PXR: a center of transcriptional regulation in cancer

Pregnane X receptor (PXR, NR1I2) is a prototypical member of the nuclear receptor superfamily. PXR can be activated by both endobiotics and xenobiotics. As a key xenobiotic receptor, the cellular function of PXR is mostly exerted by its binding to the regulatory gene sequences in a ligand-dependent manner. Classical downstream target genes of PXR participate in xenobiotic responses, such as detoxification, metabolism and inflammation. Emerging evidence also implicates PXR signaling in the processes of apoptosis, cell cycle arrest, proliferation, angiogenesis and oxidative stress, which are closely related to cancer. Here, we discussed, in addition to the characterization of PXR , the biological function and regulatory mechanism of PXR signaling in cancer, and its potential for the targeted prevention and therapeutics.

UHPLC-MS-MS high throughput analysis-based screening and evaluation of PXR/CYP3A4-induced lipid-regulating quality marker in propolis / 中草药

Objective: To screen and evaluate PXR/CYP3A4-induced lipid-regulating quality marker in propolis with precise and quantitative method. Methods: The LS174T cell was given certain amount of midazolam injection, along with different dosage of known components found in propolis, after incubation and extraction, the samples were determined for 1’-OH-midazolam, and each compound was evaluated to discover the PXR/CYP3A4 pathway regulatory activity according to the results; Then, compounds selected were used as indexes for UHPLC-MS-MS content determination, and their own values were regarded as a preliminary step of confirming PXR/CYP3A4-induced lipid-regulating quality markers of propolis. Results: In all components tested, chrysin, galangin, heterochlorogenic acid A, quercetin, and caffeic acid phenethylester significantly affected the 1’-OH-midazolam yield compared with blank and positive control, indicating their obvious influence on PXR/CYP3A4 expression; The UHPLC-MS-MS determination showed that except galangin, heterochlorogenic acid A, and quercetin, all the other compounds had adequate content in propolis to take effect. Conclusion: Chrysin, galangin, caffeic acid phenethylester, and quercetin were probably defined as PXR/CYP3A4-induced lipid-regulating quality marker in propolis, which inhibited the expression of such targets to down-regulate blood lipid level; Additionally, the method used for quality marker screening and evaluation in this study was fast, effective and quantitative, and capable of carrying out high throughput active component screening for PXR/CYP3A4 regulatory activities.

Effect of six components in Polygoni Multiflori Radix on regulation of CYP3A4 mediated by human pregnane X receptor / 中国中药杂志

This paper aimed to study the six chemical components of Polygoni Multiflori Radix (gallic acid, quercetin, luteolin, kaempferol, resveratrol, apigenin). By the established pregnane X receptor (human pregnant X receptor, PXR) CYP3A4 mediated drug induced rapid screening technique, the effect of chemical components on the cell activity was detected by MTS cell method, and the value of IC₅₀ was calculated. The dual luciferase reporter system was used to co-transfect PXR reporter gene expression vector containing transcriptional regulation and CYP3A4 with HepG2 cells, with 10 μmol·L⁻¹ rifampicin (RIF) as a positive control, and 10 μmol·L⁻¹ of ketoconazole (TKZ) as negative control. Gallic acid, quercetin, luteolin, kaempferol, apigenin, resveratrol(5, 10, 20 μmol·L⁻¹) were used to incubate for 24 h, and the luciferase activity was detected. The results showed that when plasmid pcDNA3.1 was co-transfected with pGL4.17-CYP3A4, gallic acid and resveratrol had an inhibitory effect on the regulation of CYP3A4, and quercetin, luteolin, kaempferol had an inductive effect on CYP3A4; when pcDNA3.14-PXR was co-transfected with pGL4.17-CYP3A4, quercetin, luteolin, kaempferol, apigenin, resveratrol had an inductive effect. To sum up, the 6 reported liver injury components had inhibitory or activating effects on CYP3A4. After PXR plasmid was involved, 5 components had an inductive effect on CYP3A4, and the inductive effects of 2 components were significantly different. In this experiment, we found that 2 kinds of potential liver injury components in Polygoni Multiflori Radix had been induced by CYP3A4, which was achieved through PXR regulation. It suggested that attention shall be paid to potential drug interactions when combined with Polygoni Multiflori Radix, so as to improve the safety and efficacy.

Effect of Gehua Jiejue Dizhi decoction on the liver fatty deposition and expression of PXR in themousealcoholic fatty liver / 中国比较医学杂志

Objective To explore the effect of a herbalcompound Gehua Jiejue Dizhi Decoction (GJDD) on the liver fat deposition and the expression of PXR, and the mRNA and protein expression of its target genes CYP3A11 and CYP3A25in the liver tissues of mouse models of alcoholic fatty liver.Methods Twenty-nine healthy male C57BL/6J mice were randomly divided into control group (n=5), model group (n=8), high dose GJDD group (n=8)and low dose GJDD group (n=8).The mouse model of alcoholic fatty liver was prepared according to the National Institute on Alcohol Abuse and Alcoholism (NIAAA) method.Then, the mice were treated with the high dose and low dose GJDD for 9 days.Serum glutamic-pyruvic transaminase (AST) and aspartate aminotransferase (AST) were detected by enzyme-linked immunosorbent assay (ELISA).Liver fat deposition was detected by oil red O staining.Real-time RT-PCR and immunohistochemistry were performed to examine the expressions of PXR, CYP3A11 and CYP3A25.Results Compared with the model group, the liver fat deposition in the intervention groups was significantly reduced in a dose-dependent manner, with a significant increase of the expression of PXR and CYP3A25 (P < 0.01).The serum ALT level was significantly reduced in the model group (P < 0.01), while the transcriptional levels of CYP3A11 mRNA in the groups were similar (P ≥ 0.05).Conclusions Gehua Jiejue Dizhi Decoction has obvious therapeutic effect on the AFLD in mice, which may be related to the activation of PXR and its target genes CYP3A25.

Transcriptional regulation effect of THSG and anthraquinones in tubers of Polygonum multiflorum based on human progesterone X receptor (PXR) mediated CYP3A4 rapid screening system / 中国中药杂志

The rapid screening technology was used to investigate the transcriptional regulation effect of main chemical constituents in tubers of Polygonum multiflorum, including 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucopyranoside(THSG) and anthraquinones (such as rhein, chrysophanol, aloe-emodin, emodin) on CYP3A4 drug inducers induced by human pregnancy X receptor (PXR).The effect of chemical composition on the cell activity was detected by MTS cell viability assay. IC₅₀ was calculated. The expression vector and the reporter vector were co-transfected into HepG2 cells, with 10 μmol•L⁻¹ rifampicin (RIF) as a positive control, and 10 μmol•L⁻¹ ketoconazole (TKZ) as a negative control. After treated with different concentrations of anthraquinones (2.5, 5, 10 μmol•L⁻¹) for 24 h, the cells were tested for dual luciferase activity. The results show that the inhibitory effect of THSG, chrysophanol, emodin, rhein and aloe-emodin on CYP3A4 was inhibited by co-transfection of pcDNA3.1 and pGL4.17-CYP3A4. The expressions of pcDNA3.14-PXR and pGL4.17-CYP3A4 were induced by the four compounds. Besides, emodin had a direct inducing effect. In conclusion, the four anthraquinone compounds have an inducing effect on CYP3A4 by PXR, but emodin can directly induce CYP3A4. THSG can inhibit CYP3A4, but plasmid can induce CYP3A4 after intervened with PXR.These results suggest that we should pay attention to the liver function and avoid liver damage in the combined administration of drugs.

Tanshinone IIA Protects Endothelial Cells from H₂O₂-Induced Injuries via PXR Activation

Tanshinone IIA (Tan IIA) is a pharmacologically active substance extracted from the rhizome of Salvia miltiorrhiza Bunge (also known as the Chinese herb Danshen), and is widely used to treat atherosclerosis. The pregnane X receptor (PXR) is a nuclear receptor that is a key regulator of xenobiotic and endobiotic detoxification. Tan IIA is an efficacious PXR agonist that has a potential protective effect on endothelial injuries induced by xenobiotics and endobiotics via PXR activation. Previously numerous studies have demonstrated the possible effects of Tan IIA on human umbilical vein endothelial cells, but the further mechanism for its exerts the protective effect is not well established. To study the protective effects of Tan IIA against hydrogen peroxide (H₂O₂) in human umbilical vein endothelial cells (HUVECs), we pretreated cells with or without different concentrations of Tan IIA for 24 h, then exposed the cells to 400 μM H₂O₂ for another 3 h. Therefore, our data strongly suggests that Tan IIA may lead to increased regeneration of glutathione (GSH) from the glutathione disulfide (GSSG) produced during the GSH peroxidase-catalyzed decomposition of H₂O₂ in HUVECs, and the PXR plays a significant role in this process. Tan IIA may also exert protective effects against H₂O₂-induced apoptosis through the mitochondrial apoptosis pathway associated with the participation of PXR. Tan IIA protected HUVECs from inflammatory mediators triggered by H₂O₂ via PXR activation. In conclusion, Tan IIA protected HUVECs against H₂O₂-induced cell injury through PXR-dependent mechanisms.

Association of Gene Polymorphism with Individualized Tacrolimus Dosage Regimen in Renal Transplant Recipients / 医药导报

Objective To offer a theory that supports the individualized tacrolimus dosage regimen by retrospectively investigating the influences of gene polymorphism and other clinical factors on tacrolimus concentration in renal transplant recipients. Methods A total of 280 renal transplant recipients were genotyped for CYP3A4?5, CYP3A4?6, CYP3A4?18B, CYP3A5?3, MDR1 1236C>T, MDR1 2677G>T/A, MDR1 3435C>T polymorphisms by PCR followed by restriction fragment length polymorphism (RFLP) analysis.PXR 6bp deletions (rs3842689) genotypes were determined by Allelic Special-Touch down PCR.Correlation between gene polymorphisms and tacrolimus concentrations was analyzed. Results The mutation frequency of CYP3A4?18B, CYP3A5?3, MDR1 1236C>T, 2677G>T/A, 3435C>T and PXR rs3842689 in the renal transplant recipients was 29.11%, 69.29%, 43.57%, 49.64%, 36.43% and 26.07%, respectively.Multiple regression analysis showed that, CYP3A5?3 and red blood cell count were associated with the value of C0/D of FK506, the best regression model was:D=C0/(-60.445 +95.777×CYP3A5 +34.938×RBC), and the equation could explain 38.8% of tacrolimus individual differences. Conclusion Gene polymorphism of CYP3A5?3 and red blood cell count may be responsible, in part, for the large interindividual variability of FK506 dose and concentration.

PXR Mediated Protection against Liver Inflammation by Ginkgolide A in Tetrachloromethane Treated Mice

The pregnane X receptor (PXR), a liver and intestine specific receptor,, has been reported to be related with the repression of inflammation as well as activation of cytochromosome P450 3A (CYP3A) expression. We examined the effect of PXR on tetrachloromethane (CCl4)-induced mouse liver inflammation in this work. Ginkgolide A, one main component of Ginkgo biloba extracts (GBE), activated PXR and enhanced PXR expression level, displayed both significant therapeutic effect and preventive effect against CCl4-induced mouse hepatitis. siRNA-mediated decrease of PXR expression significantly reduced the efficacy of Ginkgolide A in treating CCl4-induced inflammation in mice. Flavonoids, another important components of GBE, were shown anti-inflammatory effect in a different way from Ginkgolide A which might be independent on PXR because flavonoids significantly inhibited CYP3A11 activities in mice. The results indicated that anti-inflammatory effect of PXR might be mediated by enhancing transcription level of IkappaBalpha through binding of IkappaBalpha. Inhibition of NF-kappaB activity by NF-kappaB-specific suppressor IkappaBalpha is one of the potential mechanisms of Ginkgolide A against CCl4-induced liver inflammation.

Nuclear receptors as drug targets in cholestasis / 中国药理学通报

Drug for cholestasis therapy is extremely limited.Ur-sodeoxycholic acid is currently the only FDA approved drug to treat primary biliary cirrhosis,whereas its efficacy is limited to early stage of the disease.Therefore,developing novel drugs re-presents a major goal for both pharmaceutical industry and aca-demic researchers.Targeting nuclear receptors in cholestasis is an intriguing approach since these receptors are critically in-volved in the regulation of bile acid homeostasis.This review summarizes the roles of individual nuclear receptors in cholestasis and evaluates their potential clinical application.

Ginsenoside F1 induces CYP3 A4 expression through activation of human pregnane X receptor / 中国药理学通报

Aim To study the effect of ginsenoside F1 on the enzyme activity and expression of gene of CYP3 A4 through activation of pregnane X receptor ( PXR ) . Methods With different concentrations of ginsenoside F1 treated on LS174T cells, the expression of CYP3A4 mRNA was determined by Q-PCR, and the enzyme activity was measured by P450-GloTM CYP3A4 assay according to the manufacturer′s instructions, fur-ther PXR-CYP3 A4 stable translation HepG2 cell lines were used to test ginsenoside F1 activates PXR by re-porter gene screening assay. Results The results re-vealed that the levels of CYP3 A4 gene and protein ex-pression were significantly increased by ginsenoside F1 in a concentration-dependent manner. At the same time, reporter gene screening showed that ginsenoside F1 could also enhance the transcriptional activity of PXR. Conclusion Ginsenoside F1 can significantly up-regulate the gene expression and enzyme activity of CYP3A4 via the PXR-CYP3A4 pathway.

Establishment of cell models for PXR, FXR and LXRαagonists high-throughput screening based on reporter gene assay / 中国药理学通报

Aim To develop an in vitro high throughput drug screening system based on reporter gene assay for identification of novel compounds with PXR, FXR and LXRα agonist activity. Methods The expressions of exogenous PXR, FXR and LXRαgene in HEK293, HepG2 and LS174T cells were examined by Real-Time quantity PCR. pSG5-hPXR and pGL3-XREM-CYP3A4, pEGFP-N3-hFXR and EcRE-TK-Luc, pCMX-FLAG-hLXRα and pGL3-XREM-CYP3A4 were cotransfected into cells and the optimal ratio of three plasmids was determined. The dose-response relationship between the positive drug and the fold induction was determined. The specificity of the model was ex-amined, and the repeatability was also determined by Z′ value. Results ① The PXR, FXR and LXRα mRNA expression in HEK293 cell is low among three different cells. ②reporter gene vector and expression plasmid ratio of 1∶ 1, 2∶ 1 and 2∶ 1 were proved to be suitable for highest relative luciferase activity for PXR, FXR or LXRα agonist screening model. ③ The relative luciferase activity was induced by Rif, CDCA or T0901317 in a dose-dependent manner. ④Only Rif, CDCA or T0901317 could significantly increase the relative luciferase activity in PXR,FXR or LXRα agonist screening model, no effect of other nuclear re-ceptors agonist was observed, and the values of Z′-factor for PXR, FXR and LXRαagonist screening model were 0. 58, 0. 66 and 0. 63, respectively. Conclusion An in vitro PXR, FXR and LXRα agonist high-throughput screening models are devel-oped with acceptable specificity and repeatability, and the mod-els can be used to screen PXR, FXR and LXRα agonist.

Pregnane X Receptor(PXR) and Constitutive Androstane Receptor(CAR) in the Multidrug Resistance / 实用肿瘤学杂志

Chemotherapy is one of the three most common treatment modalities for cancer .However , chemotherapy as current firstline therapy induces significant side effects and limited efficacy ,leading to multidrug resistance and fast recurrence challenging the patient survival rate .Drug metabolizing enzymes ( DMEs) and efflux transporters promote the metabolism,clearance,and detoxification of chemotherapeutic agents .Nuclear receptors, especially pregnane X receptor (PXR,NR112)and constitutive androstane activated receptor (CAR,NR113),reg-ulate the expressions of target genes that could encode phase I DMEs ,phase II DMEs,and efflux transporters in the development of multidrug resistance ( MDR) during chemotherapy .Recent studies have revealed that PXR and CAR play pivotal roles in MDR of various human carcinomas .And their expressve levels or activation statuses could predict the risk of drug resistance in the patients subjected to chemotherapy .Accordingly ,PXR/CAR antag-onists,combining with existing chemotherapeutics that activate PXR /CAR,are promising options that could over-come MDR in cancer.

Research Progress in Effects of Herbal Medicines on PXR and CAR Nuclear Receptor Pathways / 中国药师

Pregnane X receptor ( PXR) , a member of the nuclear receptor subfamily, plays an important role in the regulation of drug metabolic enzymes and transporters. PXR can regulate the expression of downstream target genes through transforming a large number of exogenous and endogenous chemical substances, and can be activated by a variety of Chinese herbal medicines. The same as PXR, constitutive androstane receptor ( CAR) can participate in the regulation of drug metabolic enzyme CYP450 and become the targets of drug action through combining with exogenous ligands to regulate the expression of CYP2B6, CYP3A4, CYP2C19 and UGT1A1.

Pregnane X Receptor agonist Increases the Expression Levels of the Plasma Membrane Monoamine Transporter

We evaluated the effect of the pregnane X receptor agonist, pregnenolone 16 alpha-carbonitrile (PCN) on the expression levels of plasma monoamine transporter (PMAT) in the intestine. Male C57/BL6 mice were divided into two 2 groups: mice in the PCN group (n=3) were administered PCN once a day for 4 days, while those in the control group (n=3) received the same volume of vehicle once a day for 4 days. After the mice were killed 24 h after administration of the last dose of PCN or vehicle, and the expression levels of PMAT in the intestine tissues were isolated and measured the expression level of PMAT using immunohistochemical and western blotting analyses. The expression level of PMAT expression levels in the small intestine increased after PCN treatment. These results suggest that the induction of PMAT may play a clinically significant role by increasing intestinal absorption of PMAT substrates such as metformin.

Reversing effects of silybin on TAA-induced hepatic CYP3A dysfunction through PXR regulation / 中国天然药物

AIM@#Silybin (SB), a major constituent of the milk thistle, has been used to treat several liver disorders. However, liver diseases were always accompanied by CYP450 dysfunction. This study was designed to explore the relationship between the hepatoprotective effect and CYP3A regulation of SB during thioacetamide (TAA)-induced rat liver injury.@*METHODS@#Serum biochemical analysis and histopathological study were taken to evaluate the hepatoprotectinve effect of SB. α-SMA were detected by immunohistochemical analysis and cytokine release in rat liver was determined by ELISA assay. CYP3A and PXR expression were determined by RT-PCR and Western blot analysis, and CYP3A activity was based on the midazolam 4-hydroxylation reaction. Also, siRNA transfection was induced in HepG2 cells to evaluate the effect of PXR on cytotoxicity and CYP3A4 dysregulation caused by TAA.@*RESULTS@#SB showed powerful hepatoprotective effects, and anti-inflammatory and anti-fibrosis effects, and reversed the loss of CYP3A and PXR in TAA-injured rat liver, and decreased PXR translocation into the cell nucleus. PXR silencing weakened the effect of SB on cytoprotection and CYP3A regulation.@*CONCLUSIONS@#PXR was a very important factor of CYP3A regulation and might be the target of SB in TAA-induced liver disease. Also, because of the potential interactions of SB and co-administered medicines, it might be necessary to adjust the dosage in the clinical medication of liver disease.

Role of PXR and CAR in Cholestasis / 대한간학회지

Cholestatic liver diseases are characterized by impairments of bile flows and accumulations of biliary constituents such as bile acids and bilirubin. The changes of phase I and II metabolism and the hepatobiliary transport system minimize cholestatic liver injury. These adaptive responses are transcriptionally regulated by several nuclear receptors. Recent studies have revealed that the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are key nuclear receptors for regulating many of the adaptive responses noted in cholestasis. PXR and CAR coordinately regulate not only bile acid metabolism and transport, but also bilirubin clearance. PXR and CAR ligands may be useful in the future for the treatment of cholestatic liver disease.

Traditional Chinese medicine Rhizoma curcumae activates PXR and affects cytochrome P450 3A in the rat liver / 中国药理学通报

Aim To investigate whether Rhizoma curcumaecould induce pregnane X receptor(PXR)-mediated transcriptional expression of CYP3A4 and study the modulatory effects on the enzyme activity and mRNA expression of CYP3A in the rat liver.Methods Transient cotransfection reporter gene assays were performed in HepG2 cells;the rat liver microsomal cytochrome P450 and CYP3A isoenzyme-erythromycin N-demethylase(ERD)activities were determined by UV chromatography;the mRNA expression level of CYP3A was detected by reverse transcriptase-polymerase chain reaction(RT-PCR).Results In vitroinvestigation showed Rhizoma curcumaeand its four selected constituents could induce the CYP3A4 transcriptional expression by activating PXR.In vivoinvestigation showed the CYP450 content of liver microsomes and enzyme activity of CYP3A were markedly increased and induced by Rhizoma curcumaeextract;at the mRNA level, the expression of CYP3A1 and CYP3A2 gene were markedly induced by Rhizoma curcumaeextract.Conclusions Rhizoma curcumaeand its four selected constituents could induce the expression of the CYP3A4 gene transcriptional expression through activating PXR;Rhizoma curcumaeextract could increase and induce the enzyme activity and mRNA expression of CYP3A.