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Cancer Research 67, 10361, November 1, 2007. doi: 10.1158/0008-5472.CAN-06-4758
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Human Pregnane X Receptor and Resistance to Chemotherapy in Prostate Cancer

Yakun Chen1,2, Yong Tang1, Man-Tzu Wang1, Su Zeng2 and Daotai Nie1

1 Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University, School of Medicine and SimmonsCooper Cancer Institute, Springfield, Illinois and 2 Department of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China

Requests for reprints: Daotai Nie, Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine and SimmonsCooper Cancer Institute, Springfield, IL 62794-9626. Phone: 217-545-9702; Fax: 217-545-3227; E-mail: dnie{at}siumed.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Resistance to chemotherapy is a significant barrier to the effective management of prostate cancer. Human pregnane X receptor (hPXR), an orphan nuclear receptor known for its activation by many important clinical drugs, interacts with many cellular signaling pathways during carcinogenesis and is a major transcription factor regulating the expression of drug metabolism enzymes, including transporters. It is unknown whether hPXR is a determinant of drug resistance in prostate cancer. In this study, we first detected the expression of hPXR in both normal and cancerous prostate tissues. Pretreatment with SR12813, a potent and selective agonist of hPXR, led to nuclear translocation of PXR in PC-3 cells and increased expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance 1 (MDR1). SR12813 pretreatment increased resistance of PC-3 cells to Taxol and vinblastine, as assessed by viability and clonogenic survival. To further study the role of hPXR in prostate cancer drug resistance, hPXR expression was knocked down using PXR-targeting short hairpin RNAs. The activities of hPXR toward the promoter of CYP3A4 in hPXR-ablated clones decreased when compared with that of wild-type PC-3 cells. Their sensitivities to Taxol and vinblastine were enhanced by hPXR ablation. Our data here suggest that hPXR may play an important role in prostate cancer resistance to chemotherapeutics. [Cancer Res 2007;67(21):10361–7]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cancer is a major public health problem in the United States, accounting for one in four deaths (1). Chemotherapy is one of the three most common treatment modalities of cancer, but its efficacy is limited by the resistance of cancer cells to drugs. Induction of drug metabolizing enzymes (DME) and efflux transporters has been regarded as one of major mechanisms of chemoresistance (2, 3). The differential inductions or activities of DMEs may account for the variations of drug efficacy, but the precise mechanism of inductions remains unknown.

Pregnane X receptor (PXR; also called PAR, SXR, or NR1I2) is a nuclear receptor that has been identified as a master regulator of DMEs (4, 5). Upon activation with ligands, PXR translocates from cytosol into nucleus and regulates transcription of its target genes (6). The significance of PXR in drug metabolism and disposition is implicated by its activation by a variety of structurally diverse compounds (7) and by its role in regulating the transcriptions of many important DMEs (6, 8–12). Two most important DME families, cytochrome P450 3A (CYP3A) and ATP-binding cassette (ABC) transporters, are predominantly regulated by PXR. The induction of ABC transporters, including P-glycoprotein, has been known to cause multidrug resistance of cancer (13). CYP3A4 accounts for the oxidative metabolism of >60% of clinical drugs, including many chemotherapeutics (14). Recently, it has been shown that disruption of PXR activation is a mechanism involved for ketoconazole to inhibit the induction of DMEs, including CYP3A4 and multidrug resistance 1 (MDR1; ref. 15).

PXR is mainly expressed in liver, colon, and intestinal tissues (8). Although many researches have explored the characteristics and functions of PXR in normal absorption and metabolism system, little work has been done to evaluate its role in cancerous tissues. PXR expression has been detected in breast (16, 17) and endometrial cancers (18). The importance of PXR in cancer is underlined by the studies demonstrating the activation of human PXR by some commonly used chemotherapeutic agents (19–21). However, there are few studies being done or reported to examine a possible role of PXR in cancerous tissues in resistance to chemotherapy.

The present study is undertaken to test the hypothesis that PXR in cancerous tissues is a determinant of tumor responses toward chemotherapy. Here, we report that PXR expression was detected in prostate cell lines and tissues. Preactivation of PXR with SR12813 led to an enhanced resistance to chemotherapeutics. Down-regulation of PXR in prostate cancer cells resulted in sensitization toward chemotherapeutic agents. Our studies suggest an important role for PXR in determining the responses of tumor cells toward chemotherapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. The human prostate cancer cell line PC-3, DU145, and LNCap cells were obtained from American Type Culture Collection. Human prostate normal or cancerous tissue array was obtained from Biomax. All cell culture, reverse transcription (RT) and immunochemistry reagents were purchased from Invitrogen unless otherwise indicated. Fetal bovine serum (FBS) and puromycin were purchased from Sigma. Declere was supplied by Cell Marque Corporation. Anti-PXR purified rabbit antibody was available from Biolegend. PXR (H-1) mouse monoclonal antibody was from Santa Cruz Biotechnology, Inc. CYP3A4 primary antibody was from Calbiochem. SR12813 (dissolved in DMSO), Taxol (dissolved in DMSO), and vinblastine (dissolved in water) were acquired from BIOMOL International L.P. The regular PCR reagents, MTS solution, luciferase assay system, pGL3 vector, and cell culture lysis reagent were from Promega. All real-time quantitative PXR reagents were from Applied Biosystems. The GenePORTER liposome transfection reagent was from Genlantis. PXR short hairpin RNA (shRNA) constructs were from Open Biosystems.

Immunohistochemistry. For PXR tissue immunostaining, paraffin-embedded tissue sections or tissue arrays were deparaffinized, rehyrated, and antigen retrieved by placing in Declere 1x working solution in an electric pressure cooker for 15 min. After a hot rinse with boiling Declere, slides were left to cool down for 5 min. After washing in deionized water, slides were processed for immunohistochemical staining using a Zymed Histostain-SP kit according to manufacturer's instructions. The sections of negative controls were stained after the same procedure except for the nonspecific rabbit IgG used as the primary antibody. Tissue sections were scored combining both the intensity and percentage of stained cells according to previous reports (22, 23). Intensities were classified into 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). Ten percent of the groupings were used for the percentage of cells that stained positive. For each slide, a value-designated H score was derived from the formula, H score = (percentage of cells stained at intensity category 1 x 1) + (percentage of cells stained at intensity category 2 x 2) + (percentage of cells stained at intensity category 3 x 3). Three areas were scored to obtain the average H score for each tumor section. This formula produces an H score in the range of 0 to 300, wherein 0 = none of the tumor cells stained and 300 = 100% of tumor cells stained strongly.

Immunocytochemistry. PC-3 cells were seeded into six-well plates containing cover glass. Next day, cells were fixed in 3% paraformaldehyde for 15 min, incubated with 0.1% Triton X-100 for 1 min, and blocked with 1% bovine serum albumin and 2% horse serum for 30 min. Then cells were incubated sequentially with purified rabbit PXR primary antibody (Biolegend) for 1 h (1:100 dilution), Alexa Fluor 488 goat anti-rabbit IgG (H + L) secondary antibody for 1 h (1:100 dilution). The slides were then washed, mounted in Prolong Gold antifade reagent with 4',6-diamidino-2-phenylindole and visualized with a BX41 system microscope (Olympus).

RT-PCR. The expression profile of PXR or its target genes, CYP3A4 and MDR1, was evaluated by semiquantitative RT-PCR. A normal human colon tissue sample (BioChain Institute, Inc.) and a human liver cancer cell line, HepG2, were used in parallel to compare the transcript levels of PXR in prostate cancer cells with human colon tissue and liver cancer cell line. Total RNA was extracted from cell cultures with TRIzol reagent. RT was done by Superscript III First-Strand synthesis system. Genes of interest were amplified by PCR from cDNA with the following primer pairs: PXR (348 bp), 5'-TGTCATGACATGTGAAGGATG-3'/5'-TTGAAATGGGAGAAGGTAGTG-3'; CYP3A4 (325 bp), 5'-CTAGCACATCATTTGGACTG-3'/5'-ACAGAGCTTTGTGGGACT-3'; MDR1 (698 bp), 5'-TCACCTTCGTCAGCTACTTCGG-3'/5'-CAGGAGGTCACAGCCGACTTTAAAC-3'; ß-actin (294 bp), 5'-TCACCCACACTGTGCCCATCTACGA-3'/5'-CAGCGGAACCGCTCATTGCCAATGG -3'. The PCR mixtures were initially denatured at 94°C for 2 min, followed by denaturation for 20 s at 94°C, primer-annealing for 30 s at 54.5°C, and extension for 1 min at 72°C for 45 cycles with the MJ Mini Personal Thermal Cycler (Bio-Rad Laboratories). A final extension for 5 min at 72°C ensured complete extension of the PCR products. The amplified DNA fragment sequences were determined directly with agarose gel electrophoresis. The ß-actin was used for normalization.

Real-time PCR. RT–quantitative PCR for PXR target genes, CYP3A4, and MDR1 was done using an Applied Biosystems 7500 real-time PCR system (Applied Biosystems). The procedure of RNA isolation and RT was the same as above semiquantitative RT-PCR. Total RNA (300 ng) from each sample was used for cDNA synthesis. RNA quantity and quality of the different samples were determined by the 260:280 nm absorbance ratio using a BioSpec-mini DNA/RNA/protein analyzer (Shimadzu Corporation). Quantitative PCR was done using gene-specific primers: CYP3A4, 5'-CTAGCACATCATTTGGACTG-3'/5'-ACAGAGCTTTGTGGGACT-3'; MDR1, 5'-GTCTTTGGTGCCATGGCCGT-3'/5'-ATGTCCGGTCGGGTGGGATA-3'. The reference gene ß-actin (5'-TCACCCACACTGTGCCCATCTACGA-3'/5'-CAGCGGAACCGCTCATTGCCAATGG-3') was used for normalization. All reactions were done in a singleplex way. The total reaction volume was 20 µL containing 2x SYBR Green PCR master mix, 100 nmol/L of each primer, and 2 µL cDNA as template. After two initial steps of 50°C for 2 min and 95°C for 10 min, the PCR reaction was run 40 cycles of 95°C for 15 s and 60°C for 1 min. Melt curve analyses were done for all samples and only sharp melting points were observed, indicating a specific signal and no primer dimers or mispriming. No template control was always included in each run to check for the presence of exogenous contaminant DNA. Standard curve was generated by serial dilution of cDNA. With the threshold cycle values in real-time PCR obtained, the amounts of CYP3A4 or MDR1 were then determined from standard curves. After normalization with the amount of ß-actin, the expression levels in each time point were expressed as a ratio (%) compared with that of vehicle-treated cells (0 h, DMSO). Each sample was analyzed twice in triplicate, and data were analyzed using 7500 system SDS software (Applied Biosystems).

Cell culture and shRNA transfection. PC-3, DU145, and LNCap cells were grown in a RPMI 1640 containing 5% FBS, supplemented with antibiotics and antimycotics in a humidified incubator, with an atmosphere of 5% CO2, at 37°C. Cells were transfected with shRNA constructs at a 70% to 80% confluence using GenePORTER liposome transfection reagent. After the transfection, puromycin was added at final concentration of 5 µg/mL to eliminate nontransfected cells and 1 µg/mL to maintain transfected cells.

Chemotherapeutic sensitivity assay. PC-3 cells were tripsinized, resuspended, counted by Vi-cell XR cell viability analyzer (Beckman), and seeded into 96-well plates at an initial density of 10,000 cells per well. After attachment, cells were treated with Taxol or vinblastine for 72 h directly or after a 12-h or 48-h treatment of SR12813, a selective agonist of PXR (24). Cell viability was measured by MTS assay. Plates were incubated at 37°C for 2 h, and the absorbance at 490 nm was measured in a plate reader (Thermo Electron).

Colony formation assay. The clonogenic efficiency of surviving fractions of tumor cells after exposure to drugs was determined by modified colony formation assay (25). Basically, PC-3 cells were grown into 12-well plates at an initial density of 1,000 cells per well. After 12 h of pretreatment of SR12813 at different concentration (DMSO only, 0.2 and 1 µmol/L), the cells were then treated with vinblastine (1 µmol/L) or Taxol for 24 h. After treatment, the cells were then cultured in fresh RPMI media for 7 days. The colonies formed were then stained with Giemsa solutions and enumerated.

Luciferase assay. PC-3 cells were seeded into six-well plate. After attachment, cells were transiently transfected with LacZ and pGL-PXRE luciferase reporter constructs using GenePORTER liposome transfection reagents as described above. The pGL-PXRE was constructed by inserting two PXR-responsive fragments in the proximal (–362/+53) and distal (–7,836/–7,208) CYP3A4 promoters into pGL3 vector at XhoI and HindIII sites. Seventy-two hours later, cell lysates were harvested using cell culture lysis reagent, assayed for luciferase (Promega) and ß-galactosidase activities (Invitrogen). The luciferase activity was normalized with ß-galactosidase activity for transfection efficiency. All values were averaged from triplicate determinations in one individual experiment, and the experiment was repeated at least thrice.

Statistical analysis. Student's t test (two tails) was used to analyze the difference between two groups. For comparisons among three or more groups, ANOVA test was used. For statistical analysis of PXR immunoreactivities in prostate tumor specimens, nonparametric tests were used. Kruskal-Wallis test was used to test the difference in the ranks of scores of 3 or more independent groups. Dunnett's multiple comparison test was used to further compare the scores between two groups. The statistical tests were done using GraphPad Prism 4.00 for Windows (GraphPad Software). A P value, if smaller than 0.05, would reject the null hypothesis and accept the alternative hypothesis, i.e., the difference between groups is significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of PXR in prostate cancer. PXR expression has been reported in human endometrial and breast cancers (17, 18). To examine whether PXR is expressed in prostate cancer, we first evaluated the expression of PXR at protein level in normal and cancerous human prostate tissues by immunohistochemistry. Tissue slides were stained with purified rabbit anti-PXR antibody (Biolegend). The human liver tissue, which has high-PXR expression, was used as a positive control (Fig. 1A ). As shown in Fig. 1, PXR immunoreactivity was present in normal prostate tissue sample (Fig. 1A and C), whereas no positive staining was observed in the negative control (Fig. 1A). In normal prostate gland, PXR immunoreactivity was strong in the basal cells, whereas in normal differentiated epithelial cells, PXR staining was reduced (Fig. 1A and C). PXR immunoreactivities were detected in nearly all prostate adenocarcinoma examined (Fig. 1A and C). Interestingly, nuclear localization of PXR immunoreactivities was noted in some tumors (Fig. 1A). The PXR immunoreactivites in all tissues were scored and analyzed according to the tissue types and Gleason scores. As shown in Fig. 1B, in cancerous prostate tissues (n = 124), PXR immunoreactivities were significantly different from normal prostate tissues (n = 19), especially in those with Gleason score of 6 (n = 16). The H scores of prostate adenocarcinomas increased with Gleason scores until 6, but decreased when the Gleason score was >6.


Figure 1
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  		Figure 1. Immunohistochemical evaluation of PXR expression in normal and cancerous human prostate tissues. A, micrographies of representative tissue samples from normal prostate tissue and prostate adenocarcinomas. Brown or dark brown, PXR staining; blue, nucleus staining with hemotoxylin; arrows, strong immunoreactivities of PXR in the basal area of the normal gland (normal) and in the nuclei of cancer cells (cancer). Tumor section incubated with nonspecific rabbit IgG was used as negative control (Neg. Ctrl). Human liver tissue was used as positive control (Pos. Ctrl). Original magnification, 200x. B, H scores of PXR immunoreactivities as function of Gleason scores. Columns, median H score; bars, SE. **, P < 0.01 when compared with normal control. *, P < 0.05 when compared with tumors with Gleason score of 9 or higher. C, panoramic micrographies of PXR immunoreactivities in normal prostate tissue or tissues with different degrees of malignancy. Brown or dark brown, PXR staining; blue, nucleus staining with hemotoxylin. Original magnifications: 40x (left) and 100x (right).

 
Functional characterization of PXR expressed in prostate cancer cells. The detection of PXR expression in clinical specimens of prostate cancer led us to hypothesize a potential role of PXR in modulating tumor responses to chemotherapy via regulating the expression of DMEs. First, we determined the expression of PXR in established human prostate cancer cell lines. As shown in Fig. 2A , PXR expression at mRNA levels was detected in established prostate cancer LNCap, PC-3, and DU145 cells by RT-PCR. Immunocytochemical staining with a specific PXR antibody revealed that in PC-3 cells cultured in serum-containing media, PXR was mainly localized in the cytosol (Fig. 2B, left). Treatment with SR12813, a selective and potent agonist of PXR (24), stimulated nuclear localization of PXR (Fig. 2B, right), indicating that PXR expressed in PC-3 cells was responsive to ligand treatment.


Figure 2
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  		Figure 2. Functional characterization of PXR expressed in prostate cancer cell lines. A, RT-PCR detection of PXR expression. A plasmid of PXR was used as positive control. The PCR reaction mixture without template was a negative control. B, nuclear localization of PXR after SR12813 treatment. PC-3 cells were treated with SR12813 for 12 h and processed for immunocytochemical evaluation of PXR cellular localization. Original magnification, 200x. C, the time-dependent increase of mRNA levels of CYP3A4 and MDR1 in PC-3 cells after SR12813 treatment. The cells were cultured in RPMI 1640 containing 5% FBS and treated with a potent and selective activator of PXR, SR12813, for 0, 4, 8, 12, 24, 48, or 72 h. The mRNA levels of CYP3A4 and MDR1 were evaluated by RT–quantitative PCR and normalized to ß-actin. Columns, mean fold induction of mRNA levels compared with vehicle-treated group (0 h, DMSO), n = 6; bars, SE.

 
Next, we examined the levels of CYP3A4 and MDR1 expression in PC-3 cells after treatment of SR12813. The levels of CYP3A4 and MDR1 expression in PC-3 cells after exposure to SR12813 were examined using quantitative PCR. As shown in Fig. 2C, exposure of PC-3 cells to SR12813 stimulated CYP3A4 expression at mRNA level up to 72 h, with peak stimulation (26.7-fold) recorded within 4 h of treatment. MDR1 expression at mRNA level was also stimulated by SR12813 treatment with peak stimulation around 28.1-fold at 8 h after treatment (Fig. 2C). The data suggest that activation of PXR could stimulate the expression of DMEs in prostate cancer cells.

Increased resistance of PC-3 cells to chemotherapeutics after SR12813 treatment. To study whether above-described stimulation of DME expression as result of PXR activation can lead to changes in drug resistance, we examined the effects of SR12813 pretreatment on the resistance of PC-3 cells to Taxol and vinblastine, two chemotherapeutics widely used to treat a spectrum of malignancies including prostate cancer. It was found the preactivation of PXR by SR12813 led to an increased survival of PC-3 cells, as assessed by MTS viability assay, after treatment of Taxol (10 and 100 nmol/L; Fig. 3A ) or vinblastine (0.3 and 0.6 µmol/L; Fig. 3B). A similar increase in chemoresistance was observed for cells pretreated with SR12813 for 48 h (data not shown), consonant with the observations that SR12813 increased the gene expression of MDR1 and CYP3A4 at mRNA levels up to 72 h (Fig. 2C).


Figure 3
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  		Figure 3. Increased chemoresistance in PC-3 cells by PXR agonist, SR12813. Cells were cultured in RPMI 1640 containing 5% FBS and treated with 0 µmol/L (DMSO, {circ}), 0.2 µmol/L ({blacksquare}), or 1 µmol/L ({blacktriangleup}) SR12813 for 12 h followed by the 72-h treatment of Taxol (A) or vinblastine (B). *, P < 0.05, assayed by Student's t test, compared with the vehicle-treated group. Points, mean viability as a percentage of control (i.e., cells without chemotherapeutics treatment, 100%) from replicates (n = 9) from three separate experiments; bars, SE.

 
The increased resistance of prostate cancer cells after preactivation of PXR with SR12813 was also confirmed by colony formation assay. As shown in Fig. 4 , pretreatment with SR12813, which increased the gene expression of CYP3A4 and MDR1 (Fig. 2C), enhanced clonogenic survival of PC-3 cells after treatment of vinblastine (Fig. 4A) or Taxol (Fig. 4B). The results further establish a potential role of PXR in resistance of prostate cancer cells toward Taxol and vinblastine.


Figure 4
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  		Figure 4. PXR promotion of clonogenic survival of prostate cancer cells after chemotherapy. A, SR12813 stimulation of clonogenic survival of PC-3 cells from Taxol treatment. PC-3 cells were seeded into 12-well plates at the density of 1,000 cells per well. After attachment, cells were treated with SR12813 [0.2 µmol/L ({blacksquare}) or 1 µmol/L ({blacktriangleup})] or DMSO (vehicle control, {circ}) for 12 h and then Taxol for 24 h. Seven days later, colonies with >20 cells were counted and normalized with the group without Taxol treatment. *, P < 0.05, assayed by t test. Points, average from three separate repeats; bars, SE. B, increased clonogenic survival after vinblastine treatment. PC3 cells were seeded into 12-well plates at the initial density of 1,000 cells per well. After attachment, cells were pretreated with SR12813 for 12 h and then vinblastine (1 µmol/L) for 24 h. Seven days later, colonies were counted and normalized with their respective groups without vinblastine treatment. *, P < 0.05, assayed by t test. Columns, average from three separate experiments; bars, SE.

 
Enhanced sensitivity toward chemotherapy in prostate cancer cells with reduced PXR activity. To further confirm the role of PXR in PC-3 resistance to chemotherapeutics, we knocked down the expression of PXR in PC-3 cells with the shRNA constructs. Total eight PXR shRNA constructs were used in this experiment. For the wild-type and PXR non-knockdown PC-3 cells, the PXR was detected as a band of 348 bp, whereas for the knockdown clones, PXR were undetected by RT-PCR (Fig. 5A ). The knockdown of PXR expression in P1622-1, P1622-3, and Pm1623-3 clones was further confirmed by reduced immunoreactivity of PXR (Fig. 5B). In contrast, PC-3 and the P1620-4 clone retained intensive immunoreactivity of PXR with variable nuclear localization. The P1620-4 clone, in which PXR expression was not knocked down, was used as a control.


Figure 5
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  		Figure 5. Increased chemosensitivity of PC-3 cells with PXR expression knocked down. A, RT-PCR analysis of the mRNA level of PXR in wild-type or PXR non-knockdown PC-3 cells or PXR knockdown PC-3 cells. ß-actin was used as the loading control. B, immunocytochemical staining of hPXR in wild-type or PXR non-knockdown PC-3 cells or PXR knockdown PC-3 cells. The fluorescence indicates the immunoreactivity of PXR. C, reduced promoter activities of PXR in PC-3 cells with PXR expression knocked down. The wild-type, PXR non-knockdown, or PXR knockdown PC-3 cells were transiently cotransfected with a LacZ plasmid and a control vector or a pGL-PXRE vector containing two PXR-responsive fragments in CYP3A4 promoter. After 72 h, cells were harvested and lyzed. The luciferase activity was measured and normalized for transfection efficiency with ß-galactosidase activity. Calculated as the ratio of luciferase activity of transfectants to that of untransfected PC-3 cells. Columns, mean of assays done in triplicate; bars, SE. Represents one of three independent experiments. D, sensitivity of wild-type and PXR knockdown PC-3 cells to vinblastine (0.3 µmol/L) or Taxol (0.1 µmol/L). The wild-type PC-3, P1622-1, P1622-3, and Pm1623-3 were treated with chemotherapeutics for 72 h and then the cell viability was measured by MTS assay. *, P < 0.05; **, P < 0.01, when compared with the vehicle control (0.01% DMSO for Taxol; water for vinblastine).

 
A luciferase reporter gene assay was used to evaluate the ability of PXR to stimulate CYP3A4 promoter activity. Wild-type PC-3, P1620-4 clone, or various clones with PXR expression knocked down were transiently transfected with a LacZ plasmid and a pGL-PXRE plasmid, which contains two PXR-responsive fragments in the CYP3A4 promoter, or a control vector. As shown in Fig. 5C, both the wild-type PC-3 and P1620-4 clone had strong PXR promoter activities when compared with the untransfected groups, indicating a basal expression of functional PXR in PC-3 cells. The promoter activities of PXR in all PXR knockdown cells were significantly lower than the wild-type or P1620-4 PC-3 cells (Fig. 5C).

Finally, the PC-3 cells with PXR knocked down were subjected to chemotherapeutics. As shown in Fig. 5D, all three clones showed enhanced sensitivities to vinblastine or Taxol when compared with wild-type PC-3 cells. The results further suggest a role of PXR in determining the responses of prostate cancer cells toward chemotherapy.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, the expression of PXR, a master regulator for DMEs, was detected in human prostate cancerous tissues and in established cancer cells. Treatment of PC-3 cells with SR12813, a selective agonist of PXR, stimulated the expression of MDR1 and CYP3A4 and enhanced the resistance of cancer cells toward chemotherapy. Down-regulation of PXR via shRNAs, on the other hand, sensitized cancer cells to Taxol and vinblastine treatment. Our data suggest an important role for PXR at local tumor region in determining the responses of prostate tumors to chemotherapy. It is the first time that a role of PXR in chemoresistance was shown in human prostate cancer cells.

The endogenous expression of PXR in both normal and cancerous prostate tissues raises an interesting question regarding the potential role of PXR in the normal function of prostate as well as in carcinogenesis. In normal prostate gland, the highest expression of PXR was detected in the basal layer, an area in which basal stem cells give rise to transit-amplifying cells that in turn undergo terminal differentiation in the luminal area. The pattern of PXR expression in the normal gland suggests an inverse relationship between PXR expression and epithelial differentiation. However, further studies are needed to address the possible inhibitory role of PXR in the terminal differentiation of prostate epithelial cells.

In cancerous tissues of prostate, PXR immunoreactivities were generally elevated when compared with normal tissues. When the PXR immunoreactivities in cancerous tissues were scored and analyzed as a function of tumor grade or Gleason score, tumors with Gleason score of 6 or grade II presented the highest levels of PXR expression. Interestingly, when tumors progress to a more advanced stage, the expression of PXR tends to be reduced. Another interesting observation is the nuclear translocation of PXR observed in some patients. Similar observations were also made in breast cancer cases (16). Although not a precise indicator of activities in situ, the observed nuclear localization of PXR suggest that, at least in some tumors, PXR was activated, possibly due to the treatment patients received. Further studies are needed to determine how PXR is activated endogenously and whether PXR plays a physiologic role in the prostate.

A possible role of PXR in prostate biology and cancer progression is the regulation of cell death and survival pathways. A recent study using microarray analysis (26) identified >200 genes regulated by PXR, many of which concern intracellular metabolism, transport of essential small molecules, cell cycle, and survival mechanism. It remains to be studied, however, whether or not PXR activation has effects on the survival or proliferation of prostate cancer cells.

As a master regulator of many enzymes involved in drug metabolism, PXR may regulate the expression of DMEs in prostate cancer. In the past several decades, a myriad of targets have been identified in tumor resistance toward chemotherapy. MDR1 gene and its family members (27) and phase I and phase II metabolism enzymes (28) have been implicated in resistance of cancer cells toward chemotherapy. Our studies suggest that PXR is involved in drug resistance in PC-3 human prostate cancer cells. The up-regulation of drug metabolism and excretion genes by PXR can reduce the clinical efficacy of antineoplastic agents, especially at low doses or when the in situ availability of the chemotherapeutics becomes a limiting factor as in the case of metronomic dosing or due to the poor vascularization of tumors. Whereas the doses of chemotherapeutics used in the present study are comparable with those in other in vitro studies (29, 30), cautions should be exercised in extrapolation of the doses used in vitro to clinical settings. In the present studies, 10,000 tumor cells were used wherein chemotherapeutics had access to every single cell. However, in clinical settings, a gram of tumor mass may contain 109 tumor cells, and dependent on the status of vascularization, chemotherapeutics in the circulation may have limited access to some tumor cells. The amount of drugs available for each tumor cell may ultimately determine the efficacy of chemotherapy. Whereas it is plausible that the drug resistance mediated by PXR can be overcome by increasing the availability of drugs to tumors in situ through dose-intense schedule of chemotherapeutics for the objective of attaining a cure in certain malignancies (e.g., breast cancer; ref. 31) or certain tissue-selective delivery to tumor tissues (32, 33), an increase in PXR expression or activity in tumor cells may narrow the therapeutic window of chemotherapeutics due to the inherent side effects when delivered at high doses.

The PXR-CYP and PXR-MDR1 pathways might be one of the main pathways for PXR to regulate drug resistance. Both drugs used in the study are the substrates of MDR1 and might be metabolized by the CYP family (34–39), and the MDR1 or CYP3A4 are highly regulated by PXR. Interestingly, Taxol has been identified as an activator of PXR (6, 21, 40). Therefore, it may induce its own metabolism and excretion. Vincristine, an analogue of vinblastine, is a potential PXR ligand and induces ABCC2 and ABCC3 expression in carcinoma cells (20). The expression of PXR in prostate cancer raises possibility that the intratumoral regulation of MDR1 and CYP3A4, or other DMEs, by PXR may lead to alterations in the efficacy of chemotherapeutics.

Due to the big space/volume and flexibility in ligand-binding domain (7, 41), PXR can be activated by a variety of structurally diverse compounds. Known activators of PXR include many clinical drugs (42), diet components (42), pesticide (7), bile acid, and other endogenous ligands (5). Because most cancer patients use herbal supplements (43), the potential activation of PXR by components of herb may alter the clinical efficacy of chemotherapeutics. More studies are clearly needed to address the potential interaction of drug-drug and drug-herb supplements in determining the clinical outcomes of chemotherapy in prostate cancer patients.

In summary, we detected PXR expression and distribution in normal and cancerous prostate tissues. PXR activation conferred upon prostate cancer cells with increased resistance toward chemotherapy, whereas down-regulation of PXR sensitized prostate cancer cells toward chemotherapy. If the activation of human PXR (hPXR) is one of the major underlying mechanisms of drug resistance in cancer chemotherapy, inhibition of PXR will be a new approach to enhance the clinical efficacy of prostate cancer chemotherapy. Recent identification of ketoconazole (15) and sulforaphane (44) as inhibitors of PXR activation raises an exciting possibility that these compounds can be used to intercede PXR induction of drug resistance. Alternatively, chemotherapeutic drugs that do not activate hPXR should be developed to circumvent PXR-mediated drug resistance.


   Acknowledgments
 
Grant support: Southern Illinois University School of Medicine and SimmonsCooper Cancer Institute, U.S. Department of Defense Prostate Cancer Research Program New Investigator award W81XWH-04-1-0143, and Illinois Department of Public Health Prostate Cancer Research Program grant (D. Nie).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Chunna Yu and Dr. Shuqing Chen for the construction of pGL-PXRE plasmid.

Received 12/27/06. Revised 7/24/07. Accepted 9/ 4/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA Cancer J Clin 2005;55:10–30.[Abstract/Free Full Text]
  2. Das PC, Cao Y, Cherrington N, Hodgson E, Rose RL. Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes. Chem Biol Interact 2006;164:200–14.[CrossRef][Medline]
  3. Kolars JC, Schmiedlin-Ren P, Schuetz JD, Fang C, Watkins PB. Identification of rifampin-inducible P450IIIA4 (CYP3A4) in human small bowel enterocytes. J Clin Invest 1992;90:1871–8.[Medline]
  4. Blumberg B, Sabbagh W, Jr., Juguilon H, et al. SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes Dev 1998;12:3195–205.[Abstract/Free Full Text]
  5. Kliewer SA, Moore JT, Wade L, et al. An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 1998;92:73–82.[CrossRef][Medline]
  6. Synold TW, Dussault I, Forman BM. The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux. Nat Med 2001;7:584–90.[CrossRef][Medline]
  7. Moore LB, Parks DJ, Jones SA, et al. Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands. J Biol Chem 2000;275:15122–27.[Abstract/Free Full Text]
  8. Bertilsson G, Heidrich J, Svensson K, et al. Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc Natl Acad Sci U S A 1998;95:12208–13.[Abstract/Free Full Text]
  9. Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, Kliewer SA. The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J Clin Invest 1998;102:1016–23.[Medline]
  10. Anapolsky A, Teng S, Dixit S, Piquette-Miller M. The role of pregnane X receptor in 2-acetylaminofluorene-mediated induction of drug transport and -metabolizing enzymes in mice. Drug Metab Dispos 2006;34:405–9.[Abstract/Free Full Text]
  11. Courtois A, Payen L, Guillouzo A, Fardel O. Up-regulation of multidrug resistance-associated protein 2 (MRP2) expression in rat hepatocytes by dexamethasone. FEBS Lett 1999;459:381–5.[CrossRef][Medline]
  12. Geick A, Eichelbaum M, Burk O. Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin. J Biol Chem 2001;276:14581–7.[Abstract/Free Full Text]
  13. Kool M, de Haas M, Scheffer GL, et al. Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines. Cancer Res 1997;57:3537–47.[Abstract/Free Full Text]
  14. Guengerich FP. Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu Rev Pharmacol Toxicol 1999;39:1–17.[CrossRef][Medline]
  15. Huang H, Wang H, Sinz M, et al. Inhibition of drug metabolism by blocking the activation of nuclear receptors by ketoconazole. Oncogene 2007;26:258–68.[CrossRef][Medline]
  16. Miki Y, Suzuki T, Kitada K, et al. Expression of the steroid and xenobiotic receptor and its possible target gene, organic anion transporting polypeptide-A, in human breast carcinoma. Cancer Res 2006;66:535–42.[Abstract/Free Full Text]
  17. Dotzlaw H, Leygue E, Watson P, Murphy LC. The human orphan receptor PXR messenger RNA is expressed in both normal and neoplastic breast tissue. Clin Cancer Res 1999;5:2103–7.[Abstract/Free Full Text]
  18. Masuyama H, Hiramatsu Y, Kodama J, Kudo T. Expression and potential roles of pregnane X receptor in endometrial cancer. J Clin Endocrinol Metab 2003;88:4446–54.[Abstract/Free Full Text]
  19. Masuyama H, Suwaki N, Tateishi Y, Nakatsukasa H, Segawa T, Hiramatsu Y. The pregnane X receptor regulates gene expression in a ligand- and promoter-selective fashion. Mol Endocrinol 2005;19:1170–80.[Abstract/Free Full Text]
  20. Huang R, Murry DJ, Kolwankar D, Hall SD, Foster DR. Vincristine transcriptional regulation of efflux drug transporters in carcinoma cell lines. Biochem Pharmacol 2006;71:1695–704.[CrossRef][Medline]
  21. Mani S, Huang H, Sundarababu S, et al. Activation of the steroid and xenobiotic receptor (human pregnane X receptor) by nontaxane microtubule-stabilizing agents. Clin Cancer Res 2005;11:6359–69.[Abstract/Free Full Text]
  22. Nie D, Che M, Zacharek A, et al. Differential expression of thromboxane synthase in prostate carcinoma: role in tumor cell motility. Am J Pathol 2004;164:429–39.[Abstract/Free Full Text]
  23. McClelland RA, Finlay P, Walker KJ, et al. Automated quantitation of immunocytochemically localized estrogen receptors in human breast cancer. Cancer Res 1990;50:3545–50.[Abstract/Free Full Text]
  24. Watkins RE, Wisely GB, Moore LB, et al. The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity. Science 2001;292:2329–33.[Abstract/Free Full Text]
  25. Le Mee S, Chaminade F, Delaporte C, Markovits J, Saucier JM, Jacquemin-Sablon A. Cellular resistance to the antitumor DNA topoisomerase II inhibitor S16020–2: importance of the N-[2(Dimethylamino)ethyl]carbamoyl side chain. Mol Pharmacol 2000;58:709–18.[Abstract/Free Full Text]
  26. Guzelian J, Barwick JL, Hunter L, Phang TL, Quattrochi LC, Guzelian PS. Identification of genes controlled by the pregnane X receptor by microarray analysis of mRNAs from pregnenolone 16{alpha}-carbonitrile-treated rats. Toxicol Sci 2006;94:379–87.[Abstract/Free Full Text]
  27. Lage H. ABC-transporters: implications on drug resistance from microorganisms to human cancers. Int J Antimicrob Agents 2003;22:188–99.[CrossRef][Medline]
  28. Townsend DM, Tew KD. The role of glutathione-S-transferase in anti-cancer drug resistance. Oncogene 2003;22:7369–75.[CrossRef][Medline]
  29. Hu H, Jiang C, Ip C, Rustum YM, Lu J. Methylseleninic acid potentiates apoptosis induced by chemotherapeutic drugs in androgen-independent prostate cancer cells. Clin Cancer Res 2005;11:2379–88.[Abstract/Free Full Text]
  30. Lebedeva I, Rando R, Ojwang J, Cossum P, Stein CA. Bcl-xL in prostate cancer cells: effects of overexpression and down-regulation on chemosensitivity. Cancer Res 2000;60:6052–60.[Abstract/Free Full Text]
  31. Hudis CA, Schmitz N. Dose-dense chemotherapy in breast cancer and lymphoma. Semin Oncol 2004;31:19–26.[Medline]
  32. Li C, Newman RA, Wu QP, et al. Biodistribution of paclitaxel and poly(L-glutamic acid)-paclitaxel conjugate in mice with ovarian OCa-1 tumor. Cancer Chemother Pharmacol 2000;46:416–22.[CrossRef][Medline]
  33. Feng SS, Mu L, Win KY, Huang G. Nanoparticles of biodegradable polymers for clinical administration of paclitaxel. Curr Med Chem 2004;11:413–24.[CrossRef][Medline]
  34. Fujitaka K, Oguri T, Isobe T, Fujiwara Y, Kohno N. Induction of cytochrome P450 3A4 by docetaxel in peripheral mononuclear cells and its expression in lung cancer. Cancer Chemother Pharmacol 2001;48:42–6.[Medline]
  35. Cresteil T, Monsarrat B, Dubois J, Sonnier M, Alvinerie P, Gueritte F. Regioselective metabolism of taxoids by human CYP3A4 and 2C8: structure-activity relationship. Drug Metab Dispos 2002;30:438–45.[Abstract/Free Full Text]
  36. Yamamoto N, Tamura T, Kamiya Y, Sekine I, Kunitoh H, Saijo N. Correlation between docetaxel clearance and estimated cytochrome P450 activity by urinary metabolite of exogenous cortisol. J Clin Oncol 2000;18:2301–8.[Abstract/Free Full Text]
  37. Royer I, Monsarrat B, Sonnier M, Wright M, Cresteil T. Metabolism of docetaxel by human cytochromes P450: interactions with paclitaxel and other antineoplastic drugs. Cancer Res 1996;56:58–65.[Abstract/Free Full Text]
  38. Monsarrat B, Royer I, Wright M, Cresteil T. Biotransformation of taxoids by human cytochromes P450: structure-activity relationship. Bull Cancer 1997;84:125–33.[Medline]
  39. Garcia-Martin E, Pizarro RM, Martinez C, et al. Acquired resistance to the anticancer drug paclitaxel is associated with induction of cytochrome P450 2C8. Pharmacogenomics 2006;7:575–85.[CrossRef][Medline]
  40. Ferguson SS, Chen Y, LeCluyse EL, Negishi M, Goldstein JA. Human CYP2C8 is transcriptionally regulated by the nuclear receptors constitutive androstane receptor, pregnane X receptor, glucocorticoid receptor, and hepatic nuclear factor 4{alpha}. Mol Pharmacol 2005;68:747–57.[Abstract/Free Full Text]
  41. Carnahan VE, Redinbo MR. Structure and function of the human nuclear xenobiotic receptor PXR. Curr Drug Metab 2005;6:357–67.[Medline]
  42. Jacobs MN, Nolan GT, Hood SR. Lignans, bacteriocides and organochlorine compounds activate the human pregnane X receptor (PXR). Toxicol Appl Pharmacol 2005;209:123–33.[CrossRef][Medline]
  43. Nelson PS, Montgomery B. Unconventional therapy for prostate cancer: good, bad or questionable? Nat Rev Cancer 2003;3:845–58.[CrossRef][Medline]
  44. Zhou C, Poulton EJ, Grun F, et al. The dietary isothiocyanate sulforaphane is an antagonist of the human steroid and xenobiotic nuclear receptor. Mol Pharmacol 2007;71:220–9.[Abstract/Free Full Text]



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