MDR1 Phenotype Research Articles

Fungicide resistance characterized across seven modes of action in Botrytis cinerea isolated from Australian vineyards.

Botrytis bunch rot, caused by Botrytis cinerea, is an economically important disease of grapes in Australia and across grape-growing regions worldwide. Control of this disease relies on canopy management and the application of fungicides. Fungicide application can lead to the selection of resistant B. cinerea populations, which has an adverse effect on the management of the disease. Characterizing the distribution and severity of resistant B. cinerea populations is needed to inform resistance management strategies. In this study, 724 isolates were sampled from 76 Australian vineyards during 2013-2016 and were screened against seven fungicides with different modes of action (MOAs). The resistance frequencies for azoxystrobin, boscalid, fenhexamid, fludioxonil, iprodione, pyrimethanil and tebuconazole were 5%, 2.8%, 2.1%, 6.2%, 11.6%, 7.7% and 2.9%, respectively. Nearly half of the resistant isolates (43.8%) were resistant to more than one of the fungicides tested. The frequency of vineyards with at least one isolate simultaneously resistant to one, two, three, four or five fungicides was 19.7%, 7.9%, 6.6%, 10.5% and 2.6%. Resistance was associated with previously published genotypes in CytB (G143A), SdhB (H272R/Y), Erg27 (F412S), Mrr1 (D354Y), Bos1 (I365S, N373S + Q369P, I365S + D757N) and Pos5 (V273I, P319A, L412F/V). Novel genotypes were also described in Mrr1 (S611N, D616G), Pos5 (V273L) and Cyp51 (P347S). Expression analysis was used to characterize fludioxonil-resistant isolates exhibiting overexpression (6.3-9.6-fold) of the ABC transporter gene AtrB (MDR1 phenotype). Resistance frequencies were lower when compared to most previously published surveys of B. cinerea resistance in grape and other crops. Nevertheless, continued monitoring of critical MOAs used in Australian vineyards is recommended. © 2021 Society of Chemical Industry.

Mitochondria of a human multidrug-resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane.

Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P-glycoprotein (P-gp), breast cancer resistant protein and multiple resistance protein-1. The MDR phenotype is associated with the constitutive expression of COX-2 and iNOS, whereas celecoxib, a specific inhibitor of COX-2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX-2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1-positive cells, whereas COX-2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P-gp in mitochondria of MDR cancer cells independently from inhibition of COX-2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells.

Prevention of MDR development in leukemia cells by micelle-forming polymeric surfactant

Doxorubicin (Dox) incorporated in nanosized polymeric micelles, SP1049C, has shown promise as monotherapy in patients with advanced esophageal carcinoma. The formulation contains amphiphilic block copolymers, Pluronics, that exhibit the unique ability to chemosensitize multidrug resistant (MDR) tumors by inhibiting P-glycoprotein (Pgp) drug efflux system and enhancing pro-apoptotic signaling in cancer cells. This work evaluates whether a representative block copolymer, Pluronic P85 (P85) can also prevent development of Dox-induced MDR in leukemia cells. For in vitro studies murine lymphocytic leukemia cells (P388) were exposed to increasing concentrations of Dox with/without P85. For in vivo studies, BDF1 mice bearing P388 ascite were treated with Dox or Dox/P85. The selected P388 cell sublines and ascitic tumor-derived cells were characterized for Pgp expression and functional activity (RT-PCR, Western Blot, rhodamine 123 accumulation) as well as Dox resistance (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). The global gene expression was determined by oligonucleotide gene microarrays. We demonstrated that P85 prevented development of MDR1 phenotype in leukemia cells in vitro and in vivo as determined by Pgp expression and functional assays of the selected cells. Cells selected with Dox in the presence of P85 in vitro and in vivo exhibited some increases in IC 50 values compared to parental cells, but these values were much less than IC 50 in respective cells selected with the drug alone. In addition to mdr1, P85 abolished alterations of genes implicated in apoptosis, drug metabolism, stress response, molecular transport and tumorigenesis. In conclusion, Pluronic formulation can prevent development of MDR in leukemia cells in vitro and in vivo.

De Novo Expression of ATP Binding Cassette C1 (ABCC1) on T-ALL Cells Confers Resistance to Vincristine Independent of an MDR1 Phenotype.

Acquired drug resistance eventually leads to treatment failure in T-cell acute lymphoblastic leukemia (T-ALL). To define mechanisms of vincristine (VCR)-resistance, and to address whether inducible resistance in T-ALL might be mechanistically-dependent on ABC drug transporter expression, we developed VCR-resistant Sup T1 and Jurkat by incrementally growing cells in increasing drug concentrations. Jurkat (CD1a−, sCD3+) and Sup T1 (CD1a+, sCD3−) are arrested at “mature” and “cortical” stages of T-cell development and, respectively, showed 1190-fold (0.0006 μM to 0.714 μM) and 790-fold (0.0006 μM to 0.474 μM) increased resistance to VCR. Microarray analysis showed that expression of the ATP Binding Cassette C1 (ABCC1; Multidrug Resistance Protein, MDR2) was increased more than 29-fold in VCR-resistant cells as compared to the parental controls; fold increases in mRNA of ABCC1 were confirmed by RT-PCR, and cell surface expression by flow cytometric analysis. We then employed a flow cytometry-based assay that measures the ability of ABC pumps to extrude fluorescent dyes (Calcein AM). We confirmed that VCR-resistant Jurkat and Sup T1 actively extrude Calcein AM; drug extrusion was also effectively blocked by verapamil and other known ABCB1 antagonists. In contrast to our previous observations for ABCB1-mediated multidrug resistance (Estes, et al., British Journal of Haematology , in press), ABCC1-mediated drug resistance conferred a more narrow spectrum of multidrug resistance for the chemotherapeutic agents commonly used in T-ALL induction. To investigate whether ABC expression might impact patient outcome, we next examined ABCC1 and ABCB1 expression (>1.5-fold) in 80 T-ALL patients treated on COG studies 8704 and 9404. Different from our earlier findings that ABCB1 expression in diagnostic marrows conferred a 100% risk for on-therapy relapse, we found that ABCC1 expression only predicted treatment failure in approximately 25% of affected patients. These results suggest that ABC transporters are differentially regulated in Sup T1 and Jurkat, and that substrate-specific ABC protein regulation results in unique multidrug resistance phenotypes in T-ALL. This study contributes to the repertoire of substrate-specific, drug resistant T-ALL cells lines that are available for modeling treatment failure, provides mechanistically-based insights into relapse, and establishes an experimental means for investigating compounds that reverse ABCC1 mediated drug resistance.

MDR1, MRP1 and LRP expression in patients with untreated acute leukaemia: Correlation with 99mTc-MIBI bone marrow scintigraphy

Chemotherapy failure linked to multidrug-resistance (MDR) plays an important role in many cancer types, including leukaemia. It is believed that overexpression of some of membrane or intracellular proteins confers the MDR phenotype to cancer cells. (99m)Tc-sestamibi (MIBI) is a transport substrate for the Pgp pump. We assessed the bone marrow uptake of (99m)Tc-MIBI and its correlation with messenger RNA (mRNA) levels of MDR1, multidrug-resistance associated protein-1 (MRP1) and lung resistance protein (LRP) in acute leukaemia. A total of 26 patients (age range 17-72 years; mean age 51.88+/-2.52 years) with newly diagnosed acute leukaemia were included in the study. The expression of MDR1, MRP1 and LRP on mRNA levels were assessed by quantitative RT-PCR in the blast cells. The MIBI uptake in the bone marrow was evaluated using a quantitative scoring system with determination of the tumour-to-background ratios for the bone marrow areas. The correlation between the quantitative RT-PCR results and MIBI uptake was analysed by using Spearman's rank correlation coefficients with two-tailed test of significance. There was an inverse relationship between (99m)Tc-MIBI uptake of bone marrow and both mRNA levels of MDR1 and MRP1 (P=0.000, r= -0.733 and P=0.001, r= -0.610, respectively). No correlation was found between MIBI uptake and mRNA levels of LRP. Increased expression of MDR1 and MRP1 correlates with a low accumulation of (99m)Tc-MIBI in bone marrow areas in patients with acute leukaemia. (99m)Tc-MIBI bone marrow scintigraphy can identify the MDR1 and MRP1 phenotype, but not LRP, in patients with acute leukaemia.

MDR1 C3435T Polymorphism in Patients with Breast Cancer

The human multidrug-resistant gene (MDR1) encodes P-glycoprotein (Pgp), a membrane-bound efflux transporter conferring resistance to a number of natural cytotoxic drugs and potentially toxic xenobiotics. Single-nucleotide polymorphisms (SNPs) in MDR1 gene are associated with phenotypic variation in Pgp expression levels of tissue. SNPs may alter the physiological protective role of Pgp and, therefore, influence disease risk. In our study we identified the MDR1 C3435T polymorphism in breast cancer patients (n = 57) and healthy subjects (n = 50). DNA was extracted from peripheral blood samples by standard phenol/chloroform extraction method. Polymerase chain reaction-restriction fragment length polymorphism was used for the detection of C3435T single nucleotide polymorphism. We obtained CC, CT and TT genotype frequencies in breast cancer patients as 12.3%, 57.9% and 29.8%, respectively. In the control group, frequencies of genotypes were found as 36% for CC, 46% for CT and 18% for TT. We observed difference in SNPs in MDR1 gene C3435T polymorphism between breast cancer patients and healthy controls (chi(2) = 8.66, df = 2, p = 0.013). The C allele frequency was found in 41.2% and the T allele frequency was found in 58.8%. C3435T MDR1 gene allele frequencies in breast cancer patients as compared to results in control group were as follows: [OR = 1.5 (95% CI: 1.09-1.96)]. In the patient group, T allele frequency was significantly higher than controls (p <0.01). Clinicopathological parameters of patients with breast cancer were compared for C3435T polymorphism. We did not find any significant difference between clinicopathological parameters and MDR1 phenotype of breast cancer patients. The progression-free survival rate in a subgroup analysis based on MDR1 genotypes with CC genotype was 71.4%, CT genotype was 75.7%, and TT genotype was 88.2%, respectively. This difference was not statistically significant (log rank p = 0.63). Results of the present study demonstrated a 1.5-fold increased risk for development of breast cancer in T allele carriers.

Optimal Post-Remission Therapy for Flow-Cytometry Minimal Residual Disease Positive Patients with Acute Myeloid Leukemia.

Autologous (AuSCT) and allogeneic stem cell transplantation (SCT) are well established post-remissional strategies for patients with Acute Myeloid Leukemia (AML). However, there is still a debate ongoing about the relative merit of each of these options in first CR. Minimal residual disease (MRD) may be a useful tool to stratify AML patients into categories of risk which can benefit from differentiated post-remissional approaches. To address this issue, we analysed retrospectively, a series of 123 patients affected with AML in whom flow-cytometry serial determinations of MRD were available, at established time-points (post-induction, post-consolidation, post-stem cell transplantation). All were entered into the EORTC/GIMEMA protocols AML10/AML12 (age <61 yrs) or AML13 (age>61 yrs), all consisting in intensive induction and consolidation cycles, and, for patients aged <61 years, AuSCT or SCT. Median age was 52 years (range 18–78), no APL cases were included in the study. The “Maximally Selected Rank Statistic” analysis was used to select the MRD level and the time-point of analysis achieving the best prognostic significance in terms of overall survival (OS) and relapse free survival (RFS). This test was specifically developed to find out the optimal cut-off for a given biological variable correlating with clinical parameters of interest. This approach confirmed the threshold of 3.5x10−4 residual leukemic cells after consolidation therapy, as a discriminator between MRD− and MRD+ cases with different 5-years OS (64% vs. 14%, P<.001) and RFS (68% vs. 13%, P<.001). Therefore, among these 123 patients, we enucleated 2 groups of patients which underwent stem cell transplant procedure, 53 AuSCT and 11 SCT. The two subgroups were balanced in terms of FAB categories, WBC count, karyotype and expression of MDR-1 phenotype; 34/64 (53%) were MRD+ (9 SCT, 25 AuSCT) and 30/64 (47%) were MRD− (1 SCT, 28 AuSCT). Among the 53 patients submitted to AuSCT, MRD− had a significant better outcome than those MRD+, both in terms of 5-years OS (68% vs. 24%, P=0.003) and RFS (68% vs. 11%, P<.001). In the SCT group the low numbers hampered any firm conclusion; however, when the category of post-consolidation MRD+ patients was separately analysed, the use of SCT was associated with a better RFS (49% vs. 11%, P=NS); we assume that the lack of statistical significance was merely due to the few cases in the SCT group. In conclusion,the threshold of 3.5x10−4 at post-consolidation check-point is critical to predict disease outcome;MRD− patients have an excellent outcome regardless of the post-consolidation therapy;in the MRD+ group, AuSCT seems not to improve the prognosis whereas the use of SCT is associated with a superior outcome, although a larger number of patients is required to confirm this assumption. [Display omitted]

Statistical Validation of a Threshold To Discriminate Flow-Cytometry Minimal Residual Disease (MRD) Levels with Prognostic Significance in Acute Myeloid Leukemia (AML).

The current experience in the use of multiparametric flow-cytometry for immunological detection of MRD in AML, indicates that the method is feasible, rapid and relatively sensitive. In our previous studies of AML patients with aberrant phenotype (Blood 2000, Leukemia 2003 & 2006), we found that the level of MRD after consolidation therapy was the best predictor of outcome. In fact, a MRD level higher than 3.5x10−4 residual leukemic cells after consolidation was significantly correlated with a poor outcome. The major problem with using an empirically established cut-off to define thresholds is that it may not be reproducible over different populations of patients. In order to confirm the prognostic value of the threshold of 3.5x10−4 residual leukemic cells, previously selected, we used the Maximally Selected Rank Statistic analysis to determine optimal cutpoint, yielding the best separation of AML patients into two groups with different OS and/or RFS probabilities. To do this, we evaluated the trend of standardized log-rank statistics using RFS (Fig. 1, upper panels) and OS (Fig. 1, lower panels) as dependent variables, and the values of residual leukemic cells, determined both at the post-Induction (post-Ind) and post-consolidation (post-Cons) checkpoints, as independent variable (Fig. 1A and 1B, respectively). The experimental cut-off points, identified as the absolute peak in standardized log-rank statistics plots (vertical dotted lines in Fig. 1), were 3.7x10−4 and 3.4x10−4 residual leukemic cells for post-Ind and post-Cons values, respectively. According to this data we decided to utilize the 3.5x10−4 residual leukemic cells cut-off value to discriminate MRD− from MRD+ cases both after induction and consolidation. Therefore, patients with a residual leukemic cells values below the cutoff of 3.5×10−4 will be referred to as MRD−, while those with residual leukemic cells equal or exceeding the 3.5×10−4 level will be classified as MRD+. Post-Cons MRD− patients had a superior outcome in terms of relapse rate (27% vs. 78%, P<0.001), 7-years OS (64% vs. 14%, P<0.001) and RFS (68% vs. 13%, P<0.001). Moreover, post-Cons MRD− patients had a better outcome regardless of the MRD status after induction: in particular, patients entering MRD negativity only after consolidation showed the same outcome as those achieving early negativity after induction. Multivariate analysis, including karyotype, age, MDR1 phenotype, post-Ind and post-Cons MRD levels, indicated that the post-Cons MRD status independently affected relapse rate, OS and RFS (P<0.001, for all comparisons). In conclusion:the threshold of 3.5x10−4 is valid in discriminating risk categories in adult AML;post-consolidation MRD assessment is critical to predict disease outcome regardless of the MRD status after induction. [Display omitted]

The kinetics of reduction of minimal residual disease impacts on duration of response and survival of patients with acute myeloid leukemia

We assessed by multiparametric flow cytometry the levels of minimal residual disease (MRD) in 100 adult patients with acute myelogenous leukemia (AML) achieving complete remission after intensive chemotherapy. The aim of the study was to determine the optimal threshold, in terms of residual leukemic cells, and the time point of choice, that is, post-induction (post-Ind) or post-consolidation (post-Cons), able to better predict outcome. By applying the maximally selected log-rank statistics, the threshold discriminating MRD- from MRD+ cases was set at 3.5 x 10(-4) residual leukemic cells, a level that allowed the identification of distinct subgroups of patients, both at post-Ind and post-Cons time points. Post-Cons MRD- patients had a superior outcome in terms of relapse rate, overall survival (OS) and relapse-free survival (RFS) (P<0.001, for all comparisons), regardless of the MRD status after induction. In particular, patients entering MRD negativity only after consolidation showed the same outcome as those achieving early negativity after induction. Multivariate analysis, including karyotype, age, MDR1 phenotype, post-Ind and post-Cons MRD levels, indicated that the post-Cons MRD status independently affected relapse rate, OS and RFS (P<0.001, for all comparisons). (1) the threshold of 3.5 x 10(-4) is valid in discriminating risk categories in adult AML and (2) post-Cons MRD assessment is critical to predict disease outcome.

The kinetics of reduction of minimal residual disease impacts on duration of response and survival of patients with acute myeloid leukemia

6533 Background: Multiparametric flow-cytometry is frequently used to assess the levels of minimal residual disease (MRD) in acute myeloid leukemia (AML) patients achieving complete remission after intensive chemotherapy. In our previous experience, MRD negativity after consolidation cycle, as defined by a level of bone marrow residual leukemic cells ≤3.5x10−4, was associated with a significantly longer relapse free survival (RFS) and overall survival (OS). The aims of the present study were to confirm, in a larger series of patients, the prognostic relevance of the post consolidation MRD assessment and to validate the threshold of 3.5x10−4. Methods: 100 patients affected with AML were entered into the EORTC/GIMEMA protocols AML10/AML12 (age &lt;61 yrs) or AML13/AML15 (age &gt;61 yrs), consisting in intensive induction and consolidation cycles. Median age was 52 years (range 18–78), all FAB subtypes were represented with the exception of APL cases. A Maximally Selected Rank Statistic analysis was used to select the best threshold. Results: The statistical test confirmed that the level of 3.5×10−4 residual leukemic cells was the most significant in discriminating categories of risk. In fact, levels of MRD measured at the post-consolidation time-point identified 2 distinct subgroups: 36 patients MRD negative and 56 MRD positive. MRD negative patients had a better outcome in terms of relapse rate (P&lt;0.001), OS (P=0.039) and RFS (P=0.008), regardless of MRD status after induction. In fact, patients entering MRD negativity only after consolidation showed the same outcome as those achieving early negativity after induction. The multivariate analysis, including karyotype, age, MDR1 phenotype and post induction and post consolidation MRD levels, indicated that the post-consolidation MRD status was an independent factor affecting relapse rate (P&lt;0.001), OS (P=0.039) and RFS (P=0.008). Conclusions: We conclude that 1) the threshold of 3.5x10−4 is valid in discriminating risk categories in AML; 2) MRD assessment at post-consolidation check-point is critical to predict disease outcome. The incorporation in clinical trials of flow-cytometric MRD determination may allow a more accurate risk stratification of AML patients. No significant financial relationships to disclose.

Prognostic Determinants in Adult AML Patients with Intermediate Risk Karyotype.

According to the prognostic classifications of karyotypic abnormalities of AML (MRC and SWOG), the intermediate group includes patients either lacking good and poor karyotype or with normal karyotype. Therefore, it represents, by definition, a miscellaneous group for which the evaluation of the better treatment strategy is difficult due to its heterogeneity. Moreover, patients belonging to this intermediate group account for the large majority of AML cases enrolled into clinical trials. The aim of our study was to analyze the factors specifically affecting the outcome of patients bearing intermediate risk karyotypic abnormalities in a group of 94 AML cases entered into the EORTC/GIMEMA protocols AML10/AML12 (age <61yrs) or AML13/AML15 (age >61 yrs), consisting in intensive induction and consolidation cycles. The clinico-biological variables evaluated in our model included age, FAB, WBC count, MDR1 phenotype, FLT3 mutations and level of post-consolidation bone marrow residual leukemic cells (BMRCL) assessed by multiparametric flow-cytometry (MPFC). In our experience, patients with <2.0 x10−4 BMRLC at the end of consolidation therapy were considered MRDneg and showed a better outcome, patients whose level of MRD were >2.0x10−4 at the end of consolidation were considered MRDpos and showed a poor prognosis. However, the category of MRDpos patients could be divided into 2 subgroups depending on whether they had a reduction of BMRLC between induction and consolidation, even not reaching the threshold of MRD negativity. In fact, we identified patients whose BMRLC were reduced by at least 1-log between induction and consolidation, referred to as “chemosensitive MRDpos” with an intermediate prognosis, and those showing no reduction of MRD levels between induction and consolidation, referred to as “chemoresistant MRDpos” with the worst outcome. Using the MRC classification, 14/94 patients (15%) had a good-risk cytogenetics, 74/94 (79%) an intermediate-risk and 6/94 (6%) a poor-risk. When we restricted the analysis to cases with intermediate-risk karyotype we found that: 1) in multivariate model, MRD status after consolidation was the only variable significantly affecting relapse free survival (RFS)(P=0.001); 2) patients in the MRDneg, “chemosensitive MRDpos” and “chemoresistant MRDpos” group differed significantly in terms of relapse rate (25% vs. 50% vs. 78%, respectively, P<0.001); 3) the 5 years median duration of RFS was 71%, 46% and 16%, respectively (P<0.001); 4) MRDneg patients had an outcome slightly better than those bearing good risk karyotype; 5) “chemoresistant MRDpos” patients showed a dismal outcome comparable to poor-risk cytogenetic patients; 6) “chemosensitive MRDpos” patients showed a true intermediate behavior (Fig. 1). These results suggest that the inclusion of MPFC assessment of MRD in patients with intermediate risk karyotype may be particularly useful in discriminating subgroups with different outcomes, allowing to design risk-based therapeutic programs in a very heterogeneous group of AML. [Display omitted]

Inhibition of P-glycoprotein transport function and reversion of MDR1 multidrug resistance by cnidiadin

Overexpression of P-glycoprotein (Pgp) encoded by the MDR1 gene is one of the major obstacles to successful cancer chemotherapy. The goal of this study was to evaluate if, among other natural coumarins, cnidiadin, a furanocoumarin present in traditional Chinese medications and in a spice commonly used in Greek food, inhibits Pgp transport activity and has the potential to reverse MDR1 multidrug resistance. Using MDR1-transfected Madin-Darby canine kidney (MDCK-MDR1) cells as a model of cells expressing the human MDR1 phenotype, and verapamil or CsA or both as positive control, we tested the capacity of six natural coumarins (umbelliferone, esculin, esculetin, cnidiadin, angelicin and psoralen) to induce the accumulation of rhodamine-123 (R-123) and [3H]-vinblastine ([3H]-VBL) and to modulate the photolabeling of Pgp by SDZ 212-122, a diazirin cyclosporin A. The growth-inhibitory effect of cnidiadin and its capacity to enhance the cell toxicity of vinblastine (VBL) or vincristine (VCR) was then evaluated by the WST-1 assay in two cell lines overexpressing Pgp (MDCK-MDR1 and vincristine-resistant KB/VCR). Cnidiadin was the only tested coumarin capable of significantly accumulating R-123 and [3H]-VBL and inhibiting Pgp photolabeling in MDCK-MDR1 cells. The dose-dependent increase in [3H]-VBL uptake (IC50 26.5 microM) induced by cnidiadin in the dose range 1-100 microM correlated with inhibition of Pgp photolabeling. At 10 microM cnidiadin inhibited photolabeling by 59% and sensitized both MDCK-MDR1 and KB/VCR cells to vinca alkaloids. Cnidiadin is a cytotoxic agent capable in vitro of competitively inhibiting the binding and efflux of drug by Pgp and of enhancing the cell toxicity of vinca alkaloids in two cell lines (MDCK-MDR1 and mutant human carcinoma KB/VCR) overexpressing Pgp. This suggests that diet or traditional preparation containing cnidiadin may contribute to the reversal of MDR1 multidrug resistance and may affect the bioavailability of Pgp substrates orally administered. However, due to its cell toxicity, clinical interest in cnidiadin as a chemosensitizer appears to be limited.

The Effect of CYP3A5 and MDR1 Polymorphic Expression on Cyclosporine Oral Disposition in Renal Transplant Patients

Variability in CYP3A (CYP3A4/5) and P-glycoprotein (human MDR1 gene product) activity underlies interindividual differences in oral cyclosporine (CsA) bioavailability. Racial differences in polymorphic expression of CYP3A5 and MDR1 may explain observed interracial variability in oral bioavailability. Our objective was to evaluate the effect of CYP3A5 and MDR1 polymorphic expression on CsA oral disposition. Steady-state plasma concentration profiles (n = 19) were sampled in renal transplant recipients receiving concentration-adjusted CsA maintenance therapy. CsA plasma concentrations were measured by fluorescence polarization immunoassay. CYP3A5 and MDR1 genotypes were determined by real-time polymerase chain reaction. Noncompartmental pharmacokinetic analysis and nonlinear mixed-effects modeling (NONMEM) were performed to assess the effect of genotype on CsA pharmacokinetics. MDR1 C3435T genotype was identified as the best predictor of CsA systemic exposure. CsA oral clearance was significantly higher in subjects who carried at least one 3435T allele compared to homozygous wild-type individuals (40.0 ± 2.2 vs. 26.4 ± 3.1 L/h, p = 0.007). MDR1 C3435T genotype accounted for 43% of the interindividual variability of CsA oral clearance in the study population after accounting for interoccasion variability. The authors were unable to independently assess whether CYP3A5 correlated with any CsA pharmacokinetic parameter since all CYP3A5 nonexpressors were also 3435T allele carriers. MDR1 3435T allele carriers have enhanced oral clearance compared to individuals with the CC genotype. The frequency of the 3435T allele is lower in African Americans compared to Caucasians. Thus, the MDR1 C3435T genotype offers a potential mechanistic basis to explain interracial differences in CsA oral bioavailability. Further studies are needed to explore the relationship between CYP3A5 and MDR1 genotype and phenotype.

The Effect of CYP3A5 and MDR1 Polymorphic Expression on Cyclosporine Oral Disposition in Renal Transplant Patients

Variability in CYP3A (CYP3A4/5) and P-glycoprotein (human MDR1 gene product) activity underlies interindividual differences in oral cyclosporine (CsA) bioavailability. Racial differences in polymorphic expression of CYP3A5 and MDR1 may explain observed interracial variability in oral bioavailability. Our objective was to evaluate the effect of CYP3A5 and MDR1 polymorphic expression on CsA oral disposition. Steady-state plasma concentration profiles (n = 19) were sampled in renal transplant recipients receiving concentration-adjusted CsA maintenance therapy. CsA plasma concentrations were measured by fluorescence polarization immunoassay. CYP3A5 and MDR1 genotypes were determined by real-time polymerase chain reaction. Noncompartmental pharmacokinetic analysis and nonlinear mixed-effects modeling (NONMEM) were performed to assess the effect of genotype on CsA pharmacokinetics. MDR1 C3435T genotype was identified as the best predictor of CsA systemic exposure. CsA oral clearance was significantly higher in subjects who carried at least one 3435T allele compared to homozygous wild-type individuals (40.0 +/- 2.2 vs. 26.4 +/- 3.1 L/h, p = 0.007). MDR1 C3435T genotype accounted for 43% of the interindividual variability of CsA oral clearance in the study population after accounting for interoccasion variability. The authors were unable to independently assess whether CYP3A5 correlated with any CsA pharmacokinetic parameter since all CYP3A5 nonexpressors were also 3435T allele carriers. MDR1 3435T allele carriers have enhanced oral clearance compared to individuals with the CC genotype. The frequency of the 3435T allele is lower in African Americans compared to Caucasians. Thus, the MDR1 C3435T genotype offers a potential mechanistic basis to explain interracial differences in CsA oral bioavailability. Further studies are needed to explore the relationship between CYP3A5 and MDR1 genotype and phenotype.

Modulation of steady-state kinetics of digoxin by haplotypes of the P-glycoprotein MDR1 gene.

We investigated the effect of polymorphisms in the P-glycoprotein (P-gp) MDR1 gene on steady-state pharmacokinetics of digoxin in Caucasians. According to earlier data, homozygous TT of the exon 26 complementary deoxyribonucleic acid (cDNA) 3435C>T polymorphism was associated with low P-gp expression in the human intestine. Eight healthy male homozygous carriers of the wild-type exon-26 3435C>T (CC), 8 heterozygous subjects (CT), and 8 homozygous mutant (TT) subjects were selected. Seven further MDR1 polymorphisms were determined. Digoxin was administered orally twice daily on the first two study days; on days 3 to 5, 0.25 mg was given in the morning. On day 5, kinetic parameters were analyzed for genotype-phenotype and haplotype-phenotype relationships. The area under the plasma concentration-time curve from time zero to 4 hours [AUC(0-4)] (P =.042) and C(max) (P =.043) values of digoxin were higher in subjects with the 3435TT genotype than in those with the 3435CC. No influence of other single nucleotide polymorphisms (SNPs) on digoxin parameters was detected. Comparison of genotypes deduced from SNPs 2677G>T (exon 21) and 3435C>T revealed significant differences for AUC(0-4) (P =.034) and C(max) (P =.039), which were substantiated by haplotype analysis. Haplotype 12 (2677G/3435T), which had a frequency of 13.3% in a randomly drawn Caucasian sample (n = 687), was associated (Mann-Whitney test) with higher AUC(0-4) values (P =.009) than were found in noncarriers (mean +/- SD, 5.7 +/- 0.9 microg. h/L [n = 7] versus 4.8 +/- 0.9 microg. h/L [n = 17]). Haplotype 11 (2677G/3435C) had lower AUC(0-4) values (P =.013) compared with those of noncarriers (mean +/- SD, 4.7 +/- 0.9 microg. h/L [n = 16] versus 5.6 +/- 0.9 microg. h/L [n = 8]). Results of haplotype analysis match data of other MDR1 studies. Haplotype 12 codes for high values of AUC(0-4) and C(max) of orally administered digoxin. Analysis of MDR1 haplotypes is superior to unphased SNP analysis to predict MDR1 phenotype.

Clinical relevance of minimal residual disease detection in adult acute myeloid leukemia.

We have used flow cytometry to quantify minimal residual disease (MRD) in 63 patients with acute myeloid leukemia (AML). No significant correlation was found between the level of MRD after induction and disease outcome. After consolidation, a threshold of 3.5 x 10(-4) residual leukemic cells divided the 57 evaluable patients into two distinct groups: the MRDCons(+) and the MRDCons(-) group, with a relapse rate of 81% (22/27) and 27% (8/30), respectively (p = 0.000035). Although not correlated with prognosis, the level of MRD after induction course affected the degree of cytoreduction achieved with consolidation. In fact, the patients who entered a MRDCons(-) status had a median number of leukemic residual cells of 1.8 x 10(-4) after induction; at the same stage, the bone marrow of patients who were in a MRDCons(+) condition harbored a median level of 1.7 x 10(-3) malignant residual cells (p = 0.00073). The MRDCons(+) status also correlated significantly with poor/intermediate risk cytogenetics, MDR1 phenotype, short duration of overall survival, and relapse-free survival (p = 0.024, 0.021, 0.00001, and 0.00001, respectively). In multivariate analysis, the MRDCons(+) status was associated with a high probability of relapse (p < 0.00026) and short duration of relapse free survival (p = 0.008). Stem cell transplantation did not seem to alter the prognostic impact of high levels of MRD after consolidation: within the MRDCons(+) group, the relapse rate after transplant was 78%. Thus, a MRD > or = 3.5 x 10(-4) leukemic cells at the end of consolidation strongly predicts relapse, and is significantly associated with MDR1-positive phenotype and intermediate/unfavorable cytogenetics.

The role of 99mTc-MIBI scintigraphy in the assessment of MDR1 overexpression in patients with musculoskeletal sarcomas: comparison with therapy response.

The occurrence of multidrug resistance (MDR), which is in part due to the overexpression of P-glycoprotein (Pgp), is a major problem in neoadjuvant therapy of malignant musculoskeletal tumours. The aim of this study was to investigate the role of technetium-99m hexakis-2-methoxyisobutylisonitrile (99mTc-MIBI) scintigraphy for functional imaging of the MDR1 phenotype in patients with musculoskeletal sarcomas. We aimed to compare 99mTc-MIBI uptake and washout kinetics with the expression of Pgp and with chemotherapy response. Twenty-five patients (16 males and 9 females, aged between 8 and 65 years) with malignant musculoskeletal tumours were studied. After injection of 555-740 MBq 99mTc-MIBI, dynamic flow images of the involved area were obtained for 3 min, and planar images were acquired at 10 min and 1 h. From the dynamic images, a tumour perfusion index (TPI) was obtained using Patlak-Rutland analysis. Tumour to background (T/B) ratios of both early and delayed images and percent wash-out rate (WR%) of 99mTc-MIBI were calculated. Immunohistochemical analysis of Pgp was performed on biopsy specimens and the degree of expression was graded according to a semiquantitative scoring system, from 0 to 6. After neoadjuvant therapy, tumour response was assessed by examining the ratio of viable cells and by detecting percent necrosis. Scintigraphic results were compared with Pgp status and therapy response. Irrespective of the Pgp status, all patients showed significant perfusion and 99mTc-MIBI uptake in early images. There was not a significant correlation between T/B ratios of early and delayed images and Pgp expression. We observed a positive correlation between WR% and Pgp status (r=0.61, P<0.01), and the wash-out rate of 99mTc-MIBI was significantly higher in patients with high Pgp expression than in those with a low Pgp score (33% +/- 9% vs 17% +/- 9%). Therapy response was determined in 21 of 25 patients, and in only 5 of 21 cases was the percent necrosis more than 90%. Neither Pgp expression rate nor WR% was found to show a significant correlation with percent necrosis in the bulk tumour specimens. In conclusion, the initial uptake of 99mTc-MIBI in bone and soft tissue sarcomas did not correlate with Pgp expression. A relationship was found between the wash-out rate of 99mTc-MIBI and the Pgp score, with a significant difference in WR% being observed between patients with high and patients with low Pgp expression.

Dexrazoxane significantly impairs the induction of doxorubicin resistance in the human leukaemia line, K562.

Dexrazoxane combined with doxorubicin (+ 5-fluorouracil + cyclophosphamide – the FAC regime) leads to a significant decrease in doxorubicin cardiotoxicity and a significant increase in median survival time for patients with advanced breast cancer responsive to FAC. The reason for this increase in survival may be due to interference with the mechanism involved in the emergence of multidrug resistance (MDR). In order to test this hypothesis, we induced resistance to doxorubicin in the K562 cell line by growing cells in increasing concentrations of doxorubicin (10–30 nM) in the presence and absence of dexrazoxane (20 nM). The doxorubicin sensitivity of all resultant sublines was measured using the MTT assay. Flow cytometry was used to assess the MDR1 phenotype, measuring P-glycoprotein expression with MRK 16 antibody and drug accumulation in the presence and absence of PSC 833 for functional P-glycoprotein. Long-term growth in doxorubicin increased the cellular resistance (IC 50) of K562 cells in a concentration-dependent manner (r2 = 0.908). Doxorubicin resistance was not induced in the presence of dexrazoxane (P< 0.0001) for several months. In parallel, the expression of functional P-glycoprotein was delayed after concomitant addition of dexrazoxane to the selecting medium (P< 0.001). Dexrazoxane did not act as a conventional modulator of P-glycoprotein. These results suggest that dexrazoxane may delay the development of MDR1, thus allowing responders to the FAC regime to continue to respond. © 2001 Cancer Research Campaign http://www.bjcancer.com

Tissue factor expression and multidrug resistance in cancer: two aspects of a common cellular response to a hostile milieu

Tissue factor (TF) is the main physiological initiator of blood coagulation and its activation or de-encryption within plasma membranes is important for trapping, extravasation and angiogenesis in the development and spread of solid malignancies. Multidrug resistance is also an adaptive response in malignant (and normal) cells. It is often mediated by the over-expression of the P-glycoprotein (P-gP) efflux pump.Both TF and P-gP tend to be expressed together, perhaps as part of a coherent ‘crisis management’ response of cells to environmental change or challenge. An associated feature in such a response appears to be the reversal of normal phospholipid charge asymmetry in the plasma membrane bilayer.Responses to environmental stimuli affect function in normal and malignant tissue. Uniting the study of TF expression or de-encryption and MDR-1 phenotype would be biologically enlightening and might ultimately influence clinical cancer management and the control of thrombotic problems associated with treatment.

Doxorubicin-resistant LoVo adenocarcinoma cells display resistance to apoptosis induction by some but not all inhibitors of ser/thr phosphatases 1 and 2A

LoVo adenocarcinoma cells are fairly sensitive to cytostatic drugs, e.g. doxorubicin, but can develop drug resistance by expression of a P-glycoprotein-mediated MDR1 phenotype. LoVo cells respond with apoptosis to nanomolar concentrations of okadaic acid and micromolar concentrations of cantharidic acid. Interestingly, LoVoDx cells which had become about 10-fold less sensitive to doxorubicin by incubation in increasing concentrations of this cytostatic drug were also less sensitive to the toxicity of okadaic acid. Resistance to both agents was lost or significantly reduced by incubation in drug-free medium for about 4 months. On the other hand, LoVoDx cells did not lose responsiveness to the structurally different phosphatase inhibitor cantharidic acid but were about twofold more sensitive to the cytotoxic effect of this agent. Thus, MDR expression protects LoVo cells from the toxicity of phosphatase inhibitors that presumably are substrates of the P-glycoprotein, e.g. okadaic acid and its derivatives but not cantharidic acid, despite the fact that both agents are potent inducers of apoptotic cell death via ser/thr phosphatase inhibition.