| Background and purposeColon cancer is one of the most frequent malignant tumors in the world. It's worth noting that the morbidity and mortality of colon cancer have been increasing year by year in China. Chemotherapy is one of the commonest treatments for colon cancer, but its efficacy is limited by the resistance of cancer cells to drugs. The inductions of drug metabolizing enzymes and ATP-binding cassette (ABC) transporters are involved in the cancer cell multidrug resistance, however, the mechanism has not been clearly clarified.The pregnane X receptor (PXR; NR1I2; also termed PAR, SXR) is a member of the nuclear receptor superfamily that plays a central role in protecting tissues from potentially toxic exogenous and endogenous compounds (xenobiotics and endobiotics, respectively). PXR is a master regulator of pathways involving the major drug metabolic enzymes and transporters. After binding xenobiotic (e.g. drugs) or endobiotic ligands, PXR is activated and binds to the regulatory region of target genes as a heterodimer with RXRα. Its target genes include phase I enzymes such as multiple CYP enzymes(CYP3A4, CYP2C9, CYP2C19 and CYP2B6), dehydrogenases, carboxylesterases; phase II enzymes such as glutathione-S-transferases, UDP-glucuronosyl transferases and sulfotransferases; and transporters p-glycoprotein/multi-drug resistance protein (MDR1), multi-drug drug resistance associated proteins (MRPs), organic anion transporting peptide 2. Most of these target genes are involved in drug resistance. PXR regulates all stages of xenobiotic metabolism and transport and is responsible for important inductive drug interactions. So it can change the metabolism and transport of chemotherapeutics in cancer cells and affect the sensitivity of chemotherapy. In brief, PXR is involved in multiple key points of drug resistance mechanism. Therefor it may be closer to the nature of multidrug resistance and the needs of individual chemotheraphy in cancer patients. So PXR may become a potential target of drug resistance and tumor therapy. The overexpression of ABC transporters with wide substrate selectivity can result in an increased efflux of chemotherapeutic agents from within cells and is one of the primary causes of multidrug resistance in cancer cell lines and tumors. The multidrug resistance associated protein 3 (MRP3), also known as ABCC3, localizes to the basolateral membrane of polarized epithelial cells, and is expressed in liver, small intestine, colon, adrenal and pancreas. MRP3 is capable of transporting a wide range of substrates including anti-cancer drugs, organic anions, as well as glucuronide and sulfate conjugates of a number of endogenous and exogenous compounds. MRP3 was associated with the prognosis of leukemia. MRP3 also was correlated with resistance to platinum drugs in cancer cell lines. Two MRP3 polymorphisms were identified to influence response to platinum-based therapy in lung cancer patients. Furthermore, Daniele Campa et al. studied the association between MRP3 polymorphism and colon cancer risk, and provided information potentially relevant for pharmacogenetics in colon cancer chemotherapy. It suggests MRP3 may play an important role in colon cancer chemotherapy based on platinum drugs.Little is known about the mechanisms behind the regulation of MRP3 expression. Analysis of the 5'-flanking region of the human MRP3 gene revealed a TATA-less promoter and the presence of binding sites for the transcription factors SP1, AP-1, AP-2 and LRH-1. The proximal promoter region of the rat Mrp3 gene was found to contain putative binding sites for LRH-1 and SP-1 that were essential for MRP3 transcriptional activity. Recent studies have also indicated an involvement of nuclear receptors in MRP3 regulation, including PPARα, VDR and RARα. The involvement of PXR has not been determined, but the induction of MRP3 expression by PXR's ligands has been reported.In this study, we first detected the expression of metabolic nuclear receptors including PXR, PPARα, VDR and RARαin cancerous and matched nonneoplastic colon tissues to study initially on their implication in colon cancer. We also examined the expression of drug resistance related genes in tissues samples and analysised the correlation between PXR mRNA and MRP2, MRP3 mRNA. We further studied the role of PXR in proliferation and resistance to chemotheraphy in human colon cancer cell lines through PXR activiation by ligands or PXR knocking-down via RNAi and investigated the upregulation of MRP3 by PXR in colon cancer chemotherapy. Methods1. The mRNA and protein expression of nuclear receptor and drug resistance related genes in tissues samples were detected by RT-PCR and Western blot analysis. The mRNA expression of PXR, MRP2, MRP3 was semiquantitative analysised, the difference between cancerous and nonneoplastic colon tissues was analyzed by Paired-Samples T Test .The correlation between PXR and MRP2, MRP3 mRNA was assessed with Pearson correlation test.2. The expression of PXR in colon cancer cell lines or LS174T treated with 10μM rifampicin was detected by RT-PCR and Western blot analysis. The effect of PXR ligand on LS174T cell proliferation and chemotherapeutic sensitivity was analyzed via MTT assay.3. PXR shRNA products(PXRi 1# and PXRi 2#) were builded up with two different validated target sequence, and A corresponding random siRNA sequence was used as a negative control of PXR shRNA(PXRi control). LS174T cells were stably transfected, and selected for clones by G418. The expression of PXR and MRP3 was detected by RT-PCR and Western blot analysis. The effect of PXR knocking-down on clone proliferation and chemotherapeutic sensitivity were analyzed via MTT assay.4. The expression of SP1, PXR and MRP3 in LS174T cells treated with 10μM rifampicin or 5μM paclitaxel was detected by RT-PCR and Western blot analysis.5. The change and subcellular localization of PXR and MRP3 expression in LS174T cells treated with 5μM paclitaxel was determined by immunofluorescence.6. LOVO cells were cotransfected with PXR expression plasmid, MRP3 report gene plasmid and pRL-SV40 plasmid by Lipofectamin2000, and divided into four groups: transfection control group (cotransfection with vehicle vector), DMSO group (cotransfection with PXR expression plasmid+0.1%DMSO), RIF group(cotransfection with PXR expression plasmid+10μM rifampicin) and Paclitaxel group(cotransfection with PXR expression plasmid+5μM paclitaxel). After cotransfected cells were treated for 24h, the firefly luciferase activity of MRP3 report gene was assayed using Dual-Luciferase?Reporter Assay System and the renilla luciferase activity of pRL-SV40 was used for normalization. Results1. The mRNA and protein expression of RARα, PXR, MRP2 and MRP3 in colon cancer tissues was remarkable increased, and the mRNA expression of VDR and PPARαalso was increased, but the expression of FXR, LRH-1, SP1, ABCG2, MDR1 and CYP3A4 mRNA was not increased.2. The mRNA levels of PXR (0.6794±0.2623 vs 0.3894±0.2712, P=0.000), MRP2 (0.703±0.459 vs 0.351±0.234, P=0.000) and MRP3 (0.6682±0.2032 vs 0.4412±0.1932, P=0.000) were all significantly higher in colon cancer tissues than in the matched nonneoplastic tissues with Paired-Samples T Test. The increase of MRP2 mRNA was the most significant among the three genes. The MRP3 mRNA was significantly positively correlated with the PXR mRNA in colon cancer tissues (r=0.735, P=0.001), in matched nonneoplastic colon tissues (r=0.759, P=0.001) and in C/N tissues(r=0.629, P=0.007), but the MRP2 mRNA was not correlated with the PXR mRNA in tissue samples (P=0.842; P=0.342; P=0.443).3. The expression of PXR was the highest in LS174T cells, but hardly detected in LOVO, HT29 and HCT116 cells. In contrast, the mRNA expression of LRH-1 was weak or hardly detected in LS174T cells. At the different time of rifampicin treatment, MRP3 mRNA changed with PXR mRNA. The expression of PXR, SP1 and MRP3 was all increased remarkably in LS174T cells treated with 10μM rifampicin or 5μM paclitaxel when compared with 0.1% DMSO group.4. With the treatment of 10μM rifampicin, LS174T cell grew more quickly, the optical density of 490nm in 3d, 5d, 7d followed as: 0.682±0.048, 1.183±0.156, 1.817±0.095, which in DMSO group was 0.528±0.035, 0.920±0.036, 1.402±0.092, respectively. Compared with PXRi control group, the difference was significant(P<0.05). No matter exposure to oxaliplatin (0.8-80μg/ml) or 5-fluorouracil (1-250μg/ml), we found an increased survival of LS174T cells with rifampicin pretreatment compared with the vehicle-treated group. For oxaliplatin, IC50 without or with rifampicin pretreatment were 8.80±0.26 and 19.37±4.22μg/ml (P=0.012), respectively, and for 5-fluorouracil, the corresponding IC50 were 32.27±2.92 and 62.3±9.08μg/ml (P=0.005).5. The expression of PXR in in PXRi 1# and PXRi 2# clones was markedly knocked down compared with that in the wild-type LS174T and PXRi control cells. Corresponding with the expression of PXR, the protein level of MRP3 also was decreased in PXRi 1# and PXRi 2# clones.6. Silencing PXR gene suppressed cell proliferation, OD mean of the wild-type LS174T and PXRi control cells in 3d, 5d, 7d followed as: 0.313±0.019, 0.640±0.040, 0.782±0.073 and 0.284±0.009, 0.680±0.026, 0.835±0.059 , respectively. Which in PXRi 1# and PXRi 2# clones as: 0.313±0.019, 0.640±0.040, 0.782±0.073 and 0.284±0.009, 0.680±0.026, 0.835±0.059, respectively. Compared with PXRi control group, the difference was significant (P<0.05). The survival percentage of PXRi 1# and PXRi 2# clones((8.0±1.1)%; (11.4±2.2)% or (57.6±7.0)%; (62.3±6.7)%) decreased significantly when compared with the wild-type LS174T and PXRi control((36.8±5.4)%; (37.9±6.9)% or (78.9±5.3)%; (80.5±5.6)%)with treatment of 20μg/ml oxaliplatin or 5μg/ml 5-fluorouracil (both P<0.05).7. As shown in LCM, the expression of PXR in LS174T was increased by 5μM paclitaxel, and localized in cell nucleus, and the expression of MRP3 in LS174T cell membrane also was increased clearly. 8. The relative luciferase activity of DMSO group was 2 folds as one of Control group, but the difference was not significant((4.67±0.60)×10-2 vs (2.23±0.31)×10-2, P=0.095);After cotransfected cells were treated with10μM rifampicin or 5μM paclitaxel for 24h, the relative luciferase activity of RIF group and Paclitaxel group both increased significantly((7.93±0.91)×10-2, P=0.025; (12.30±1.85)×10-2, P=0.000, respectively) when compared with DMSO group.Conclusions1. The differential upregulation of PXR, MRP2 and MRP3 in colon cancer tissues may be associated with colon cancer chemoresistance.2. The expression of MRP3 mRNA is positively correlated with PXR in both cancerous and nonneoplastic colon tissues.3. PXR, activated by rifampicin, can promote cell proliferation and reduce the chemotherapeutic sensitivity of colon cancer cells.4. PXR, knocked-down via shRNA, can inhibit cell proliferation and enhance the chemotherapeutic sensitivity of colon cancer cells.5. The upregulation of MRP3 by PXR is one part of PXR-mediated colon cancer chemoresistance.
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