MDR1 Gene Product Research Articles

Suppression of αvβ6 downregulates P-glycoprotein and sensitizes multidrug-resistant breast cancer cells to anticancer drugs.

Multidrug resistance (MDR) in breast cancer treatment is the major cause leading to the failure of chemotherapy. P-glycoprotein (P-gp), the product of the human MDR1 gene, plays a key role in resistance to chemotherapy and confers cross-resistance to many structurally unrelated anticancer drugs. We have previously reported that integrin αvβ6 plays a critical role in breast cancer invasion and metastasis. However, whether and how αvβ6 is associated with P-gp and regulated by potential genetic mechanisms in breast cancer remains unclear. In the present study, we further investigated the reversal effect and underlying mechanisms of MDR in breast cancer. Two small interfering RNA constructs (pSUPER-β6shRNAs) targeting two different regions of the β6 gene have been designed to inhibit αvβ6 expression by transfecting them into adriamycin-resistant MCF-7/ADR cell lines. Suppression of αvβ6 dramatically downregulated the levels of MDR1 gene mRNA and P-gp. In particular, β6shRNA-mediated silencing of αvβ6 gene increased significantly the cellular accumulation of Rhodamine 123 and markedly decreased drug efflux ability, suggesting that β6shRNAs indeed inhibit P-gp mediated drug efflux and effectively overcome drug resistance. In addition, inhibition of integrin αvβ6 suppressed the expression of ERK1/2. Interestingly, our data demonstrate that suppression of integrin αvβ6 caused significant downregulation of Bcl-2, Bcl-xL and upregulation of caspase 3, Bad, accompanied by increasing activity of cytochrome C. A possible connection between αvβ6 and P-gp in drug resistance biology is suggested. Taken together, β6shRNA could efficiently inhibit αvβ6 and MDR1 expression in vitro and these findings may offer specifically useful means to reverse MDR in breast cancer therapy.

SUN-113 COMBINATION OF *1/*1 WILD VARIANT OF CYP3A5 AND TT/AA MUTANT OF MDR G2677T/A GENE POLYMORPHISM REQUIRES HIGHER TACROLIMUS DOSE TO ACHIEVE TARGET TROUGH LEVEL

Tacrolimus (Tac) is the most commonly used immunosuppressive drug in transplantation. Tac disposition has very narrow therapeutic range and great inter-individual variability in its pharmacokinetics. Achieving the target blood concentration is of crucial importance in the early stage after transplantation and to attain good transplant outcome. This differential bioavailability in trough Tac concentration may be due to expression of P-gp varying between individuals and/or differential metabolizing capacity of CYP450.Tac share CYP3A5 enzymes for their hepatic elimination and P-gp, a product of MDR-1 gene expressed on gut wall, renal tubular cells, hepatocytes, lymphocytes etc and works as transmembrane efflux pump which exports xenobiotics from inside the cell to outside. Hence we aimed this study to analyse the influence of the combination of MDR-1 and CYP3A5 SNPs on daily Tac requirement to achieve trough level post-transplantation. We recruited 248 renal transplant recipients (mean age 36.48±10.54; male (83.47%),all on Tac; MMF and prednisone in the study. Of the 248 patients; 110 had CGN; 65 had CIN; 7 had ADPKD; 21 had DKD; 8 had CKDu; 7 had DGGS; 3 had Alport's syndrome and 27 had other native kidney diseases. The mean creatinine level at the time of discharge was 1.15±0.27(0.48-2.31). P-gp expression was analyzed by flow cytometry and polymorphism was performed by PCR-RFLP. Of the 248; 123(49.59%) were CYP3A5*3/*3(non-expressers homozygous); 94(37.90%) CYP3A5*1/*3; and 31(12.5%) were CYP3A5*1/*1 (expressers). In MDR G2677T/A gene of 248 patients; 99(39.91%) were GG (wild type homozygous); 104(41.92%) were GT/GA (heterozygous) and 45(18.13%) were TT/AA (mutant homozygous) of MDR Gene. Tac trough concentration was high in *3/*3 as compare to *1*1(p=0.001) and *1*3 (p=0.022) respectively. CYP3A5*1/*1 patients had lower levels of dose-adjusted TAC (55.69±26.84 ng/mL/mg/kg/day) to achieve target blood level and required higher daily dose per weight (0.142±0.028mg/kg/day) than CYP3A5*1/*3 patients; 111.12±8.93ng/mL/mg/kg/day and 0.121±0.035mg/kg/day(p=0.022) and CYP3A5*3/*3 patients; 142.21±75.23ng/mL/mg/kg/day and 0.109±0.032mg/kg/day(p<0.001). Tac Co was low in TT/AA as compare to GG(p<0.001) and GT/GA(p<0.001) respectively and daily Tac dose requirement was high in TT/AA as compare to GG(p<0.001) and GT/GA(p=0.027) genotype to achieve target blood concentration of MDR G2677T/A gene. The P-gp expression was also high in TT/AA as compare to GG(p<0.001) and GT/GA(p<0.001). Genotype C1236T and C3435T of MDR-1 gene had no significant effect on Tac dose requirement. Patients having CYP3A5*1/*1(expresser) and TT/AA (homozygous mutant) of MDRG2677T/A genotype had highest Tac daily dose requirement (0.147mg/kg/day) and having CYP3A5*3/*3 (non-expresser) and GG of MDRG2677T/A (wild type) genotype requires lowest Tac dose (0.101 mg/kg/day) to achieve target Tac tough concentration (8-12ng/ml). Daily Tac dose requirement is highest in patients having CYP3A5 expresser and MDR G2677T/A homozygous mutant gene together. Genetic variants may predicts the optimal dose of Tac in renal transplant recipients.

Nuclear Transcription Factor Kappa B Downregulation Reduces Chemoresistance in Bone Marrow-derived Cells Through P-glycoprotein Modulation

Nuclear transcription factor kappa B (NF-κB) is associated with many types of refractory cancer. However, despite multiple strategies to treat cancer and novel target drugs, multidrug resistance still causes relapses. The best-characterized mechanism responsible for multidrug resistance involves the expression of the MDR-1 gene product, P-glycoprotein (P-gp). Because the direct inhibition of this protein is very toxic, other methods of multidrug resistance (MDR) regulation have been proposed. The MDR-1 promoter sequence contains a κB site, which is recognized by NF-κB. The aim of this work was to characterize whether NF-κB modulation changes the response of bone marrow-derived cells (BMDCs) to chemotherapy. We exposed BMDCs to etoposide and doxorubicin, two of the most used antineoplastic drugs. BMDCs presented high tolerance to these drugs, which correlated with high intrinsic P-gp activity and strong protein expression of NF-κB. To determine the mechanism behind the poor sensitivity of BMDCs to chemotherapy, we blocked the activity of the heterodimer protein NF-κB using the pharmacological inhibitor Bay 11-7085 and through the transfection of an adenovirus negative mutant of I kappa B alpha. The multidrug resistance phenotype of BMDCs was reversed by inhibiting the NF-κB pathway, and this change was accompanied by a decrease in P-gp activity. NF-κB is a possible target for improving the antineoplastic response.

Recurrence rate and progression of chondrosarcoma is correlated with heat shock protein expression.

Heat shock proteins (HSPs) are involved in tumour immunity, and are correlated with survival and drug resistance in numerous types of cancer. The present study investigated the expression of HSPs and multiple drug resistance (MDR) in human chondrosarcoma. HSP and P-glycoprotein (the MDR1 gene product) expression was evaluated by immunohistochemical analysis of paraffin-embedded sections obtained from 37 patients with chondrosarcoma (19 male and 18 female; aged 33–85 years; mean age, 48.5 years). HSP73 and 90 were significantly overexpressed in patients with local recurrence: HSP73 was expressed in 7/7 patients (100%) with local recurrence and 9/18 patients (50%) without recurrence (P<0.02), while HSP90 was expressed in all patients with recurrence but only 8/18 (44%) without recurrence (P<0.02). A marked association was also identified between HSP expression and survival. HSP72 and 73 were significantly overexpressed in tumours from patients who succumbed to the disease (all positive for HSP72 and 73; P<0.05). No differences were observed between HSP27, 73 or 90-positive or -negative tumours according to age or gender. In addition, HSP72 expression was correlated with differentiation of the tumours (P<0.02). These results indicate that HSP72, 73 and 90 may function as novel prognostic markers for chondrosarcoma, and initiate further studies regarding the use of such markers for the identification of patients with poor prognosis.

Abstract 3594: P-glycoprotein attenuates Src activation and DNA repair activity via increased C-terminal Src kinase-binding protein, a negative regulator of Src, in multidrug-resistant cells

Abstract Multidrug resistance (MDR) remains a significant obstacle to the success of cancer chemotherapy. MDR is often associated with increased expression of ATP-binding cassette transporter family members, which extrude anticancer drugs out of cells. The MDR1 gene product, P-glycoprotein (P-gp) is one of the most well-known ABC transporters and expels a broad range of anticancer drugs, such as vinblastine, vincristine, doxorubicin and paclitaxel. Overexpression of P-gp in tumor tissues is thus a prognostic indicator associated with poor response to chemotherapy and poor clinical outcome. Our previous study has shown that P-gp overexpression attenuates the repair activity of DNA interstrand crosslinks (ICLs) and hence contributes the susceptibility of MDR cells to DNA ICL agents [1]. In the present study, we explored the molecular mechanisms underlying which P-gp interferes with Src-activated DNA repair activity. We first confirmed that ectopic expression of P-gp attenuates DNA ICL repair activity and becomes more susceptible to DNA ICL agents than vector control cells. Our present results showed that the C-terminal Src kinase-binding protein (Cbp), a negative regulator of Src, is significantly enhanced in MDR cells. Cbp silencing by siRNA technique apparently enhances cisplatin-induced Src activation and DNA repair activity, and hence increases the resistance of MDR cells to ICL agents. By aid of immuneprecipitation and immunofluorescence staining techniques, we demonstrated that P-gp not only interacts with Cbp and Src but also enhances the formation of inhibitory Csk-Cbp complex that reduces phosphorylation of Src activation residue Tyr416 and increases phosphorylation of Src negative regulatory Tyr527 residue. Taken together, the current results indicate that P-gp overexpression in MDR cells attenuates DNA repair activity by mediating Cbp dependent mechanism to interfere with Src activation. These results implicate that increasing the expression of Cbp is potentially a therapeutic strategy to increase the susceptibility of cancer cells to chemotherapeutic agents. 1. Lee PC, Lee HJ, Kakadiya R, Sanjiv K, Su TL, Lee TC: Multidrug-resistant cells overexpressing P-glycoprotein are susceptible to DNA crosslinking agents due to attenuated Src/nuclear EGFR cascade-activated DNA repair activity. Oncogene 2013, 32:1144-1154. Citation Format: Li-Fang Lin, Ming-Hsi Wu, Tsann-Long Su, Te-Chang Lee. P-glycoprotein attenuates Src activation and DNA repair activity via increased C-terminal Src kinase-binding protein, a negative regulator of Src, in multidrug-resistant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3594. doi:10.1158/1538-7445.AM2015-3594

Potential implications of CYP3A4, CYP3A5 and MDR-1 genetic variants on the efficacy of Lopinavir/Ritonavir (LPV/r) monotherapy in HIV-1 patients.

Introduction Several genetic single nucleotide polymorphisms (SNPs) in biotransformation enzymes (CYP3A4, CYP3A5) or transporter proteins (multidrug resistance MDR1 gene product, P-gp) are involved in PI metabolism so that PI pharmacokinetics is characterized by a large inter-individual variability. The aim of this study was: (i) to develop an in-house PCR/direct sequencing, based on DNA purification of full-length CYP3A4 and CYP3A5 genes (SNPs) and MDR1 C3435T variant; (ii) to investigate association of CYP3A4 and CYP3A5 reported or unreported genetic polymorphisms and MDR1-C3435T (CC homozygote, CT heterozygote, TT homozygote) with clinical outcome of HIV-1 infected subjects treated with PI.Methods Overall, 39 HIV-1 infected patients receiving boosted Lopinavir (LPV/r) monotherapy after virological suppression were genotyped and analyzed through PCR and direct sequencing of full-length CYP3A4 and CYP3A5 gene sequences [1] and MDR1 gene (C3435T). CD4+T-cell counts and plasma viral load were analyzed before and after LPV/r initiation; LPV/r therapeutic drug monitoring (TDM) was determined at 12-hours.Results LPV/r TDM (ng/ml) did not show significant differences among CYP3A4 or CYP3A5 SNPs, although a mean lower level of LPV/r was associated with detection of several SNPs: CYP3A5*3 rs776746; CYP3A5 rs28365088, CYP3A5 rs15524, CYP3A4 rs2687116, and a not already described polymorphism CYP3A4 nt20338. In follow-up analysis, <90% adherence was the main factor associated with virological failure of LPV/r monotherapy (83.3% of failure vs 34.4%, p<0.001 at log-rank test). Adjusting for adherence, the detection of a single CYP3A5*3 rs776746 and CYP3A5 rs15524 SNPs was associated with higher probability of LPV/r monotherapy failure (p<0.01), and in general, detection of any CYP3A5 SNP was associated with failure (26.2% vs 58.3%, p=0.067). No-association with detection of any CYP3A4 SNPs was found. MDR1 TT variants showed significant lower frequency of treatment failure (0.0% vs 47.7%, p=0.026), since non-TT homozygote patient failed LPV/r monotherapy.Conclusions Efficacy of PI monotherapy is strongly dependent from patient adherence, but, in adherent patients, genetic factors, such as CYP3A5 and MDR1-C3435T gene variants, may affect the response to treatment, though their role, as well of other genetic variants, need further investigation.

Abstract 3125: Fisetin inhibits p90RSK/YB-1 signaling and downregulates chemoresistance associated P-glycoprotein in A375 melanoma cells

Abstract Malignant melanoma is notoriously difficult to treat due to lack of targeted systemic therapies and emergence of resistance to BRAF inhibitors, currently the treatment of choice for metastatic melanoma. The p90 ribosomal S6 kinase (RSK), an important effector of RAF/ERK signaling promotes melanoma growth and contributes to melanoma chemoresistance by altering response to chemotherapeutic agents. Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor involved in multiple cellular functions including proliferation, migration, and epithelial mesenchymal transition (EMT). Recent studies show that RSK1/2 can activate YB-1 through phosphorylation at its Ser102 residue. We found YB-1 expression to be significantly upregulated in metastatic melanoma cell lines when compared to normal human melanocytes. Treatment of A375 human melanoma cells with the dietary flavonoid fisetin (20-60µM) resulted in inhibition of phosphorylation of YB-1 at Ser102, with subsequent decrease in nuclear translocation and activation. We further studied the effect of fisetin on YB-1 signaling in a 3-D melanoma skin equivalent model, comprising of A375 melanoma cells, cultured with epidermal keratinocytes and dermal fibroblasts that closely simulates the human skin. Studies in the 3-D model showed that fisetin treatment resulted in reduction in melanocytic lesions and tumor progression (p&amp;lt; .01) associated with decreased expression of YB-1, and its downstream targets c-Myc and MTA-1. Fisetin suppressed the expression of EMT markers N-cadherin and Vimentin, with a concomitant increase in E-cadherin in melanoma cells. In addition, a significant decrease in the protein expression of matrix-metalloproteinases -2 and -9 was observed. Next we investigated whether fisetin interfered with RSK signaling involved in YB-1 activation. Immunoblot studies revealed that fisetin inhibited the phosphorylation of p90RSK at the Thr359/Ser363 residue but not at Ser380. Notably, the MDR-1 gene product, P-glycoprotein (P-gp), was markedly decreased in fisetin-treated cells in a dose-dependent manner. Fisetin-mediated increase in the phosphorylation of ERK1/2 at Thr202/Tyr204 residues suggests that diminished P-gp expression is linked to suppression of RSK/YB-1 signaling in fisetin treated cells. Together, our results suggest that fisetin is a potent inhibitor of the p90RSK/YB-1 pathway and may be used to modulate chemosensitivity, which is one of the major obstacles to melanoma treatment. Citation Format: Deeba N. Syed, Rahul K. Lall, Mohammad Imran Khan, Maria Shabbir, Hasan Mukhtar. Fisetin inhibits p90RSK/YB-1 signaling and downregulates chemoresistance associated P-glycoprotein in A375 melanoma cells. [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 3125. doi:10.1158/1538-7445.AM2014-3125

PW01-040 – Definition of polymorphism C3435T MDR1 gene in JIA

Juvenile idiopathic arthritis (JIA) is one of the most common rheumatic disease in children. Brug of choice is methotrexate (MTX) - cytostatic drug from the group of antimetabolites, folic acid antagonists. According to various authors, about 70% of patients receiving therapy with MTX are in remission for the disease. In 1988, MTX was approved FDA (Food and Drug Administration, USA) for the treatment of rheumatoid arthritis. Currently, MTX is the main drug in the treatment of JIA. Preferred therapeutic recommendations on the number of MTX based on a randomized, double-blind, placebo-controlled study conducted in 1992. MDR1-gene product - the P-glycoprotein (P-gp), the protein acts as a trans-membrane pump, and affects the activity of many drugs. Polymorphism in the gene MDR1, may affect the pharmacokinetics of many drugs, including anticancer drugs. According to the authors of Jinwei Chen (2011) in adult patients with rheumatoid arthritis (RA) in the Chinese population C3435T MDR1 gene polymorphism may be associated with susceptibility to RA, but may influence the effectiveness of antirheumatic therapy of RA, and CC genotype may be associated with immune rheumatoid arthritis (RRA).

AB1144 Influence the level of PGP activity (product of MDR1GENE) on the efficiency of basic therapy in children with juvenile idiopathic arthritis

BackgroundStudies of the MDR1 gene started from the early 80s of the XX century, when first studied protein expression Pgp-170. In 2000, for the first time it was found that...

MTOR inhibition in MDR acute myeloid leukemia.

7085 Background: Signaling through the PI3K/PTEN/AKT/mTOR pathway is aberrantly activated in several human cancers, including Acute Myeloid Leukemia (AML) patients where it contributes to leukemic cell proliferation, survival and drug resistance. In leukemia patients treated with drugs such as anthracyclines, vinca alkaloids, chemo-resistance one of the major problems contributing to therapeutic failure, where all this drugs are modulated in their intracellular retention by a 170 kDa Phospho-glycoprotein, an mdr-1 gene product. The role of mTOR in various processes such as proliferation, growth, has been comprehensively described in many reviews, following its inhibition by rapamycin. Thus inhibition of mtor signaling in AML blasts could account for enhancing their sensitivity to cytotoxic agents. This study is to embrace the effect of Rapamycin, an allosteric mTOR inhibitor on MDR AML patients. Methods: We selected samples of 25 MDR AML patients isolated from bone marrow or peripheral blood and cultured in vitro. The effects of Rapamycin on fresh AML samples were analyzed by treating the cells with intermediate drug concentrations (100 nM) for 24 h, 48 h, 72 h, 96 h respectively followed by cytotoxicity assay (MTT assay). The effects of the drug on proliferation of cells were analyzed also by cell counting. Results: A marked reduction in cell viability at 96 h was detected in 25 samples treated with rapamycin. Out of the 25 analyzed samples, 16 were sensitive to the drug where it was observed that the leukemic cell count came down to almost 2% whereas 9 samples displayed various degree of resistance to the drug. Overall, these findings demonstrated that rapamycin has a cytotoxic activity against primary cells from AML patients with up-regulated mTOR signaling. Conclusions: We have evaluated the in vitro effects of Rapamycin on AML patient samples where reduced cell viability primary cells from AML patients were observed. However, further studies of the additional effects of rapamycin will be of the supreme importance, as they could help in designing more efficient curative protocols, for the treatment of acute leukemia patients.

FLT3-ITD and MLL-PTD Mutations Influence the Expression of MDR1, MRP1 and BCRP mRNA in Adult Acute Myeloid Leukemia

Abstract 1334 BackgroundA number of molecular aberrations have been described in acute myeloid leukemia (AML). They disturb normal haematopoiesis and are response for leukaemogenic mechanism and drug resistance. Therefore, they seem to be of prime importance for treatment outcome. FMS-like tyrosine kinase 3- internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are leukemia associated aberrations which confer worse prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined.The over-expression of ABC transport proteins that function as a membrane pump responsible for the efflux of a range of chemotherapeutic drugs are known mechanism behind multidrug drug resistance (MDR) in AML. The p-glycoprotein (Pgp)-product of MDR1 gene and multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP) encoded by the relevant genes negatively influence the results of AML therapy. AimsThe aim of this study was to analyze the expression of the MDR-1, MRP-1, BCRP messenger RNA (mRNA) in relation to the response to induction chemotherapy and relapse and with pretreatment laboratory and clinical characteristics such as age (<60, ≥60 years), disease status (de novo/secondary), cytogenetics, FLT3-ITD and MLL-PTD mutational status, white blood cell count (WBC) and the type of AML according to the French-American-British (FAB) classification. Moreover, the influence of MDR genes on disease free survival (DFS) and overall survival (OS) were estimated. MethodsA total of 185 adult consequent patients (88 females and 97 males) at a median age of 53,1 years (range 18,5–86,6 years) with newly diagnosed, previously untreated AML, as well as 40 healthy donors were included in this study. DNA and RNA were extracted from mononuclear cells of bone marrow (147 patients) or peripheral blood (38 patients) samples collected after approval from the ethics committee and informed consent from the patients. FLT3-ITD and MLL-PTD were detected with PCR method. The RQ-PCR method was performed for the assessment of expression of the MDR-1, MRP-1, BCRP and LRP mRNA and the results were presented as coefficients calculated with an intermediate method according to Pfaffl’s rule. The relative expression ratio of a target gene was calculated based on efficiency and the Ct deviation of the healthy donor′s sample versus the patient′s sample, and expressed in comparison to a reference gene: ResultsIn univariate analysis, the high expression of MDR-1 mRNA (>0,13) was associated with outcome of induction therapy (p=0,06) and of BCRP mRNA (> 1,14) with high relapse rate (RR) (p=0,013). We found that high expression of MDR-1 (>0,13), MRP-1 mRNA (> 0,84) and BCRP mRNA (> 1,14) significantly influence DFS (p=0,059, 0,032 and 0,009, respectively) and OS (0,048, 0,014 and 0,059, respectively). Interestingly, high BCRP mRNA expression (>1,14) proved to be an independent prognostic factor for RR (p=0,01) and DFS (p=0,002) in the multivariate analysis. FLT3-ITD and MLL-PTD were detected in 53 (29%) and 44 (25%), respectively.Interestingly, we found correlation between expression of MDR1, MRP1 and BCRP mRNA and FLT3-ITD and MLL-PTD mutational status in presented group of patients. The expression of MDR-1 mRNA was significantly higher in patients no harboring FLT3-ITD mutation (0,20 vs. 0,05, p= 0,0001) and ≥60 years old (0,30 vs. 0,10, p= 0,007). The higher expression of MRP-1 mRNA was found in patients with FLT3-ITD mutation (0,96 vs. 0,70, p= 0,002) and the expression of BCRP mRNA was significantly higher in patients with MLL-PTD (0,61 vs 0,38, p=0,03). ConclusionsThe combined analysis of MDR genes allowed different classes of AML to be identified, with distinct clinico-biological features. The high expression of MDR genes correlates with worse treatment outcome of patients with AML. It seems that especially BCRP is associated with clinical resistant disease in AML and influence the RR and DFS. Our study also shows that MDR gene expression in AML patients should be considered together with leukemia associated aberrations. The interesting correlation found between expression of MDR1, MRP1 and BCRP mRNA and FLT3-ITD and MLL-PTD in AML patients needs further investigation to determine its clinical value and made final conclusions. Disclosures:No relevant conflicts of interest to declare.

Abstract 4330: Transcriptional regulation of gp170 by YY1 and prognostic significance in childhood acute lymphoblastic leukemia (ALL)

Abstract Leukemia is the most common cancer among children under the age of 15 years in the United States and many developed countries. In recent years, there has been the emergence of novel therapeutic drugs for this disease. However, the development of drug resistance by the tumor cells remains the most important obstacle in the treatment of leukemia. A widely studied mechanism of tumor resistance to chemotherapeutic drugs is through the expression of the multi-drug resistance (MDR) genes. P-glycoprotein 170 (gp170) is an MDR1 gene product which serves as an ATP-dependent cell membrane transporter; it facilitates the efflux of xenobiotics from the cells to prevent damage for prolonged drug activity, including chemotherapeutics agents. In addition to MDR1, several reports in different cancers have indicated that the transcription factor Yin Yang 1 (YY1) is over-expressed and regulates tumor cell response to chemotherapeutic drugs. TESS analysis demonstrated that the gp170 proximal promoter contains four putative binding sites for the YY-1 protein. We also hypothesized that the YY1 may too be over-expressed in ALL and that its expression may be correlated with the expression of gp170. These hypotheses were examined first by reporter systems analyze, the gp170 protein was cloned and we developed a luciferase reporter assay. Examination of the putative YY1 binding sites that were individually mutated (deleted), showed revealed that mutation at the site –1860 abolished the activity. Mutation at the site –1920 abolished approximately 50% and mutation at –1230 site abolished approximately 25%. Further, CHIP analysis demonstrated that YY1 transcription factor binds directly to the gp170 gene MDR1 promoter. In addition, we examined the expression of YY1 and gp170 in childhood ALL (n= 84). The expression of YY1 and gp170 was determined evaluated by IHC in tumor tissues and the frequency of positive cells was determined. For comparison, normal controls were also analyzed (n=53). The findings demonstrate that were a significant increase in the frequency of positive cells in ALL compared to controls for both YY1 (38% vs. 8%, p=0.001) and for gp170 (42% vs. 12%, p=0.001). There was a good correlation between the expression of YY1 and gp170 (Pearson's test, r=0.4, p=0.001). The findings suggest that YY1 transcriptionally regulates the mdr1 and may be a therapeutic target in MDR positive ALL and its inhibition may reverse resistance to chemotherapeutic drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4330. doi:1538-7445.AM2012-4330

Co-Overexpression of YY1 and Gp-170 (MDR1) in Childhood Acute Lymphocytic Leukemia (ALL): Transcription Regulation of Gp-170 by YY1 and Prognostic Significance,

Abstract 3567Leukemia is the most common cancer among children under the age of 15 years in the United States and many developed countries. In recent years, there has been the emergence of novel therapeutic drugs for this disease. However, the development of drug resistance by the tumor cells remains the most important obstacle in the treatment of leukemia. A widely studied mechanism of tumor resistance to chemotherapeutic drugs is through the expression of the multi-drug resistance (MDR) genes. P-glycoprotein 170 (gp-170) is an MDR1 gene product which serves as an ATP-dependent cell membrane transporter; it facilitates the efflux of xenobiotics (including chemotherapeutic drugs) from the cells to prevent damage for prolonged drug activity, including chemotherapeutics agents. In addition to MDR1, several reports in different cancers have indicated that the transcription factor Yin Yang 1 (YY1) is over-expressed and regulates tumor cell response to chemotherapeutic drugs. Thus, we hypothesized that YY1 may also be over-expressed in ALL and that its expression may be correlated with the expression of gp-170. We also hypothesized that the co-expression of YY1 and gp-170 may be of prognostic significance in high risk ALL patients. These hypotheses were examined with childhood ALL (n= 88) (of median age 8.2 years, range 0.21–16). The expression of YY1 and gp-170 was determined by IHC in tumor tissues and the frequency of YY1 positive cells was determined. For comparison, normal controls were also analyzed (n= 53). The findings demonstrate that were a significant increase in the frequency of positive cells in ALL compared to controls for both YY1 (38% vs. 8%, p= 0.0001) and for gp-170 (42% vs. 12%, p= 0.001). There was a good correlation between the expression of YY1 and gp-170 using Pearson’s test, r=0.4, p=0.0001. We then examined if the co-expression of YY1 and gp-170 is also associated with decreased risk. Indeed, patients with low risk (L1) had significantly less frequency of positive cells compared to patients with high risk for both YY1 (40% vs. 30%, p=0.045) and for gp-170 (48% vs. 35%, p=0.0003). The strong correlation betweenYY1 and gp-170 expression suggested that there may be a cross-talk. TESS analysis demonstrated that the gp-170 proximal promoter contains four putative binding sites for the YY-1 protein. The gp-170 protein was cloned and we developed a luciferase reporter assay. Examination of the putative YY1 binding sites that were individually mutated revealed that mutation at the sites -1860 and -270 abolished activity. Mutation at the site -1420 abolished approximately 50% and mutation at -1230 abolished approximately 75%. Further, CHIP analysis demonstrated that YY1 binds directly to the gp-170 gene. Transfection of cells with YY1 siRNA inhibited the efflux of adriamycin from the cells confirming the regulation of gp-170 expression by YY1. Overall, the findings above demonstrate that both gp-170 and YY1 are over-expressed in childhood ALL and further over-expressed in patients with high risk. The findings suggest that YY1 may be a therapeutic target in MDR positive ALL and its inhibition may reverse resistance to chemotherapeutic drugs. Disclosures:No relevant conflicts of interest to declare.

Imaging of multidrug resistance in cancer

Primary intrinsic and/or acquired multidrug resistance (MDR) is the main obstacle to successful cancer treatment. Functional molecular imaging of MDR in cancer using single photon or positron emitters may be helpful to identify multidrug-resistant tumours and predict not only those patients who are resistant to treatment, with a clinically unfavourable prognosis, but also those who are susceptible to the development of drug toxicity or even certain tumours . Variations in the mdr1 gene product may directly affect the therapeutic effectiveness, and single nucleotide polymorphisms for the mdr1 gene may be associated with altered oral bioavailability of MDR1 substrates, drug resistance, and a susceptibility to some human diseases. The challenge of translating the concept of MDR modulation in vivo involves a complex cellular interplay between both malignant and normal cells. Integration and correlation of functional single photon emission tomography or positron emission tomography imaging findings with mdr1 genotype and clinical data may contribute to efficient management by selecting cancer patients with the appropriate molecular phenotype for maximal individual therapeutic benefit, as well as those who are non-responders. This review describes a role for functional imaging of classical mechanisms of MDR with an emphasis on readily available [99mTc]MIBI scintigraphy. MIBI scintigraphy has been shown to be a non-invasive cost-effective in vivo assay of ATP-binding cassette transporters associated with MDR in cancer, including P-glycoprotein, multidrug-resistant protein 1 and breast cancer resistant protein. New imaging agents for molecular targets such as vascular endothelial growth factor and HER2 receptors, may potentially be combined with MDR imaging substrates to more accurately predict the therapeutic response to anticancer drugs, guiding individualised treatment while minimising the economic health costs of ineffective therapy in an era of personalised medicine.

Abstract P2-09-21: Effect of MDR1 Gene Polymorphisms and Haplotypes on Taxanes-Based Chemotherapy Responses in Chinese Han Patients with Breast Cancer

Abstract Background: In recent years, taxanes play an important role in chemotherapy of breast cancer patients, and its response rate is about 50%. Taxanes is primarily metabolized by CYP450 in liver microsome, and it also is a substrate for P-glycoprotein (the coded product of MDR1 gene). The interindividual heterogeneity in drug-metabolizing enzymes and transporters has contributed to the variability on anti-cancer efficacy of taxanes in breast cancer patients. Several studies showed that the pharmacokinetics of taxanes was influenced by the MDR1 polymorphisms. The aim of this study is to investigate the relationship between MDR1 gene polymorphisms and taxanes-based chemotherapy responses, and provides useful information for clinical personalized medicine and prognostic judgment. Material and Methods: Genotyping of MDR1 variants was determined in the blood samples from 142 Chinese Han patients with breast cancer, by PCR-RFLP. The association of the gene polymorphisms of MDR1 C1236T at exon 12, G2677T/A at exon 21 and C3435T at exon 26 with chemotherapy responses was analyzed on the 63 patients, who received taxanes-based neoadjuvant chemotherapy [56 cases with TA (taxanes and anthracycline), 7 cases with TAC (C: cyclophosphamide)] and had the intact treatment follow-up information. Results: In the 142 breast cancer patients, the frequencies of C1236T, G2677T, G2677A and C3435T variant alleles was 70.7%,52.1%,2.5% and 46.5%, respectively, and the frequency of 3435T allele was significantly higher in comparison with that in healthy subjects (34.7%). In the 63 patients with neoadjuvant chemotherapy, the overall clinical response rate (CR+PR) to chemotherapy was 66.7%. 3435TT genotype was showed to be related to taxanes-based chemotherapy, and its efficient rate was 23.1%, significantly lower than that of patients carrying C allele (73.5%; x2 = 9.125, P=0.003). The 3 SNPs were in linkage disequilibrium, and the 4 haplotypes TTT, TGC, CGC and TTC were 90.6 % of all. The patients who carrying 2677G-3435C haplotype showed better response to chemotherapy compared with those with the other haplotypes combined (72.1% vs 40.0%;x2=9.125, P=0.003). The response rate of the patients with 2677T-3435T haplotype or 1236T-3435T haplotype (54.3% or 54.9%) were lower than that of the patients harboring the other haplotypes (82.4% or 91.7%;x2=4.128 and x2=4.118, P =0.042 for both) respectively, and the efficient rate of the patients with 1236T-2677T-3435T (54.3%) were also lower than that of the patients with other haplotypes combined (82.4%;x2=4.128, P=0.042). Conclusion: C1236T, G2677T/A and C3435T of MDR1 gene were polymorphic with different frequency in breast cancer patients. The analyses of MDR1 C3435T genotype and its haplotypes with C1236T or G2677T/A are valuable for predicting the taxanes-based chemotherapy responses in breast cancer patients, which deserve the further study. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-21.

Treatment of advanced extranodal NK/T cell lymphoma, nasal-type and aggressive NK-cell leukemia

Extranodal NK/T cell lymphoma, nasal type (ENKL) with advanced stage and aggressive NK-cell leukemia (ANKL) are highly aggressive neoplasms with a dismal clinical outcome. It is well known that P-glycoprotein, which is a product of MDR1 gene and related to multi-drug resistance, is expressed on tumor cells of ENKL or ANKL. This is a major reason for the refractoriness to conventional chemotherapeutic regimens for malignant lymphoma containing anthracycline. However, recent studies have identified that several drugs including L: -asparaginase, methotrexate and alkylators show excellent effect for these tumors. The SMILE (steroid, methotrexate, ifosfamide, L: -asparaginase and etoposide) regimen is one of the promising regimens for advanced or relapsed/refractory ENKL, but its myelotoxicity is strong. ANKL needs another treatment strategy because of a systemic disease progression and extensive organ insufficiency. Optimal treatment scheme using such effective agents for these unfavorable NK-cell tumors should further be explored.

Reversal of multidrug resistance by magnetic Fe3O4 nanoparticle copolymerizating daunorubicin and MDR1 shRNA expression vector in leukemia cells

In many instances, multidrug resistance (MDR) is mediated by increasing the expression at the cell surface of the MDR1 gene product, P-glycoprotein (P-gp), a 170-kD energy-dependent efflux pump. The aim of this study was to investigate the potential benefit of combination therapy with magnetic Fe(3)O(4) nanoparticle [MNP (Fe(3)O(4))] and MDR1 shRNA expression vector in K562/A02 cells. For stable reversal of "classical" MDR by short hairpin RNA (shRNA) aiming directly at the target sequence (3491-3509, 1539-1557, and 3103-3121 nucleotide) of MDR1 mRNA. PGC silencer-U6-neo-GFP-shRNA/MDR1 called PGY1-1, PGY1-2, and PGY1-3 were constructed and transfected into K562/A02 cells by lipofectamine 2000. After transfected and incubated with or without MNP (Fe(3)O(4)) for 48 hours, the transcription of MDR1 mRNA and the expression of P-gp were detected by quantitative real-time PCR and Western-blot assay respectively. Meanwhile intracellular concentration of DNR in K562/A02 cells was detected by flow cytometry (FCM). PGC silencer-U6-neo-GFP-shRNA/MDR1 was successfully constructed, which was confirmed by sequencing and PGY1-2 had the greatest MDR1 gene inhibitory ratio. Analysis of the reversal ratio of MDR, the concentration of daunorubicin (DNR) and the transcription of MDR1 gene and expression of P-gp in K562/A02 showed that combination of DNR with either MNP (Fe(3)O(4)) or PGY1-2 exerted a potent cytotoxic effect on K562/A02 cells, while combination of MNP (Fe(3)O(4)) and PGY1-2 could synergistically reverse multidrug resistance. Thus our in vitro data strongly suggested that a combination of MNP (Fe(3)O(4)) and shRNA expression vector might be a more sufficient and less toxic anti-MDR method on leukemia.

Compatibility risks between drugs and herbal medicines or botanical supplements

Herbal supplements along with prescribed medicines raise concerns of therapeutic activity due to possible drug–herbal interactions. MDR gene products: P-gp, MRP and BCRP and metabolizing enzyme (cytochrome P450) together constitute a highly effective barrier for many orally absorbed drugs. Therapeutic agents and active herbal constituents are substrates, inhibitors and inducers of both P-gp and CYP3A4. Inhibition of P-gp by pure herbal constituents such as hypericin, kaempferol and quercetin or extract of St. John's wort resulted in higher absorption of ritonavir and erythromycin in vitro. Similarly, inhibition of CYP by hypericin, kaempferol and quercetin also caused a remarkable reduction in cortisol metabolism in vitro. Conversely, an enhanced expression of both CYP3A4 and MDR-1 mRNA in Caco-2 cells was observed after chronic exposure of hyperforin, kaempferol, quercetin and hypericin, kaempferol, quercetin, silibinin respectively. Chronic exposure (96h) of African herbal (Sutherlandia frutescens) extract (300μg/mL) resulted in elevation of both P-gp and CYP3A4 expressions in LS180V cells. Cellular accumulation of [3H]Digoxin, a model P-gp substrate was significantly reduced indicating higher efflux. These in vitro results were further confirmed in rats. Administration of Sutherlandia-extract for 4 days significantly affected pharmacokinetics of nevirapine – a CYP3A4 (CYP3A2) substrate. Plasma concentration of nevirapine was diminished by 51% (Cmax) and 45% (AUC0-inf) compared to controls. Similar pharmacokinetics of fexofenadine, a P-gp substrate was also observed. Plasma concentration of fexofenadine was declined by 49.7% (Cmax) and 34.5% (AUC0-inf) compared to controls. Hepatic CYP3A4 and P-gp mRNA levels were enhanced by 3-fold and 1.75-fold respectively. Intestinal CYP3A4 (2-fold) and P-gp expressions (3-fold) was also elevated. Such reduction of steady state plasma concentrations of nevirapine and fexofenadine after concomitant administration of Sutherlandia in rats suggests medicinal agents that are substrates for P-gp and/or CYP are likely to be potential candidates for drug–herbal interactions.

Reversal ofMDR1Gene-Dependent Multidrug Resistance in HL60/HT9 Cells Using Short Hairpin RNA Expression Vectors

Multidrug resistance (MDR) is a serious obstacle to cancer chemotherapy. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers MDR to tumor cells. This study explored the possibility of reducing drug resistance by targeting the mdr1 gene using short hairpin RNA (shRNA). Two different shRNAs were designed and constructed in a pSilencer 2.1-U6 neo plasmid. The shRNA recombinant plasmids were transfected into HT9 leukemia cells. Real-time polymerase chain reaction and Western blotting were used to characterize the inhibited expression of MDR1 mRNA and P-gp, and the drug sensitivity of the transfected cells was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The results indicated that the inhibition of P-gp expression by small interfering RNA selectively restored sensitivity to the drugs transported by P-gp. Evaluation of chemosensitivity showed 52.58% reversal by p2.1-shRNA1 and 73.07% reversal by p2.1-shRNA2 in drug resistance for harringtonine, and 84.87% reversal by p2.1-shRNA1 and 94.23% reversal by p2.1-shRNA2 in drug resistance for curcumin in the transfected cells. The results demonstrated the efficacy and selectivity of shRNA in reversing MDR in drug-resistant HT9 cells in vitro.

SiRNA-Mediated Knock-Down of P-Glycoprotein Expression Reveals Distinct Cellular Disposition of Anticancer Tyrosine Kinases Inhibitors

Studies on the cellular disposition of targeted anticancer tyrosine kinases inhibitors (TKIs) have mostly focused on imatinib while the functional importance of P-glycoprotein (Pgp) the gene product of MDR1 remains controversial for more recent TKIs. By using RNA interference-mediated knockdown of MDR1, we have investigated and compared the specific functional consequence of Pgp on the cellular disposition of the major clinically in use TKIs imatinib, dasatinib, nilotinib, sunitinib and sorafenib. siRNA-mediated knockdown in K562/Dox cell lines provides a unique opportunity to dissect the specific contribution of Pgp to TKIs intracellular disposition. In these conditions, abrogating specifically Pgp-mediated efflux in vitro revealed the remarkable and statistically significant cellular accumulation of imatinib (difference in cellular levels between Pgp-expressing and silenced cells, at high and low incubation concentration, respectively: 6.1 and 6.6), dasatinib (4.9 and 5.6), sunitinib (3.7 and 7.3) and sorafenib (1.2 and 1.4), confirming that these TKIs are all substrates of Pgp. By contrast, no statistically significant difference in cellular disposition of nilotinib was observed as a result of MDR1 expression silencing (differences: 1.1 and 1.5), indicating that differential expression and/or function of Pgp is unlikely to affect nilotinib cellular disposition. This study enables for the first time a direct estimation of the specific contribution of one transporter among the various efflux and influx carriers involved in the cellular trafficking of these major TKIs in vitro. Knowledge on the distinct functional consequence of Pgp expression for these various TKIs cellular distribution is necessary to better appreciate the efficacy, toxicity, and potential drug-drug interactions of TKIs with other classes of therapeutic agents, at the systemic, tissular and cellular levels.