Search

Purpose: We aimed to investigate the association between the polymorphism and expression patterns of multiple drug resistance genes (MDR) in breast cancer (BC)., Materials and Methods: The MDR gene expression levels were measured in tumor tissues of 106 breast cancer patients using quantitative real-time PCR. Affymetrix CytoScan™ HD Array chips were used to assess genotypes. Pairwise correlation analysis for ABCB1, ABCC1, ABCC2 and ABCG2 gene expression levels was carried out to reveal co-expression clusters. Associations between SNPs of MDR genes and their preoperative expression levels were assessed using analysis of covariance adjusting for covariates., Results: The SNPs associated with the expression of the ABCB1, ABCC1, ABCC2 and ABCG2 genes before NAC were detected. In addition, 21 SNPs associated with the expression of four ABC-transporter genes and involved in the expression regulation were identified. Validation in an independent sample confirmed the association between the MDR cluster genes and 11 SNPs., Conclusions: Four MDR genes: ABCB1, ABCC1, ABCC2 and ABCG2 were shown to form the functional expression cluster in breast tumor. Further studies are required to discover precise mechanisms of the cluster regulation, thereby providing new approaches and targets to combat the development of the MDR phenotype during chemotherapy.

Purpose: We aimed to examine the association between alterations in multidrug resistance (MDR) gene expression, measured before and after neoadjuvant chemotherapy (NAC), and short-term response in a cohort of stage IIA-IIIC breast cancer patients (n = 84)., Methods: All patients were treated with two to four preoperative cycles of FAC (5-fluorouracil-adriamycin-cyclophosphamide), CAX (cyclophosphamide-adriamycin-xeloda) or taxane regimes. The expression levels of key MDR genes (ABCB1, ABCC1, ABCC2, ABCC3, ABCC5, ABCG1, ABCG2, GSTP1, and MVP) were evaluated in both tumor tissues obtained pre-therapy and in specimens removed by final surgery, using TaqMan-based quantitative reverse transcriptase PCR., Results: No significant difference in the average level of MDR gene expression in paired breast tumors before and after NAC was found when analyzed in both responsive and non-responsive patients. There was no correlation between the expression levels of MDR genes in pre-NAC tumors and immediate NAC response. In the group with tumor responses, we found a statistically significant downregulation of expression of ABCB1, ABCC1, ABCC2, ABCC5, ABCG1, ABCG2, GSTP1, and MVP genes following NAC in FAC and CAX-treated patients (67-93% of cases). In contrast, we found that expression of these genes was upregulated after NAC, mostly in non-responsive patients (55-96% of cases). Responsiveness to taxotere was related to reduced levels of ABCB1, ABCC2, ABCG1, ABCG2, and MVP mRNA in tumor samples collected after chemotherapy., Conclusion: Our results suggest that reductions in MDR gene expression in post-NAC samples in comparison with pre-NAC are associated with tumor response to FAC and CAX as well as taxotere-based NAC, while patients displaying MDR gene upregulation had resistance to therapy.

Purpose: P-glycoprotein (P-gp), encoded by MDR1 (or ABCB1), is important in anticancer drug delivery and resistance. We evaluated alterations in P-gp-mediated transport of anticancer agents due to the MDR1 G1199A polymorphism., Methods: Using stable recombinant epithelial cells expressing wild-type (MDR1 (wt)) or G1199A (MDR1 (1199A)), anticancer drug sensitivity and transepithelial permeability were evaluated., Results: The recombinant cells MDR1 (wt) and MDR1 ( 1199A ) displayed comparable doxorubicin resistance. However, MDR1 (1199A) cells displayed greater resistance to vinblastine, vincristine, paclitaxel, and VP-16 (11-, 2.9-, 1.9-, and 2.9-fold, respectively). Alterations in transepithelial permeability paralleled these changes. Efflux of doxorubicin was similar between MDR1 wt - and MDR1 (1199A)-expressing cells, while P-gp-mediated transport was greater for vinblastine and vincristine in MDR1 (1199A) cells (2.9- and 2.0-fold, respectively)., Conclusions: The occurrence and magnitude of the MDR1 G1199A effect is drug specific. Overall, the MDR1 G1199A polymorphism may impact anticancer efficacy through modulation of drug distribution and delivery to target tumor cells.

Purpose: The anti-tumoral activity of bisphosphonates (BPs) has been reported in leukemia. In this study, we attempted to evaluate the apoptotic effects of the BPs pamidronate (PAM) and zoledronic acid (ZOL) in chronic lymphocytic leukemia (CLL) samples, and to correlate it with clinical parameters and multidrug resistance (MDR) phenotype (P-glycoprotein and multidrug resistance-related protein expression and/or their functional activity)., Results: Both BPs were able to induce apoptosis significantly. No correlation was observed between BP-induced apoptosis and clinical parameters or MDR phenotype., Conclusion: Our data suggest that concurrent or sequential administration of BPs with conventional chemotherapeutic drugs may have significant therapeutic potential for CLL patients.

Purpose: One of the mechanisms responsible for the multidrug resistance (MDR) phenotype of cancer cells is overexpression of so-called ATP-dependent drug efflux proteins: the 170-kDa P-glycoprotein (P-gp) encoded by the MDR1 gene and the 190-kDa multidrug resistance-associated protein 1 encoded by the MRP1 gene. The purpose of the present study was to verify the hypothesis postulating that P-gp expression, apart from enabling drug efflux, confers on the cells resistance to apoptosis by inhibiting caspase-8 and caspase-3., Materials and Methods: Human HL-60 cells, either drug-sensitive or with the MDR phenotype caused by overexpression of P-gp (HL-60/Vinc) or MRP1 (HL-60/Adr), were treated with the natural dye curcumin at 50 micro M or with UVC to induce apoptosis. Symptoms of cell death were assessed by morphological observation after Hoechst staining, DNA fragmentation was measured by flow cytometry and the TUNEL method, and caspase-8 and caspase-3 activation and cytochrome c release from mitochondria were measured by Western blotting., Results: Curcumin induced cell death in HL-60 cells, both sensitive and with the MDR phenotype, which could be classified as caspase-3-dependent apoptosis, together with cytochrome c release, activation of caspase-3 and oligonucleosomal DNA fragmentation. No active caspase-8 was detected. Also UVC caused caspase-3 activation in both the sensitive and the MDR HL-60 cells., Conclusions: Our findings show that there was no correlation between P-gp expression and resistance to caspase-3-dependent apoptosis induced by curcumin and UVC, at least in HL-60 cells. However, we cannot exclude the possibility of parallel P-gp expression and caspase-3 inhibition in some other cell lines, as cancer cells can acquire many different apoptosis-resistance mechanisms.

Purpose: The aim of this study was to investigate the multidrug resistance (MDR) pattern, MDR gene and P-glycoprotein (P-gp) expression, and P-gp function in drug-induced human T-lymphoblastoid leukemia MOLT-4 sublines., Methods: The MDR sublines were developed by exposing the parental MOLT-4 cells to stepwise increasing concentrations of anticancer drugs daunorubicin (DNR), vinblastine (VBL) and doxorubicin (DOX). Degrees of resistance were assessed in terms of IC(50) values in an MTT assay and the P-gp function was evaluated in terms of rhodamine 123 (Rh123) accumulation and efflux. The percentage of cells undergoing apoptosis was determined by flow cytometry after staining with annexin V-FITC and propidium iodide. The levels of P-gp and MDR mRNA expression were estimated using the PE-conjugated anti-P-gp monoclonal antibody 17F9 and quantitative real-time reverse transcription-polymerase chain reaction., Results: Three MOLT-4 sublines were established and revealed a 2- to 115-fold resistance to the anticancer reagents DNR, VBL and DOX as compared to the parental cell line. The highest MDR was expressed in MOLT-4/DNR cells, which was overcome by the P-gp modulator, cyclosporin A (CsA). The resistant sublines showed a decreased accumulation and an increased efflux of Rh123 in proportion to the degree of resistance, and these were completely reversed in the presence of 8 microM CsA. The decreased apoptotic response in these cell lines was clearly associated with the degree of drug resistance. P-gp antigen and MDR1 mRNA were highly expressed in both the MOLT-4/DNR and MOLT-4/DOX sublines. Less-resistant MOLT-4/VBL cells expressed lower levels of MDR1 mRNA and P-gp, even though the cell line was established by exposing the parental MOLT-4 cells to VBL for longer (5 months) than to the other two reagents (3 months)., Conclusions: MOLT-4 cells were able to acquire a high level of drug resistance by culturing the cells in the presence of certain anticancer drugs, and acquisition of the resistance was relatively reagent-specific. The degrees of resistance to the anticancer drugs were well correlated with the expressions of MDR1 mRNA and functional P-gp, and were also associated with a decreased response to apoptosis.

The potential clinical use of technetium-99m labeled sestamibi (Tc-MIBI) and tetrofosmin (Tc-Tfos) to image tumours is currently being evaluated. In this study. the accumulation and efflux of Tc-MIBI and Tc-Tfos in the nasopharyngeal carcinoma cell line CNE-1 were examined in the presence or absence of various inhibitors of P-glycoprotein (PGP) and/or multidrug resistance associated protein (MRP) activity [GG918, PSC833, verapamil (Vrp), cyclosporin A (CsA) and buthionine sulfoximine (BSO)]. Reverse-transcriptase polymerase chain reaction analysis and immunodetection of the CNE-1 cells detected expression of MRP, MRPI and MRP2 but not PGP. Tc-MIBI and Tc-Tfos accumulation was increased (P < 0.0001) and efflux decreased (P < 0.05) in the presence of BSO, CsA, Vrp and PSC833 but not GG918, which is a specific inhibitor of PGP. The absolute accumulation of Tc-MIBI was approximately twofold higher than that seen with Tc-Tfos, whereas the addition of inhibitors caused a much greater suppression of Tc-Tfos transport (>2 times greater than for Tc-MIBI). However, no qualitative differences in inhibitors were seen between Tc-MIBI and Tc-Tfos. These results suggest that both Tc-MIBI and Tc-Tfos are substrates for the MRP transporter and that PSC833, Vrp, CsA and BSO but not GG918 can inhibit MRP activity. These results indicate that Tc-MIBI and Tc-Tfos may be suitable imaging agents for detecting MRP-mediated drug resistance in human cancers.

Purpose: The consequences of using cyclosporine (CsA) therapy to modulate P-glycoprotein-mediated multidrug resistance include increased myelosuppression, hyperbilirubinemia, and altered disposition of the cytotoxin. The purpose of this study was to analyze further the relationship between the degree of leukopenia, and etoposide pharmacokinetic factors., Methods: Each patient initially received intravenously-administered etoposide alone (150-200 mg/m2/d x 3). Later it was given in combination with CsA administered at escalating loading doses (range 2-7 mg/kg) as a 2 hour intravenous (IV) infusion followed by a 3 day continuous infusion, at doses ranging from 5 to 21 mg/ kg/day. Serial plasma etoposide concentration-time samples were assayed by high-performance liquid chromatography (HPLC). The area under the curve (AUC) of unbound etoposide was calculated from the total plasma etoposide AUC using a previous published equation [22] where % unbound etoposide = (1.4 x total bilirubin) - (6.8 x serum albumin) + 34.4. The percent decrease in white blood cell (WBC) count and the total or unbound etoposide AUC relationship was fitted to a sigmoid Emax model adapted for paired observations, where: % Decrease in WBC count =E(max) x PDRV(H+Z x delta)/(PDRV50 + Z x beta) + PDRVH + Z x delta In this equation, Z was the variable describing the two treatment groups (0 = no CsA and 1 = CsA). The fitted parameters were PDRV50, the pharmacodynamic response variable (PDRV) producing 50% of the maximal response; parameter beta, which describes the effect of the treatment group on the PDRV50; parameter H (Hill constant), which defines the slope of the response curve and parameter delta, which describes the effect of the treatment group on parameter H., Results: CsA at a median concentration of 1,938 microg/ml resulted in a median increase in the total plasma etoposide AUC by 103% and the calculated unbound plasma etoposide AUC by 104%. This paralleled a 12% greater median percent decrease in WBC count during etoposide + CsA treatment (72% vs. 84%, P = 0.03). The percent decrease in WBC count and total or unbound etoposide AUC relationship was fitted to the sigmoid Emax model. The model using the unbound etoposide AUC described the data adequately (r = 0.790) and was precise, with a mean absolute error of 6.4% (95% confidence interval: -4.9, 7.8). The fitted parameter-estimates suggested that at equivalent unbound etoposide AUC values above 10 microg x h/ml, the sigmoid Emax model predicted a 5% greater WBC count suppression when CsA was added to the treatment regimen., Conclusion: These findings suggest that a small degree of the enhanced myelosuppression observed with CsA combined with etoposide might be attributable to inhibition of P-glycoprotein in bone marrow precursor cells. However, the majority of the effect observed appears to be due to pharmacokinetic interactions, which result in increases in unbound etoposide.

Purpose: Among flavonoids, chalcones have been identified as interesting compounds having chemopreventive and antitumor properties. We studied a panel of newly developed chalcone analogues (S1-S10) using MDA-MB 231 and MCF-7 ADRr breast cancer cells and the T-leukemic Jurkat cell line. Quercetin was used as the reference compound., Methods: Antiproliferative activity was evaluated by cell counts performed after 72 h of exposure to the drugs. DNA analysis and redox activity were evaluated using flow cytometry. Apoptosis was assessed by morphological analysis, using YOYO-1 as DNA dye; p-glycoprotein function was ascertained by quantitating the efflux of rhodamine 123., Results: All cells were sensitive to chalcone analogues yielding IC50 in micromolar concentrations with the following order regardless of the multidrug resistance (MDR) status: S1 > S2 > quercetin. S1 and S2, the most active compounds, were selected to evaluate their effect on the cell cycle, apoptosis, redox activity, and modulation of the p-glycoprotein function. No significant perturbation in cell cycle was seen with concentration up to 1 microM after 24 h. After 72 h a slight increase in G2/M block and DNA fragmentation occurred at 10 microM. Morphological analysis of apoptosis showed that chalcone analogues induced apoptosis to a higher extent than quercetin. Redox analysis demonstrated that all substances were able to increase intracellular thiol levels, which returned to baseline value after 24 h for all drugs except quercetin. Production of reactive oxygen species was essentially unaffected by all compounds. Finally, in MDR-positive MCF-7 ADRr cells chalcone analogues were unable to modulate p-glycoprotein function while quercetin was able to., Conclusions: Newly developed S1 and S2 chalcones have a different but higher antitumor activity than quercetin and could be considered as potential new anticancer drugs.

In order to investigate the phenomenon of multidrug resistance as a possible mechanism for poor response to treatment in patients with acute lymphoblastic leukemia (ALL) from India, a series of 32 cases of de novo untreated ALLs were analyzed by a cDNA-PCR approach to estimate the relative mRNA levels of the MDR-associated genes encoding MDR1, MRP, GSTpi, and GSTmu. The expression of beta2 microglobulin served as an internal standard. Quantifiable transcripts were observed in 20 patients for MRP, in 5 for MDR1, in 24 for GSTpi, and in 19 for GSTmu. The values ranged from undetectable to 132% of the control A549 cell line for MRP, undetectable to 49% of the HL60/DNR control cell line for MDR1, undetectable to 268% of A549 control cell line for GSTpi, and undetectable to 247% of A549 control cell line for GSTmu mRNA. Increased MRP levels were associated with increased GSTpi and GSTmu levels (p<0.01 for both), and increased levels of MDR1 were associated with increased GSTpi levels (p<0.05). The present observations showed no correlation between the MDR1 and MRP values with treatment outcome, in terms of either achieving a complete remission or predilection to early relapse. In view of some recent studies that envisage MRP as an energy-dependent pump involved in the efflux of GSH conjugates, the simultaneous up-regulation of transcription of all these genes might well be part of an integrated detoxification response that has been switched on after exposure to an environmental stress.

Peripheral blood samples from 61 patients (36 male, 25 female) with all stages of B-type chronic lymphocytic leukemia (CLL) were studied for MDR1 phenotype using monoclonal antibodies and rhodamine-123 dye exclusion, a functional assay of MDR1 expression. The duration of the disease varied from 1 month to 22 years at the time of initial study. Overall, 74% of the patients were positive for rhodamine-123 exclusion. When analyzed by gender, significantly more men than women were positive (89% versus 48%, p<0.001). There were more positive men than women for every stage of the disease. Female patients were found to be either MDR1 phenotype positive or negative at any stage of the disease. In contrast, all male patients with early (stages 0-II) disease were MDR1 phenotype positive. One early-stage (stage II) male patient converted from rhodamine-efflux positive to rhodamine-efflux negative as he progressed from stage-II to stage-IV disease. We suggest that some of the differences in disease biology of male versus female CLL patients (women having a more benign course) may be due to gender-dependent differences in drug-resistance gene activity, including MDR1. Our results also emphasize the need to take into account gender in evaluating the clinical course of patients with CLL.

Up till now, clinical data on the possible prognostic influence of multidrug resistance (MDR) in hematological malignancies have been inconsistent, probably due to technical pitfalls. Moreover, in most studies qualitative information on the presence/absence of MDR-1 expression has been used instead of quantitative results. In addition, results usually refer to the total BM population and not specifically to blast cells. In the present study we analyzed the expression of MDR-1 in a series of 50 newly diagnosed de novo AML using a double-staining technique: (a) monoclonal antibodies for the specific identification of blast cells and (b) the rhodamine-123 efflux assay, which allows a quantitative and calibrated measurement of MDR-1 function. Expression of MDR-1 was correlated with clinical, biological, and immunophenotypical disease characteristics. All patients were uniformly treated according to the AML 87/91 protocols of the Spanish Pethema Cooperative Group; the median age was 51 +/- 19 years and the FAB distribution was as follows: 2 M0, 9 M1, 9 M2, 12 M3, 11 M4, 5 M5, and 2 M6 cases. Upon grouping the 50 AML patients analyzed according to the level of rh123 elimination, it was observed that those cases with > or = 30% decrease in rh123 fluorescence displayed higher WBC counts (9 +/- 12 vs 37 +/- 73 x 10(9)/l, p = 0.02) and platelet numbers (94 +/- 11 vs 35 +/- 25 x 10(9)/l, p = 0.02), together with a higher incidence of extramedullary involvement (35% vs 24%, p = 0.02). Half of the patients (47%) displaying a low rh123 elimination (< 30%) showed M3 morphology, while among the 33 patients with a higher rate of rh123 elimination (> or = 30%), only four (12%) corresponded to the M3 morphological subtype (p = 0.0006). From the immunophenotypic point of view, a low rate of rh123 elimination was associated with a lower expression of HLADR antigen (p = 0.003) and a higher expression of CD117 (p = 0.01). Regarding the possible prognostic influence of MDR1 expression, we found that a high rate of rh123 elimination (> 30%) was associated with a tendency towards poor disease outcome, illustrated by both a lower complete remission rate with the first cycle of chemotherapy (36% vs 56%) and a lower median disease-free survival (22 months vs median DFS not reached), although differences did not reach statistical significance (p = 0.1 in both comparisons). This data shows that although MDR-1 can be a relevant parameter in the evaluation of AML patients, larger series of patients using appropriate techniques for specifically analyzing the MDR of blast cells will be necessary in order to establish the final clinical value of this parameter.

The clinical significance of the multidrug resistance (MDR 1) gene phenotype was investigated in newly diagnosed AML and was compared with other clinical and biological prognostic factors in patients who received at least one course of induction therapy with intercalating agents and conventional doses of Ara-C. MDR 1 gene was overexpressed in 40% of the 110 cases of AML at presentation, MRP in 15% of the 48 patients tested for both markers. Both gene expressions were closely linked (p = 0.008). Except for a lower frequency in the "good risk" cytogenetic group, MDR 1 overexpression was not associated with other prognostic factors. In univariate analysis, MDR 1 overexpression, age over 50 years, and cytogenetic were associated with a higher rate of resistance to induction treatment. The overall survival was shorter in the case of intermediate or poor cytogenetics, high leukocytosis, MDR 1 overexpression, age over 50 years, secondary AML, and poor cytologic differentiation. Using multivariate analysis on 64 patients receiving intensive treatment, MDR 1 overexpression was the first significant prognostic factor for resistance to the first course of induction treatment. Cytogenetic analysis maintained its prognostic value only in MDR 1-negative patients. These data underline the value of MDR 1 gene expression as a powerful prognostic factor in AML for response to the first induction treatment and overall survival, sustaining the use of MDR 1 modulators for first-line therapy in this disease.