Cellular Daunorubicin Accumulation Research Articles

Effect of D, L‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol and tetrandrine on the reversion of multidrug resistance in K562/A02 cells

In this study, we applied D, L‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP) hydrochloride as a chemical inhibitor for glucosylceramide synthase (GCS) and tetrandrine (Tet) for P‐glycoprotein (P‐gp) to reverse daunorubicin (DNR) resistance of human leukemia cell line K562/A02. Cytotoxicity assays showed that either PDMP or Tet enhanced cytotoxic effect of DNR on K562/A02 cells, while cotreatment of these two drugs had a more significant effect on chemosensitization. Using flow cytometric analysis, we confirmed that the enhancement effect was accompanied by elevated cellular DNR accumulation and DNR‐induced apoptosis. According to reverse transcription‐polymerase chain reaction and western blot, the reversal effect of that composite might owe to the significant downregulation of mdr1 and GCS gene expressions. Importantly, PDMP diminished mdr1 gene expression and Tet also downregulated GCS gene expression. Moreover, a positive correlation was observed between GCS and P‐gp. Thus, our results suggest that a potential clinical application of PDMP in combination with Tet may enhance chemosensitivity in leukemia.

Effect of magnetic nanoparticles of Fe3O4 and 5-bromotetrandrine on reversal of multidrug resistance in K562/A02 leukemic cells

This study aims to evaluate the multidrug resistance (MDR) reversal activity by magnetic nanoparticles of Fe3O4 (MNPs-Fe3O4) and 5-bromotetrandrine (BrTet) MDR cell line K562/A02 solitarily or symphysially. The proliferation of K562 and K562/A02 cells and the cytotoxicity on peripheral blood mononuclear cells (PMBCs) were evaluated by MTT assay. Cellular accumulation of daunorubicin (DNR) was analyzed by flow cytometry. Real-time polymerase chain reaction and Western blotting analyses were performed to examine the mRNA and protein levels of mdr1, respectively. The results showed that the combination of MNPs-Fe3O4 and BrTet with effective concentrations significantly increased cytotoxicity against MDR cell line K562/A02. Both BrTet and MNPs-Fe3O4 increased the intracellular DNR accumulation in the K562/A02 cell line, and downregulated the level of mdr1 gene and expression of P-glycoprotein. Furthermore, the combination did not have significant cytotoxicity in PMBCs. We propose that MNPs-Fe3O4 conjugated with DNR and BrTet probably have synergetic effects on MDR reversal.

Macelignan: A New Modulator of P-Glycoprotein in Multidrug-Resistant Cancer Cells

The effect of macelignan, a phytoestrogen, on P-gp function was investigated using multidrug resistant cancer cells overexpressing P-gp (NCI/ADR-RES) and the fluorescent P-gp substrates, daunorubicin and rhodamine 123. Macelignan (40 μM) increased the cellular accumulation of daunorubicin by approximately threefold in NCI/ADR-RES cells, whereas it did not alter the cellular accumulation of daunorubicin in MCF-7/sensitive cells. Similarly, the presence of macelignan also enhanced significantly (P < 0.05) the cellular accumulation of rhodamine 123 in a concentration-dependent manner in NCI/ADR-RES cells. Furthermore, cancer cells were more susceptible to the cytotoxicity of vinblastine, a P-gp substrate, in the presence of macelignan. Those results suggest that macelignan has inhibitory effects on P-gp mediated cellular efflux. However, P-gp activity did not affect the cellular accumulation of macelignan itself. Taken all together, macelignan was identified as a novel inhibitor of P-gp activity and may be a promising lead compound for the rational design of more efficacious drugs to reverse multidrug resistance in cancer.

Chemotoxicity of doxorubicin and surface expression of P-glycoprotein (MDR1) is regulated by the Pseudomonas aeruginosa toxin Cif

P-glycoprotein (Pgp), a member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily, is a major drug efflux pump expressed in normal tissues, and is overexpressed in many human cancers. Overexpression of Pgp results in reduced intracellular drug concentration and cytotoxicity of chemotherapeutic drugs and is thought to contribute to multidrug resistance of cancer cells. The involvement of Pgp in clinical drug resistance has led to a search for molecules that block Pgp transporter activity to improve the efficacy and pharmacokinetics of therapeutic agents. We have recently identified and characterized a secreted toxin from Pseudomonas aeruginosa, designated cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif). Cif reduces the apical membrane abundance of CFTR, also an ABC transporter, and inhibits the CFTR-mediated chloride ion secretion by human airway and kidney epithelial cells. We report presently that Cif also inhibits the apical membrane abundance of Pgp in kidney, airway, and intestinal epithelial cells but has no effect on plasma membrane abundance of multidrug resistance protein 1 or 2. Cif increased the drug sensitivity to doxorubicin in kidney cells expressing Pgp by 10-fold and increased the cellular accumulation of daunorubicin by 2-fold. Thus our studies show that Cif increases the sensitivity of Pgp-overexpressing cells to doxorubicin, consistent with the hypothesis that Cif affects Pgp functional expression. These results suggest that Cif may be useful to develop a new class of specific inhibitors of Pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs, and at improving the bioavailability of Pgp transport substrates.

P-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP) expression in acute promyelocytic leukaemia.

We analysed the expression of three drug transporter proteins [p-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP1)] involved in anthracycline resistance that are frequently overexpressed in poor-risk adult acute non-lymphocytic leukaemia (ANLL), in 23 acute promyelocytic leukaemia (APL) patients at onset managed at a single institution. Cellular daunorubicin accumulation was also evaluated. At onset, no case had PGP or MRP1 expression that exceeded that of non-multidrug-resistant (MDR) cell lines. Only one case showed LRP overexpression. No peculiar MDR features distinguished the seven patients who relapsed from those who maintained complete remission. In the onset vs. first relapse, only one patient showed an increased (threefold) PGP expression at relapse. At second relapse, three out of four patients showed a PGP expression two- to threefold higher than baseline values. These results are consistent with the view that low PGP, LRP and MRP1 expression and the absence of defects in intracellular drug accumulation may account for the peculiarly high sensitivity of APLs to anthracycline. It does not support the screening of MDR markers in APL patients at onset as predicting factors of early relapse. The results suggest that no significant changes in PGP, LRP or MRP1 expression are likely to occur at first relapse. In contrast, PGP expression is likely to increase later in the patient history as a result of additional chemotherapy courses.

P-glycoprotein-independent decrease in drug accumulation by phorbol ester treatment of tumor cells

The effect of a change in the phosphorylation state of the drug transporter P-glycoprotein (P-gp) on its drug transport activity was studied for the substrates daunorubicin (DNR), etoposide (VP-16), and calcein acetoxymethyl ester (Cal-AM). Phorbol ester (PMA), added to stimulate phosphorylation of P-gp by protein kinase C (PKC), caused a decrease in the cellular accumulation of DNR and VP-16, both in multidrug-resistant (MDR) P-gp-oversxpressing cells and in wild-type cells. Since treatment of cells with kinase inhibitor staurosporine (ST) reversed this effect of PMA and the non-PKC-stimulating phorbol ester 4cn-phorbol, 12,13-didecanoate (4αPDD) did not result in a decreased DNR accumulation, we conclude that this effect is the result of kinase activity. The concentration dependence of the inhibition of P-gp by verapamil (Vp) was not influenced by PMA. Accumulation of the P-gp substrate Cal-AM was not influenced by PMA in wild-type cells. Therefore, Cal-AM was used to study the effect of PMA-induced phosphorylation of P-gp on its transport activity. Activation of PKC with PMA or inhibition of protein phosphatase 1/2A (PP1/PP2A) with okadaic acid (OA) did not affect the accumulation of Cal-AM in the MDR cells or wild-type cells. The kinase inhibitor ST increased the Cal-AM accumulation only in the MDR cells. Neither stimulating PKC with PMA nor inhibiting PP1/PP2A with OA led to a decreased inhibition of P-gp by ST, indicating that ST inhibits P-gp directly. From these experiments, we conclude that PKC and PP1/PP2A activity do not regulate the drug transport activity of P-gp. However, these studies provide evidence that PMA-induced PKC activity decreases cellular drug accumulation in a P-gp-independent manner.

Flow cytometric analysis of P-glycoprotein expression and drug efflux in human soft tissue and bone sarcomas.

Twenty-two fresh surgical specimens of human sarcomas (soft tissue and bone) from 20 patients were analyzed by flow cytometry for the expression of drug resistance-related P-glycoprotein (P-gp) and cellular daunorubicin (DNR) accumulation with or without the presence of DNR efflux blockers. Single-cell suspensions prepared from the tumor specimens were analyzed by dual-color flow cytometry after reaction with MRK-16 (anti-P-gp) and anti-CD45 (pan-leukocyte) antibodies. MRK-16 reactivity of tumor cells was evaluated after exclusion of CD45-positive cells by electronic gates. Parallel samples were incubated with DNR alone or in combination with DNR efflux blockers, verapamil (VPL), or dipyridamole (DPD) for determination of cellular DNR accumulation and the effect of the efflux blockers. Extensive heterogeneity was observed in both P-gp expression and DNR accumulation of the tumor specimens examined. Eight of the 22 tumor specimens had significant numbers of P-gp-positive cells. In three of the eight P-gp-positive tumors, cellular DNR accumulation was significantly increased by co-incubation with the efflux blockers VPL or DPD. These results indicate that both quantitative and functional analysis of P-gp expression may be essential in determining the cellular drug resistance phenotype of tumor cells and its correlation with therapeutic outcome.

Levels of mdr1 and mrp mRNA in leukaemic cell populations from patients with acute myelocytic leukaemia are heterogenous and inversely correlated to cellular daunorubicin accumulation.

Multidrug resistance gene (mdrl) expression is associated with a poor prognosis in acute myelocytic leukaemia (AML). Whether expression of the recently described multidrug resistance-associated gene (mrp) has any prognostic importance in AML is still unclear. The aim of the present study was to investigate the functional role of the mdr1 and mrp mRNA levels in peripheral leukaemic cell populations from patients with AML. Peripheral leukaemic cells from 10 patients with AML were incubated with daunorubicin (DNR). Cellular DNR content was analysed with a fluorescence-activated cell sorter (FACS). From each cell population the 20-25% cells with the lowest and highest DNR content were sorted out, and mdr1 and mrp RNA were quantified in these subpopulations with competitive polymerase chain reaction. The ratio between the mean DNR content in the cell populations with high and low DNR content varied between 1.9 and 6.6. the cell fraction with low DNR content had higher (3.8-40 times)mdr1 mRNA levels in 10/10 patients and higher (1.4-26 times) mrp mRNA levels in 8/10, as compared to the cell fraction with high DNR accumulation. In conclusion, mdr1 and mrp mRNA expressions are heterogenous in leukaemic cell populations from patients with AML. The mdr1 expression, and to some extent mrp expression, is inversely correlated to DNR accumulation in vitro.

Resistance‐associated factors in human small‐cell lung‐carcinoma GLC4 sub‐lines with increasing adriamycin resistance

Previous studies have shown that the in vitro-selected adriamycin-resistant human small-cell lung-carcinoma cell line GLC4-ADR150 displays multidrug resistance as the result of 3-fold decreased DNA-topoisomerase II (topo II) activity and a 6-fold reduction in adriamycin accumulation. Not the MDR1 gene, but the MRP gene, was over-expressed in this cell line. The aim of our study was to establish which of these drug-resistance-associated factors are already involved in drug resistance occurring at early steps of selection with adriamycin. To address this question, changes in expression of topo II alpha/topo II beta, MRP and drug accumulation were measured along with adriamycin resistance (from 2- to 10- to 150-fold) and in a partial revertant cell line (10-fold resistant). Topo II alpha and II beta mRNA and protein levels were decreased in the resistant sub-lines, except in the 10-fold-resistant cell line. Cellular daunorubicin accumulation was decreased 1.2- to 5-fold with increasing resistance. MRP mRNA was over-expressed in all resistant sub-lines, with a marked increase in the 10-fold-resistant cells (level of expression as high as in the GLC4-ADR150 cells). Expression of an ATP-binding 190-kDa membrane protein and Western-blot analysis with anti-MRP anti-serum ASPKE, was in accordance with the expression of MRP mRNA in all cell lines. Expression of MRP mRNA and protein, however, was not proportional with the decrease in drug accumulation in all resistant sub-lines. This study also shows that drug accumulation, topo II and MRP expression were all changed at the earliest stage of resistance development of GLC4 cells upon adriamycin selection.

Predictive Value for Treatment Outcome in Acute Myeloid Leukemia of Cellular Daunorubicin Accumulation and P-Glycoprotein Expression Simultaneously Determined by Flow Cytometry

To evaluate the clinical relevance of multidrug resistance (MDR) phenotype, the intracellular daunorubicin accumulation (IDA) and P-glycoprotein (P-gp) expression were investigated in 87 adult patients with acute leukemia: 69 patients with de novo acute myeloid leukemia (AML), 10 with AML at relapse, and eight with secondary leukemia to myelodysplastic syndromes (MDS-AML). IDA and P-gp expression were determined by double-labeling flow cytometry analysis. Of 87 patients, 36 expressed P-gp (41%). P-gp expression was more frequently observed in AML at relapse and MDS-AML as compared with de novo AML (P = .0001). P-gp expression was significantly associated with CD34 expression (P = .0003) and chromosome 7 abnormalities (P = .027). A significantly reduced IDA was observed in P-gp+ as compared with P-gp- patients (P = .0007). Of the 87 patients, 51 achieved complete remission (CR). A reduced IDA was observed in patients in failure as compared with patients in CR (22% +/- 17% v 42% +/- 21%; P = 10(-4). Twelve of 36 P-gp+ patients as compared with 40 of 51 P-gp- patients achieved CR (33% v 78%; P = 10(-4). The prognostic value of IDA and P-gp expression was confirmed in multivariate analysis. These data suggest that the determination of IDA and P-gp expression may be useful in designing therapy for patients with AML.

Modification by brefeldin A, bafilomycin A1 and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) of cellular accumulation and intracellular distribution of anthracyclines in the non-P-glycoprotein-mediated multidrug-resistant cell line COR-L23/R.

We have investigated the effects of H(+)-ATPase inhibitors, bafilomycin A1 and 7-chloro-4-nitro-benz-2-oxa-1,3 diazole (NBD), and the Golgi inhibitor, brefeldin A, on daunorubicin accumulation and doxorubicin intracellular distribution in the non-P-glycoprotein-mediated multidrug-resistant cell line COR-L23/R. This cell line overexpress a 190 kDa protein which is probably the product of the MRP gene and shows an anthracycline accumulation defect and a drastically altered intracellular anthracycline distribution from the parental cell line COR-L23/P. We found that all three agents could selectively increase the cellular accumulation of daunorubicin in resistant cells. However, these effects were only seen at doses of the modifiers which were equal to or greater than the IC50 of the modifier alone. Effects of the modifiers on the intracellular distribution of doxorubicin fluorescence could, however, be seen at doses lower than those required to produce significant effects on daunorubicin accumulation. However, when used in a continuous MTT chemosensitivity assay none of the agents, used at maximum non-toxic doses, was able to sensitise COR-L23/R cells to doxorubicin or to colchicine. Although these lead compounds are unlikely to be useful as clinical modifiers, development of more selective analogues may prove useful in the modification of non-P-glycoprotein-mediated multidrug resistance.

Effects of verapamil on the cellular accumulation of daunorubicin in blast cells and on the chemosensitivity of leukaemic blast progenitors in acute myelogenous leukaemia.

Verapamil, a calcium channel blocker, was studied for its effects on the cellular daunorubicin (DNR) accumulation in blast cells and on the sensitivity of the blast progenitors to DNR in 30 acute myelogenous leukaemia (AML) patients. Using flow cytometry, verapamil was shown to increase the accumulation of DNR in blast cells. The effect was more prominent in the patients who showed poorer response to chemotherapy including DNR. The per cent increases of DNR content by verapamil were 6.4 +/- 6.3% and 19.5 +/- 23.1% in the 16 responders and the 14 nonresponders, respectively (P less than 0.05). The data suggest the presence of enhanced efflux of DNR in nonresponders. Marked variation in the effects of verapamil among nonresponders suggests the heterogeneity of the mechanisms of drug resistance involved. Verapamil also enhanced the sensitivity of blast progenitors to DNR. The degree of increase of cellular DNR accumulation by verapamil correlated with the degree of increase in chemosensitivity of blast progenitors (nonresponders, P less than 0.005; responders, P less than 0.05). We conclude that enhanced efflux of DNR in blast progenitors may be related to remission induction failure in at least some of resistant AML patients.

A rapid and sensitive flow cytometric method for the detection of multidrug-resistant cells.

Multidrug-resistant (MDR) cells are characterized by a defect in drug accumulation caused by activity of an energy-dependent rapid drug efflux pump. The action of this drug pump can be inhibited by specific agents, referred to as membrane transport modulating agents (MTMAs), resulting in a restoration of the intracellular drug accumulation. This paper presents a flow cytometric assay for the detection of MDR cells, which is based on the ability of these cells to respond to MTMAs. Daunorubicin net-uptake kinetics were measured of anthracycline-sensitive (A2780/S) and -resistant (A2780/R) human ovarian carcinoma cells in vitro. A2780/R cells accumulated significantly less (about a factor of 5) daunorubicin as compared to A2780/S cells. Addition of verapamil or cyclosporin A to A2780/R cells at steady-state daunorubicin uptake led to a dose-dependent increase in cellular daunorubicin accumulation. The sensitivity of the assay was determined by testing mixtures of A2780/S and A2780/R cells. Analysis of A2780/S cells contaminated with A2780/R cells showed that as few as 2.5% MDR cells could readily be detected in the mixture. In conclusion, this functional assay enables the detection of MDR cells in a heterogeneous cell suspension and is ideally suited for the study of the occurrence of typical MDR in human cancer.

Effect of cyclosporin A on daunorubicin accumulation in multidrug-resistant P388 leukemia cells measured by real-time flow cytometry.

We investigated the mode of action of cyclosporin A (Cy-A) as a modifier of multidrug resistance in P388 mouse leukemia cells. A fluorescence-activated flow cytometer (FCM) was modified with a flow-through cuvette to allow continuous on-line monitoring of daunorubicin uptake in vitro. The addition of Cy-A to multidrug-resistant P388/R cells at steady-state daunorubicin uptake, led to a dose-dependent increase in cellular daunorubicin accumulation, as measured by FCM and high-performance liquid chromatography (HPLC). A linear relationship was found between the daunorubicin concentration in the incubation medium and the Cy-A concentration required for optimal stimulation of cellular anthracycline accumulation. The results of a cytotoxicity assay indicated that Cy-A completely restored the chemosensitivity of the P388/R cells. Intracellular Cy-A measurements in P388/S and P388/R cells showed that P388/R cells accumulated significantly less Cy-A than P388/S cells. Relatively high daunorubicin concentrations could not restore that accumulation defect. These results suggest that Cy-A promotes cellular anthracycline accumulation by competing for an outward drug-transport system that operates in multidrug-resistant cells.

Cyclosporin A reverses vincristine and daunorubicin resistance in acute lymphatic leukemia in vitro.

The development of drug resistance by tumor cells is a major obstacle to the cure of human malignancy. Cyclosporin A (CsA) completely reverses primary resistance to vincristine and cross resistance to daunorubicin in a pleiotropic drug-resistant subline of human T cell acute lymphatic leukemia. This subline is over 50-fold resistant to vincristine and fivefold resistant to daunorubicin. CsA has little effect on vincristine or daunorubicin activity in drug-sensitive parental leukemia and corrects daunorubicin resistance without altering cellular daunorubicin accumulation.