MDR1 Gene Promoter Research Articles

Down-regulation of MDR1 gene expression by CRISPRi to enhance the sensitivity of lung adenocarcinoma A549/DDP cells to cisplatin

To investigate the effects of down-regulating MDR1 gene expression by CRISPRi on enhancing the sensitivity of lung adenocarcinoma A549/DDP cells to cisplatin. The potential CRISPRi interference sites on the MDR1 gene promoter were predicted by bioinformatics software, and the interference fragments were designed and constructed. The mRNA and protein expression levels of MDR1 gene in each group of cells were detected by qRT-PCR and Western blot methods, and the recombinant vectors with high interference efficiency were screened. Human lung cancer A549/DDP cells were divided into three groups: A549/DDP, Scrambed and sgRNA-MDR1-1, with three multiple holes in each group. After each vector was transfected into the cells for 48 h, the efflux of cells in each group was detected by flow cytometry, the IC50 value of cells in each group was detected by MTT method, and the cell morphology of cells treated with cisplatin was observed under laser confocal microscope. After sequencing and comparison, two kinds of CRISPRi recombinant vectors interfering with MDR1 gene transcription were constructed successfully. After transfection of A549/DDP cells, the mRNA and protein levels of MDR1 gene in all transfection groups were decreased significantly (P＜ 0.01). Among them, the interference efficiency of sgRNA-MDR1-1 was the highest, and the interference efficiency of mRNA and protein was 60% and 51%, respectively. After transfection of sgRNA-MDR1-1 vector, compared with the control group, the efflux ability of cells was decreased (P＜0.01), the IC50 value of cells to cisplatin was decreased significantly (P＜0.01), and the intracellular chromatin gathered and marginalized, and apoptotic bodies appeared. CRISPRi interference with MDR1 gene in drug-resistant A549/DDP cells can significantly enhance the sensitivity to cisplatin.

Sesquiterpene lactone 6-O-angeloylplenolin reverses vincristine resistance by inhibiting YB-1 nuclear translocation in colon carcinoma cells.

Multidrug resistance (MDR) is a major obstacle to cancer chemotherapy efficacy. In the present study, 6-O-angeloylplenolin repressed the overexpression of ATP binding cassette subfamily B member 1 (MDR1) and increasing the intracellular concentration of anticancer drugs. A reduction in P-glycoprotein expression (encoded by MDR1) was observed in parallel with a decline in mRNA expression in vincristine-resistant HCT (HCT-8/VCR) cells treated with 6-O-angeloylplenolin. In addition, 6-O-angeloylplenolin suppressed the activity of the MDR1 gene promoter. Treatment with 6-O-angeloylplenolin also decreased the amount of the specific protein complex that interacted with the MDR1 gene promoter in HCT-8/VCR cells, potentially leading to the suppression of MDR1 expression. Treatment with 6-O-angeloylplenolin inhibited the nuclear translocation of Y-box binding protein-1 in HCT-8/VCR cells treated with 6-O-angeloylplenolin, contributing to the negative regulation of MDR1. Finally, 6-O-angeloylplenolin reversed VCR resistance in an HCT/VCR xenograft model. In conclusion, 6-O-angeloylplenolin exhibited a MDR-reversing effect by downregulating MDR1 expression and could represent a novel adjuvant agent for chemotherapy.

MiR-138 indirectly regulates the MDR1 promoter by NF-κB/p65 silencing

MicroRNAs (miRNAs) are known to mediate post-transcriptional gene silencing in the cytoplasm and recent evidence indicates that may also possess nuclear roles in regulating gene expression. A previous study showed that miR-138 is involved in the multidrug resistance of leukemia cells through down-regulation of the drug efflux pump P-glycoprotein (P-gp), the protein encoded by the human multidrug-resistant ABCB1/MDR1 gene. However, the transcriptional regulatory mechanisms responsible remain to be elucidated. To deepen the description of the mechanism of transcriptional gene silencing on the MDR1 promoter, we initially performed a bioinformatics search for potential miR-138 binding sites in the MDR1 gene promoter sequence. Interestingly, we did not find miR-138 binding sites in this region, suggesting an indirect regulation. From six representative transcriptional factors involved in MDR1 gene regulation, an in silico analysis revealed that NF-κB/p65 has a specific binding site for miR-138. The results of luciferase reporter assay, western blot and flow cytometry shown here suggest that miR-138 might modulate the human MDR1 expression by inhibiting NF-κB/p65 as an indirect mechanism of MDR1 regulation. Furthermore, employing the human macrophage-like cell line U937 we observed comparable results with NF-κB/p65 down-regulation and we also observed a significant reduction in the IL-6 and TNF-α mRNA, as well as in their secreted pro-inflammatory cytokines following miR-138 expression, suggesting that canonical NF-κB target genes might also be potential targets for miR-138 in leukemia cells.

Influence of MDR1 methylation on the curative effect of interventional embolism chemotherapy for cervical cancer.

BackgroundMulti-drug resistance (MDR) is the main cause of tumor failure to chemotherapy. This study aims to explore the influence of MDR1 methylation on curative effect of interventional embolism chemotherapy for cervical cancer.MethodsSixty-seven patients with cervical cancer receiving embolism chemotherapy were selected, and 45 normal cervical tissues were included as a control. Immunohistochemistry was used to detect the level of P-glycoprotein (P-gp) in cervical cancer, and to make an analysis compared with normal tissues. The methylation status of the MDR1 gene promoter region 16 CpG units was analyzed by using kilobase-specific cracking and matrix-assisted laser desorption ionization time of flight mass spectrometry.ResultsThe results indicated that the positive expression rates of P-gp were 0% (0/45) in normal cervical tissue, and 61.19% (41/67) and 77.61% (52/67) before and after interventional embolism chemotherapy in cervical cancer tissues, respectively. There were significant differences compared with normal cervical tissues (χ2=4.2523, 0.0392). The positive expression rate of P-gp before chemotherapy was negatively correlated with efficacy of chemotherapy (r=−0.340, P=0.005). Methylation rate of 13 CpG units in normal tissues was significantly greater than cervical tissues (P<0.05). In cervical cancer tissue, methylation rate of six CpG units before interventional embolism chemotherapy was higher than after chemotherapy, but that of one CpG unit was lower than after chemotherapy (P<0.05). The methylation rate of one CpG unit with effective chemotherapy before chemotherapy was significantly higher than ineffective chemotherapy (P<0.05), and the other CpG units were similar (P>0.05).ConclusionP-gp expression level coded by MDR1, methylation status of partial MDR1 gene promoter regions CpG island, is closely related to the efficacy of interventional embolism chemotherapy for cervical cancer before the operation.

Abstract 4701: Acquisition of Oct-4 upregulation transactivates MDR1 and is associated with increased tumor recurrence in bladder cancer

Abstract Platinum-based drug like cisplatin is the standard first-line drug to treat patients with bladder cancer. However, tumors may recur and develop into multiple-drug resistant characteristics. The acquisition of resistance to conventional chemotherapy is a challenge in the treatment of bladder cancer relapse. Cancer stem cell (CSC) hypothesis has been an attractive theory. CSCs can resist therapeutic assaults, giving rise to multiple drug resistance and promotion of tumor relapse and metastasis. It has been shown that the fraction of cancer cells expressing MDR1 that functions as an energy-dependent drug efflux pump may be associated with Oct-4 expression. We have demonstrated that Oct-4 expression in bladder cancer predicts tumor progression. In the present study, we proposed that Oct-4 expression in bladder cancer increases MDR1 gene expression and thereby results in multiple drug resistance. We found a positive correlation between Oct-4 and MDR1 expression levels in clinical samples of bladder cancer. Furthermore, overexpression of Oct-4 increased MDR1 expression, which resulted in poor response to cisplatin in human bladder transitional cell carcinoma cells. Conversely, knockdown of Oct-4 reduced MDR1 expression and rendered cells hypersensitive to cisplatin. We also verified that Oct-4 transactivated the MDR1 gene promoter by binding to the Oct-4 response element (ORE). More importantly, Oct-4 and MDR1 gene expression could be induced by treatment with cisplatin, suggesting that Oct-4 is a member of the MDR1 enhanceosome. Our data can explain, in part, the high recurrence rate and drug resistance of bladder cancer. For clinical implication, we demonstrated that reduction of Oct-4 expression by all-trans retinoic acid, a derivative of vitamin A, improved sensitivity of bladder cancer cells to gemcitabine and cisplatin. In addition, we found that high expression levels of Oct-4 and MDR1 were associated with high tumor recurrence. Taken together, our results provide evidence that Oct-4 plays an important role in cisplatin-acquired resistance in bladder cancer. They also implicate Oct-4 as a therapeutic target. Note: This abstract was not presented at the meeting. Citation Format: Chia-Sing Lu, Ai-Li Shiau, Gia-Shing Shieh, Bing-Hua Su, Wu-Chou Su, Wen-Horng Yang, Chao-Liang Wu. Acquisition of Oct-4 upregulation transactivates MDR1 and is associated with increased tumor recurrence in bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4701. doi:10.1158/1538-7445.AM2014-4701

HIF-1α Inhibition Reverses Multidrug Resistance in Colon Cancer Cells via Downregulation of MDR1/P-Glycoprotein

BackgroundMultidrug resistance (MDR) is one of the major reasons chemotherapy-based treatments fail. Hypoxia is generally associated with tumor chemoresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1) and the multidrug resistance (MDR1) gene/transporter P-glycoprotein (P-gp) remains unclear. This study aims to explore the molecular mechanisms of reversing colon cancer MDR by focusing on the target gene HIF-1α.MethodsA chemotherapeutic sensitivity assay was used to observe the efficiency of MDR reversal in LoVo multicellular spheroids (MCS). The apoptotic level induced by different drugs was examined by flow cytometry (FCM). Binding of HIF-1α to the MDR1 gene promoter was evaluated by Chromatin immunoprecipitation (ChIP). The relationship between HIF-1α/P-gp expression and sensitivity to chemotherapy was analyzed.ResultsThe sensitivity of LoVo MCS to all four chemotherapy drugs was decreased to varying degrees under hypoxic conditions. After silencing the HIF-1α gene, the sensitivities of LoVo MCS to all four chemotherapy drugs were restored. The apoptotic levels that all the drugs induced were all decreased to various extents in the hypoxic group. After silencing HIF-1α, the apoptosis level induced by all four chemotherapy drugs increased. The expression of HIF-1α and P-gp was significantly enhanced in LoVo MCS after treatment with hypoxia. Inhibiting HIF-1α significantly decreased the expression of MDR1/P-gp mRNA or protein in both the LoVo monolayers and LoVo MCS. The ChIP assay showed that HIF-1α was bound to the MDR1 gene promoter. Advanced colon carcinoma patients with expression of both HIF-1α and P-gp were more resistant to chemotherapy than that with non expression.ConclusionsHIF-1α inhibition reverses multidrug resistance in colon cancer cells via downregulation of MDR1/P-gp. The expression of HIF-1α and MDR1/P-gp can be used as a predictive marker for chemotherapy resistance in colon cancer.

Temozolomide down-regulates P-glycoprotein in human blood-brain barrier cells by disrupting Wnt3 signaling.

Low delivery of many anticancer drugs across the blood-brain barrier (BBB) is a limitation to the success of chemotherapy in glioblastoma. This is because of the high levels of ATP-binding cassette transporters like P-glycoprotein (Pgp/ABCB1), which effluxes drugs back to the bloodstream. Temozolomide is one of the few agents able to cross the BBB; its effects on BBB cells permeability and Pgp activity are not known. We found that temozolomide, at therapeutic concentration, increased the transport of Pgp substrates across human brain microvascular endothelial cells and decreased the expression of Pgp. By methylating the promoter of Wnt3 gene, temozolomide lowers the endogenous synthesis of Wnt3 in BBB cells, disrupts the Wnt3/glycogen synthase kinase 3/β-catenin signaling, and reduces the binding of β-catenin on the promoter of mdr1 gene, which encodes for Pgp. In co-culture models of BBB cells and human glioblastoma cells, pre-treatment with temozolomide increases the delivery, cytotoxicity, and antiproliferative effects of doxorubicin, vinblastine, and topotecan, three substrates of Pgp that are usually poorly delivered across BBB. Our work suggests that temozolomide increases the BBB permeability of drugs that are normally effluxed by Pgp back to the bloodstream. These findings may pave the way to new combinatorial chemotherapy schemes in glioblastoma.

HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast

BackgroundInvasive micropapillary carcinoma (IMPC) of the breast is a distinct and aggressive variant of luminal type B breast cancer that does not respond to neoadjuvant chemotherapy. It is characterized by small pseudopapillary clusters of cancer cells with inverted cell polarity. To investigate whether hypoxia-inducible factor-1 (HIF-1) activation may be related to the drug resistance described in this tumor, we used MCF7 cancer cells cultured as 3-D spheroids, which morphologically simulate IMPC cell clusters.MethodsHIF-1 activation was measured by EMSA and ELISA in MCF7 3-D spheroids and MCF7 monolayers. Binding of HIF-1α to MDR-1 gene promoter and modulation of P-glycoprotein (Pgp) expression was evaluated by ChIP assay and FACS analysis, respectively. Intracellular doxorubicin retention was measured by spectrofluorimetric assay and drug cytotoxicity by annexin V-FITC measurement and caspase activity assay.ResultsIn MCF7 3-D spheroids HIF-1 was activated and recruited to participate to the transcriptional activity of MDR-1 gene, coding for Pgp. In addition, Pgp expression on the surface of cells obtained from 3-D spheroids was increased. MCF7 3-D spheroids accumulate less doxorubicin and are less sensitive to its cytotoxic effects than MCF7 cells cultured as monolayer. Finally, HIF-1α inhibition either by incubating cells with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (a widely used HIF-1α inhibitor) or by transfecting cells with specific siRNA for HIF-1α significantly decreased the expression of Pgp on the surface of cells and increased the intracellular doxorubicin accumulation in MCF7 3-D spheroids.ConclusionsMCF7 breast cancer cells cultured as 3-D spheroids are resistant to doxorubicin and this resistance is associated with an increased Pgp expression in the plasma membrane via activation of HIF-1. The same mechanism may be suggested for IMPC drug resistance.

Celecoxib enhanced the sensitivity of cancer cells to anticancer drugs by inhibition of the expression of P‐glycoprotein through a COX‐2‐Independent Manner

The P-glycoprotein (p170, P-gp) encoded by human MDR1 gene functions as a pump to extrude anticancer drugs from cancer cells. Over-expression of p170 is closely related to primary and induced drug resistance phenotype of tumor cells. Recent studies have demonstrated that expression of cyclooxygenase-2 (COX-2) is positively correlated with the p170 level, suggesting a potential of COX-2 specific inhibitors in regulation of cytotoxicity of anticancer agents. Celecoxib is one of the specific inhibitors of COX-2 and has been widely used in clinic. However, its function in the response of cancer cells to anticancer drugs and the related mechanism are still waiting to be investigated. To explore the correlation of celecoxib and the p170-mediated drug resistance, the role of celecoxib in drug response of cancer cells was analyzed with flow cytometry, high performance liquid chromatography (HPLC), and colony formation experiments. Celecoxib (50 microM) was found to significantly enhance the sensitivity of MCF-7 and JAR/VP16 cells to tamoxifen and etoposide, respectively, by inhibition of p170 expression and increase in intracellular accumulation of the drugs. However, celecoxib did not affect pump function of p170. Enzyme activity and methylation analyses demonstrated that the inhibitory effect of celecoxib on p170 was independent on COX-2 but closely related to hypermethylation of MDR1 gene promoter. Our study suggested that celecoxib was a potential agent for enhancement of the sensitivity of cancer cells to anticancer drugs. It also provided a links between epigenetic change of MDR1 and drug response of cancer cells.

Effect of MDR1 gene promoter methylation in patients with ulcerative colitis

Altered MDR1 expression and/or function contribute to the pathogenesis of inflammatory bowel disease (IBD). DNA methylation was shown as an important mechanism in gene silencing. We investigated DNA methylation of the MDR1 gene in ulcerative colitis (UC) and its relation to MDR1 C3435T genotypes. Eighty-three UC patients were enrolled. Methylation of MDR1 promoter was determined by methylation specific polymerase (MSP) for rectal inflammatory mucosa from all patients and normal terminal ileum from 17 patients. Promoter methylation of MDR1 gene was also quantified by digital densitographic analysis following MSP. MDR1 methylation was detected in 51 (61.4%) out of 83 patients in rectal inflammatory mucosa. Mean methylation level of MDR1 gene in rectal inflammatory mucosa was significantly higher than in normal terminal ileum (p=0.021). MDR1 methylation occurred more frequently in total colitis, and total+left side colitis, compared to rectal colitis (p=0.001, 0.013, respectively). Higher methylation levels were also associated with chronic continuous type (p=0.034) and earlier onset of disease (p=0.038). The 3435 CC+CT genotype of MDR1 was associated with more than 6-fold increased risk of MDR1 methylation, especially in UC patients with 9 years and shorter duration. Both frequency and level of MDR1 methylation were higher in UC onset at younger or in middle age with the same genotype. MDR1 methylation frequently occurred in inflammatory rectal mucosa from UC patients and was influenced by MDR1 C3435T polymorphism, especially in patients with shorter duration and younger onset.

U7 snRNA acts as a transcriptional regulator interacting with an inverted CCAAT sequence-binding transcription factor NF-Y

An inverted CCAAT sequence is recognized by both transcription factors NF-Y and DNA/RNA binding protein YB-1. In the process of examining the effect of nuclear RNA on an inverted CCAAT-containing promoter of MDR1 gene, we found that U7 snRNA inhibits NF-Y and suppresses the promoter activity both in vitro and in NG108-15 tumor cells. Analysis using a designed RNA, which was structurally unrelated to U7 snRNA, revealed that RNA binding by YB-1 is not specific and that the protein is not involved in the transcription. Furthermore, we demonstrated that in the nucleus of doxorubicin-treated cells, DNA binding by NF-Y and transcriptional activity of the promoter were inhibited without either a decrease of NF-Y or an increase of the p53 tumor suppressor, which is known to inhibit DNA binding by NF-Y. In these cells, U7 snRNA was specifically increased and associated with NF-Y, and treatment with RNase A eliminated the inhibition of NF-Y activity. These results suggest that U7 snRNA has a novel function as a transcriptional regulator to control NF-Y.

Examination of Glucocorticoid Receptor α-Mediated Transcriptional Regulation of P-glycoprotein, CYP3A4, and CYP2C9 Genes in Placental Trophoblast Cell Lines

The placental trophoblast at different stages of pregnancy contains some drug transporters and xenobiotic-metabolising enzymes, as well as ligand-activated nuclear receptors, which control their inducible transcriptional regulation. Glucocorticoid receptor α (GRα) is expressed in both placental syncytiotrophoblast and cytotrophoblast. GRα was shown to control inducible expression of several enzymes of the cytochrome P-450 family (CYP) and the drug transporter P-glycoprotein in the liver. However, GRα-mediated transcriptional regulation of drug transporters and CYPs has not been studied in the placental trophoblast. In this study, we examined the expression and activity of GRα in the transcriptional regulation of P-glycoprotein, CYP3A4, and CYP2C9 in placental trophoblast cell lines. Employing RT–PCR, Western blotting, and luciferase gene reporter assay, we detected the expression and activity of GRα in JEG3 and BeWo cell lines. However, we observed that only MDR1 mRNA was up-regulated after treatment of placental cells with dexamethasone. Accordingly, only the promoter of the MDR1 gene was activated by dexamethasone in gene reporter assays in placental cells and the activation was abolished by RU486, an antagonist of GRα. CYP3A4 and CYP2C9 promoters were activated in placental cells only after co-transfection with hepatocyte nuclear factor 4α (HNF4α), which indicates the hepatocyte-specific character of GRα-mediated regulation of the genes. On the other hand, coexpression of HNF4α had no effect on the activation of the MDR1 gene promoter, suggesting HNF4α-independent regulation via GRα. We conclude that GRα may be involved in the transcriptional regulation of P-glycoprotein in the placental trophoblast. We also indicate that the CYP3A4 and CYP2C9 genes are not inducible through GRα in placental cell lines, due to the lack of HNF4α expression and possibly some additional hepatocyte-specific transcriptional factors.

Tryptanthrin inhibits MDR1 and reverses doxorubicin resistance in breast cancer cells

Development of agents to overcome multidrug resistance (MDR) is important in cancer chemotherapy. Up to date, few chemicals have been reported to down-regulate MDR1 gene expression. We evaluated the effect of tryptanthrin on P-glycoprotein (P-gp)-mediated MDR in a breast cancer cell line MCF-7. Tryptanthrin could depress overexpression of MDR1 gene. We observed reduction of P-gp protein in parallel with decreases in mRNA in MCF-7/adr cells treated with tryptanthrin. Tryptanthrin suppressed the activity of MDR1 gene promoter. Tryptanthrin also enhanced interaction of the nuclear proteins with the negatively regulatory CAAT region of MDR1 gene promoter in MCF-7/adr. It might result in suppression of MDR1 gene. In addition, tryptanthrin decreased the amount of mutant p53 protein with decreasing mutant p53 protein stability. It might contribute to negative regulation of MDR1 gene. In conclusion, tryptanthrin exhibited MDR reversing effect by down-regulation of MDR1 gene and might be a new adjuvant agent for chemotherapy.

MDR1 Hypermethylation contributes to the progression of neuroblastoma

MDR1 hypermethylation plays an important role in pathogenesis and progression of neuroblastoma tumors. This hypothesis was tested by studying the methylation status of MDR1 gene promoter in neureoblastoma biopsy specimens during the progression of tumor from stage-1 to stage-4. Results of our findings demonstrate an inverse correlation between the methylation status of MDR1 promoter and MDR1 expression during the progression of disease from stage-1 to stage-4 as determined by methylation specific PCR (MSP) and RT/PCR analyses. The results of the RT/PCR and MSP analyses were validated by quantitative Real Time PCR analysis. Data from present study suggest that transcriptional inactivation of MDR1 gene due to increased MDR1 promoter methylation may be a contributing factor in pathogenesis and progression of neuroblastoma tumors, and may be used in designing an effective treatment therapy to neuroblastoma patients.

Cobalamin Potentiates Vinblastine Cytotoxicity Through Downregulation of mdr-1 Gene Expression in HepG2 Cells

Background: P-glycoprotein (Pgp), produced by multidrug resistance-1 gene (mdr-1), is a main mechanism developed by cancer cells to guard against anti-cancer drugs. Alterations of DNA methylation of the mdr-1 gene promoter are known to be linked to mdr-1 gene expression and are probably related to intracellular S-adenosyl-methionine. We here used HepG2 cells to determine the role of the methionine cycle (through the use of the Methionine-Synthase (MS) cofactor, cobalamin) on mdr-1 gene expression. Methods: Semiquantitative RT-PCR of mdr-1 gene, cellular retention of rhodamine-123, and vinblastine cytotoxicity were carried out on cells cultivated with and without cobalamin. Methylation status of the mdr-1 gene promoter was determined by methylation-specific PCR. Results: Addition of cobalamin to the cells led to an increase in MS activity, to a significant decrease in mdr-1 gene expression which is correlated to an increase in retention of the Pgp substrate Rhodamine 123. Furthermore, cobalamin potentiated cell sensitivity to vinblastine to the same range as that of the Pgp blocker verapamil and prevented methotrexate-induced up-regulation of mdr-1 gene expression. However, no modification in methylation of the mdr-1 gene promoter was observed. Conclusion: Cobalamin downregulates mdr-1 gene expression, as well as Pgp expression and function, and significantly increases cytotoxicity of vinblastine. The identification of this novel way of diminishing cellular resistance to the chemotherapeutic agent vinblastine holds promises of leading to better treatments for cancer patients.

The endocrine disrupting chemical, diethylhexyl phthalate, activates MDR1 gene expression in human colon cancer LS174T cells

Resistance to anticancer drugs is often mediated by the overexpression of P-glycoprotein encoded by the multi-drug resistance (MDR1) gene. The nuclear receptor, steroid and xenobiotic receptor (SXR), is one of the key transcriptional regulators of MDR1 gene expression. A variety of xenobiotics bind to SXR, and stimulate transcription on xenobiotic-response elements (XREs), located in the MDR1 gene promoter. Diethylhexyl phthalate (DEHP) is widely used as a plasticizer for polyvinyl chloride (PVC) medical devices. Previous studies have shown that a significant amount of DEHP leaches from PVC infusion bags and lines during interventions, such as total parenteral nutrition, blood transfusion, and cancer chemotherapy. Thus, the leaching of DEHP during parenteral chemotherapy for cancer patients may facilitate MDR1 expression in various tissues, including cancer cells, which may promote drug resistance. To examine such a hypothesis, the effect of DEHP on SXR-mediated transcription of the MDR1 gene was studied in the human colon adenocarcinoma-derived cell line, LS174T cells, which endogenously express SXR. DEHP increased the SXR-mediated transcription of the MDR1 gene in luciferase-reporter assays. The induction by DEHP was abrogated when a reporter plasmid containing mutated DR+4 motif in the XRE was used. In a mammalian two-hybrid assay, DEHP recruited steroid receptor co-activator-1 to the ligand-binding domain of SXR. Finally, using real-time reverse transcriptase-PCR, we showed that DEHP increased MDR1 gene expression in a dose-dependent manner. We conclude that DEHP is an inducer of the MDR1 gene in this cell line. As such, the leaching of DEHP from the PVC medical devices may influence the MDR1 expression, which may induce resistance to drugs in certain populations of cancer cells.

New invMED1 element cis-activates human multidrug-related MDR1 and MVP genes, involving the LRP130 protein.

The MDR1 gene is a key component of the cytotoxic defense network and its overexpression results in the multidrug resistance (MDR) phenotype. However, the molecular mechanisms that regulate the MDR1 gene and coordinate multiple MDR-related genes expression are poorly understood. In a previous study, we identified a new 12 bp cis-activating region in the 5'-flanking region of the human MDR1 gene, which we called inverted MED1. In the present study, we characterized the precise binding element, which we named invMED1, and revealed the presence of the LRP130 protein as the nuclear factor. Its binding intensity increases with the endogenous MDR1 geneexpression and with the MDR level of CEM leukemia cells. Interestingly, the LRP130 level did not vary with the chemoresistance level. We observed the involvement of LRP130 in the transcriptional activity of the MDR1 gene promoter, and moreover, in that of the MDR-related, invMED1-containing, MVP gene promoter. We used siRNAs and transcriptional decoys in two unrelated human cancer cell lines to show the role of the invMED1/LRP130 couple in both MDR1 and MVP endogenous genes activities. We showed that invMED1 was localized in the -105/-100 and -148/-143 regions of the MDR1 and MVP gene promoters, respectively. In addition, since the invMED1 sequence is primarily located in the -160/-100 bp region of mammalian MDR-related genes, our results present the invMED1/LRP130 couple as a potential central regulator of the transcription of these genes.

Association of C3435T and G2677T/A polymorphisms of multidrug resistance (MDR1) gene with colorectal cancer risk

The human multidrug-resistance (MDR1) gene encodes an integral cell membrane protein, P-glycoprotein (PGP), which generates the ATP-dependent cellular transport of substances. The activation of responsive elements in MDR1 gene promoter occurs in earlier colorectal carcinogenesis. In combination with cell proliferative activities of c-myc and cyclin D1, MDR1 may initiate colorectal tumorogenesis by suppressing cell death pathways. The failure of correct cell death mechanisms disturbs the epithelium homeostasis and may induce tumorogenesis. We have studied if the hereditarily determined activity of MDR1 could modulate the risk of developing of colorectal cancer and have investigated genetic variant 3435C>T in exon 26 and triple polymorphism 2677G>T/A in exon 21 in a group of 289 colorectal cancer patients and sex- and age-matched controls. Genotyping was performed by PCR/RFLP and Real-Time PCR assays. Homozygous carriers of 3435C allele had 1.65 (95% C. I., 1.1-2.5) higher risk (p=0.01) than carriers of 3435T allele. Presence of at least one 3435T protects significantly against colorectal cancer. Similar effect was shown for the mutation in exon 21. Carries of one 2677T allele had a lower risk of colorectal cancer (OR=0.65, 95% C. I., 0.45–0.94, p=0.02). The 2677G>T/A polymorphism and the 3435C>T polymorphism displayed tight allelic association (p<0.001). Our data suggest that carries of active MDR1 alleles are at the higher risk to develop the colorectal cancer. Clinical Pharmacology & Therapeutics (2004) 75, P17–P17; doi: 10.1016/j.clpt.2003.11.065

Sequence and structural analysis of the presumed downstream promoter of the canine mdr1 gene

The product of the canine mdr1 gene, P-glycoprotein (P-gp), plays an important role in chemotherapeutic drug resistance of several canine tumours. Increased expression of P-gp by tumour cells is associated with the multidrug-resistant phenotype. Because of its importance in cancer chemotherapy, a great deal is known about the regulation of mdr1 gene expression in human cancer patients and rodent cancer models. In contrast, there is no information regarding the regulation of P-gp expression in dogs. Initial information regarding the regulation of mdr1 gene expression can be gained by evaluating the mdr1 promoter. The downstream promoter of the canine mdr1 gene was sequenced. Several regulatory elements were identified, including an AP-1 site, AP-2 site and SP-1 site. The presumed canine mdr1 promoter was similar to that of other species; however, low overall sequence homology may suggest that aspects of P-gp regulation are distinctive in dogs.

The basic and clinical implications of ABC transporters, Y-box-binding protein-1 (YB-1) and angiogenesis-related factors in human malignancies.

In our laboratories, we have been studying molecular targets which might be advantageous for novel cancer therapeutics. In this review, we focus on how ATP-binding cassette (ABC) transporter superfamily genes, Y-box-binding protein-1 (YB-1), and tumor angiogenesis-associated factors could contribute to the development of novel strategies for molecular cancer therapeutics. ABC transporters such as P-glycoprotein/MDR1 and several MRP family proteins function to protect cells from xenobiotics, drugs and poisons, suggesting that ABC transporters are a double-edged sword. In this regard, P-glycoprotein/MDR1 is a representative ABC transporter which plays a critical role in the efflux of a wide range of drugs. We have reported that gene amplification, gene rearrangements, transcription factor YB-1 and CpG methylation on the promoter are involved in MDR1 gene overexpression in cultured cancer cells. Among them, two mechanisms appear to be relevant to the up-regulation of MDR1 gene in human malignancies. We first reported that MDR1 gene promoter is activated in response to environmental stimuli, and is modulated by methylation/demethylation of CpG sites on the MDR1 promoter. We also demonstrated that YB-1 modulates not only transcription of various genes associated with cell growth, drug resistance and DNA synthesis, but also translation, mRNA stabilization and DNA repair/self-defense processes. Angiogenesis is also involved in tumor growth, invasion and metastasis of various malignancies, and so angiogenesis-related molecules also offer novel molecular targets for anticancer therapeutics.