Sesamin: A Naturally Occurring Lignan Inhibits CYP3A4 by Antagonizing the Pregnane X Receptor Activation

Inconsistent expression and regulation of drug-metabolizing enzymes (DMEs) are common causes of adverse drug effects in some drugs with a narrow therapeutic index (TI). An important cytochrome, cytochrome P450 3A4 (CYP3A4), is predominantly regulated by a nuclear receptor, pregnane X receptor (PXR). Sesamin, a major lignan constituent in sesame seeds and oil, exhibits a variety of biological functions; however, the effect of sesamin on the modulation of CYP3A4 is not well understood. In this study, the effects of sesamin on the PXR-CYP3A4 pathway were characterized, as well as the underlying mechanisms of those effects. Sesamin potently attenuated CYP3A4 induction in a dose-dependent manner by blocking the activation of PXR. The PXR inducer-mediated inhibition of CYP3A4 was further evidenced by the ability of sesamin to attenuate the effects of several PXR ligands in the CYP3A4 reporter assay. Further mechanistic studies showed that sesamin inhibited PXR by interrupting the interacting with coregulators. These results may lead to the development of new therapeutic and dietary approaches to reduce the frequency of inducer-drug interaction. Sesamin was established as a novel inhibitor of PXR and may be useful for modulating DMEs expression and drug efficacies. Modification of CYP3A4 expression and activity by consumption of sesamin may have important implications for drug safety.


Introduction
Sesame seeds (Sesamum indicum) and their oil have been used in human diets for thousands of years and are believed to provide health benefits. A major constituent of sesame is a lignan called sesamin. Sesamin has a variety of biological functions including reduction of serum and hepatic cholesterol levels [1][2][3][4][5] as well as serum triglycerides [6] by increasing hepatic fatty acid oxidation [1,2]. Sesamin's involvement in the suppression of hypertension [7,8] and stroke prevention has been extensively studied by many researchers [9]. Moreover, sesamin has been shown to elevate the levels of γ-tocopherol [10]; decrease production of endotoxininduced interleukin (IL)-1β, prostaglandin E2 (PGE2), and thromboxane B2 [11]; elevate the production of IL-6 [12], thus inhibiting endotoxin-mediated shock [11]. Some studies also suggest that sesamin has an inhibitory effect on 7,12dimethylbenz[a]anthracene-induced mammary carcinogenesis [13] and inhibits the growth of a variety of neoplastic cells through different mechanisms [14][15][16][17][18]. Thus, sesamin is believed to protect against cancer and other chronic diseases.
Cytochrome P450-(CYP450-) dependent monooxygenase occupies a pivotal role in the metabolism and detoxification process of endogenous and exogenous compounds [19]. CYP3A4 is the most abundant CYP450 and has clinical importance because it metabolizes numerous pharmaceutical agents [19]. It is highly expressed in the liver and intestine and represents 40% of the total hepatic and 80% of the total 2 Evidence-Based Complementary and Alternative Medicine intestinal CYPs [20,21]. CYP3A4 is also involved in the metabolism of endogenous substrates (retinoic and bile acids) and steroidal hormones (testosterone and estrogen) [22]. In addition, dietary and environmental chemicals such as aflatoxin B1 and some herbicides are also CYP3A4 substrates [23,24]. The expression and activity of CYP3A4 are greatly impacted by many drugs and dietary chemicals such as rifampin (an antibiotic), carbamazepine (an anticonvulsant), glucocorticoids, and hyperforin (a major component of St. John' wort) [25]. Individual variations in drug metabolism may be due to the differential expression of the CYP3A4 enzyme, induced by some of these compounds [20]. CYP3A4 protein expression in hepatocytes varies more than 50 folds among individuals, and the enzymatic activity varies by at least 20 folds [26]. Changes caused by drug-metabolizing enzymes (DMEs) are common and undesirable, and these enzymes influence the therapeutic effects of drugs, particularly those having a narrow therapeutic index [27]. Since CYP3A4 activity may be affected by some xenobiotics, it plays a crucial role in drug-drug interactions. For example, coadministration with rifampin, phenytoin, or carbamazepine may reduce plasma concentrations of a broad range of CYP3A4 drug substrates to less than a half [28].
The PXR pathway is also activated by a large number of prescription drugs designed to treat infection, cancer, convulsions, and hypertension and is believed to play an important role in drug metabolism and efflux as well as inducerdrug interactions [40]. PXR increases the transcription of CYP3A4 as well as other DMEs and transporters after exposure to its agonists [41,42]. Because many anticancer agents have narrow therapeutic indices, modulation of the PXR-CYP3A4 pathway may cause unpredictable efficacy.
Because the PXR-CYP3A4 pathway is extremely important in drug efficacy, it is very important to find an agent that may reduce the effect of inducer-drug interaction. PXR antagonists may be useful for the study of the molecular basis of receptor function. Although many PXR agonists have been reported, comparatively few PXR compounds antagonizing the PXR-CYP3A4 pathway have been identified [43][44][45][46][47].
In this study, the effects of sesamin on the PXR-CYP3A4 pathway and the underlying mechanisms were characterized. Sesamin potently attenuated CYP3A4 induction in a dosedependent manner by blocking the activation of nuclear receptors, especially PXR through CYP3A4 reporter assay. The PXR inducer-mediated inhibition of CYP3A4 was further evidenced by the ability of sesamin to attenuate the effects of rifampin, paclitaxel, and SR12813 in the CYP3A4 reporter assay. Further mechanistic studies showed that sesamin inhibited PXR by interrupting the binding of steroid receptor coactivator-1 (SRC-1) and hepatocyte nuclear factor 4α (HNF4α). Our results may lead to the development of important new therapeutic and dietary approaches to reduce the frequency of undesirable drug interactions. Here, we established sesamin as a novel and natural potent inhibitor of PXR and proved that it can be a useful tool for modulating DMEs expression. Modification of CYP3A4 by consumption of sesamin could have important implications for drug safety.

Assessment of Cell Cytotoxicity.
To verify ≥80% cell viability following xenobiotic exposure, cell viability was evaluated using the modified acid-phosphatase (ACP) assay, with p-nitrophenyl phosphate (PNPP) disodium salt as a substrate. The cell culture media were aspirated, and the cells were washed with phosphate-buffered saline (PBS). Following the wash, 100 μL of the ACP reagent [(0.1 M sodium acetate (pH 5.5), 0.1% Triton X-100, and 10 mM PNPP)] were added. After 1 h of incubation at 37 • C, the enzyme activity was stopped by adding 10 μL of 1 N NaOH, and the extent of activity was determined photometrically at a wavelength of 405 nm [50].

Western
Blotting. Protein expressions of CYP3A4, PXR, 9-cis retinoic acid receptor (RXRα), and HNF4α were measured by using western blotting. HepG2 cells were seeded at a density of 2 × 10 6 cells/10-cm dish, before drug treatment. Various concentrations of sesamin, alone or in combination with 20 μM rifampin, were added to the HepG2 cell culture for 48 h. Following drug treatment, the medium was removed, and the cells were rinsed twice with ice-cold PBS. After adding 200 μL of ice-cold RIPA buffer with protease inhibitors, the cells were scraped from the surface of the culture dish and allowed to stand on ice for 20 min, and the cell lysates were centrifuged (14,000 rpm for 30 min at 4 • C). Total protein (50 μg) from the supernatant was resolved by using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose (NC) paper. These blots were exposed to primary antibodies against CYP3A4, PXR, RXRα, HNF4α, and βactin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The relative density of the immunoreactive bands was quantitated using densitometry (ImageQuant LAS 4000, GE Healthcare, Waukesha, WI, USA) following detection using an enhanced chemiluminescence detection system (Millipore, Billerica, MA, USA). For the mammalian two-hybrid assays, transfection was carried out by mixing 0.10 μg of the pG5luc reporter gene, 0.04 μg each of the pBIND-GAL4 and pACT-VP16 constructs, and 0.02 μg of the control β-galactosidase plasmid per well. After 6 or 7 h, the cells were exposed to rifampin, sesamin, CITCO, PCN, SR12813, paclitaxel, or a similar volume of DMSO. After a further 20-24 h of incubation, the cells were lysed in situ with 80 μL of cell culture lysis reagent (Promega). The cleared lysate (30 μL) was used for the βgalactosidase assay. A 50-μL aliquot of each cleared lysate was used for the reporter assay, following which 80 μL of luciferase assay reagent (Promega) was added to the lysates. Luminescent signals were measured using a luminescence multi-mode microplate reader. Luciferase activities were normalized to the corresponding β-galactosidase activity. Data shown are means of triplicate or quadruplicate transfections ± standard deviations, as indicated.

Statistical Analysis.
All experiments were repeated at least thrice independently. Values, expressed as mean ± SD, were analyzed using one-way ANOVA followed by Tukey's test for multiple comparisons. All statistical analyses were performed using SPSS for Windows, version 17.0 (SPSS, Inc., Chicago, IL, USA). A P value < 0.05 was considered statistically significant.

Cell Viability of HepG2 and LS174T Cells following
Exposure to Sesamin. Since sesamin ( Figure 1) has been shown to inhibit proliferation of multiple types of malignant cells [13][14][15][16][17], a cell viability test was performed to rule out cytotoxic effects due to sesamin. As shown in Figure 2, HepG2 ( Figure 2(a)) and LS174T ( Figure 2(b)) cells were exposed to a range of concentrations of sesamin alone and in combination with rifampin for 48 h, and the cell viability was assessed using the ACP assay. Rifampin did not show cytotoxicity toward either cell line. Sesamin caused mild cytoxicity as compared to DMSO-treated cells. However, even after exposure to 40 μM sesamin (the highest concentration tested), cell viability remained approximately 80%.
To evaluate CYP3A4 protein expression, HepG2 cells were treated with 20 and 40 μM sesamin alone or in combination with 20 μM rifampin for 48 h. Protein expression levels were determined using western blotting. We found that sesamin (20 μM) significantly decreased the basal level of CYP3A4 protein expression (48.5%, P < 0.05) as compared to the DMSO-treated group (Figures 3(c) and 3(d)). Rifampin significantly enhanced CYP3A4 protein expression by 55.5% (P < 0.05). Coincubation of the cells with 20 and 40 μM sesamin with 20 μM rifampin significantly decreased rifampin-induced CYP3A4 protein expression by 57.7% (P < 0.05) and 66.5% (P < 0.05), respectively, as compared to rifampin-induced cells. The effects were concentration dependent (Figures 3(c) and 3(e)) and consistent with those from the mRNA expression assays. Thus, sesamin inhibits CYP3A4 enzyme levels by reducing expression of both mRNA and protein.

Sesamin Inhibits PXR-Mediated CYP3A4
Promoter Activity in HepG2 and LS174T Cells. PXR is a main ligand-activated transcription factor of CYP3A4. PXR transactivation reporter assays were conducted to assess the inhibitory effect of sesamin alone or with rifampin-induced PXR transactivation activity, on the CYP3A4 promoter. Cells were cotransfected with a β-galactosidase expression vector, a reporter gene construct containing a responsive element for PXR in the CYP3-A4 promoter, and a human PXR expression plasmid or vector control. Cells were treated with different concentrations of sesamin, with or without 20 μM rifampin. The luciferase activity was measured after 24 h of drug treatment. The results are shown in Figure 4. In HepG2 cells, sesamin (20,30, and 40 μM) strongly attenuated the transactivation of the CYP3A4 promoter induced by rifampin in a concentrationdependent manner when cotransfected with human PXR. As shown in Figure 4(a), transactivation decreased by 31% (P < 0.05), 40.6% (P < 0.01), and 64.8% (P < 0.001), respectively, as compared to the rifampin-induced groups. Similar experiments were also performed using a colon adenocarcinoma cell line (LS174T) to exclude the possibility of a cell-typespecific effect. As shown in Figure 4(b), similar results were found when these cells were transfected with PXR in the presence of rifampin and sesamin. Treatment of LS174T cells with sesamin for 24 h significantly attenuated PXR-mediated activation of the CYP3A4 promoter induced by rifampin; the effect of sesamin was concentration dependent, with 40 μM sesamin producing the greatest inhibitory effect [80% decrease, (P < 0.001), as compared to rifampin treatment alone]. Endogenous PXR and CAR have been identified in HepG2 and LS174T cells [51,52]; thus, rifampin could activate the CYP3A4 reporter response in cells carrying the control vector only. As shown in Figures 4(a) and 4(b), exposure of vector-transfected groups to 40 μM sesamin significantly decreased rifampin-induced CYP3A4 promoter activity in HepG2 cells (a decrease of 37%, P < 0.01; Figure 4(a)) and in LS174T cells (a decrease of 55.7%, P < 0.001; Figure 4(b)), as compared to the rifampin-treated controls. In contrast, cells without rifampin induction did not show a significant reduction of CYP3A4 reporter activity in the presence of sesamin. These results suggest a PXR-dependent mechanism of regulation.

Inhibition of Human CAR-and Rat PXR-Mediated CYP3-A4
Induction. CYP3A4 expression is also regulated by CAR at the same response elements in the CYP3A4 promoter [30]. Similar reporter assays were performed in HepG2 cells to evaluate the effect of sesamin on human CAR-mediated CYP3A4 transactivation. Sesamin (20 and 40 μM) was found to markedly attenuate CITCO-induced CYP3A4 promoter activation through CAR (26.6%, P < 0.05, and 56.2%, P < 0.001, as compared to CITCO treatment alone, at 20 and 40 μM sesamin, resp.; Figure 5(a)).
CYP3A4 inhibition is predominantly modulated by the effect of PXR on CYP3A4 expression and partly modulated by CAR. Previous reports have shown that the differences in the ligand-binding domain of human and rat PXR may lead to ligand-induced species-specific effects [53]. Therefore, sesamin inhibition of rat PXR activation was also tested. As shown in Figure 5(b), when rat PXR-transfected cells were treated with the rodent-specific ligand, PCN, CYP3A4 activity was enhanced by 322%, as compared to DMSO-treated controls. Sesamin strongly attenuated rat PXR-mediated CYP3A4 promoter activity with PCN induction by either 20 μM sesamin (decreased by 28.9%, P < 0.05) or 40 μM sesamin (decreased by 54.3%, P < 0.001), as compared to the PCN-treated cells. The results indicate that sesamin activity is not highly species specific.

Effects of Sesamin on PXR, RXRα, and HNF4α Protein
Expression. Based on the finding that sesamin reduces PXR activity in cell-based assays, in vitro, the impact of sesamin on PXR protein expression was investigated. PXR binds to DNA as a heterodimer with RXRα. The heterodimer can bind to and induce transcription from response elements located in the CYP450 genes [29][30][31][32][33]. In addition, rifampininduced CYP3A4 requires PXR cross-talk with HNF4α on the CYP3A4 promoter transcription complex [54]. Western blots were performed to evaluate the PXR, RXRα, and HNF4α protein levels in sesamin-treated cells. HepG2 cells were treated LS174T cells were cotransfected with a vector control (pcDNA3) or PXR expression plasmid (pcDNA3-PXR), a CYP3A4 luciferase reporter plasmid, and a pRC-CMV-β-galactosidase vector. Subsequently, the transfected cells were exposed to sesamin and/or rifampin for 24 h. The results are expressed as mean ± SD (n = 4); * P < 0.05; * * P < 0.01; * * * P < 0.001, as compared to rifampin-treated cells.  Figure 5: Transactivation of the CYP3A4 promoter by human CAR and rat PXR in the presence of sesamin and/or rifampin. HepG2 cells were cotransfected with a vector control (pcDNA3) and (a) human CAR expression plasmids (pcDNA3-CAR) or (b) rat PXR expression plasmids (pcDNA3-rPXR), a CYP3A4 luciferase reporter plasmid, and pRC-CMV-β-galactosidase vectors. Subsequently, the transfected cells were exposed to sesamin and/or rifampin for 24 h. The results are expressed as mean ± SD (n = 4); * P < 0.05 and * * * P < 0.001, as compared to rifampin-treated cells.
with 20 and 40 μM sesamin alone or in combination with 20 μM rifampin and lysed after 48 h of exposure. As shown in Figure 6, none of the treatment conditions resulted in an alteration of protein expression of PXR (Figure 6(a)), RXRα ( Figure 6(b)), or HNF4α ( Figure 6(c)). The results indicated that the reduction of CYP3A4 activity was not due to decreased expression of the upper control elements.

Inhibition of Coregulation of Human SRC-1 and HNF4α
with PXR by Sesamin. Earlier studies showed that cotransfection of SRC-1 and HNF4α into cells produced a synergistic increase in CYP3A4 promoter activity [49,54,55]. This part of the study sought to determine whether the effects of these two CYP3A4 promoter coregulators were altered by the presence of sesamin. Full-length human SRC-1 or HNF4α expression plasmids were cotransfected with full-length human PXR/vector control and CYP3A4 reporter constructs into HepG2 cells. The cells were then exposed to sesamin, alone or in combination with rifampin, followed by measurement of luciferase activity after 24 h of incubation. Since HepG2 cells contain endogenous PXR, cells transfected with the vector control and SRC-1 demonstrated a 98% increase in CYP3A4 promoter activity in the presence of rifampin (Figure 7(a)). Sesamin (40 μM) decreased the promoter activity to 24.9% (P < 0.001) of the level observed in rifampintreated cells. Transfection with PXR and SRC-1 expression plasmids resulted in a CYP3A4 promoter activity enhancement of 243.6 folds, in the presence of rifampin. Again, sesamin (20 and 40 μM) strongly disrupted the enhancement of the promoter activity; activity was decreased to 64.2% (P < 0.001) or 18.5% (P < 0.001), respectively, of the level observed in rifampin-treated cells.  Figure 7: Coregulation of SRC-1 and HNF4α with PXR was disrupted by sesamin. HepG2 cells were cotransfected with (a) a vector control (pcDNA3) or PXR expression plasmid (pcDNA3-PXR) and SRC-1 expression plasmid (pCR3.1-SRC-1), or (b) a vector control (pcDNA3) or PXR expression plasmid (pcDNA3-PXR) and HNF4α expression plasmid (pcDNA3-HNF4α), in combination with the CYP3A4 luciferase reporter plasmid and the pRC-CMV-β-galactosidase vector. Subsequently, the transfected cells were exposed to sesamin and/or rifampin for 24 h. Luciferase activity was measured and normalized to the corresponding β-galactosidase activity. The results are expressed as mean ± SD (n = 4); * * * P < 0.001, as compared to rifampin-treated cells.
The coregulation of HNF4α and PXR was also evaluated. When cells were cotransfected with the vector control and full-length HNF4α prior to rifampin exposure, CYP3A4 activity was enhanced 23.2 folds. This effect was disrupted by sesamin, with the greatest inhibition at a sesamin concentration of 40 μM (decreased by 89.3%, P < 0.001, as compared to rifampin-treated groups). The coregulation strength was not as great as that observed in the SRC-1/PXR cells; the presence of HNF4α and PXR enhanced CYP3A4 promoter activity by 53.6 folds. As Figure 7(b) shows, 20 and 40 μM sesamin significantly reduced CYP3A4 promoter activity in these cells when exposed to rifampin (decreases of 44.8%, P < 0.01, and 88.3%, P < 0.001, resp., as compared to rifampin-treated groups). These results indicated that sesamin disrupts the coregulation effects of SRC-1/PXR and HNF4α/PXR on CYP3A4 promoter activity.

Sesamin Disrupts the Interaction of SRC-1/PXR and
HNF4α/PXR in LS174T Cells. Ketoconazole has previously been shown to inhibit the expression of CYP3A4 by disrupting the interaction of SRC-1 and PXR as well as that of HNF4α and PXR [50]. To determine whether sesamin could inhibit SRC-1/PXR or HNF4α/PXR interaction, a GAL4/ VP16 mammalian two-hybrid reporter system was used. The RID of SRC-1 and the full-length PXR gene were fused with the GAL4 DNA-binding domain to become pBIND-SRC-1 and pBIND-PXR, respectively. Similarly, the full-length PXR and HNF4α were fused with the VP-16 vector to become pACT-PXR and pACT-HNF4α, respectively. As shown in Figure 8(a), in the absence of rifampin, 20 and 40 μM sesamin decreased the interaction between PXR and SRC-1 by 27% and 49.1% (P < 0.05 and P < 0.001), as compared to control groups in LS174T cells. In contrast, rifampin strongly promoted the specific interaction between PXR and SRC-1 by 8.1 folds; sesamin attenuated the interaction between PXR and SRC-1 concentration-dependent manner, with a 25.8% (P < 0.05) and 67.9% (P < 0.001) inhibition at 20 and 40 μM sesamin, respectively.
The interaction between HNF4α and PXR was also disrupted by sesamin. As shown in Figure 8(b), the interaction of cells transfected with pBIND-PXR and pACT-HNF4α, without rifampin induction, decreased by 45.2% (P < 0.001) compared to DMSO-treated cells. Rifampin enhanced the protein-protein interaction of PXR and HNF4α by 2.2 folds. As expected, sesamin disrupted this interaction in a concentration-dependent manner. A decrease of 31.3% was seen in cells exposed to 20 μM sesamin (P < 0.01), and a decrease of 57.6% was seen in cells exposed to 40 μM sesamin (P < 0.001), as compared to rifampin-treated groups which were not exposed to sesamin. These data suggest that sesamin inhibits SRC-1/PXR and HNF4α/PXR interactions, thus transactivation in the regulation of CYP3A4 gene expression occurs through the transcriptional control.

CYP3A4 Promoter Activity in the Presence of Sesamin and
Paclitaxel or SR12813. Because many antineoplastic drugs have a narrow therapeutic index, the ability of drugs (e.g., paclitaxel) to activate PXR may be limited because of autoinduced metabolism [56]. This implies that sesamin antagonism of PXR activity might improve the pharmacokinetic properties of these drugs. As shown in Figure 9(a), 2.5 μM paclitaxel enhanced CYP3A4 promoter activity in LS174T cells up to 3 folds as compared to the controls. When the cells were cotreated with paclitaxel and 20 or 40 μM sesamin, CYP3A4 promoter activity reduced by 39.1% (P < 0.05) and 60.5% (P < 0.001), respectively, when compared with paclitaxel-induced controls. These results indicated that sesamin may rescue PXR agonists from autoinduced metabolism.
SR12813, the most efficient PXR agonist discovered to date [57], was used in some evaluations to exclude the possibility of rifampin-specific effects. In the presence of 10 μM SR12813, CYP3A4 promoter activity in LS174T cells was enhanced by 46.1 folds, as compared to DMSO-treated cells (Figure 9(b)). The presence of sesamin (20 or 40 μM) resulted in 62.8% (P < 0.001) or 78.4% (P < 0.001) inhibition, respectively, of transcriptional activity induced by SR12813. The results showed that inhibition of CYP3A4 by sesamin was not a rifampin-specific effect.

Sesamin Increases Gene Expression of Enzymes Involved
in Phase II Metabolism. The discovery that sesamin inhibits CYP3A4 expression led to an investigation of sesamin's effect on the expression of UGT1A1 mRNA in HepG2 cells by RT-PCR. UGT1A1 is an enzyme involved in hepatic phase II metabolism that is also regulated by PXR [58]. As shown in Figure 10 Figure 9: Downregulation of CYP3A4 reporter activity on PXR agonist, paclitaxel-and SR12813-mediated induction by sesamin. LS174T cells were cotransfected with a vector control (pcDNA3) or PXR expression plasmid (pcDNA3-PXR), a CYP3A4 luciferase reporter plasmid, and a pRC-CMV-β-galactosidase vector. Subsequently, the transfected cells were exposed to sesamin and/or (a) 2.5 μM paclitaxel or (b) 10 μM SR12813 for 24 h. Luciferase activity was measured and normalized to the corresponding β-galactosidase activity. The results are expressed as mean ± SD (n = 4); * P < 0.05 and * * * P < 0.001, as compared to rifampin-treated cells, as indicated. increased 11.7 folds in the presence of 20 μM rifampin, consistent with the results of previous studies [59]. With the addition of 20 to 40 μM sesamin, in combination with 20 μM rifampin, expression of UGT1A1 mRNA increased with increasing concentrations of sesamin. This increase was particularly notable at sesamin concentrations of 30 μM, where mRNA levels increased by 65.6% (P < 0.05), and 40 μM, where mRNA levels increased by 98% (P < 0.01). Presumably, sesamin induced UGT1A1 gene expression through a PXR-independent pathway and may be a nuclear factor E2-related factor 2-(Nrf2-) dependent pathway [60]. Sesamin induced the expression of another Nrf2 target gene, NAD(P)H quinone oxidoreductase 1 (NQO1), in HepG2 cells (Figure 10(b)). In this case, mRNA expression was increased by 1.4 folds (P < 0.05) in the presence of 20 μM sesamin, and 2-fold (P < 0.001) at a 30 μM sesamin concentration. The net inductive effect of sesamin on UGT1A1 gene expression is interesting because UGT1A1 is also regulated, in part, by PXR.

Discussion
In this study, sesamin was demonstrated to antagonize the induction of CYP3A4 in HepG2 and LS174T cells. It was also determined to be an inhibitor of rat and human PXR in nuclear receptor transactivation assays. Additionally, sesamin was demonstrated to suppress human CAR activation. Furthermore, the underlying mechanisms of human PXR inhibition were explored. Sesamin was found to inhibit coregulation between SRC-1 and HNF4α by disrupting their recruitment and interaction with PXR. These changes were not due to reduced protein levels of those nuclear receptors or coregulators. The inductive effects of SR12813 and paclitaxel could be attenuated by coadministration with sesamin. These results suggest a potential mechanism for preventing inducer-drug interactions between PXR ligands and minimizing the potential for P450 inducer-drug interactions. Although numerous human PXR activators have been identified, to date there are few reports of potent inhibitors of PXR. Sesamin is a novel functional inhibitor of PXR and may be a useful chemical tool for modulating PXR-regulated gene expression in vitro or in vivo. Inappropriate PXR activation leads to important and undesirable pathophysiologic consequences [56]. One of PXR's main target genes is CYP3A4, a cytochrome that is variably expressed in the liver and small intestine cells [19]. Druginduced activation of CYP3A4 may affect the safety and effective dosing of narrow therapeutic index chemotherapeutic agents, if they are CYP3A4 substrates. The genetics of CYP3A4 have not explained the variable expression of this cytochrome. This has raised the possibility that individual differences in exposure to dietary or endogenous agents may modulate CYP3A4 transcription, contributing to functional CYP3A4 variability [33]. In this study, sesamin was found to decrease constitutive CYP3A4 activity, as well as mRNA and protein expression. Moreover, it attenuated rifampin-, paclitaxel-, and SR12813-mediated CYP3A4 induction in human hepatoma and colon adenocarcinoma cells. These results suggest that dietary exposure to sesamin could potentially contribute to the large variability in basal CYP3A4 expression between individuals.
Extensive cross-talk occurs between members of the nuclear receptor superfamily. Both PXR and CAR are closely related nuclear receptors belonging to the nuclear receptor subfamily (NR1) that are involved in CYP3A4 regulation and are endogenously expressed in HepG2 cells [55]. Their ligandbinding domains (LBDs), which share 40% amino acid sequence similarity, bind overlapping areas of the CYP3A4 promoter, but with differential preference [34]. Results from cell-based nuclear receptor transactivation assays indicated that sesamin not only strongly antagonizes PXR but also greatly attenuates CAR-mediated CYP3A4 promoter activity induced by CITCO. Moreover, previous reports have shown that PXR and CAR exhibit promiscuous, low-affinity ligand binding characteristics [53]. Hence, PXR and CAR appear to share similar mechanisms of CYP3A4 regulation.
The full activity of NRs depends on a large number of cellular factors that called coactivator, that do not bind DNA directly, but are selectively recruited to the promoter by the NRs through protein-protein interactions [61]. This suggests the potential activation of NR-regulated genes at the transcriptional levels. A probable mechanism by which sesamin attenuates rifampin-induced CYP3A4 expression is through inhibition of the interaction between PXR and SRC-1. In the present study, sesamin was found to significantly attenuate rifampin-induced interactions between PXR and SRC-1 in a concentration-dependent manner. This suggests that sesamin may inhibit PXR coactivator recruitment, thereby potentially preventing ligand-mediated PXR transcriptional activation of target genes.
HNF4α also plays an essential role in the PXR-dependent induction of human CYP3A4 [55,62]. Thus, an HNF4α element adjacent to the PXR-and CAR-binding sites in the human CYP3A4 promoter is an obligatory component of a xenobiotic-responsive distal enhancer [55]. Previous studies revealed that ketoconazole inhibition of CYP3A4 expression not only involves the recruitment of SRC-1, but also HNF4α [50,63]. Analysis of the molecular mechanism of gene transcription inhibition demonstrated that sesamin specifically disrupts the interaction of PXR with HNF4α and the coactivator, SRC-1.
Differential activation of PXR orthologs between species has been well reported [64]. Moreover, PXR and CYP3A display cell-type and species-specific differences. Due to the structural differences in its ligand-binding pockets, ligandmediated activation of PXR can occur through a wide variety of compounds in a species-dependent manner [64]. Sesamin was tested in transient transfection assays using rat PXR and was found to strongly attenuate rat PXR-mediated CYP3A4 promoter activity induced by PCN. The results indicate that sesamin does not have a species-specific effect on human and rat PXR. The inductive effects of paclitaxel and SR12813 on CYP3A4 were also attenuated by coadministration with sesamin, indicating it was not a rifampin-specific effect. The protein expression of PXR and the coregulators RXRα and HNF4α were not affected by sesamin.
Literature reports indicate that UGT1A1 is transcriptionally regulated by both PXR and CAR through DR-3 and NR-1 elements in the 5 -promoter region [58,65]. Results from the present study indicated that although sesamin antagonized PXR activity, its induction of UGT1A1 expression appeared to be through a PXR-independent pathway. The expression of UGT1A1 enzymes is regulated by Nrf2, which binds to AREs present within the promoter [66]. The result of this binding is an upregulation of the transcription of those genes. We examined the expression of another Nrf2 target gene, NQO1, as well [66]. Sesamin also significantly induced the mRNA expression of this gene, adding further evidence that sesamin induced UGT1A1 expression primarily through an Nrf2-dependent pathway.
Aside from its important role in xenobiotic defense, evidence suggests that during carcinogenesis, PXR promotes tumor cell growth [67]. Cancer chemotherapy normally involves the coadministrated of multiple drugs, and these may have somewhat unpredictable therapeutic outcomes as a result of individual differences in PXR-mediated transcriptional effects. Therefore, a PXR inhibitor can be used to develop a unique method for controlling drug-induced metabolism during chemotherapy, especially if the inhibitor is comparatively nontoxic. Studies have shown that the antineoplastic agent trabectedin ET-743 [45], the antifungal agent ketoconazole(s) [50,63], the dietary isothiocyanate sulforaphane (SFN) [44], the human HIV protease inhibitor, A-792611 [68], the phytoestrogen coumestrol [46], a topoisomerase I inhibitors camptothecin (CPT) [47], and metformin [69] are all functional inhibitors of PXR.
A genotoxicity report from Hori et al. [70] included analysis of the plasma levels of sesamin in Crlj:CD1 (ICR) mice or approximately 6, 23.1, and 22.6 μM, respectively. Our results indicate that 20 μM sesamin is sufficient to decrease CYP3A4 activity and mRNA and protein expression levels significantly. Therefore, a plasma concentration of 20 μM sesamin, which is sufficient to modulate CYP3A4 according to our in vitro data, may well be achievable by oral ingestion, at least in animal studies. Differences in genetics, geographical origin, growing conditions, seed size, and capsule position may contribute to the variable amounts of sesamin in seeds. Rangkadilok et al. [71] showed ranges of sesamin content of 0.05-4.71 mg/g of seed in the landrace black seed lines and 0.74-7.23 mg/g of seed in white sesame seeds. The sesamin content of Taiwanese sesame oil is 9.47 ± 2.28 mg/g of oil [72]. Here, we describe a novel, effective PXR inhibitor from a naturally occurring substance, sesamin, and we believe that another PXR inhibitor with higher efficiency but lower toxicity can be found on the same lines.
HepaRG cells, which were derived from a female hepatocarcinoma patient, are capable of differentiating into biliary epithelial cells and hepatocytes [73]. They retain many DMEs activities and upregulate mRNA expression of such enzymes in the presence of xenobiotics. This ability is in stark contrast to that of the frequently used HepG2 cells, and HepaRG cells have, therefore, been touted as better surrogates for primary human hepatocytes for drug metabolism and disposition studies. In this study, we continued to use HepG2 cells as a simple model system that avoids the interindividual and interspecies variations and subsequent interpretation difficulties that complicate in vivo approaches. Moreover, these cells are known to constitutively express CYP1A1/2, 2A6, 2B6, 2C9/19, 2D6, 2E1, and 3A4/5 [74]. Researchers using a cell-based PXR reporter assay to measure possible CYP3A4 induction/inhibition in HepG2 cells showed CYP3A4 mRNA expression and enzymatic activity levels similar to those in cultured primary human hepatocytes [74]. Therefore, this cell line remains an excellent surrogate for primary human hepatocytes for studying induction of CYP450s. Our results demonstrate that PXR inducers still significantly enhance the activity and mRNA and protein expression levels of CYP3A4 in HepG2 cells, implying that, for several compounds, the CYP activities in HepG2 cells are high enough to activate xenobiotic receptors and thereby induce further CYP expression.
In conclusion, we have identified a naturally occurring lignan, sesamin, that is a potent inhibitor of human and rat PXR. The study provides evidence that sesamin attenuates the induction of CYP3A4 by inhibiting agonist-activated PXR and CAR. Sesamin disrupts the interaction of PXR with SRC-1 and HNF4α without affecting expression of NR-related modulators. These findings suggest a complementary mechanism by which ingestion of this naturally occurring phytochemical may reduce the frequency of adverse drug reactions secondary to PXR-mediated induction of drug clearance via CYP3A4. This may improve the therapeutic efficacy of certain drugs and may also play a role in reducing the formation of CYP3A4-mediated reactive metabolites.