be enriched in tumor cells by binding to CYP1B1. During in vivo and ex vivo imaging in a xenograft mouse model, probe 19 with the best binding affinity was proven to be able to identify tumor sites in both fluorescence imaging and photoacoustic imaging modes.

Differential Effect of ITE on the Aryl Hydrocarbon Receptor Signaling in Breast Cancer Tissue: A Histopathological Study / Efecto Diferencial de ITE en la Señalización del Receptor de Hidrocarburo de Arilo en Tejido de Cáncer de Mama: Un Estudio Histopatológico

SUMMARY: Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in various types of cancers including breast cancer. However, the role of AhR with its endogenous ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on the progression of breast cancer remains poorly understood. We aimed to investigate cell proliferation and migration states in breast cancer after activating AhR with the endogenous ligand ITE. Breast cancer tissue was evaluated by cell lines, immunohistochemistry, reverse transcription-polymerase chain reaction, cell proliferation, flow cytometry, migration assays and western blot techniques. We found that AhR was widely expressed in breast cancer tissues and metastasis lymph node tissues, but not in normal tissues. The expression AhR was independent between the age, grades and TNM classifications for breast cancer tissues. ITE treatment significantly induced the activation of AhR in a time-dependent manner in both MCF-7 and T47D breast cancer cell lines. Meanwhile, ITE did not affect the cell migration but significantly suppressed the cell proliferation in estrogen receptor positive (ER+) MCF-7 andT47D cells, which probably attribute to the induction of cell cycle arrest in G1 phase and shortened S phase. Further mechanism study showed that ERK1/2 and AKT signaling were required for the activation of AhR in MCF-7 cells. These data suggest that AhR is a potential new target for treating patients with breast cancer. ITE may be more potentially used for therapeutic intervention for breast cancer with the kind of ER(+).

El receptor de hidrocarburo de arilo (AhR) es un factor de transcripción activado por ligando que se expresa en gran medida en varios tipos de cáncer, incluido el cáncer de mama. Sin embargo, el papel de AhR con su ligando endógeno 2- (1'H-indol-3'-carbonil)-tiazol-4-ácido carboxílico metil éster (ITE) en la progresión del cáncer de mama sigue siendo poco conocido. Nuestro objetivo fue investigar la proliferación celular y los estados de migración en el cáncer de mama después de activar AhR con el ligando endógeno ITE. El tejido de cáncer de mama se evaluó mediante líneas celulares, inmunohistoquímica, reacción en cadena de la polimerasa con transcriptasa inversa, proliferación celular, citometría de flujo, ensayos de migración y técnicas de transferencia Western. Descubrimos que AhR se expresó ampliamente en tejidos de cáncer de mama y en linfonodos con metástasis, pero no en tejidos normales. La expresión AhR fue independiente entre la edad, grados y clasificaciones TNM para tejidos de cáncer de mama. El tratamiento con ITE indujo significativamente la activación de AhR de manera dependiente del tiempo en las líneas celulares de cancer de mama MCF-7 y T47D. Mientras tanto, ITE no afectó la migración celular, pero suprimió significativamente la proliferación celular en células MCF-7 y T47D con receptor de estrógeno positivo (ER+), lo que probablemente se atribuye a la inducción de la detención del ciclo celular en la fase G1 y la fase S acortada. Un estudio adicional del mecanismo mostró que las señales de ERK1/2 y AKT eran necesarias para la activación de AhR en las células MCF-7. Estos datos sugieren que AhR es un nuevo objetivo potencial para el tratamiento de pacientes con cáncer de mama. ITE puede ser utilizado más potencialmente en la intervención terapéutica para el cáncer de mama con el tipo de ER (+).

Genotyping of the rs1800440 Polymorphism in CYP1B1 Gene and the rs9258883 Polymorphism in HLA-B Gene in a Spanish Cohort of 223 Patients with Frontal Fibrosing Alopecia.

The pathogenesis of frontal fibrosing alopecia has been linked to specific genetic variants. CYP1B1 codes for a component of the cytochrome p450 machinery that is involved in the metabolism of xenobiotic oestrogens. The study of the prevalence of polymorphisms in this gene may help to understand their role in the development of frontal fibrosing alopecia. The aim of this study is to describe the frequency of genetic variations in the alleles HLA-B*07:02 and CYP1B1 in patients with frontal fibrosing alopecia. A cross-sectional study was designed to evaluate blood samples from patients with frontal fibrosing alopecia who attended the Dermatology Department at University Hospital Ramón y Cajal (Madrid, Spain), in search of the polymorphisms rs9258883 and rs1800440 in the alleles HLA-B*07:02 and CYP1B1, respectively. A total of 223 patients were included in the study. Among the 83.8% of patients who carried the rs9258883 polymorphism in HLA-B*07:02, 58.7% were heterozygous for this variant and it was not present in 14.8% of the cases. The majority of patients with frontal fibrosing alopecia lacked the protective rs1800440 polymorphism in CYP1B1 (75.2%). This suggests a relevant role of this variant in development of frontal fibrosing alopecia. The genetic approach to this condition might influence patient prognosis and therapy options.

FOXO1-regulated lncRNA CYP1B1-AS1 suppresses breast cancer cell proliferation by inhibiting neddylation.

PURPOSE: Overactivated neddylation is considered to be a common event in cancer. Long non-coding RNAs (lncRNAs) can regulate cancer development by mediating post-translational modifications. However, the role of lncRNA in neddylation modification remains unclear. METHODS: LncRNA cytochrome P450 family 1 subfamily B member 1 antisense RNA 1 (CYP1B1-AS1) expression in breast cancer tissues was evaluated by RT-PCR and TCGA BRCA data. Gain and loss of function experiments were performed to explore the role of CYP1B1-AS1 in breast cancer cell proliferation and apoptosis in vitro and in vivo. Luciferase assay, CHIP-qPCR assay, transcriptome sequencing, RNA-pulldown assay, mass spectrometry, RIP-PCR and Western blot were used to investigate the regulatory factors of CYP1B1-AS1 expression and the molecular mechanism of CYP1B1-AS1 involved in neddylation modification. RESULTS: We found that CYP1B1-AS1 was down-regulated in breast cancer tissues and correlated with prognosis. In vivo and in vitro functional experiments confirmed that CYP1B1-AS1 inhibited cell proliferation and induced apoptosis. Mechanistically, CYP1B1-AS1 was regulated by the transcription factor, forkhead box O1 (FOXO1), and could be upregulated by inhibiting the PI3K/FOXO1 pathway. Moreover, CYP1B1-AS1 bound directly to NEDD8 activating enzyme E1 subunit 1 (NAE1) to regulate protein neddylation. CONCLUSION: This study reports for the first time that CYP1B1-AS1 inhibits protein neddylation to affect breast cancer cell proliferation, which provides a new strategy for the treatment of breast cancer by lncRNA targeting neddylation modification.

ALKBH5 facilitates CYP1B1 mRNA degradation via m6A demethylation to alleviate MSC senescence and osteoarthritis progression.

Improving health and delaying aging is the focus of medical research. Previous studies have shown that mesenchymal stem cell (MSC) senescence is closely related to organic aging and the development of aging-related diseases such as osteoarthritis (OA). m6A is a common RNA modification that plays an important role in regulating cell biological functions, and ALKBH5 is one of the key m6A demethylases. However, the role of m6A and ALKBH5 in MSC senescence is still unclear. Here, we found that the m6A level was enhanced and ALKBH5 expression was decreased in aging MSCs induced by multiple replications, H2O2 stimulation or UV irradiation. Downregulation of ALKBH5 expression facilitated MSC senescence by enhancing the stability of CYP1B1 mRNA and inducing mitochondrial dysfunction. In addition, IGF2BP1 was identified as the m6A reader restraining the degradation of m6A-modified CYP1B1 mRNA. Furthermore, Alkbh5 knockout in MSCs aggravated spontaneous OA in mice, and overexpression of Alkbh5 improved the efficacy of MSCs in OA. Overall, this study revealed a novel mechanism of m6A in MSC senescence and identified promising targets to protect against aging and OA.

Hydration and Structural Adaptations of the Human CYP1A1, CYP1A2, and CYP1B1 Active Sites by Molecular Dynamics Simulations.

Cytochromes CYP1A1, CYP1A2, and CYP1B1, the members of the cytochrome P450 family 1, catalyze the metabolism of endogenous compounds, drugs, and non-drug xenobiotics which include substances involved in the process of carcinogenesis, cancer chemoprevention, and therapy. In the present study, the interactions of three selected polymethoxy-trans-stilbenes, analogs of a bioactive polyphenol trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene) with the binding sites of CYP1 isozymes were investigated with molecular dynamics (MD) simulations. The most pronounced structural changes in the CYP1 binding sites were observed in two substrate recognition sites (SRS): SRS2 (helix F) and SRS3 (helix G). MD simulations show that the number and position of water molecules occurring in CYP1 APO and in the structures complexed with ligands are diverse. The presence of water in binding sites results in the formation of water-protein, water-ligand, and bridging ligand-water-protein hydrogen bonds. Analysis of the solvent and substrate channels opening during the MD simulation showed significant differences between cytochromes in relation to the solvent channel and the substrate channels 2c, 2ac, and 2f. The results of this investigation lead to a deeper understanding of the molecular processes that occur in the CYP1 binding sites and may be useful for further molecular studies of CYP1 functions.

Mitochondrial cytochrome P450 1B1 is involved in pregnenolone synthesis in human brain cells.

Neurosteroids, which are steroids synthesized by the nervous system, can exert neuromodulatory and neuroprotective effects via genomic and nongenomic pathways. The neurosteroid and major steroid precursor pregnenolone has therapeutical potential in various diseases, such as psychiatric and pain disorders, and may play important roles in myelination, neuroinflammation, neurotransmission, and neuroplasticity. Although pregnenolone is synthesized by CYP11A1 in peripheral steroidogenic organs, our recent study showed that pregnenolone must be synthesized by another mitochondrial cytochrome P450 (CYP450) enzyme other than CYP11A1 in human glial cells. Therefore, we sought to identify the CYP450 responsible for pregnenolone production in the human brain. Upon screening for CYP450s expressed in the human brain that have mitochondrial localization, we identified three enzyme candidates: CYP27A1, CYP1A1, and CYP1B1. We found that inhibition of CYP27A1 through inhibitors and siRNA knockdown did not negatively affect pregnenolone synthesis in human glial cells. Meanwhile, treatment of human glial cells with CYP1A1/CYP1B1 inhibitors significantly reduced pregnenolone production in the presence of 22(R)-hydroxycholesterol. We performed siRNA knockdown of CYP1A1 or CYP1B1 in human glial cells and found that only CYP1B1 knockdown significantly decreased pregnenolone production. Furthermore, overexpression of mitochondria-targeted CYP1B1 significantly increased pregnenolone production under basal conditions and in the presence of hydroxycholesterols and low-density lipoprotein. Inhibition of CYP1A1 and/or CYP1B1 via inhibitors or siRNA knockdown did not significantly reduce pregnenolone synthesis in human adrenal cortical cells, implying that CYP1B1 is not a major pregnenolone-producing enzyme in the periphery. These data suggest that mitochondrial CYP1B1 is involved in pregnenolone synthesis in human glial cells.

Thrombospondin Mutations and Patients With Primary Congenital Glaucoma in a United States Population.

Mutations in the thrombospondin 1 ( THBS1 ) gene have been previously reported in primary congenital glaucoma (PCG) pedigrees that exhibit autosomal dominant inheritance with low penetrance. We sought to determine the role of THBS1 mutations in a cohort of 20 patients with PCG and 362 normal controls from Iowa using a combination of Sanger sequencing and whole exome sequencing. We detected 16 different THBS1 variants, including 4 rare, nonsynonymous variants (p.Thr611Met, p.Asn708Lys, p.Gln1089His, and p.Glu1166Lys). However, none of these variants were judged to be disease-causing mutations based on: 1) prevalence in cases and controls from Iowa, 2) prevalence in the public database gnomAD, 3) mutation analysis algorithms, and 4) THBS1 DNA sequence conservation. These results indicate THBS1 mutations are not a common cause of PCG in patients from Iowa and may be a rare cause of PCG overall.