Induction of the ABC-transporters Mdr1/P-gp (Abcb1), mrpl (Abcc1), and bcrp (Abcg2) during establishment of multidrug resistance following exposure to mitoxantrone

Resistance to mitoxantrone is often associated with enhanced drug efflux mediated by members of the superfamily of adenosinetriphosphate-binding cassette (ABC) transporters, i.e. MDR1/P-gp (ABCB1), MRP1 (ABCC1), or BCRP (ABCG2). So far it is unclear whether the same ABC-transporter is always activated from the beginning of mitoxantrone treatment to the end of drug exposure. Here, we demonstrate that the expression of all three extrusion pumps is induced by increasing levels of mitoxantrone resistance, but in the end, merely the overexpression of a dominant single drug transporter, i.e. Mdr1/P-gp, is realized. This upregulation of Mdr1/P-gp was reflected by amplification of the Mdr1/P-gp encoding gene. Short mitoxantrone exposure demonstrated that upregulation of two different transporters, Mdr1/P-gp and Bcrp, was induced. The data indicate that mitoxantrone treatment influences the expression of several ABC-transporters, but in the end, merely a single extrusion pump will be dominant.     

P-glycoprotein and Mrp1 collectively protect the bone marrow from vincristine-induced toxicity in vivo

ABC transporter proteins may protect haematopoietic progenitor cells from chemotherapy-induced toxicity. By using an in vitro colony-forming assay, we found that bone marrow of Mdr1ab, Mrp1, Mdr1ab/Mrp1 knockout (KO) mice was two-, five- to 10- and 25-fold, respectively, more sensitive to vincristine than wild-type mice bone marrow. To study the impact of ABC transporters on in vivo bone marrow sensitivity without the added complication of altered pharmacokinetics, we created chimeras of wild-type mice transplanted with bone marrow from wild-type, Mrp1, Mdr1ab or Mdr1ab/Mrp1 KO donor mice. Following a single bolus injection of vincristine, the chimeras transplanted with wild-type or Mdr1ab KO marrow cells showed no reductions in WBC. A significant reduction was observed in Mrp1 KO chimeras, but the most pronounced effect was observed in mice receiving bone marrow from Mdr1ab/Mrp1 KO mice. A pharmacokinetic analysis in wild-type and KO mice showed that the absence of P-gp reduced the body clearance of vincristine, but that no further reduction occurred when Mrp1 was also absent. However, the tissue accumulation of vincristine in tissues of these Mdr1ab/Mrp1 KO mice was further increased. This study demonstrates that the presence of multiple drug transporters protects the bone marrow, and probably other tissues as well, against chemotherapeutic insults.     

P-glycoprotein and breast cancer resistance protein: two dominant transporters working together in limiting the brain penetration of topotecan.

PURPOSE: The brain is a pharmacologic sanctuary site, due to the presence of the blood-brain barrier (BBB). Whereas the effect of P-glycoprotein (P-gp) at the BBB is well established, the role of breast cancer resistance protein (BCRP) that is also expressed at the BBB is not. EXPERIMENTAL DESIGN: We have studied the effect of BCRP by administering topotecan to wild-type (WT), single Mdr1a/b(-/-) and Bcrp1(-/-), and compound Mdr1a/b(-/-)Bcrp1(-/-) knockout mice. Drug levels in plasma and tissues were determined by high-performance liquid chromatography. RESULTS: The area under the plasma and tissue concentration-time curve (AUC) of topotecan in brains of Mdr1a/b(-/-) and Bcrp1(-/-) mice was only 1.5-fold higher compared with WT mice, but in Mdr1a/b(-/-)Bcrp1(-/-) mice, where both transporters are absent, the AUC increased by 12-fold. The AUC in plasma was approximately 0.75-, 2.4-, and 3.7-fold higher in Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)Bcrp1(-/-) mice, respectively, resulting in 2.0-fold (P < 0.01), 0.65-fold (P, not significant), and 3.2-fold (P < 0.01), respectively, higher brain-to-plasma AUC ratios. Results using Mrp4(-/-) mice showed that this transporter had no effect on the brain penetration of topotecan. The P-gp/BCRP inhibitor elacridar fully inhibited P-gp-mediated transport of topotecan, whereas inhibition of Bcrp1-mediated transport by elacridar was minimal. CONCLUSIONS: Our results using Mdr1a/b(-/-)Bcrp1(-/-) mice clearly show the effect of Bcrp1 at the BBB and also show how two drug transporters act in concert to limit the brain penetration of topotecan. We expect that this finding will also apply to other drugs that are substrates of both P-gp and BCRP. Consequently, to improve the brain penetration of such compounds for targeting intracranial malignancies in patients, it will be essential to use potent inhibitors of both drug transporters.     

Extensive contribution of the multidrug transporters P-glycoprotein and Mrp1 to basal drug resistance

Despite accumulating evidence that multidrug resistance transporter proteins play a part in drug resistance in some clinical cancers, it remains unclear whether the relatively low levels of multidrug resistance transporter expression found in most untreated tumors could substantially affect their basal sensitivity to antineoplastic drugs. To shed light on this problem, the drug sensitivities of wild-type mouse cell lines were compared with those of lines in which the Mdr1a and Mdr1b genes encoding P-glycoprotein (P-gp) were inactivated and lines in which the Mrp1 gene was inactivated in addition to Mdr1a and Mdr1b. These models permit a clean dissection of the contribution of each transporter to drug resistance at expression levels similar to those in normal tissues and avoid complications that might arise from previous exposure of cell lines to drug selection. For substrate drugs, we found that these contributions can indeed be very substantial. Lines lacking functional P-gp were, on average, markedly more sensitive to paclitaxel (16-fold), anthracyclines (4-fold) and Vinca alkaloids (3-fold). Lines lacking both P-gp and Mrp1 were (compared with wild-type lines) hypersensitive to an even broader array of drugs, including epipodophyllotoxins (4-7-fold), anthracyclines (6-7-fold), camptothecins (3-fold), arsenite (4-fold) and Vinca alkaloids, especially vincristine (28-fold). Thus, even very low levels of P-gp and Mrp1 expression that may be difficult to detect in tumors could significantly affect their innate sensitivity to a wide range of clinically important substrate drugs. An implication is that the use of resistance reversal agents to sensitize drug-naive tumors may be appropriate in more cases than is presently appreciated.     

Individual and combined roles of CYP3A, P-glycoprotein (MDR1/ABCB1) and MRP2 (ABCC2) in the pharmacokinetics of docetaxel

Docetaxel is one of the most widely used anticancer drugs. A major problem with docetaxel treatment, however, is the considerable interpatient variability in docetaxel exposure. Another disadvantage of the drug is that it has a very low oral bioavailability and can, therefore, only be administered intravenously. The drug-metabolizing enzyme CYP3A and the drug transporter MDR1 (P-glycoprotein) are major determinants of docetaxel pharmacokinetics. In vitro studies have indicated that docetaxel is also a substrate for the drug transporter MRP2, but the in vivo importance of MRP2 for docetaxel is currently unknown. We, therefore, investigated the role of MRP2 in the pharmacokinetics of docetaxel by utilizing Mrp2(-/-) mice. We also generated and characterized Cyp3a/Mdr1a/b/Mrp2(-/-) combination knockout mice to get more insight into how these drug-handling systems work together in determining docetaxel pharmacokinetics. The systemic exposure in Mrp2(-/-) mice was not significantly different from wild-type, after either oral or intravenous administration. Strikingly, however, in Cyp3a/Mdr1a/b/Mrp2(-/-) mice, systemic docetaxel exposure was increased 166-fold after oral administration when compared with wild-type mice, and 2.3-fold when compared with Cyp3a/Mdr1a/b(-/-) mice. Interestingly, this 166-fold increase was disproportionate compared with that for the separate Cyp3a (12-fold) or Mdr1a/b/Mrp2 (4-fold) knockouts. The oral bioavailability was increased to 73% in the Cyp3a/Mdr1a/b/Mrp2(-/-) strain, versus only 10% in wild-type mice. Our data thus indicate that in the absence of CYP3A and Mdr1a/b activity, Mrp2 has a marked impact on docetaxel pharmacokinetics. These findings could have important implications for improving the oral bioavailability and reducing the variability in docetaxel exposure.     

Breast cancer resistance protein in drug resistance of primitive CD34+38- cells in acute myeloid leukemia.

PURPOSE: Acute myeloid leukemia (AML) is considered a stem cell disease. Incomplete chemotherapeutic eradication of leukemic CD34+38- stem cells is likely to result in disease relapse. The purpose of this study was to investigate the role of the breast cancer resistance protein (BCRP/ATP-binding cassette, subfamily G, member 2) in drug resistance of leukemic stem cells and the effect of its modulation on stem cell eradication in AML. EXPERIMENTAL DESIGN: BCRP expression (measured flow-cytometrically using the BXP21 monoclonal antibody) and the effect of its modulation (using the novel fumitremorgin C analogue KO143) on intracellular mitoxantrone accumulation and in vitro chemosensitivity were assessed in leukemic CD34+38- cells. RESULTS: BCRP was preferentially expressed in leukemic CD34+38- cells and blockage of BCRP-mediated drug extrusion by the novel fumitremorgin C analogue KO143 resulted in increased intracellular mitoxantrone accumulation in these cells in the majority of patients. This increase, however, was much lower than in the mitoxantrone-resistant breast cancer cell line MCF7-MR and significant drug extrusion occurred in the presence of BCRP blockage due to the presence of additional drug transport mechanisms, among which ABCB1 and multiple drug resistance protein. In line with these findings, selective blockage of BCRP by KO143 did not enhance in vitro chemosensitivity of leukemic CD34+38- cells. CONCLUSIONS: These results show that drug extrusion from leukemic stem cells is mediated by the promiscuous action of BCRP and additional transporters. Broad-spectrum inhibition, rather than modulation of single mechanisms, is therefore likely to be required to circumvent drug resistance and eradicate leukemic stem cells in AML.     

Impact of BCRP/MXR, MRP1 and MDR1/P-Glycoprotein on thermoresistant variants of atypical and classical multidrug resistant cancer cells

The impact of the ABC transporters breast cancer resistance protein/mitoxantrone resistance associated transporter (BCRP/MXR), multidrug resistance-associated protein 1 (MRP1) and multidrug resistance gene-1/P-glycoprotein (MDR1/PGP) on the multidrug resistance (MDR) phenotype in chemoresistance and thermoresistance was investigated in the parental human gastric carcinoma cell line EPG85-257P, the atypical MDR subline EPG85-257RNOV, the classical MDR subline EPG85-257RDB and their thermoresistant counterparts EPG85-257P-TR, EPG85-257RNOV-TR and EPG85-257RDB-TR. Within the atypical MDR subline EPG85-257RNOV expression of BCRP/MXR and of MRP1 were clearly enhanced (vs. parental and classical MDR lines). MDR1/PGP expression was distinctly elevated in the classical MDR subline EPG85-257RDB (vs. parental and atypical MDR sublines). In all thermoresistant counterparts basal expression of BCRP/MXR, MRP1 and MDR1/PGP was increased relative to thermosensitive sublines. Although it could be shown that the overexpressed ABC transporters were functionally active, however, no decreased drug accumulations of doxorubicin, mitoxantrone and rhodamine 123 were observed. Thus, expression of BCRP/MXR, MRP1 and MDR1/PGP was found to be dependent on the appropriate type of chemoresistance; correlating with a classical or atypical MDR phenotype. Within the thermoresistant variants, however, the increase in ABC transporter expression did obviously not influence the MDR phenotype.     

Tet调控的乳腺癌耐受蛋白表达细胞系的建立及功能研究

背景与目的:乳腺癌耐受蛋白(breastcancerresistanceprotein,BCRP)是1998年发现的新的ATP结合盒式(ATPbindingcassette,ABC)跨膜转运蛋白超家族成员。本研究拟建立Tet调控的具有功能的BCRP表达细胞系,为研究BCRP介导的药物耐受机制和探寻耐受逆转方法提供理想的实验平台。方法:利用Tet-on基因表达调控系统,先后将调控质粒pTet-on和反应质粒pTRE-BCRP转染入PA317细胞,并用G418和潮霉素分别进行二轮筛选,挑取6个单细胞克隆并扩增培养;不同浓度的强力霉素(doxycycline,Dox)诱导后,用RT-PCR和Westernblot检测并选取BCRP表达量与Dox剂量具有较好量-效关系的PA317/Tet-on/TRE-BCRP细胞克隆;采用MTT方法检测米托蒽醌(mitoxantrone)对不同浓度Dox诱导下的PA317/Tet-on/TRE-BCRP克隆细胞的杀伤作用;采用BCRP特异性抑制剂Ko143的干扰试验来检测PA317/Tet-on/TRE-BCRP细胞对米托蒽醌的药物敏感性;米托蒽醌处理不同BCRP表达量的细胞后,利用流式细胞仪检测细胞内存留米托蒽醌所释放出来的荧光强度。结果:发现5号PA317/Tet-on/TRE-BCRP克隆细胞BCRP表达量与Dox诱导剂量具有较好的量-效关系;其药物耐受性与BCRP表达量呈正相关(r=0.995,P=0.002);Ko143能显著增强PA317/Tet-on/TRE-BCRP细胞对米托蒽醌的药物敏感性(P<0.05     

PARP-1在米托蒽醌诱导的MCF-7耐药细胞中的表达及作用分析

目的：研究抗癌药物米托蒽醌诱导乳腺癌细胞系MCF-7耐药过程中,聚ADP-核糖聚合-1（poly ADP-ribosepolymerase-1,PARP-1）的表达变化,探讨MCF-7耐药细胞株中多药耐药的形成机制。方法：分别设米托蒽醌6个不同浓度实验组（0.005、0.01、0.02、0.04、0.08、0.16μmol/L）,先以0.005μmol/L的米托蒽醌持续作用MCF-7细胞30d后,改用下一个剂量0.01μmol/L继续作用30d,依次按由低到高的浓度顺序进行,诱导产生耐药细胞。并设未用米托蒽醌处理的MCF-7为对照组。采用实时荧光定量RT-PCR和蛋白免疫印迹杂交分别检测米托蒽醌各浓度组细胞中PARP-1mRNA和蛋白的表达水平。结果：与对照组相比,随着米托蒽醌浓度的增加,PARP-1表达水平呈增高趋势,并且有剂量-反应效应,其中0.02、0.04和0.08μmol/L等3个浓度组的表达量显著高于对照组（P〈0.05）。结论：米托蒽醌持续染毒可诱导MCF-7细胞中PARP-1的表达,其表达参与了肿瘤多药耐药的形成。     

Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts.

A high incidence of relapses following induction chemotherapy is a major hindrance to patient survival in acute myelogenous leukemia (AML). There is strong evidence that activation of the phosphoinositide 3 kinase (PI3K)/Akt signaling network plays a significant role in rendering AML blasts drug resistant. An important mechanism underlying drug resistance is represented by overexpression of membrane drug transporters such as multidrug resistance-associated protein 1 (MRP1) or 170-kDa P-glycoprotein (P-gp). Here, we present evidence that MRP1, but not P-gp, expression is under the control of the PI3K/Akt axis in AML blasts. We observed a highly significant correlation between levels of phosphorylated Akt and MRP1 expression in AML cells. Furthermore, incubation of AML blasts with wortmannin, a PI3K pharmacological inhibitor, resulted in lower levels of phosphorylated Akt, downregulated MRP1 expression, and decreased Rhodamine 123 extrusion in an in vitro functional dye efflux assay. We also demonstrate that wortmannin-dependent PI3K/Akt inhibition upregulated p53 protein levels in most AML cases, and this correlated with diminished MRP1 expression and enhanced phosphorylation of murine double minute 2 (MDM2). Taken together, these data suggest that PI3K/Akt activation may lead to the development of chemoresistance in AML blasts through a mechanism involving a p53-dependent suppression of MRP1 expression.     

Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytes

The poor prognosis of glioma patients is partly based on the minor success obtained from chemotherapeutic treatments. Resistance mechanisms at the tumor cell level may be, in addition to the blood–brain barrier, involved in the intrinsic chemo-insensitivity of brain tumors. We investigated the expression of the drug-transporter proteins P-glycoprotein (P-gp) and multidrug-resistance protein 1 (MRP1) in cell lines (N=24) and primary cell cultures (N=36) from neuroectodermal tumors, as well as in brain tumor extracts (N=18) and normal human astrocytes (N=1). We found that a considerable expression of P-gp was relatively rare in glioma cells, in contrast to MRP1, which was constitutively overexpressed in cells derived from astrocytomas as well as glioblastomas. Also, normal astrocytes cultured in vitro expressed high amounts of MRP1 but no detectable P-gp. Meningioma cells frequently co-expressed P-gp and MRP1, while, most of the neuroblastoma cell lines express higher P-gp but lower MRP1 levels as compared to the other tumor types. Both, a drug-exporting and a chemoprotective function of P-gp as well as MRP1 could be demonstrated in selected tumor cells by a significant upregulation of cellular ³H-daunomycin accumulation and daunomycin cytotoxicity via administration of transporter antagonists. Summing up, our data suggest that P-gp contributes to cellular resistance merely in a small subgroup of gliomas, but frequently in neuroblastomas and meningiomas. In contrast, MRP1 is demonstrated to play a constitutive role in the intrinsic chemoresistance of gliomas and their normal cell counterpart.     

The mouse Bcrp1/Mxr/Abcp gene: amplification and overexpression in cell lines selected for resistance to topotecan, mitoxantrone, or doxorubicin.

Mouse fibroblast cell lines lacking functional Mdr1a, Mdr1b, and Mrp1 genes were selected for resistance to topotecan, mitoxantrone, or doxorubicin. Each of the resulting drug-resistant lines showed marked gene amplification of Bcrp1, the mouse homologue of the human ATP-binding cassette transporter gene BCRP/MXR/ABCP, and greatly elevated expression of Bcrp1 mRNA. All three of the resistant cell lines were highly cross-resistant to topotecan and mitoxantrone and, to a variable extent, doxorubicin. All showed greatly reduced cellular accumulation and greatly increased efflux of mitoxantrone that was dependent on cellular ATP and efficiently reversed by the compound GF120918. The mouse Bcrp1 cDNA encodes a 657-amino-acid protein with 81% identity (86% similarity) to the human breast cancer resistance protein (BCRP) and a virtually superimposable hydrophobicity profile. Our data argue strongly that mouse Bcrp1 is functionally comparable with human BCRP, conferring multidrug resistance to topotecan, mitoxantrone, doxorubicin, and related compounds. Mouse models and cell lines should, therefore, be highly informative in understanding the clinical, pharmacological, and physiological roles of BCRP.     

Induction of P-glycoprotein expression on the plasma membrane of human melanoma cells

Melanoma cells exhibit, both in vivo and in vitro, intrinsic drug resistance to various chemotherapeutic agents. Cultured human melanoma cells (M14) intrinsically express significant amounts of multidrug resistance-related protein (MRP1) and P-glycoprotein (P-gp) in the Golgi apparatus, but do not express these drug transporters on the plasma membrane. A panel of multidrug resistant (MDR) melanoma cell lines (M14Dx), showing different degrees of resistance to doxorubicin (DOX), were isolated. In M14Dx lines, the appearance of surface P-gp, but not of MRP1 or lung resistance related protein (LRP), occurred in cells grown in the presence of DOX concentrations higher than 60 nM. Furthermore, P-gp levels appeared to be dose-dependent. Flow cytometry, laser scanning confocal microscopy and cytotoxicity studies demonstrated that the activity of the drug extrusion system was related to both surface P-gp expression and resistance to DOX. In conclusion, P-gp, but not MRP1 or LRP, might play a pivotal role in the pharmacologically-induced MDR phenotype of melanoma cells.     

Human OATP1B1, OATP1B3 and OATP1A2 can mediate the in vivo uptake and clearance of docetaxel

Organic anion transporting polypeptides (human: OATPs and mouse: Oatps) are uptake transporters with important roles in drug pharmacokinetics and toxicity. We aimed to study the in vivo impact of mouse and human OATP1A/1B transporters on docetaxel plasma clearance and liver and intestinal uptake. Docetaxel was administered to Oatp1a/1b knockout and liver‐specific humanized OATP1B1, OATP1B3 and OATP1A2 transgenic mice. Experiments were conducted with a low polysorbate 80 (2.8%) formulation, as 8% polysorbate somewhat inhibited docetaxel plasma clearance after intravenous administration. After intravenous administration (10 mg/kg), Oatp1a/1b knockout mice had an approximately threefold higher plasma area under the curve (AUC). Impaired liver uptake was evident from the significantly reduced (approximately threefold) liver‐to‐plasma AUC ratios. Absence of mouse Oatp1a/1b transporters did not affect the intestinal absorption of orally administered docetaxel (10 mg/kg), while the systemic exposure of docetaxel was again substantially increased owing to impaired liver uptake. Most importantly, liver‐specific expression of each of the human OATP1B1, OATP1B3 and OATP1A2 transporters provided a nearly complete rescue of the increased plasma levels of docetaxel in Oatp1a/1b‐null mice after intravenous administration. Our data show that one or more of the mouse Oatp1a/1b transporters and each of the human OATP1A/1B transporters can mediate docetaxel uptake in vivo. This might be clinically relevant for OATP1A/1B‐mediated tumor uptake of docetaxel and for docetaxel clearance in patients in whom the transport activity of OATP1A/1B transporters is reduced owing to genetic variation or pharmacological inhibition, leading to potentially altered toxicity and therapeutic efficacy of this drug.     

Mdr1反义寡核苷酸联合磁性载药微球逆转胰腺癌耐药实验研究

目的：探讨辐射促细胞转染的多药耐药（multidrug resistance,MDR）基因Mdr1反义寡核苷酸（ASON）逆转肿瘤细胞SW1990／Fu的耐药效果。方法应用RT-PCR方法和流式细胞仪,检测2种不同的转染方法对SW1990／Fu细胞的Mdr1-mRNA及其表达产物P-糖蛋白（ P-gp）的调控情况。结果反转录RT-PCR和流式细胞仪的结果显示,联合辐射的阳性脂质体介导ASON组的SW1990／Fu细胞Mdr1-mRNA的表达水平及细胞膜糖蛋白（ P-gp）的阳性率均明显低于反义寡核苷酸（ASON）组（P＜0．01）。结论辐射促转染的Mdr1ASON联合磁性载药微球对肿瘤细胞具有较好的耐药逆转作用。     

Safety and efficacy of the MDR inhibitor Incel (biricodar,VX-710) in combination with mitoxantrone and prednisone in hormone-refractory prostate cancer

PURPOSE: VX-710 (biricodar, Incel) restores drug sensitivity to cells expressing P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP1). MRP1 is expressed in a high proportion of prostate tumors while P-gp expression is variable. Since mitoxantrone (M) and prednisone (P) are substrates for MDR transporters, we initiated a study to evaluate the safety, pharmacokinetics, and efficacy of VX-710 plus M/P in patients with hormone-refractory prostate cancer (HRPC). PATIENTS AND METHODS: Eligible patients had progressive HRPC (defined as new lesions, new disease-related pain, or 50% increase in PSA within 6 weeks of entry), testosterone <30 ng/ml, no prior chemotherapy, ECOG performance status of 0-3, and adequate organ function. Patients received VX-710 (120 mg/m(2) per h) as a 72-h continuous intravenous infusion with intravenous bolus mitoxantrone (12 mg/m(2)) administered 4 h after VX-710 was started and prednisone (5 mg twice daily) administered throughout the study treatment. Endpoints included serum PSA response, PSA response duration, time to PSA progression, pain reduction, and quality of life measures. RESULTS: Enrolled in the study were 40 patients and 184 courses of VX-710 plus M/P were administered. Intensive pharmacokinetics, which were performed on six patients who received one cycle of M/P alone, followed by VX-710 plus M/P for all other cycles, showed that VX-710 did not alter mitoxantrone clearance. VX-710 blood concentration at the time of mitoxantrone administration averaged 4.52 microg/ml. VX-710 plus M/P was well tolerated. Transient nausea/vomiting and mild neutropenia were the principal treatment toxicities. Five patients experienced an uncomplicated febrile neutropenic episode (12%), three had severe nausea/vomiting, and two experienced transient moderate to severe ataxia. Of the 40 patients, 12 (30%, 95% confidence interval 16-44%) had a reduction in PSA of >/=50% and 9 of the 12 patients (23% overall, 95% CI 10-35%) achieved a reduction in PSA of >/=80% that was sustained for the duration of treatment with M/P plus VX-710. The median time to PSA progression was 41 weeks (95% CI 34-68 weeks). Of the 40 patients, 15 completed treatment with stable disease and 13 had progressive disease with increasing serum PSA during study treatment. Median survival was 48 weeks for the intent-to-treat population of 40 patients. CONCLUSIONS: The addition of VX-710 to M/P therapy did not appear to increase the proportion of patients with significant serum PSA reductions compared to M/P alone. However, the duration of PSA response observed for the 12 PSA responders suggests that MDR inhibition may benefit some patients with HRPC. In addition to MRP1 or P-gp expression, other mechanisms of drug resistance are probably associated with the relative insensitivity of HRPC to cytotoxic therapy.     

MDR-1, but not MDR-3 gene expression, is associated with unmutated IgVH genes and poor prognosis chromosomal aberrations in chronic lymphocytic leukemia

Two P-glycoprotein (P-gp) genes, MDR-1 (ABCB1) and MDR-3 (ABCB4), have been identified in humans. This study was designed to investigate whether associations exist between expression of MDR-1 and MDR-3 P-gp and other markers of poor prognosis and/or prior exposure to therapeutic agents in chronic lymphocytic leukemia (CLL). IgV_H mutational status, gene usage, CD38 positivity, FISH analysis and clinical information were available on all patients. Twenty-one of 101 patients tested showed MDR-3 P-gp positivity. Associations with markers of poor prognosis or prior chemotherapy did not reach statistical significance, but MDR-3 P-gp positive patients had significantly shorter survivals than MDR-3 P-gp negative patients. MDR-1 P-gp expression (18/25) showed a strong association with unmutated IgV_H genes and adverse prognosis cytogenetics (p = 0.015, p = 0.014, respectively), but was independent of prior exposure to chemotherapeutic agents. These results suggest a role for MDR-1 and MDR-3 in chemoresistant disease. This study highlights the value of determining MDR phenotype in CLL patients prior to treatment, to allow the design of novel drug regimens containing agents that reverse MDR function.     

Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference

Multidrug resistance (MDR) is the major cause of failure of effective chemotherapeutic treatment of disseminated neoplasms. The "classical" MDR phenotype of human malignancies is mediated by drug extrusion by the adenosine triphosphate binding cassette (ABC)-transporter P-glycoprotein (MDR1/P-gp). For stable reversal of "classical" MDR by RNA interference (RNAi) technology, an H1-RNA gene promoter-driven expression vector encoding anti-MDR1/P-gp short hairpin RNA (shRNA) molecules was constructed. By introduction of anti-MDR1/P-gp shRNA expression vectors into the extremely high drug-resistant human gastric carcinoma cell line EPG85-257RDB, the MDR phenotype was completely reversed. The reversal of MDR was accompanied by a complete suppression of MDR1/P-gp expression on mRNA and protein level, and by a considerable increased intracellular anthracyline accumulation in the anti-MDR1/P-gp shRNA-treated cells. The data indicate that stable shRNA-mediated RNAi can be tremendously effective in reversing MDR1/P-gp-mediated MDR and is therefore a promising strategy for overcoming MDR by gene therapeutic applications.