The traditional Chinese herbal remedy tian xian activates pregnane X receptor and induces CYP3A gene expression in hepatocytes

Abstract

The pregnane X receptor (PXR, NR1I2) is a member of the nuclear receptor superfamily that is activated by a myriad of clinically used compounds and natural products. Activation of PXR in liver regulates the expression genes encoding proteins that are intimately involved in the hepatic uptake, metabolism, and elimination of toxic compounds from our bodies. PXR-mediated herb-drug interactions can have undesirable effects in patients receiving combination therapy. This can be especially important in cancer patients who self-administer over-the-counter herbal remedies together with conventional anticancer chemotherapeutics. Tian xian is a traditional Chinese herbal anticancer remedy that activates human PXR in cell-based reporter gene assays. Moreover, tian xian alters the strength of interaction between the human PXR protein and transcriptional cofactor proteins. A novel line of humanized PXR mice are described and used here to show that tian xian increases expression of Cyp3a11 in primary cultures of rodent hepatocytes. Tian xian also induces expression of CYP3A4 in primary cultures of human hepatocytes. Taken together, these data indicate that coadministration of tian xian is probably contraindicated in patients undergoing anticancer therapy with conventional chemotherapeutic agents. These data are of particular importance due to the fact that this herbal remedy is currently marketed as an adjunct therapy that reduces the side effects of conventional chemotherapy and is available without a prescription. Future studies should be conducted to determine the extent to which coadministration of this Chinese herbal remedy alters the pharmacokinetic and pharmacodynamic properties of conventional anticancer therapy.

Figure 1. Tian Xian Induces PXR Activity in XREM-Luc Reporter Gene Assays

(A) CV-1 cells were transfected with the expression vector for human PXR and the CYP3A4-derived XREM-Luc reporter gene. Cells were treated with vehicle (Veh, 0.1% Ethanol) or 10 microM rifampicin. Two-fold serial dilutions of a stock extract of tian xian (250 mg/ml) were used to perform the concentration-response analysis. All compounds were added as 1000X stock to each individual well. All cells were treated for 24 h. The data represent the mean of replicates ± SD (n=8) and are normalized against β-galactosidase activity, and are expressed as fold induction over vehicle control. * = Statistically different from vehicle control (p<0.05).

Figure 2. Differential Modulation of PXR-SRC-1/2 and PXR-NCoR Interactions by Tian Xian

Receptor-interaction domains fused to the GAL4-DNA-binding domain were used to determine whether tian xian altered PXR association with (A) SRC1/2 and (B) NCoR in the mammalian two-hybrid system. Transient transfection of CV-1 cells was performed as described in materials and methods. Twenty-four hr post-transfection, CV-1 cells were treated with vehicle (Veh, 0.1% Ethanol) or 10 microM rifampicin. A stock extract (250 mg/ml) was used to treat cells with three different dilutions of tian xian (1:64,000; 1:8,000, and 1:1,000). All compounds were delivered as 1000X (1 μl/ml) and all wells were treated for 24 hr. The data represent the mean of replicates ± SD (n=8) and are normalized against β-galactosidase activity, and are expressed as percent full reporter gene activity. In (A) * = Statistically different from GAL4 fusion alone control (p<0.05). In (B) * = Statistically different from GAL4-NCoR + VP16-hPXR control (p<0.05).

Figure 2. Differential Modulation of PXR-SRC-1/2 and PXR-NCoR Interactions by Tian Xian

Receptor-interaction domains fused to the GAL4-DNA-binding domain were used to determine whether tian xian altered PXR association with (A) SRC1/2 and (B) NCoR in the mammalian two-hybrid system. Transient transfection of CV-1 cells was performed as described in materials and methods. Twenty-four hr post-transfection, CV-1 cells were treated with vehicle (Veh, 0.1% Ethanol) or 10 microM rifampicin. A stock extract (250 mg/ml) was used to treat cells with three different dilutions of tian xian (1:64,000; 1:8,000, and 1:1,000). All compounds were delivered as 1000X (1 μl/ml) and all wells were treated for 24 hr. The data represent the mean of replicates ± SD (n=8) and are normalized against β-galactosidase activity, and are expressed as percent full reporter gene activity. In (A) * = Statistically different from GAL4 fusion alone control (p<0.05). In (B) * = Statistically different from GAL4-NCoR + VP16-hPXR control (p<0.05).

Figure 3. Humanized PXR Transgenic Mouse Production and Expression Profiling

(A). A 3,000 bp upstream fragment of the transthyretin promoter was used to drive expression of the FLAG-tagged human PXR cDNA. The location of the PCR primers used for genotyping is shown with the forward primer (fp) located in the transthyretin promoter region. The reverse primer (rp) is derived from the human PXR cDNA sequence. The sequence of each primer is listed in Materials and Methods. The resulting transgenic mouse line was crossed to the PXR knockout mice and then bred to homozygosity for both the Pxr knockout allele as well as the transgenic allele. Nine successive backcrosses were performed into the C57Bl6 strain of mice to obtain a congenic line of mice that are homozygous for the transgene and nullizygous for the wild type Pxr allele. (Inset). The anti-FLAG M2 monoclonal antibody was used to precipitate immuno-reactive proteins from wild type and humanized PXR transgenic livers. Proteins were resolved using SDS-PAGE on identical gels. One was stained with coomassie blue (left) and the other was transferred to PVDF membrane. The membrane was subsequently probed using anti-human PXR antibodies (right). (B). Total RNA was isolated from liver, heart, and lung tissue. The RNA was DNase-treated and reverse transcribed as described in Materials and Methods. Real-time quantitative PCR analysis was used to detect expression levels of the human PXR transgene. Data are expressed as relative transgene expression over wild type liver control and are normalized to 18S values obtained as described in Materials and Methods.

Figure 3. Humanized PXR Transgenic Mouse Production and Expression Profiling

(A). A 3,000 bp upstream fragment of the transthyretin promoter was used to drive expression of the FLAG-tagged human PXR cDNA. The location of the PCR primers used for genotyping is shown with the forward primer (fp) located in the transthyretin promoter region. The reverse primer (rp) is derived from the human PXR cDNA sequence. The sequence of each primer is listed in Materials and Methods. The resulting transgenic mouse line was crossed to the PXR knockout mice and then bred to homozygosity for both the Pxr knockout allele as well as the transgenic allele. Nine successive backcrosses were performed into the C57Bl6 strain of mice to obtain a congenic line of mice that are homozygous for the transgene and nullizygous for the wild type Pxr allele. (Inset). The anti-FLAG M2 monoclonal antibody was used to precipitate immuno-reactive proteins from wild type and humanized PXR transgenic livers. Proteins were resolved using SDS-PAGE on identical gels. One was stained with coomassie blue (left) and the other was transferred to PVDF membrane. The membrane was subsequently probed using anti-human PXR antibodies (right). (B). Total RNA was isolated from liver, heart, and lung tissue. The RNA was DNase-treated and reverse transcribed as described in Materials and Methods. Real-time quantitative PCR analysis was used to detect expression levels of the human PXR transgene. Data are expressed as relative transgene expression over wild type liver control and are normalized to 18S values obtained as described in Materials and Methods.

Figure 4. The Expression of Cyp3a11 is Induced by Tian Xian in a PXR-dependent Manner and in Humanized PXR Mouse Hepatocytes

(A). Primary cultures of hepatocytes were isolated from transgenic humanized PXR, PXR knockout, and wild type mice. Cultures were treated with vehicle (Veh, 0.1% DMSO) or 10 microM of Rifampicin or PCN. All cells were treated for 48 h before RNA isolation. Total RNA was isolated and used in real time quantitative PCR analysis. The data are normalized to 18S levels and are expressed as average values (n=3) ± SD. * = Statistically different from vehicle control group (p<0.05). (B). Primary cultures of hepatocytes were isolated from wild type and PXR-KO mice. Cultures were treated with vehicle (Veh, 0.1% DMSO), 10 microM PCN, and increasing concentrations of tian xian extract. All cells were treated for 48 h before RNA isolation. Total RNA was isolated and used in real time quantitative PCR analysis. The data are normalized to 18S levels and are expressed as average values (n=3) ± SD. * = Statistically different from vehicle control group. (C). Primary cultures of hepatocytes were isolated from transgenic humanized PXR mice. Cultures were treated with vehicle (Veh, 0.1% Ethanol), 10 μιχρoM of Rifampicin, or with three different dilutions of tian xian (1:64,000; 1:8,000, and 1:1,000). All compounds were delivered as 1000X (1 μl/ml) and all wells were treated for 24 hr. All cells were treated for 24 h before RNA isolation. Total RNA was isolated and used in real time quantitative PCR analysis. The data are normalized to 18S levels and are expressed as average values (n=3) ± the SD. * = Statistically different from vehicle control group.

Figure 4. The Expression of Cyp3a11 is Induced by Tian Xian in a PXR-dependent Manner and in Humanized PXR Mouse Hepatocytes

(A). Primary cultures of hepatocytes were isolated from transgenic humanized PXR, PXR knockout, and wild type mice. Cultures were treated with vehicle (Veh, 0.1% DMSO) or 10 microM of Rifampicin or PCN. All cells were treated for 48 h before RNA isolation. Total RNA was isolated and used in real time quantitative PCR analysis. The data are normalized to 18S levels and are expressed as average values (n=3) ± SD. * = Statistically different from vehicle control group (p<0.05). (B). Primary cultures of hepatocytes were isolated from wild type and PXR-KO mice. Cultures were treated with vehicle (Veh, 0.1% DMSO), 10 microM PCN, and increasing concentrations of tian xian extract. All cells were treated for 48 h before RNA isolation. Total RNA was isolated and used in real time quantitative PCR analysis. The data are normalized to 18S levels and are expressed as average values (n=3) ± SD. * = Statistically different from vehicle control group. (C). Primary cultures of hepatocytes were isolated from transgenic humanized PXR mice. Cultures were treated with vehicle (Veh, 0.1% Ethanol), 10 μιχρoM of Rifampicin, or with three different dilutions of tian xian (1:64,000; 1:8,000, and 1:1,000). All compounds were delivered as 1000X (1 μl/ml) and all wells were treated for 24 hr. All cells were treated for 24 h before RNA isolation. Total RNA was isolated and used in real time quantitative PCR analysis. The data are normalized to 18S levels and are expressed as average values (n=3) ± the SD. * = Statistically different from vehicle control group.

Figure 4. The Expression of Cyp3a11 is Induced by Tian Xian in a PXR-dependent Manner and in Humanized PXR Mouse Hepatocytes

(A). Primary cultures of hepatocytes were isolated from transgenic humanized PXR, PXR knockout, and wild type mice. Cultures were treated with vehicle (Veh, 0.1% DMSO) or 10 microM of Rifampicin or PCN. All cells were treated for 48 h before RNA isolation. Total RNA was isolated and used in real time quantitative PCR analysis. The data are normalized to 18S levels and are expressed as average values (n=3) ± SD. * = Statistically different from vehicle control group (p<0.05). (B). Primary cultures of hepatocytes were isolated from wild type and PXR-KO mice. Cultures were treated with vehicle (Veh, 0.1% DMSO), 10 microM PCN, and increasing concentrations of tian xian extract. All cells were treated for 48 h before RNA isolation. Total RNA was isolated and used in real time quantitative PCR analysis. The data are normalized to 18S levels and are expressed as average values (n=3) ± SD. * = Statistically different from vehicle control group. (C). Primary cultures of hepatocytes were isolated from transgenic humanized PXR mice. Cultures were treated with vehicle (Veh, 0.1% Ethanol), 10 μιχρoM of Rifampicin, or with three different dilutions of tian xian (1:64,000; 1:8,000, and 1:1,000). All compounds were delivered as 1000X (1 μl/ml) and all wells were treated for 24 hr. All cells were treated for 24 h before RNA isolation. Total RNA was isolated and used in real time quantitative PCR analysis. The data are normalized to 18S levels and are expressed as average values (n=3) ± the SD. * = Statistically different from vehicle control group.

Figure 5. The Expression of CYP3A4 is Induced Tian Xian in Hepatocytes Isolated from the Transgenic Humanized PXR Mice

Primary cultures of hepatocytes were obtained from XenoTech, LLC. Cultures were treated with vehicle (Veh, 0.1% Ethanol), or with three different dilutions of tian xian (1:64,000; 1:8,000, and 1:1,000). All compounds were delivered as 1000X (1 μl/ml) and all wells were treated for 24 hr. All cells were treated for 24 h before RNA isolation. Total RNA was isolated and used in real time quantitative PCR analysis. The data are normalized to 18S levels and are expressed as average values (n=3) ± SD. * = Statistically different from vehicle control group.