Overexpression Of MDR1 Research Articles

An MDR1 promoter allele with higher promoter activity is common in clinically isolated strains of Candida albicans

In the opportunistic fungal pathogen Candida albicans, up-regulation of MDR1, encoding an efflux transporter, leads to increased resistance to the antifungal drug fluconazole. Antifungal resistance has been linked to several types of genetic change in C. albicans, including changes in genome structure, genetic alteration of the drug target, and overexpression of transporters. High-level over-expression of MDR1 is commonly mediated by mutation in a trans-acting factor, Mrr1p. This report describes a second mechanism that contributes to up-regulation of MDR1 expression. By analyzing the sequence of the MDR1 promoter region in fluconazole-resistant and fluconazole-susceptible strains, we identified sequence polymorphisms that defined two linkage groups, corresponding to the two alleles in the diploid genome. One of the alleles conferred higher MDR1 expression compared with the other allele. Strains in which both alleles were of the higher activity type were common in collections of clinically isolated strains while strains carrying only the less active allele were rare. As increased expression of MDR1 confers higher resistance to drugs, strains with the more active MDR1 promoter allele may grow or survive longer when exposed to drugs or other selective pressures, providing greater opportunity for mutations that confer high-level drug resistance to arise. Through this mechanism, higher activity alleles of the MDR1 promoter could promote the development of drug resistance.

The Transcription Factor Ndt80 Does Not Contribute to Mrr1-, Tac1-, and Upc2-Mediated Fluconazole Resistance in Candida albicans

The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis, by the overexpression of genes encoding multidrug efflux pumps or ergosterol biosynthesis enzymes. Zinc cluster transcription factors play a central role in the transcriptional regulation of drug resistance. Mrr1 regulates the expression of the major facilitator MDR1, Tac1 controls the expression of the ABC transporters CDR1 and CDR2, and Upc2 regulates ergosterol biosynthesis (ERG) genes. Gain-of-function mutations in these transcription factors result in constitutive overexpression of their target genes and are responsible for fluconazole resistance in many clinical C. albicans isolates. The transcription factor Ndt80 contributes to the drug-induced upregulation of CDR1 and ERG genes and also binds to the MDR1 and CDR2 promoters, suggesting that it is an important component of all major transcriptional mechanisms of fluconazole resistance. However, we found that Ndt80 is not required for the induction of MDR1 and CDR2 expression by inducing chemicals. CDR2 was even partially derepressed in ndt80Δ mutants, indicating that Ndt80 is a repressor of CDR2 expression. Hyperactive forms of Mrr1, Tac1, and Upc2 promoted overexpression of MDR1, CDR1/CDR2, and ERG11, respectively, with the same efficiency in the presence and absence of Ndt80. Mrr1- and Tac1-mediated fluconazole resistance was even slightly enhanced in ndt80Δ mutants compared to wild-type cells. These results demonstrate that Ndt80 is dispensable for the constitutive overexpression of Mrr1, Tac1, and Upc2 target genes and the increased fluconazole resistance of strains that have acquired activating mutations in these transcription factors.

Subchronic arsenic exposure induces MDR1 overexpression in breast cancer MCF-7 cells and alters its response to tamoxifen

Subchronic arsenic exposure induces MDR1 overexpression in breast cancer MCF-7 cells and alters its response to tamoxifen

Abstract 1721: Resistance to silvestrol is mediated by MDR1/Pgp over-expression in a lymphoblastic leukemia cell line and is reversible by treatment with rapamycin

Abstract We previously showed that silvestrol, a translation initiation inhibitor, has potent cytotoxic activity in chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) B-cells in vitro and in vivo (Blood. 2009; 113(19):4656-66). To study potential mechanisms of silvestrol resistance, we generated a silvestrol-resistant B-leukemia cell line as a model system for B-cell leukemias. The 697 ALL cell line was incubated with step-wise increasing concentrations of silvestrol to generate a cell line (697-R) resistant to 80 nM silvestrol. In contrast, the LC50 (concentration lethal to 50%) of silvestrol in the parental 697 cell line is approximately 5 nM. 697-R cells retained genotypic and immunophenotypic features of the parental 697 cell line. One common mechanism of drug resistance in cancer is the over-expression of the multidrug resistance gene ABCB1 and its protein product, multidrug resistance-1/permeability glycoprotein (MDR1/Pgp). Using RT-PCR and flow cytometry, we confirmed increased MDR1 mRNA and protein expression in 697-R cells compared to parental 697 cells. Consistent with MDR1 upregulation, 697-R cells also showed resistance to the MDR1 substrates vincristine and depsipeptide, but not to the non-MDR1 substrate flavopiridol. We next assessed the functional activity of MDR1 in 697-R by measuring efflux of the fluorescent MDR1 substrate rhodamine 123 by flow cytometry. Rhodamine 123 was nearly completely eliminated from 697-R cells within 2 hours, but was fully retained in parental 697 cells. The common MDR1 inhibitors verapamil and cyclosporine inhibited rhodamine efflux from 697-R cells. As this type of multidrug resistance can be mediated by several genes, we used MDR1-specific siRNA to determine whether over-expression of MDR1 is responsible for rhodamine efflux. At 48 hours post-transfection, MDR1 siRNA-transfected cells showed significantly reduced efflux of rhodamine 123 compared to scrambled and irrelevant controls. It has previously been reported that the mTOR inhibitor rapamycin can also block MDR1 function. We therefore tested this using the 697-R cell line. Similar to verapamil, rapamycin was also able to inhibit the efflux of rhodamine 123 from these cells. Furthermore, rapamycin was also able to restore silvestrol sensitivity to 697-R cells. In conclusion, we show that MDR1/Pgp over-expression is at least one mechanism of silvestrol resistance in cell lines, and known MDR1 inhibitors including rapamycin can reverse this resistance in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1721. doi:10.1158/1538-7445.AM2011-1721

Abstract 2524: Targeting microtubule-associated protein 4 with novel small molecule CH4938056 confers antitumor activity under quasi in vivo conditions and in xenograft models

Abstract Tumor microenvironment is a major factor influencing treatment resistance to conventional anticancer therapies. Indeed, under “quasi in vivo” conditions, with low oxygen (1%), low pH (6.5) and low glucose concentration (0.01%) to mimic the environments in grafted tumors in mice or in clinical tumors, anti-proliferative activities of some conventional anticancer agents were diminished. Here, we describe the small molecule CH4938056, which has a novel chemical structure and was identified through cell-based screening under the “quasi in vivo” conditions followed by chemical modification. Our initial phenotypic profiling revealed that CH4938056 specifically arrests cells at the M phase and that it overcomes multiple resistance mechanisms to conventional anticancer agents including over-expression of MDR1 and BCRP. Then, after designing and identifying a water-soluble phosphate prodrug which successfully converts to CH4938056 after injection, we demonstrated antitumor efficacy of CH4938056 in a HCT116 xenograft model and a MDR1-overexpressing cancer model. Chemo-proteomic studies and consequent biochemical analysis revealed that CH4938056 binds to microtubule-associated protein 4 (MAP4). Moreover, CH4938056 inhibited MAP4-dependent microtubule assembly in a cell free system. siRNA-mediated knockdown of MAP4 induced chromosomal misalignment in metaphase cells, which closely resembles the primary phenotype of the CH4938056-treated cells. Under the “quasi in vivo” conditions, MAP4 expression turned out to be down-regulated (since MAP4 transcription is known to be negatively regulated by p53 which is up-regulated under these conditions) and when we knocked down MAP4 with siRNA, cancer cells became sensitive to CH4938056, which altogether is consistent with the fact that CH4935056 has antitumor activity even under the “quasi in vivo” conditions. From these observations, we conclude that CH4938056 inhibits proliferation of cancer cells by targeting MAP4. Targeting MAP4 with CH4938056 offers a novel approach for the treatment of cancer, especially for patients resistant to conventional anticancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2524. doi:10.1158/1538-7445.AM2011-2524

Correlation between the Type of BCR-ABL Transcripts and Blood Cell Counts in Chronic Myeloid Leukemia—A Possible Influence of MDR1 Gene Expression

The impact of BCR-ABL mRNA type (b3a2 vs. b2a2) on chronic myeloid leukemia (CML) phenotype is still a subject of controversies. We searched for a correlation between the BCR-ABL transcripts type and CML patients' characteristics, including MDR1 gene expression. Ninety-eight untreated chronic phase CML patients were studied. The type of BCR-ABL fusion transcripts and MDR1 gene expression were determined by reverse transcriptase polymerase chain reaction. B3a2 and b2a2 transcripts were found in 53 [54%] and 44 [45%] patients, respectively. One patient co-expressed b3a2/b2a2 and was excluded from analysis. The only difference in the clinical characteristics between the two groups was the platelets count, that was higher in b3a2(+) patients [791.3±441.3×109/L vs. 440.4±283.4×109/L in b2a2(+); P=0.007]. MDR1 over-expression [MDR1(+)] was observed in 48 patients (49.5%), more frequently in older patients >60 years [71% (24/34) vs. 38% (24/63) in younger; P=0.008], and was associated with a lower white blood cells (WBC) count [105.5±79.8× 109/L vs. 143.6±96.5×109/L in MDR1(−) cases; P=0.047]. On performing the analysis only within the MDR1(+) group, the b3a2(+) cases were characterized with a significantly higher platelets count [908.7±470.1×109/L vs. 472.9±356.1×109/L; P=0.006] and a lower WBC count [85.4±61.2×109/L vs. 130±93.9×109/L; P=0.004) compared to b2a2(+) patients. No similar differences were found between b3a2(+) and b2a2(+) groups with normal MDR1 levels. These results indicate that the type of BCR-ABL transcripts correlates with the hematological parameters of CML, however only in the subgroup of patients characterized by MDR1 over-expression.

Different Transport Activity of Human Triallelic MDR1893Ala/Ser/Thr Variant and its Association with Herb Extracts

Individual pharmacokinetic differences for herb-drug interaction have been associated with genetic variations of the multidrug resistance (MDR) gene. A high level expression of MDR protein increases cellular efflux and might decrease drug sensitivity. This study investigated the drug efflux activity difference of human MDR1 triallelic variant 2677G/T/A (rs2032582), as a nonsynonymous 893Ala/Ser/Thr, using Xenopus laevis oocytes and MDR1 overexpressing LLC-PK1 cells. Two MDR1 variants (2667T/893Ser and 2667A/893Thr) were generated using human MDR1 cDNA (2677G/893Ala). No significant difference in the expression of MDR1 893Ala/Ser/Thr was found in X. laevis oocytes. However, the MDR1 2667A/893Thr variant interestingly showed a significant decrease of efflux activity for both digoxin and daunorubicin compared with those of 893Ala and 893Ser variants. In further investigation assessing the inhibitory effects of three herbal extracts on MDR1, 893Ala and 893Ser showed significant decreases of efflux activities in treatments with P. cocos (p = 0.005 for 893Ser) and D. dasycarpus (p = 0.0009 for 893Ala; p = 0.002 for 893Ser) in X. laevis oocytes. The results in this study suggest that herbal medicines could interact with other drugs and change the therapeutic effects depending on the genetic polymorphisms of individuals.

Over-expression of MDR1 in amrubicinol-resistant lung cancer cells

Amrubicin, a totally synthetic 9-aminoanthracycline anticancer drug, has shown promising activity for lung cancer, but little is known about the mechanism of resistance for this agent. This study was aimed to clarify the role of P-glycoprotein (P-gp) in amrubicinol, an active metabolite of amrubicin, resistance in lung cancer cells. Amrubicinol-resistant cell line PC-6/AMR-OH was developed by continuously exposing the small-cell lung cancer cell line PC-6 to amrubicinol. Gene expression level of MDR1, which encodes P-gp, and intracellular accumulation of amrubicinol were evaluated by PC-6 and PC-6/AMR-OH cells. The involvement of MDR1 in amrubicinol resistance was evaluated by treatment with P-gp inhibitor verapamil and small interfering RNA (siRNA) against MDR1. Also, expression levels and single-nucleotide polymorphisms (SNPs) of MDR1 in 22 lung cancer cell lines were examined, and the relationships between these factors and sensitivity to amrubicinol were evaluated. The MDR1 gene was increased approximately 4,500-fold in PC-6/AMR-OH cells compared with PC-6 cells, and intracellular accumulation of amrubicinol in PC-6/AMR-OH cells was decreased to about 15 percent of that in PC-6 cells. Treatment with verapamil and siRNA against MDR1 significantly increased the sensitivity to amrubicinol in PC-6/AMR-OH cells with increased cellular accumulation of amrubicinol. Meanwhile, neither MDR1 gene expression levels nor SNPs of the gene were associated with amrubicinol sensitivity. Results of this study indicate that increased MDR1 expression and P-gp activity confer acquired resistance to amrubicinol. In contrast, neither expression level nor SNPs of MDR1 are likely to be predictive markers for amrubicin activity.

Probing Multidrug Resistance P-glycoprotein Transporter Activity with SPECT Radiopharmaceuticals

Multidrug resistance (MDR) mediated by overexpression of MDR1 (ABCB1) P-glycoprotein (Pgp) is one of the best characterized transporter-mediated barriers to successful chemotherapy in cancer patients and is also a rapidly emerging target in the progression of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Therefore, molecular imaging probes capable of imaging noninvasively Pgp and closely related transporter activities in tissues as well as tumors would be expected to contribute to personalized medicine. Interrogation of Pgp-mediated transport activity in vivo via noninvasive SPECT imaging could be beneficial for stratification of patient populations likely to benefit from a given therapeutic treatment, assist in the management of chemotherapy and aid the study of neurodegenerative diseases.

Recovery of Motor Spontaneous Activity After Intranasal Delivery of Human Recombinant Erythropoietin in a Focal Brain Hypoxia Model Induced by CoCl2 in Rats

Stroke is a major human health problem inducing long-term disability without any efficient therapeutic option being currently available. Under hypoxia, hypoxia-inducible factor-1α (HIF-1α) activates several genes as erythropoietin receptor (Epo-R) related with O(2) supply, and the multidrug-resistance gene (MDR-1) related with drug-refractory phenotype. Brain cortical injection of CoCl(2) produces focal hypoxia-like lesion with neuronal and glial alterations, as well as HIF-1α stabilization and MDR-1 overexpression. Intranasal (IN) drug delivery can by-pass blood-brain barrier (BBB) where MDR-1 is normally expressed. We evaluated the effects of IN-rHu-Epo administration on spontaneous motor activity (SMA) and the brain pattern expression of HIF-1α, MDR-1, and Epo-R in our cobalt-induced hypoxia model. Adult male Wistar rats were injected by stereotaxic surgery in frontoparietal cortex, with CoCl(2) (2μl-50mM; n=20) or saline (controls; n=20). Ten rats of each group were treated with IN-rHu-Epo 24U or IN-saline. In addition, erythropoietic stimulation was evaluated by reticulocytes (Ret) account during three consecutive days, after intraperitoneal (i.p.)-recombinant-human Epo (rHu-Epo) (950U; n=6) or IN-rHu-Epo (24U; n=6) administration. SMA was evaluated by open field and rotarod tests, before and after surgical procedures during five consecutive days. Histological and immunostaining studies of HIF-1α, MDR-1, and Epo-R were performed on brain slides. A significant difference in SMA was observed in the hypoxic rats of IN-rHu-Epo-administered group as compared with Co-Saline-treated subjects and controls (p<0.001). HIF-1α, EPO-R, and MDR-1 were overexpressed in the hypoxic cortex areas, while in contralateral hemisphere or controls, they were negatives. Reticulocytes were only increased in intraperitoneal (i.p.)-rHu-Epo-administered group. In spite of MDR-1 overexpression being detected in neurons, the coexpression of Epo-R could explain the positive effects observed on SMA of IN-rHu-Epo-administered group.

Co-expression of CD147 (EMMPRIN), CD44v3-10, MDR1 and monocarboxylate transporters is associated with prostate cancer drug resistance and progression

Background:The aim of this study is to seek an association between markers of metastatic potential, drug resistance-related protein and monocarboxylate transporters in prostate cancer (CaP).Methods:We evaluated the expression of invasive markers (CD147, CD44v3-10), drug-resistance protein (MDR1) and monocarboxylate transporters (MCT1 and MCT4) in CaP metastatic cell lines and CaP tissue microarrays (n=140) by immunostaining. The co-expression of CD147 and CD44v3-10 with that of MDR1, MCT1 and MCT4 in CaP cell lines was evaluated using confocal microscopy. The relationship between the expression of CD147 and CD44v3-10 and the sensitivity (IC50) to docetaxel in CaP cell lines was assessed using MTT assay. The relationship between expression of CD44v3-10, MDR1 and MCT4 and various clinicopathological CaP progression parameters was examined.Results:CD147 and CD44v3-10 were co-expressed with MDR1, MCT1 and MCT4 in primary and metastatic CaP cells. Both CD147 and CD44v3-10 expression levels were inversely related to docetaxel sensitivity (IC50) in metastatic CaP cell lines. Overexpression of CD44v3-10, MDR1 and MCT4 was found in most primary CaP tissues, and was significantly associated with CaP progression.Conclusions:Our results suggest that the overexpression of CD147, CD44v3-10, MDR1 and MCT4 is associated with CaP progression. Expression of both CD147 and CD44v3-10 is correlated with drug resistance during CaP metastasis and could be a useful potential therapeutic target in advanced disease.

Expression of MDR1 and KIT in imatinib-resistant gastrointestinal stromal tumor cells

To explore the relationship between imatinib resistance and genes MDR1 and KIT in gastrointestinal stromal tumor (GIST) cells. The MDR1 and KIT mRNA level in GIST882-R and GIST882-S cells were detected by RT-PCR. Immunocytochemistry and Western blot were employed to detect P-gp and CD117 expression in GIST882-R and GIST882-S cells. The relative expression of MDR1 mRNA was 0.321 + or - 0.033 in GIST882-R and 0.157 + or - 0.056 in GIST882-S cells, and the difference was statistically significant (P<0.05). The relative expression of KIT mRNA was 0.389 + or - 0.063 in GIST882-R and 0.339 + or - 0.067 in GIST882-S, and the difference was not statistically significant (P>0.05). The relative density of P-gp was 0.443 + or - 0.058 in GIST882-R and 0.237 + or - 0.094 in GIST882-S, and the difference was statistically significant (P<0.05). The relative density of CD117 was 0.744 + or - 0.123 in GIST882-R and 0.704 + or - 0.094 in GIST882-S, and the difference was not statistically significant (P>0.05). Over-expression of gene MDR1 may be associated with imatinib resistance in GIST. KIT may not be involved in imatinib resistance.

An anti-apoptotic pattern correlates with multidrug resistance in acute myeloid leukemia patients: a comparative study of active caspase-3, cleaved PARPs, Bcl-2, Survivin and MDR1 gene

Bone marrow samples of 17 acute myeloid leukemia (AML) patients were analyzed for apoptosis-related markers. The levels of active caspase-3 (aC-3), Bcl-2 and cleaved poly(ADP-ribose) polymerase (cPARP) were measured by flow cytometry and compared with survivin and MDR1 gene expression as defined by reverse transcriptase polymerase chain reaction (RT-PCR). The results showed heterogeneous patterns of intracellular levels of the studied proteins in AML patients: aC-3 (mean 34·6±52·5 U/ml), Bcl-2 (mean 3268·4±2055·2 U/ml), and cPARPs (mean 24·59±29·97 U/ml). Survivin and MDR1 genes were overexpressed in 9 and 10 patients, respectively. Patients with high levels of survivin mRNA showed significantly lower cPARPs (11·8±14·3 versus 53·9±31·9 U/ml P=0·005) and a tendency towards higher aC-3 (49·3±70·0 versus 18·1±9·9 U/ml), and MDR1 overexpression (7/9 patients versus 3/8 patients), as well as poorer therapeutic response and survival. Our data support the potential relevance of apoptosis-related markers in AML for further understanding the disease; however, the heterogeneity and complexity of molecular interactions warrants further prospective studies.

Mdr1 gene expression as a marker of treatment response in non-small cell lung cancer (NSCLC).

e13151 Background: NSCLC is frequently chemoresistant. Multidrug resistance phenotype is involved in the development of chemoresistance. It has been related to several proteins expression, like P-glycoprotein (mdr1 gene). We examined whether elevated mdr1 expression levels in peripheral blood mononuclear cells (PBMC) predict treatment response. Methods: We studied mdr1 gene expression in PBMC of 23 patients with NSCLC, advanced stages in most of the cases, who had not received treatment previously. Blood extraction of 5ml was made to each patient before (hour 0) and after paclitaxel-carboplatin chemotherapy (hour 6). The mdr1 gene expression levels were assessed by quantitative reverse transcription polymerase chain reaction. Results: After treatment, we found mdr1 overexpression in 22 patients. Expression levels were randomly distributed, so that overexpression under 3 was considered low level of expression, and overexpression above 4 was considered high level of expression. High levels were found in 11 patients, and low levels were observed in the other 11 patients. The relationship between mdr1 expression and the treatment response was found to be not significant (p 0,68). No correlation was found between mdr1 expression and survival rate (p 0,54). No significant differences in expression were found in the other variables: gender, tobacco habit, weight loss at the diagnosis, tumor staging, and tumor histology. Conclusions: Our results suggest that the combined treatment with paclitaxel-carboplatin induces mdr1 overexpression in PBMN of patients with NSCLC, but mdr1 expression is not a predictive factor for treatment response nor a prognostic factor of the disease. No significant financial relationships to disclose.

Abstract 2985: DNA-damage related histone marks can distinguish the anti-tumor effects of established and novel histone deacetylase inhibitors in ovarian cancer cells

Abstract The majority of epithelial ovarian carcinomas harbor molecular alterations in DNA-damage response genes. Epithelial ovarian cancers are surprisingly sensitive to DNA-damaging platinum-based (cisplatin or carboplatin) chemotherapy, suggesting that the dependencies of ovarian cancers on impaired DNA-damage response pathways can be harnessed for therapeutic purposes. Our group and others have shown that histone deacetylase (HDAC) inhibitors are promising anti-tumor agents, particularly when combined with DNA-damaging drugs in ovarian cancer cells. In this study, H2AX phosphorylation (pH2Ax), a known histone mark of DNA damage and other histone marks related to DNA damage and repair pathways, including the newly recognized acetylated lysine K56 on histone 3 (H3K56ac) were used to distinguish the biological effects of a unique and diverse panel of clinically utilized and novel HDAC inhibitors. Ovarian cancer cell lines with diverse molecular alterations, including mutations in p53, Brca1, Akt, Ras, and overexpression of MDR1, were utilized in these screens. The HDAC inhibitors that induced the highest levels of pH2Ax produced the greatest anti-tumor effects (growth inhibition and apoptosis). Furthermore, a screen of DNA-damage response histone marks led to the selection of novel HDAC inhibitors that exerted synergistic anti-tumor effects when combined with cisplatin and other DNA-damaging drugs. Taken together, our results support a role of HDAC inhibitors in the treatment of ovarian cancer and suggest that pH2Ax is a potential surrogate biomarker of the efficacy of HDAC inhibitor combination therapy with DNA-damaging agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2985.

Abstract 3536: The biphasic mechanism of TNF-alpha mediated modulation of mdr1 expression and chemosensitization of colon cancer cells is linked to the NFkappaB pathway

Abstract Multidrug resistance (MDR) is one major cause for cancer chemotherapy failure. Therefore, numerous overcoming strategies have been tested for tumor chemosensitization. Among these, persistent action of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF) showed great potential for downregulation of MDR-associated genes, particularly for the ABC-transporter P-glycoprotein Pgp, encoded by the mdr1 gene. These cytokine-mediated effects are still under controversial discussion, since transient action of TNF was shown to increase mdr1 expression, whereas persistent TNF action reduces mdr1 expression. Key mediator for this is the transcription factor NFkappaB/p65. This factor is acting via binding to NFkappaB/p65 consensus sequences within the mdr1 promoter. To determine the cellular mechanisms of TNF mediated modulation of mdr1 expression, we analyzed transient and persistent effects in regard to biphasic NFkappaB signaling in colon carcinoma cells. The mdr1 overexpressing, multidrug resistant human HCT15 colon carcinoma cells were treated for transient 30 to 120 min or long-term, 24 to 96 h incubations with TNF. The TNF mediated modulation of mdr1 expression was analyzed by real-time RT-PCR and Western-blot. Involvement of TNF-receptor I or II and time-dependent analysis of NFkappaB/p65 signaling was determined by Western blot, luciferase cell signaling reporter assay and EMSA. Our studies revealed that the persistent long-term action of TNF leads to down-regulation of mdr1 at mRNA and protein level. We showed, that this effect is mediated by TNF-receptor I, which is known to trigger the NFkappaB/p65 pathway. More importantly, the persistent TNF action on the colon cancer cells abolishes TNF mediated induction of the NFkappaB/p65 signaling, associated with reduced nuclear accumulation of NFkappaB/p65 and also its impaired binding to the consensus sequence of the mdr1 promoter. This study supports the biphasic nature of TNF action and points to the contrary effects of transient or persistent action of this cytokine. It provides insights into mechanism of MDR overcoming in colon cancer and potential clues for effects associated with chronic inflammation of the intestine Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3536.

Multidrug resistance P‐glycoprotein/ABCB1/MDR1 protects against apoptosis by reducing cellular ceramides (Cer) induced by cadmium (Cd)

Cd damages the kidney by apoptosis, but chronic Cd exposure also leads to cancer. Previously, we showed Cd apoptosis mediated by ceramide‐calpain signaling (Lee, W.‐K., Am J Physiol Cell Physiol 2007), but also Cd‐induced upregulation of the multidrug resistance transporter P‐glycoprotein (MDR1) to promote kidney proximal tubule cell (PTC) survival (Thévenod, F., J Biol Chem 2000). The aim of the study was to investigate mechanisms of MDR1 dependent survival of Cd exposed cells. In MDR1 overexpressing kidney cells (MDR1‐MDCK), toxicity of 10–20 μM Cd and 10–50 μM C6 Cer (by MTT assay and nuclear staining) was significantly reduced by up to 75% and 60%, respectively, which was abolished by the MDR1 blocker PSC833. Electrical measurements (ECIS) of MDCK and MDR1‐MDCK monolayers ± Cd showed similar effects. Efflux of 109Cd was not enhanced in MDR1‐MDCK and unaffected by MDR1 inhibitors (PSC833, C219). A diacylglycerol kinase assay revealed lower basal total Cer levels in MDR1‐MDCK (0.31 ± 0.04 μM) than controls (0.49 ± 0.07 μM). Cer levels were increased by 67.2 ± 16.0% in MDCK but only a 21.9 ± 4.8% increase in MDR1‐MDCK by 3 h Cd. Furthermore, induction of MDR1 expression by Wnt signalling (transfection of β‐catenin and TCF4) reduced cell death by Cd in PTC. Rather than exporting Cd, MDR1 seems to confer its protective effects by reducing cellular Cer levels or by efflux of Cer metabolites.Funded by DFG TH 345/10‐1 and TH 345/11‐1

Bcr–Abl-independent mechanism of resistance to imatinib in K562 cells: Induction of cyclooxygenase-2 (COX-2) by histone deacetylases (HDACs)

Our previous studies have shown that overexpression of MDR1 and cyclooygenase-2 (COX-2) resulted in resistance development to imatinib in chronic myelogenous leukemia (CML) K562 (IR-K562) cells. In the present study, the regulatory mechanism of MDR1 induction by COX-2 was investigated. A gradual overexpression of MDR1 and COX-2 during the process of development was observed. Furthermore, down regulation of MDR1 upon COX-2 knockdown by siRNA showed a decrease in the PKC levels and activation of PKC by addition of PGE 2 to K562 cells, suggesting a role for PKC in the COX-2 mediated induction of MDR1. The present study demonstrates COX-2 induction by HDACs and MDR1 induction by COX-2 via PGE 2–cAMP–PKC-mediated pathway.

Glucosylceramide synthase upregulates MDR1 expression in the regulation of cancer drug resistance through cSrc and β-catenin signaling

BackgroundDrug resistance is the outcome of multiple-gene interactions in cancer cells under stress of anticancer agents. MDR1 overexpression is most commonly detected in drug-resistant cancers and accompanied with other gene alterations including enhanced glucosylceramide synthase (GCS). MDR1 encodes for P-glycoprotein that extrudes anticancer drugs. Polymorphisms of MDR1 disrupt the effects of P-glycoprotein antagonists and limit the success of drug resistance reversal in clinical trials. GCS converts ceramide to glucosylceramide, reducing the impact of ceramide-induced apoptosis and increasing glycosphingolipid (GSL) synthesis. Understanding the molecular mechanisms underlying MDR1 overexpression and how it interacts with GCS may find effective approaches to reverse drug resistance.ResultsMDR1 and GCS were coincidently overexpressed in drug-resistant breast, ovary, cervical and colon cancer cells; silencing GCS using a novel mixed-backbone oligonucleotide (MBO-asGCS) sensitized these four drug-resistant cell lines to doxorubicin. This sensitization was correlated with the decreased MDR1 expression and the increased doxorubicin accumulation. Doxorubicin treatment induced GCS and MDR1 expression in tumors, but MBO-asGCS treatment eliminated "in-vivo" growth of drug-resistant tumor (NCI/ADR-RES). MBO-asGCS suppressed the expression of MDR1 with GCS and sensitized NCI/ADR-RES tumor to doxorubicin. The expression of P-glycoprotein and the function of its drug efflux of tumors were decreased by 4 and 8 times after MBO-asGCS treatment, even though this treatment did not have a significant effect on P-glycoprotein in normal small intestine. GCS transient transfection induced MDR1 overexpression and increased P-glycoprotein efflux in dose-dependent fashion in OVCAR-8 cancer cells. GSL profiling, silencing of globotriaosylceramide synthase and assessment of signaling pathway indicated that GCS transfection significantly increased globo series GSLs (globotriaosylceramide Gb3, globotetraosylceramide Gb4) on GSL-enriched microdomain (GEM), activated cSrc kinase, decreased β-catenin phosphorylation, and increased nuclear β-catenin. These consequently increased MDR1 promoter activation and its expression. Conversely, MBO-asGCS treatments decreased globo series GSLs (Gb3, Gb4), cSrc kinase and nuclear β-catenin, and suppressed MDR-1 expression in dose-dependent pattern.ConclusionThis study demonstrates, for the first time, that GCS upregulates MDR1 expression modulating drug resistance of cancer. GSLs, in particular globo series GSLs mediate gene expression of MDR1 through cSrc and β-catenin signaling pathway.

Establishment of a human hepatoma multidrug resistant cell linein vitro

To establish a multidrug-resistant hepatoma cell line (SK-Hep-1), and to investigate its biological characteristics. A highly invasive SK-Hep-1 cell line of human hepatocellular carcinoma, also known as malignant hepatoma was incubated with a high concentration of cisplatin (CDDP) to establish a CDDP-resistant cell subline (SK-Hep-1/CDDP). The 50% inhibitory dose (IC(50)) values and the resistance indexes [(IC(50) SK-Hep-1/CDDP)/(IC(50) SK-Hep-1)] for other chemotherapeutic agents and the growth curve of cells were all evaluated using cell counting kit-8 assays. The distribution of the cell cycles were detected by flow cytometry. Expression of acquired multidrug resistance P-glycoprotein (MDR1, ABCB1) and multidrug resistance-associated protein 1 (MRP1, ABCC1) was compared with that in parent cells by Western blotting and immunofluorescence combined with laser scanning confocal microscopy. The SK-Hep-1/CDDP cells (IC(50) = 70.61 +/- 1.06 microg/mL) was 13.76 times more resistant to CDDP than the SK-Hep-1 cells (IC(50) = 5.13 +/- 0.09 microg/mL), and CDDP-resistant cells also demonstrated cross-resistance to many anti-tumor agents such as doxorubicin, 5-fluorouracil and vincristine. Similar morphologies were determined in both SK-Hep-1 and SK-Hep-1/CDDP groups. The cell cycle distribution of the SK-Hep-1/CDDP cell line exhibited a significantly increased percentage of cells in S (42.2% +/- 2.65% vs 27.91% +/- 2.16%, P < 0.01) and G2/M (20.67% +/- 5.69% vs 12.14% +/- 3.36%, P < 0.01) phases in comparison with SK-Hep-1 cells, while the percentage of cells in the G0/G1 phase decreased (37.5% +/- 5.05% vs 59.83% +/- 3.28%, P < 0.01). The levels of MDR1 and MRP1 were overexpressed in the SK-Hep-1/CDDP cells exhibiting the MDR phenotype. Multiple drug resistance of multiple drugs in the human hepatoma cell line SK-Hep-1/CDDP was closely related to the overexpression of MDR1 and MRP1.