OBJECTIVETo screen active components in Compound Danshen (CD) based on pregnane X receptor-cytochrome P450 3A4 (PXR-CYP3A4).

METHODSBy using PXR-CYP3A stable transfection human hepatoblastoma G2 (HepG2) cell lines engineering cell strain combined reporter genes technology, active components that induce or inhibit PXR-CYP3A4 paths in CD were screened, and confirmed at the level of enzymic activities. The experiment was divided into the positive control group (RIF 10 micro mol/L), the DMSO group (DMSO 0.1%), each dose of treatment groups (ginsenoside Rc, Rf, Rb2, Rg2, F2, F1, tanshinone I , isoborneol 5, 10, 25, 50, 100, and 200 micro mol/L; each with six duplicates). Cells medium was removed 36, 48, and 60 h after treatment. The activity of CYP3A4 was then determined in the supernant and the fold induction was calculated.

RESULTSCompared with the DMSO group, the fold induction increased when ginsenoside Rc, Rf, Rb2, Rg2, F2, F1, tanshinone I , and isoborneol 50 and 100 micro mol/L was respectively intervened for 36, 48, and 60 h (P <0.05). When cells were treated with isoborneol 200 micro mol/L for 48 and 60 h,the fold induction of ginsenoside Rb2, Rg2, and F1 was significantly higher than that of the RIF group (P <0.05). Enzymic activity results showed that ginsenoside Rc, Rf, Rb2, F2, and F1 could increase the enzyme activity of CYP3A4 at 48 h (P <0.05).

CONCLUSIONGinsenoside Rc, Rf, Rb2, F2, F1, tanshinone I, and isoborneol in DC could induce CYP3A4 enzymes.

Cytochrome P-450 CYP3A ; metabolism ; Diterpenes, Abietane ; Drugs, Chinese Herbal ; chemistry ; Genes, Reporter ; Ginsenosides ; metabolism ; Hep G2 Cells ; Humans ; Receptors, Steroid ; metabolism ; Salvia miltiorrhiza ; Transfection

In this study, we investigated the role of Nur77, an orphan nuclear receptor, in HIF-alpha transcriptional activity. We found that Nur77 associates and stabilizes HIF-1alpha via indirect interaction. Nur77 was found to interact with pVHL in vivo via the alpha-domain of pVHL. By binding to pVHL, Nur77 competed with elongin C for pVHL binding. Moreover, Nur77-binding to pVHL inhibited the pVHL-mediated ubiquitination of HIF-1alpha and ultimately increased the stability and transcriptional activity of HIF-1alpha. The ligand-binding domain of Nur77 was found to interact with pVHL and the expression of this ligand-binding domain was sufficient to stabilize and transactivate HIF-1alpha. Under the conditions that cobalt chloride was treated or pVHL was knocked down, Nur77 could not stabilize HIF-alpha. Moreover, Nur77 could not further stabilize HIF-2alpha in A498/VHL stable cells, which is consistent with our finding that Nur77 indirectly stabilizes HIF-alpha by binding to pVHL. Thus, our results suggest that an orphan nuclear receptor Nur77 binds to pVHL, thereby stabilizes and increases HIF-alpha transcriptional activity under the non- hypoxic conditions.
Animals ; DNA-Binding Proteins/chemistry/*metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics ; Models, Biological ; PC12 Cells ; Protein Binding ; *Protein Processing, Post-Translational ; Protein Structure, Tertiary ; Rats ; Receptors, Cytoplasmic and Nuclear/chemistry/*metabolism ; Receptors, Steroid/chemistry/*metabolism ; Thermodynamics ; Transcription Factors/chemistry/*metabolism ; Transcriptional Activation/genetics ; Ubiquitination ; Up-Regulation/*genetics ; Von Hippel-Lindau Tumor Suppressor Protein/*antagonists & inhibitors/chemistry/*metabolism

OBJECTIVETo define the effect of Curculiginis Rhizoma and its active ingredient orcinol glucoside on PXR-CYP3A of L02 cells in normal and deficiency-cold states, in order to lay a foundation for studies on the mechanism of efficacy expression differentiation of Curculiginis Rhizoma in different states.

METHODSerums of normal and deficiency-cold rats were adopted to culture L02 cells and induce cells in normal and deficiency-cold states. After aqueous extracts from Curculiginis Rhizoma and its active ingredient orcinol glucoside were used in cells in different states, PXR protain expression and CYP3A activity of L02 cells in normal and deficiency-cold states were observed.

RESULTMTT results showed that aqueous extracts from Curculiginis Rhizoma and orcinol glucoside could significantly enhance viability of L02 cells. Aqueous extracts from Curculiginis Rhizoma could significantly reduce PXR protein expression of L02 cells in normal state, while orcinol glucoside could significantly reduce CYP3A activity and PXR protein expression of L02 cells in normal state. Meanwhile, aqueous extracts from Curculiginis Rhizoma could significantly increase CYP3A activity and PXR protein expression of L02 cells in deficiency-cold state, while orcinol glucoside could significantly reduce CYP3A activity and increase PXR protein expression of L02 cells in deficiency-cold state.

CONCLUSIONCurculiginis Rhizoma can activate PXR and induce CYP3A activity of L02 cells in deficiency-cold state, but with no effect or even counteraction on PXR and its induced CYP3A of L02 cells in normal state.

Animals ; Cell Line ; Cytochrome P-450 CYP3A ; metabolism ; Gene Expression ; drug effects ; genetics ; Glucosides ; pharmacology ; Humans ; Male ; Plant Extracts ; chemistry ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Steroid ; metabolism ; Resorcinols ; pharmacology ; Rubiaceae ; chemistry

It is essential for a successful drug to possess two basic characteristics: satisfactory pharmacological action with sufficient potency and selectivity; good druggability with eligible physicochemical, pharmacokinetic and safety profiles, as well as structural novelty. Promiscuity is defined as the property of a drug to act with multiple molecular targets and exhibit distinct pharmacological effects. Promiscuous drugs are the basis of polypharmacology and the causes for side effects and unsuitable DMPK. Drug promiscuity originates from protein promiscuity. In order to accommodate, metabolize and excrete various endo- and exogenous substances, protein acquired the capability during evolution to adapt a wide range of structural diversity, and it is unnecessary to reserve a specific protein for every single ligand. The structures of target proteins are integration of conservativity and diversity. The former is represented by the relatively conservative domains for secondary structures folding, which leads to overlapping in ligand-binding and consequent cross-reactivity of ligands. Diversity, however, embodies the subtle difference in structures. Similar structural domain may demonstrate different functions due to alteration of amino acid sequences. The phenomenon of promiscuity may facilitate the "design in" of multi-target ligands for the treatment of complicated diseases, whereas it should be appropriately handled to improve druggability. Therefore, one of the primary goals in drug design is to scrutinize and manipulate the "merits and faults" of promiscuity. This review discusses the application of promiscuity in drug design for receptors, enzymes, ion channels and cytochrome P450. It also briefly describes the methods to predict ligand promiscuity based on either target or ligand structures.
Cytochrome P-450 Enzyme System ; chemistry ; Drug Design ; Drug Discovery ; Drug Resistance, Multiple ; Drug-Related Side Effects and Adverse Reactions ; Enzyme Inhibitors ; chemistry ; Ion Channels ; chemistry ; Ligands ; Pharmaceutical Preparations ; chemistry ; metabolism ; Pharmacokinetics ; Pharmacology ; Protein Binding ; Protein Conformation ; Receptors, G-Protein-Coupled ; chemistry ; Receptors, Steroid ; agonists ; antagonists & inhibitors

OATP1B3, a member of SLC superfamily, is specifically expressed on the sinusoidal membrane of hepatocytes and is considered to be important in hepatic drug elimination. The overexpression of OATP1B3 was found recently in tumor tissues such as prostate, colon, and pancreatic tumors. Sequence variations in SLCO1B3 gene, such as SNPs, have been described and a common haplotype consisting of 334T>G and 699G>A SNPs is related to altered transport characteristics of OATP1B3. OATP1B3 is of relevance to drug metabolism through affecting alteration of hepatic concentration of endo- and xenobiotic compounds that interact with nuclear receptors such as PXR and CAR, and thereby directly alter the extent of target gene transcription, including major CYP isoenzymes such as CYP3A4. This review will provide an overview of substrates and inhibitors of OATP1B3 and subsequently to assess the effect of genetic mutation on transport activity. The studies linking OATP1B3 with cancer clinical outcomes are also discussed in this review.
Animals ; Biological Transport ; Cytochrome P-450 CYP3A ; metabolism ; Drug Interactions ; Gene Expression Regulation, Neoplastic ; Gene Frequency ; Hepatocytes ; metabolism ; Humans ; Liver ; metabolism ; Neoplasms ; metabolism ; Organic Anion Transporters, Sodium-Independent ; antagonists & inhibitors ; chemistry ; genetics ; Polymorphism, Single Nucleotide ; RNA, Messenger ; metabolism ; Receptors, Cytoplasmic and Nuclear ; metabolism ; Receptors, Steroid ; metabolism ; Solute Carrier Organic Anion Transporter Family Member 1B3

OBJECTIVETo test the effect on human pregnane X receptor (hPXR)-mediated transcription regulation of CYP3A4 by five selected phytochemicals.

METHODSTransient cotransfection reporter gene assays in HepG(2) cells were performed with the hPXR expression plasmid and the reporter gene plasmid which contains XRE in the promoter of CYP3A4 linked to luciferase.

RESULTSIn the dose-effect study, soybean isoflavone, luteolin and curcumin induced the CYP3A4 transcription via PXR in an evident dose-dependent manner, but isorhamnetin and rutin did not. The inducibility of soybean isoflavone, luteolin and curcumin was also increased in concentrations between 1 micromol/L and 50 micromol/L, 24 h after induction, 50 micromol/L soybean isoflavone, luteolin and curcumin exhibited a 5.46-fold, 2.87-fold, and 2.07-fold increase respectively, compared with 0.1% DMSO treated cells. In the time-effect study, 10 micromol/L and 50 micromol/L soybean isoflavone, luteolin and curcumin induced CYP3A4 transcription between 12 h and 48 h, the strongest induction appeared in 48 h. 48 h after induction, 50 micromol/L soybean isoflavone, luteolin and curcumin exhibited a 6.72-fold, 3.24-fold, and 2.13-fold increase respectively, compared with 0.1% DMSO treated cells.

CONCLUSIONThree phytochemicals, i.e. soybean isoflavone, luteolin and curcumin stimulate the PXR-mediated transcription of CYP3A4. Isorhamnetin and rutin have no effect on the CYP3A4 transcription via PXR.

Carcinoma, Hepatocellular ; pathology ; Curcumin ; pharmacology ; Cytochrome P-450 CYP3A ; Cytochrome P-450 Enzyme System ; biosynthesis ; genetics ; Humans ; Isoflavones ; pharmacology ; Liver Neoplasms ; pathology ; Luteolin ; pharmacology ; Plant Preparations ; pharmacology ; Receptors, Steroid ; metabolism ; Soybeans ; chemistry ; Transcription, Genetic ; Transfection ; Tumor Cells, Cultured

This paper is to report the development of a high-throughput in vitro system to screen hPXR/CAR mediated CYP2B6 drug inducers, and the application of it into the quick determination of induction activity toward CYP2B6 by various commonly used traditional Chinese medicines (TCMs) extract. Dual reporter gene assays were performed. The hPXR/CAR expression vectors and the reporter vector pGL3-CYP2B6-Luc involved in the distal and proximal promoters of CYP2B6 were co-transfected into HepG2 cells. Relative luciferase activities in cell lysate were analyzed after 48 h treatment of blank vehicle or drugs to determine the induction activity toward CYP2B6 by various commonly used TCMs extract. The positive hPXR/hCAR activators rifampicin and CITCO were applied to make sure that the reporter gene model was successfully established. Then 5 kinds of commonly used TCM extracts and 1 herbal compound were successfully investigated, some were found to activate hPXR or hCAR and therefore have the potential to induce CYP2B6 enzyme. This is the first domestic article to report the hCAR3-mediated CYP2B6 induction model and the establishment of a reporter gene system for hPXR/CAR-mediated CYP2B6 induction can be an effective and systemic in vitro method to investigate the drug inducers of CYP2B6 and to explain the mechanism involved.
Aryl Hydrocarbon Hydroxylases ; genetics ; metabolism ; Cytochrome P-450 CYP2B6 ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Genes, Reporter ; Genetic Vectors ; Hep G2 Cells ; High-Throughput Screening Assays ; Humans ; Luciferases ; genetics ; metabolism ; Oximes ; pharmacology ; Plants, Medicinal ; chemistry ; Plasmids ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Receptors, Steroid ; genetics ; metabolism ; Rifampin ; pharmacology ; Thiazoles ; pharmacology ; Transfection

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.
Animals ; Chemical and Drug Induced Liver Injury ; drug therapy ; enzymology ; Cytochrome P-450 CYP3A ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Liver ; drug effects ; enzymology ; metabolism ; Male ; Milk Thistle ; chemistry ; Pregnane X Receptor ; Rats ; Rats, Sprague-Dawley ; Receptors, Steroid ; genetics ; metabolism ; Signal Transduction ; drug effects ; Silybin ; Silymarin ; administration & dosage ; Thioacetamide ; adverse effects