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. Author manuscript; available in PMC: 2012 Jul 9.
Published in final edited form as: Cancer Biol Ther. 2009 Jul;8(13):1265–1272. doi: 10.4161/cbt.8.13.8696

Regulation of drug resistance by human pregnane X receptor in breast cancer

Yakun Chen 1, Yong Tang 1, Shuqing Chen 2, Daotai Nie 1,*
PMCID: PMC3392171  NIHMSID: NIHMS389783  PMID: 19746521

Abstract

Drug resistance is a significant barrier to an effective treatment of breast cancer. Human pregnane X receptor (hPXR), an orphan nuclear receptor known for its activation by many important clinical drugs, is a major transcription factor of drug metabolism enzymes (DMEs), such as cytochrome P450 3A4 (CYP3A4), and efflux transporters such as multi-drug resistance gene (MDR1). hPXR has been detected in human breast cancers but its role in responses of cancers toward drugs remains unknown. In this study, hPXR expression was confirmed in breast cancer cell lines and in normal and cancerous human breast specimens. Preactivation of hPXR by SR12813 in MDA-MB-231 cells led to an increased resistance to Taxol at concentrations of 20 and 50 nmol/L. A significant increase in resistance toward tamoxifen was also observed in MCF-7 with hPXR preactivation. Activation of hPXR led to an increased expression of CYP3A4 and MDR1, two possible mediators for hPXR-mediated drug resistance in breast cancers. Furthermore, knockdown of hPXR via small hairpin RNA (shRNA) sensitized MDA-MB-231 and MCF-7 cells to the treatment of Taxol, vinblastine or tamoxifen. The reduction in resistance of hPXR knockdown cells was further confirmed by reduced colony formation under the pressure of cancer treatment drugs. Taken together, our data suggest a potential role of hPXR in breast cancer resistance to drug treatments.

Keywords: pregnane X receptor, steroid and xenobiotic receptor, breast cancer, chemotherapy, drug resistance, taxol, tamoxifen, vinblastine, drug metabolism enzymes, transcriptional regulation

Introduction

Breast cancer is one of the most common malignancies inflicted on women.1 The efficacies of drug treatments on breast cancers are often limited by the resistance of tumor cells.2 Differential inductions or activities of drug metabolism enzymes (DMEs)3 and ATP binding cassette (ABC) efflux transporters,4 which are able to prevent the intracellular accumulation of drugs by increasing excretion or efflux mechanisms, were associated with drug resistance in a number of cancers.5

Human pregnane X receptor (hPXR; also termed steroid and xenobiotic receptor, SXR), an orphan nuclear receptor, plays a central role in protecting tissues from potentially toxic exogenous and endogenous compounds. In 1998, it was reported that hPXR could be activated by nearly all of the structurally diverse compounds that induce CYP3A gene expression.6 hPXR has since been seen as a xenobiotic defender regulating the expressions of numerous drug metabolism genes and efflux transporters, an endobiotic sensor regulating cholesterol homeostasis and bile acid metabolism, and a possible trigger in the development of some cancers.7 The activation of hPXR can stimulate the expression of many important DMEs and ABC transporters, such as cytochrome P450 3A4 (CYP3A4),6 an enzyme that is involved in the metabolism of more than 60% of all marketed drugs,8 multi-drug resistance gene (MDR1; protein: p-gp)9 and breast cancer resistance protein (BCRP).10 Consequently, hPXR may represent a potential regulator of drug resistance in breast cancer.

hPXR’s ligand binding domain (LBD) can accommodate an array of structurally diverse compounds, including cholesterol and its metabolites,11 statins,12 endocrine disruptors,13 herbal components and plant extracts,14 carotenoids,15 vitamins,16 HIV protease inhibitors,17 calcium channel modulators,17 steroids,18 pesticides,19 peroxisome proliferator-activated receptor antagonists,20 agonists for other nuclear receptors,21 as well as other diverse xenobiotics and endobiotics. The importance of hPXR in cancer has hence been highlighted by the activation of hPXR by some commonly used cancer treatment agents or supplementary agents.22,23 In the absence of agonists or in the presence of antagonists, hPXR is thought to be retained in the cytoplasm as a complex with corepressors. The binding of a lipophilic agonist to the LBD of hPXR elicits conformational changes, nuclear translocations and recruitments of coactivators.24 The receptor-agonist complex forms a heterodimer with retinoid X receptor-α (RXRα) upon binding to the specific repeats of AG(G/T)TCA in target genes’ promoters, then regulates the transcription of target genes.25

The expressions of hPXR have been detected in several types of cancers.26,27 Mensah-Osman and coworkers characterized the expression and function of hPXR in drug resistance in osteosarcoma cell lines.28 Recently we demonstrated that hPXR was expressed in prostate tumor specimens and regulated the responses of prostate cancer cells toward chemotherapeutics.29 Although hPXR expressions were detected in breast cancer cell lines and breast tumor tissues,30,31 the role of hPXR in the responses of breast cancer cells toward drugs remains unknown.

Since the responses of hPXR to drugs and consequential functions can be cell- and tissue-specific,28 in the present study we determined the role of hPXR in drug resistance of breast cancer cells. Herein we report that pre-activation of hPXR with SR12813, a selective activator of hPXR,32 led to an enhanced resistance to breast cancer treatment drugs. Downregulation of hPXR resulted in sensitizations of breast cancer cells toward cancer therapeutic agents. Our studies suggest a potential role for hPXR in determining the responses of breast cancer cells toward drug treatments.

Results

Expression and distribution of hPXR in breast cancer

Using RT-PCR and western blot, we detected hPXR expressions at both mRNA level and protein level in two established breast cancer cell lines, MCF-7 and MDA-MB-231 (Fig. 1). Immunohistochemical staining detected hPXR expressions in both cancerous human breast tissue specimens (n = 51, eleven of grade I nonspecific infiltrating duct carcinoma, 36 of grade II nonspecific infiltrating duct carcinoma, and four of grade III nonspecific infiltrating duct carcinoma) and matched normal adjacent breast tissues (n = 37). Strong hPXR stainings were observed in luminal epithelial cells in the lobules or ducts. The myoepithelial cells lining the lobules or ducts or the smooth muscle cells in the blood vessel showed negative hPXR expression and were used as internal controls of staining procedure. No consistent quantitative differences were observed between breast tumor samples and their matched normal breast tissues. The representative stainings are shown in Figure 2A. hPXR was usually presented in the cytosol, but in some cases, nuclear localizations of hPXR were noted (Fig. 2B, part i).

Figure 1.

Figure 1

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The expression of hPXR in breast cancer cell lines, MCF-7 and MDA-MB-231. (A) RT-PCR detection of hPXR in MCF-7 and MDA-MB-231. Pos. Ctrl: a pcDNA3.1-hPXR plasmid. β-actin was used as the internal control. (B) Western blot detection of hPXR in MCF-7 and MDA-MB-231. Cells lysates were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride PVDF membranes. The membranes were incubated with either anti-hPXR monoclonal antibody H-11 (expected size: 50 kd) or anti-β-actin monoclonal antibody AC-15 (expected size: 42 kd). The immunoblots were visualized and quantified by infrared imaging system. Pos. ctrl: 293T cells transiently transfected with a pcDNA3.1-hPXR plasmid.

Figure 2.

Figure 2

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Immunohistochemical analysis of hPXR expression and cellular localization. (A) Immunohistochemical staining of hPXR in normal and cancerous breast tissues. (i) normal breast; (ii) nonspecific infiltrating duct carcinoma (Grade I); (iii) nonspecific infiltrating duct carcinoma (Grade II); (iv) nonspecific infiltrating duct carcinoma (Grade III); (v) Pos. Ctrl: normal liver tissue; (vi) Neg. Ctrl: breast tissue stained with mouse IgG. Brown, hPXR staining; blue, nucleus staining with hematoxylin. (B) Representative nuclear localizations of hPXR in breast cancer tissues and cells. (i) Immunohistochemical staining of hPXR nuclear localizations in breast nonspecific infiltrating duct carcinoma; (ii) immunocytochemical staining of hPXR nuclear translocation in MDA-MB-231 cells. The DMSO-treated (left) or SR12813-treated (0.2 μmol/L, right) MDA-MB-231 cells were stained with H-11 mouse monoclonal PXR antibody. Green, immunoreactivity of hPXR, indicated by white arrows; red, cytoskeleton staining with phalloidin.

Increased CYP3A4 and MDR1 expression after hPXR activation in breast cancer cells

To determine the possible functionalities of hPXR expressed in breast cancer cells, we first examined cellular localization of hPXR after activation. As shown in Figure 2B, part ii, a nuclear translocation was observed in MDA-MB-231 cells after SR12813 treatment. The nuclear hPXR immunoreactivity was also noted in some clinical specimens (Fig. 2B, part i).30 The cytotoxicity of SR12813 was evaluated before experiments. SR12813 did not significantly affect cell viabilities at concentrations up to 1 μmol/L for treatment of 3 d (Fig. 3A).

Figure 3.

Figure 3

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(A) The cytotoxicity of SR12813 to breast cancer cells. MCF-7 or MDA-MB-231 cells were cultured in 96-well plates at an initial density of 10,000 cells/well and treated with SR12813 (DMSO, 0.1 μmol/L, 0.2 μmol/L, 0.5 μmol/L or 1 μmol/L) for 24 h (day 1), 48 h (day 2) or 72 h (day 3). The cell viability was measured by MTS assay. Columns, mean viability as a percentage of DMSO-treated cells from n = 4 replicates; bars, SE. (B) Regulation of hPXR in breast cancer cell lines to CYP3A4. Cells in 12-well plates were cotransfected with a LacZ plasmid and a pGL3-hPXRE plasmid. To overexpress PXR, a pcDNA3.1-PXR plasmid was cotransfected (left, MCF-7 cells). Twenty-four h later, cells were treated with SR12813 at different concentrations for another 24 h. The luciferase activity was normalized with β-gal activity. Columns, mean of assays done in triplicate; bars, SE.

A luciferase assay was used to evaluate the regulation ability of hPXR on the promoter of its target gene, CYP3A4. hPXR overexpressing MCF-7 cells showed a dose-dependent induction by SR12813 (Fig. 3B, left). The luciferease activities in wild-type MCF-7 cells and MDA-MB-231 cells increased with the doses of SR12813 until the concentrations were more than 0.2 μmol/L (Fig. 3B, middle and right). The data suggest that at a concentration of 0.2 μmol/L, SR12813 was able to active all endogenous hPXR in breast cancer cells.

To determine whether hPXR in breast cancer cells had the ability to regulate gene expressions of target DMEs and transporters, levels of CYP3A4 and MDR1 were examined by RT-PCR after 4 h, 8 h, 12 h, 24 h, 48 h or 72 h exposure of cells to SR12813. Exposure of MDA-MB-231 cells or MCF-7 cells to SR12813 induced the expressions of CYP3A4 and MDR1 in a time-dependent manner (Fig. 4A). The maximal induction of CYP3A4 was obtained at 8 h post-treatment. The MDR-1 levels increased with time until 8 h. During the course of treatment, the levels of hPXR had no significant changes (Fig. 4B). Taken together, the activation of hPXR in breast cancer cells stimulated the expression of DMEs and efflux transporters, which in turn may significantly altered the responses of tumor cells toward cancer therapeutic agents.

Figure 4.

Figure 4

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(A) The time-dependent increase of mRNA levels of CYP3A4 and MDR1 after the SR12813 treatment. Cells were treated with SR12813 for 0, 4, 8, 12, 24, 48 or 72 h. The mRNA levels were evaluated by RT- PCR using β-actin as the loading control. (B) Consistent hPXR protein levels after SR12813 treatment for 0, 4, 8, 12, 24, 48 or 72 h. The hPXR protein levels were evaluated by western-blot and normalized by β-actin. The hPXR were detected with anti-hPXR mono clonal antibody H-11. Columns, mean results from three independent experiments; bars, SE.

Increased resistance of MCF-7 and MDA-MB-231 to cancer therapeutic agents after SR12813 treatment

To study whether hPXR can alter the responses of tumor cells toward cancer therapeutic agents, we examined the effects of hPXR activation on the resistance of breast cancer cells to cancer treatment agents. Cells were pretreated with SR12813 for 12 h and then subjected to graded levels of cancer therapeutic agents for 72 h. As shown in Figure 5A, SR12813 treatment on MDA-MB-231 cells led to an increased resistance to Taxol at low concentrations (20 nmol/L and 50 nmol/L) but not at higher concentrations. No significant change on resistance of MDA-MB-231 cells to vinblastine was observed. The hPXR preactivation also conferred upon MCF-7 cells with a significantly increased resistance to tamoxifen (Fig. 5C). Colony formations of SR12813-pretreated MDA-MB-231 and MCF-7 were significantly higher than that of no SR12813 pretreatment cells after a short-time exposure to cancer treatment drugs (Fig. 5B and D). When tamoxifen concentrations were equal to or higher than 10 μmol/L, almost all MCF-7 cells perished, and few clones were formed. No significant differences could be observed among the groups with or without SR12813 pretreatment (Fig. 5D).

Figure 5.

Figure 5

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Increased drug resistance in breast cancer cells after the pretreatment of SR12813. (A and C) MTS assay. Cells were treated with DMSO or 0.2 μmol/L SR12813 for 12 h followed by the 72 h treatment of Taxol (A, MDA-MB-231) or tamoxifen (C, MCF-7) at different concentrations. The viability percentages of cells with no cancer therapeutic agents treatment were deemed as 100%. (B and D) colony formation assay. Cells were seeded into 12-well plates at the initial density of 500 cells/ well. After attachment, cells were treated with DMSO or 0.2 μmol/L SR12813 for 12 h and then Taxol (B, MDA-MB-231) or tamoxifen (D, MCF-7) at different concentrations for 36 h. Seven to fourteen days later, absolute colonies were counted, and the colony numbers in wells with no cancer therapeutic agents treatment were deemed as 100%. *p < 0.05, **p < 0.01, assayed by Student’s t-test, compared with the DMSO-treated group. Columns, average percentage from three separate experiments; bars, SE.

Enhanced sensitivity toward drug treatments in breast cancer cells with hPXR expression silenced by shRNA

To further study the role of hPXR in drug resistance, we examined the effects of hPXR knockdown on the responses of tumors toward cancer therapeutic agents. In MCF-7 cells, three different hPXR shRNA constructs were used. hPXR knockdown was observed in all three sublines (1619, 1620 and 1621, Fig. 6A). The abilities of hPXR activators to regulate the CYP3A4 expression were significantly compromised in the hPXR-knockdown clones when compared to the control scramble shRNA constructs (Fig. 6B). The sensitivity of hPXR knockdown cells to tamoxifen was enhanced when compared with the control cells (Fig. 6C). The reduced resistance of hPXR knockdown cells was further confirmed by reduced colony formation ability after a short-time exposure to tamoxifen (Fig. 6D). In MDA-MB-231 cells, only one construct produced significant transient hPXR knockdown (T1622, Fig. 7A). Both T1622 and an unknockdown subline, T1621, were used for drug sensitivity assays. A subline of MDA-MB-231 cells with hPXR stably knocked down (v3014-2, Fig. 7B) was also used due to the low transient transfection efficiency. A significant increase in sensitivity toward vinblastine was observed at all tested concentrations in hPXR knocked down MDA-MB-231 when compared to the cells of control or parent cells (Fig. 7C). In addition, knockdown of hPXR also sensitized MDA-MB-231 cells toward Taxol (Fig. 7D). The results, taken together, suggest that knockdown of hPXR expression sensitized breast cancer cells toward cancer therapeutic agents.

Figure 6.

Figure 6

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Knockdown of hPXR in MCF-7 cells. 1619, 1620 and 1621 were three sublines of hPXR knockdown MCF-7 cells. (A) Western blot of hPXR knockdown. The hPXR was probed with an anti-hPXR monoclonal antibody H-11. (B) The regulation of hPXR to CYP3A4 promoter. The MCF-7 cells were cotransfected with an hPXR shRNA vector/scramble shRNA vector, a lacZ plasmid and a pGL3-hPXRE vector containing two hPXR-responsive fragments in CYP3A4 promoter. After 24 h, cells were treated with 0.2 μmol/L SR12813, and another 24 h later, the luciferase activity was measured and normalized with β-gal activity. (C) Increased sensitivity of hPXR-knockdown MCF-7 cells to tamoxifen. Cells were treated with SR12813 for 12 h followed by tamoxifen treatment for another 72 h, then the cell viability was measured by MTS assay. The viability percentages of cells with no cancer therapeutic agents treatment were deemed as 100%. (D) Colony formation assay. Cells were seeded into 24-well plates at the initial density of 500 cells/well. After attachment, cells were treated with SR12813 (0.2 μmol/L) for 12 h and then tamoxifen at different concentrations for 36 h. Seven to fourteen days later, absolute colonies were counted, and the colony numbers in wells with no cancer therapeutic drugs treatment were deemed as 100%. *p < 0.05; **p < 0.01, assayed by Student’s t-test, compared with scramble shRNA control. Columns, the mean of assays performed in triplicate; bars, SE.

Figure 7.

Figure 7

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hPXR knockdown in MDA-MB-231 cells. (A) Western blot. The hPXR was probed with an anti-PXR monoclonal antibody H-11. T1622, transient hPXR knockdown MDA-MB-231 cells; T1621 and T1623, hPXR unknockdown MDA-MB-231. (B) RT-PCR analysis of the mRNA level of hPXR in stable hPXR knockdown MDA-MB-231 cells. v3014, stable hPXR knockdown MDA-MB-231 cells. (C) Increased sensitivity of hPXR knockdown MDA-MB-231 cells to vinblastine. (D) Increased sensitivity of hPXR knockdown MDA-MB-231 cells to Taxol. The cell viability was measured by MTS assay. The viability percentages of cells with no cancer therapeutic agents treatment were deemed as 100%. *p < 0.05; **p < 0.01, assayed by Student’s t-test, compared with scramble shRNA control. Columns, the mean of assays performed in triplicate; bars, SE.

Discussion

Drug resistance remains a major obstacle to the successful treatment of breast cancer. Due to the inherent side effects of many cancer therapeutic drugs, the resistance presented by tumor cells cannot be overcome by simply increasing doses to tumor sites. Many anticancer drugs have a narrow therapeutic window and a steep dose-toxicity curve. Therefore, changes in the metabolism and/ or disposition of drugs will likely have significant impacts on the clinical responses to drug treatments.33,34

Proposed as a sensor for xenobiotics, hPXR was found to be expressed mainly in human liver, colon and small intestine, sites associated with detecting and combating xenobiotics and potentially toxic endobiotics.6,25 hPXR was also expressed in several types of cancers, such as sarcoma cells and endometrial cancer,27,28 raising an interesting question regarding the potential roles of hPXR in neoplastic tissues. As a dominant transcription factor of DMEs and transporters, hPXR might play a role in the drug resistance in cancer. Recently we have demonstrated that hPXR is expressed in prostate tumor specimens and further it regulates the responses of prostate cancer cells to chemotherapeutics.29 It should be noted that the responses of hPXR to drugs and consequential function are cell- and tissue-specific.28 Although hPXR expression was detected in breast cancer cell lines, breast tumors and surrounding normal breast tissues, the role of hPXR in breast drug resistance remained obscure.30,31

hPXR and many of its target genes have been characterized in human breast carcinoma specimens30 and breast cancer cells.35 In the present study, hPXR expression at the protein level was detected in established MCF-7 cells and MDA-MB-231 cells. MCF-7 had a higher level of hPXR than MDA-MB-231 cells, and its resistance to drugs elicited by SR12813 in the cell viability assay (Fig. 5) was more pronounced than MDA-MB-231 cells, which indicates a PXR-specific correspondence. As a master regulator of many enzymes involved in drug metabolism, the hPXR-DMEs/hPXR-transporters pathway might be one of the mechanisms in hPXR-mediated drug resistance. In this study, increased levels of a predominant DMEs, CYP3A4 and ABC transporter, MDR1, were observed after SR12813 treatment in breast cancer cells. The upregulation of drug metabolism and excretion genes by hPXR might reduce the clinical efficacy of anti-cancer agents, especially at low doses or when the in situ availability of drugs becomes a limiting factor, due to the metronomic dosing or the poor vascularization of tumors. While the baseline levels of CYP3A4 and MDR1 in current breast cancer cell lines were low, hPXR activation can stimulate the expression of CYP3A4 and MDR1 (Fig. 4A). Other DMEs or transporters, especially those highly expressed in breast cancer cells, such as organic anion transporting polypeptide1,30 multidrug resistance associated protein 2,36 might be also subjected to the regulation of hPXR. However, further studies are required to discern the involvement of DMEs and efflux transporters in the hPXR-mediated drug resistance.

Another possible mechanism for hPXR-mediated drug resistance is the regulation of cell death and survival pathways by hPXR. A microarray analysis identified over 200 genes regulated by rat PXR, many of which were related to molecules involved in cell cycle and survival mechanisms.37 Cell growth inhibition and apoptosis has been observed in endometrial cancer cells by the downregulation of hPXR.38 In breast carcinomas, hPXR may play an important role as a hormone sensitivity factor for in situ estrogen actions and may be correlated with progression or de differentiation of breast carcinoma.30 However, further studies are required to determine whether PXR also regulates the growth and survival of breast cancer cells.

Due to the large volume of its ligand binding domain, hPXR can be activated by diverse compounds.32 This is especially pertinent for cancer patients as many of them often take over-the-counter drugs, nutritional supplements or other complementary alternative medicines, which may activate hPXR and reduce the efficacy of anti-cancer drugs.39 In some tumor specimens, we and other groups30 observed a nuclear localization of hPXR, indicative for hPXR activation in clinical settings. In addition, several anticancer drugs themselves can activate hPXR at clinically relevant concentrations. Taxol (paclitaxel), a commonly used chemotherapeutic agent, had been reported to activate hPXR and self-regulate its own clearance.17 Vincristine, an analogue of vinblastine, is a potential hPXR ligand and induces ABCC2 and ABCC3 expression in carcinoma cells. Altered uptake and retention of tamoxifen and possible altered local metabolism have been suggested as mechanisms of tamoxifen resistance in some human breast cancers.40,41 In addition, many endogenous compounds in normal or neoplastic breast tissues can also activate hPXR.42 Clearly more future studies are needed to address the potential interactions of drug-drug and drug-supportive medicines in determining the clinical outcomes of drug treatment in cancer patients.

In summary, we detected hPXR expression in normal and cancerous breast tissues. Activation of hPXR rendered breast cancer cells with increased resistance toward drugs, whereas downregulation of hPXR sensitized breast cancer cells toward drugs. If the activation of hPXR is one of the major underlying mechanisms of drug resistance or drug-drug interactions during chemotherapy, hPXR will be a new target of intervention to modulate the clinical efficacy of breast cancer drug treatment. Alternatively, cancer therapeutic drugs that do not activate hPXR should be developed to circumvent hPXR-mediated drug resistance.

Materials and Methods

Materials

MDA-MB-231 cells and MCF-7 cells were obtained from ATCC (Manassas, VA). Normal or cancerous human breast tissue arrays were obtained from US Biomax, Inc., (Rockville, MD). Puromycin was purchased from Sigma (St. Louis, MO). hPXR (H-11) sc-48340 mouse monoclonal antibody was from Santa Cruz biotechnology Inc., (Santa Cruz, CA). SR12813 [dissolved in Dimethyl Sulfoxide (DMSO)], vinblastine (dissolved in water), Taxol (dissolved in DMSO) and tamoxifen (dissolved in DMSO) were acquired from BIOMOL International L.P. (Plymouth Meeting, PA). The PCR reagents, MTS solution, luciferase assay system, pGL3 vector and β-galactosidase assay system were from Promega (Madison, WI). hPXR shRNA constructs were from Open Biosystems (Huntsville, AL). 293TN producer cell line and a pPACKH1 lentivector packaging kit were from System Biosciences (Mountain View, CA). Phoenix A cell line was a gift from Dr. Randolph Elble in Southern Illinois University.

Immunohistochemistry

For hPXR tissue immunostaining, paraffin-embedded tissues were deparaffinized, rehyrated and antigen retrieved by Declere® working solution. After washing in deionized water, slides were processed for immunohistochemical staining using a Zymed® Histostain-SP kit according to manufacturer’s instructions. An hPXR (H-11) sc-48340 mouse monoclonal antibody was used for hPXR detection.

Immunocytochemistry

The day before staining, cells were seeded into six-well plates containing cover glass. The next day, cells were fixed in 3% paraformaldehyde, incubated with 0.1% Triton X-100 and blocked with 1% BSA and 2% horse serum. Following blocking, cells were incubated sequentially with H-11 mouse monoclonal hPXR antibody for 1 h (1:100 dilution), Alexa Fluor® 488 goat anti-mouse IgG(H + L) secondary antibody for 1 h (1:100 dilution). The slides were then washed, mounted in Prolong® gold antifade reagent with DAPI.

Semi-quantitative RT-PCR

Total RNA was extracted from cell cultures with TRIzol® reagent. Reverse transcription was performed by SuperscriptTM III First-Strand synthesis system. Genes of interest were amplified by PCR from cDNA with the following primer pairs: hPXR (348 bp): 5′-TGT CAT GAC ATG TGA AGG ATG-3′/5′-TTG AAA TGG GAG AAG GTA GTG-3′; CYP3A4 (325 bp): 5′-CTA GCA CAT CAT TTG GAC TG-3′/5′-ACA GAG CTT TGT GGG ACT-3′; MDR1 (698 bp): 5′-TCA CCT TCG TCA GCT ACT TCG G-3′/5′-CAG GAG GTC ACA GCC GAC TTT AAA C-3′; β-actin (294 bp): 5′-TCA CCC ACA CTG TGC CCA TCT ACG A-3′/5′-CAG CGG AAC CGC TCA TTG CCA ATG G-3′. The PCR mixtures were initially denatured at 94°C for 2 min, followed by denaturation for 20 s at 94°C, primer-annealing at 53°C (for CYP3A4 and β-actin) or 55°C (for MDR1 and hPXR) for 30 s, and extension for 1 min at 72°C for 30 cycles (for CYP3A4, MDR1 and hPXR) or 25 cycles (for β-actin) with the MJ MiniTM personal Thermal Cycler (Bio-Rad laboratories). A final extension for 5 min at 72°C ensured the complete extension of PCR products.

Western blot

Cells were directly lysed with 2X SDS-PAGE electrophoresis sample buffer (Invitrogen), boiled and sonicated. After centrifuging, supernatants were separated on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride PVDF membrane at 100 V for 2 h. Then the membranes were incubated with either anti-hPXR monoclonal antibody H-11 (diluted 1:500 in blocking buffer) or anti-β-actin monoclonal antibody AC-15 (diluted 1:500 in blocking buffer) overnight at 4°C. For staining, goat anti-mouse fluorescently-labeled secondary antibody (diluted 1:5,000 in blocking buffer) was used. The immunoblots were visualized and quantified by Infrared imaging system.

hPXR knockdown

Four hPXR shRNAs in a pSM2c retroviral vector and four hPXR shRNAs in a pLKO.1 lentiviral vector were used. Lentiviral constructs were packaged into lentivirus first in 293TN cells with a pPACK lentivector packaging system according to the manufacturer’s instructions. Forty-eight hours later, the viral supernatants were collected, centrifuged and filtered for further transduction. Retroviral vectors were transfected into Phoenix A cells using DNAfectinTM transfection reagents. The cells were selected in 1 μg/ml puromycin medium for 3–5 days. After untransfected phoenix A cells died, the fresh medium was added. After 12 h, the retroviral supernatants were collected, centrifuged and filtered for further transduction. The MDA-MB-231 and MCF-7 cells were transduced by shRNA viral stocks with 4 μg/ml polybrene at a 70–80% confluence. Twenty-four hours post-transduction, puromycin was added at final concentration of 5 μg/ml to eliminate noninfected cells and 1 μg/ml to maintain infected cells. The scramble shRNA was used as control.

Drug sensitivity assay

The cells were seeded into 96-well plates at an initial density of 10,000 cells per well. After attachment, cells were treated with Taxol, tamoxifen or vinblastine for 72 h directly or following a 12 h treatment of SR12813. For the evaluation of SR12813 cells cytotoxicity, cells were treated with SR12813 (DMSO, 0.1 μmol/l, 0.2 μmol/l, 0.5 μmol/l or 1 μmol/L) for 24 h, 48 h or 72 h. The cell viability was measured by MTS assay according to the manufacturers’ instructions. In brief, 100 μl of fresh serum-free medium and 20 μl of MTS reagent were added into each well after decanting the old medium. Plates were incubated at 37°C for 2 h and the absorbance at 490 nm was taken.

Colony formation assay

The clonogenic efficiency of surviving fractions of tumor cells after exposure to drugs was determined by modified colony formation assay. Briefly, cells were grown into 12-well plates at an initial density of 500 cells per well. After 12 h of pretreatment of SR12813 at 0.2 μmol/L or DMSO, cells were treated with tamoxifen or Taxol for 36 h. After treatment, the cells were cultured in fresh RPMI media for 7–14 d. The colonies formed were then stained with Giemsa solutions and enumerated.

Luciferase assay

Cells were seeded into six-well plate. After attachment, cells were transiently transfected with LacZ and pGL-hPXRE luciferase reporter constructs. To overexpress hPXR, a pcDNA3.1-hPXR plasmid was cotransfected. The pGL-hPXRE was constructed by inserting two hPXR-responsive fragments in the proximal (-362/+53) and distal (-7836/-7208) CYP3A4 promoters into pGL3 vector at Xho I and Hind III sites. Twenty-four hours later, cells were treated with SR12813 at different concentrations for another 24 h. Then cell lysates were harvested, assayed for luciferase and β-galactosidase activities. The luciferase activity was normalized for transfection efficiency with β-galactosidase activity.

Statistical analysis

Student’s t-test (two-tails) was used to analyze the difference between two groups. A p value between groups, if smaller than 0.05, is considered statistically significant.

Acknowledgements

This work was partially supported by the Penny Severn Postdoctoral Fellowship from Offices of Women’s Health, Illinois Department of Public Health (Y.C.), Start-up fund from Southern Illinois University School of Medicine and SimmonsCooper Cancer Institute (D.N.), an award from Southern Illinois University School of Medicine Central Research Committee (D.N.), National Institute of Health Grant R01CA131445 and R15CA133776 (D.N.), and United States Department of Defense Breast Cancer Research Program Idea Award No. W81XWH-08-1-0540 (D.N.).

Abbreviations

hPXR

human pregnane X receptor

CYP3A4

cytochrome P450 3A4

MDR1

multi-drug resistance gene

ABC

ATP binding cassette

SXR

steroid and xenobiotic receptor

RXR

retinoid X receptor

BCRP

breast cancer resistance protein

DMEs

drug metabolism enzymes

LBD

ligand binding domain

FBS

fetal bovine serum

RT

reverse transcription

DMSO

dimethyl sulfoxide

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