Intracellular Daunorubicin Accumulation Research Articles

The New Targeted Nanoparticles: Tf-Peg-PLL-PLGA Combined with Daunorubicin Overcome Hypoxia Induced Drug Resistance of K562 Cells

Drug resistance is a major obstacle in cancer chemotherapy, especially in the treatment of leukemia. Hypoxia is an inseparable component of the solid tumor microenvironment as well as the bone marrow (BM) microenvironment. In this study, we investigated the effect of the new nanoparticles polyethylene glycol (PEG)-poly L-lysine (PLL)-poly lactic-co-glycolic acid (PLGA) (PEG-PLL-PLGA) modified by transferrin (Tf) combined with daunorubicin (DNR) on K562 cells under hypoxia. We found the IC50 of DNR and DNR-PEG-PLL-PLGA in K562 cells increased under hypoxia, while the IC50 of DNR-Tf-PEG-PLL-PLGA in K562 cells decreased under hypoxia according to the results of MTT assay. The apoptosis of cells and the intracellular concentration of DNR were detected by flow cytometry (FCM). From the datas, we found that the new targeted nanoparticles can overcome hypoxia induced drug resistance of K562 cells. By targeting the transferrin receptor (TfR) on the surface of K562 cells, the new targeted drug led to an increase in intracellular accumulation of DNR. Transcription and Protein of P-glycoprotein (P-gp) and TfR increased in K562 cells under hypoxia, which probably had a relationship with the decreased degradation of the Hypoxia-Inducible Factor-1α (HIF-1α). We further detected the levels of HIF-1α, Bcl-2, Bax and Caspase-3 of K562 cells in transcription and protein under hypoxia regulated by DNR, DNR-PEG-PLL-PLGA and DNR-Tf-PEG-PLL-PLGA, respectively. DNR-Tf-PEG-PLL-PLGA had the best effect when compared with other groups. Furthermore, intravenous injection of DNR-Tf-PEG-PLL-PLGA expressed the best effect of suppressing proliferation of K562 cells injected to nude mice among the three drugs. Therefore, the new drug DNR-Tf-PEG-PLL-PLGA may be a potential cancer therapy for human leukemia. DisclosuresNo relevant conflicts of interest to declare.

Involvement of c-Jun N-terminal kinase in reversal of multidrug resistance of human leukemia cells in hypoxia by 5-bromotetrandrine

5-Bromotetrandrine (BrTet), a candidate multidrug resistance (MDR) modulator, is a potential compound for use in cancer therapy when combined with anticancer agents such as daunorubicin (DNR) and paclitaxel. The purposeof this study was to investigate the mechanism of reversal of P-glycoprotein (P-gp)-mediated MDR by BrTet and the involvement of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway in both adriamycin-sensitive K562 and adriamycin-resistant K562 (KA) leukemia cells in hypoxia. The combination of BrTet and DNR decreased both phosphorylated JNK1/2 and MDR1/P-gp levels under hypoxic conditions. Furthermore, a pharmacological inhibitor of JNK, SP600125, or small interfering RNA (siRNA) oligonucleotides to both JNK1 and JNK2 reversed BrTet- or DNR-induced JNK phosphorylation and MDR1/P-gp levels. We further demonstrated that the decreased JNK phosphorylation and MDR1/P-gp levels were associated with a significant increase in intracellular accumulation of DNR, which dramatically enhanced the sensitivity of drug-resistant KA cells to DNR, and led to cellular apoptosis through activation of the caspase-3 pathway. It is concluded that using BrTet in combination with other chemotherapeutic agents and pharmacological inhibitors of JNK can abrogate the P-gp-induced MDR in adriamycin-resistant K562 cells, which has potential clinical relevance in cancer therapy for chemotherapeutic-resistant human leukemia.

Induction of dendritic cells with multidrug resistance from K562/MDR1 cells

To induce the differentiation of K562/MDR1 cells into dendritic cells (DC) with multidrug resistance property. K562/MDR1 cells and K562 cells were cultured in the presence of GM-CSF and IL-4 to generate DC and matured by TNF-α. On d14 K562/MDR1-DC and K562-DC cells were harvested and the expressions of CD1a, CD83, CD80, CD86, HLA-ABC and HLA-DR were assessed by flow cytometry (FCM). The antigen presentation function of K562/MDR1-DC and K562-DC was determined by allogenic mixed lymphocyte reaction (Allo-MLR). The expression of P-glycoprotein and the intracellular accumulation of daunorubicin (DNR) were detected by FCM. The sensitivity of K562/MDR1-DC and K562-DC cell to vincristine, adriamycin was measured using MTT assay. Both K562/MDR1 and K562 cells were differentiated into dendritic cells in the presence of cytokine cocktails, showing the morphologic and immunophenotypic characteristics of DC. K562/MDR1-DC more markedly enhanced proliferation of allogeneic lymphocytes in MLR than K562-DC. High level expression of P-glycoprotein and efflux of DNR were demonstrated in K562/MDR1-DC. K562/MDR1-DC showed multidrug resistance property, with higher IC(50) to VCR and ADM than that of K562-DCs. K562/MDR1 cells can be differentiated into DC with the presence of cytokines, the induced K562/MDR1-DC cells express high level of P-glycoprotein and acquire the multidrug resistance property.

Liposomal encapsulation of a synergistic molar ratio of cytarabine and daunorubicin enhances selective toxicity for acute myeloid leukemia progenitors as compared to analogous normal hematopoietic cells

To evaluate the possibility of improved selective killing of acute myeloid leukemia (AML) cells with CPX-351 (a liposomal formulation of cytarabine and daunorubicin). CPX-351 and the same molar ratio of free drugs were compared for cytotoxicity against colony-forming cells (CFCs) and subpopulations of cells enriched for primitive progenitors from AML patients and normal granulocyte colony-stimulating factor-mobilized peripheral blood (PB) and bone marrow (BM) donors. AML blasts (n = 13) and normal PB and BM cells (n = 7) were incubated for 24 hours in various concentrations of CPX-351 or free drugs before plating in CFC assay or staining with anti-CD34 and anti-CD38 antibodies, Annexin-V, and propidium iodide followed by fluorescence-activated cell sorting analysis. High performance liquid chromatography was used to measure intracellular daunorubicin accumulation. AML blasts and progenitors from patients who achieved complete remission were more sensitive to both CPX-351 and free drugs than the same cells from patients with chemotherapy refractory leukemia. However, AML CFCs and CD34(+)CD38(-) AML blasts (enriched for candidate leukemia stem cells) from the same patient showed similar sensitivity to the liposomal or free drug formulations. In contrast, CFCs and CD34(+)CD38(-) cells from normal PB and BM were fivefold more sensitive to the free drugs than to CPX-351. Consistent with these observations, preferential intracellular accumulation of CPX-351 in AML over normal cells was observed, while there was little difference in drug uptake between AML and normal cells with the free drug cocktail. CPX-351, as compared to free cytarabine:daunorubicin, shows enhanced selective in vitro cytotoxicity for AML rather than normal progenitors.

Apoptotic mechanism of human leukemia K562/A02 cells induced by magnetic iron oxide nanoparticles co-loaded with daunorubicin and 5-bromotetrandrin

The purpose of this study was to assess the induced apoptosis of self-assembled iron oxide magnetic nanoparticles (MNPs) co-loaded with daunorubicin (DNR) and 5-bromotetrandrin (Br Tet) (DNR/Br Tet-MNPs), acting as a drug depot system for the sustained release of the loaded DNR and BrTet, in the drug resistant human leukemia K562/A02 cells and further to explore potential mechanisms. After being incubated for 48 hours, K562/A02 cells were treated with DNR/Br Tet-MNPs or DNR and Br Tet in solution (DNR/Br Tet-Sol). Morphologic characteristics of K562/A02 cells were observed under a fluorescence microscope; cell apoptosis and intracellular accumulation of DNR were analyzed by FACS Calibur flow cytometry. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting analyses were performed to study the apoptosis associated gene transcription and protein expression, respectively. Typical apoptotic characteristics, including chromatin condensation and fragmentation of nuclei, were observed and a high rate of apoptosis was detected in K562/A02 cells treated with DNR/Br Tet-MNPs and DNR/Br Tet-Sol. Detection of relative fluorescence intensity of intracellular DNR demonstrated that intracellular DNR was higher in K562/A02 cells treated with DNR/Br Tet-MNPs than that of DNR/Br Tet-Sol. Further study demonstrated that both DNR/Br Tet-MNPs and DNR/Br Tet-Sol reduced the gene transcriptions and protein expressions of bcl-2 and survivin and enhanced that of bax and caspase 3. It is concluded that self-assembled DNR/Br Tet-MNPs, as one of the potential antitumor agents for hematologic malignancies, may effectively induce apoptosis of K562/A02 cells through elevating the ratio of bax/bcl-2, activating caspase 3, and inactivating survivin.

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.

Probing Cellular Binding of Dendrofullerene by in‐situ Electrochemical Contact Angle Measurement

AbstractDendrofullerene (C60DF) is a novel fullerene derivative with potential and promising biomedical applications. In this work, electrochemical/contact angle behavior of C60DF in the cellular system has been explored by in‐situ electrochemical contact angle measurement. This measuring system is a newly developed technique which can provide electrochemical and contact angle detection simultaneously. The electrochemical results indicate that dendrofullerene may effectively bind and permeate the tumor cell membrane and then distribute into the cancer cells. Our observations of in‐situ electrochemical contact angle measurement also illustrate that the permeation and interaction of C60DF with target cancer cells may lead to some variation of the configurational structure of the relative cell membrane and thus result in the change of hydrophilic/hydrophobic properties of target cellular system. Furthermore, through confocus fluorescence microscopy study we found that, upon application of C60DF, the intracellular accumulation of anticancer drug daunorubicin in leukemia K562 cells could be remarkably enhanced by C60DF. Therefore fullerene derivatives were demonstrated to be a good candidate that can play an important role in improving the intracellular drug uptake in the target cancer cells.

Biological Evaluation, Structure−Activity Relationships, and Three-Dimensional Quantitative Structure−Activity Relationship Studies of Dihydro-β-agarofuran Sesquiterpenes as Modulators of P-Glycoprotein-Dependent Multidrug Resistance

Multidrug resistance (MDR) is one of the main challenges in the chemotherapy of cancer, malaria, and other important diseases. Here, we report the inhibitory activity of a series of 76 dihydro-beta-agarofuran sesquiterpenes, tested on NIH-3T3 cells expressing the human P-glycoprotein (Pgp) multidrug transporter, to establish quantitative comparisons of their respective abilities to block the drug transport activity. The screening was performed on the basis of the ability of sesquiterpenes to modulate the intracellular accumulation of the classical Pgp substrate daunorubicin. To understand the structural basis for inhibitory activity and guide the design of more potent Pgp inhibitors, we have performed a three-dimensional quantitative structure-activity relationship model using the comparative molecular similarity indices analysis (CoMSIA). The most salient features of these requirements are in the region of the substituents at the C-2, C-3, and C-8 positions, which seem to be critical for determining the overall effectiveness of sesquiterpenes as Pgp inhibitors.

The small GTPases Rab5 and RalA regulate intracellular traffic of P-glycoprotein

P-glycoprotein (P-gp) is a plasma membrane glycoprotein that can cause multidrug resistance (MDR) of cancer cells by acting as an ATP-dependent drug efflux pump. The regulatory effects of the small GTPases Rab5 and RalA on the intracellular trafficking of P-gp were investigated in HeLa cells. As expected, overexpressed enhanced green fluorescent protein (EGFP)-tagged P-gp (P-gp-EGFP) is mainly localised to the plasma membrane. However, upon cotransfection of either dominant negative Rab5 (Rab5-S34N) or constitutively active RalA (RalA-G23V) the intracellular P-gp-EGFP levels increased approximately 9 and 13 fold, respectively, compared to control P-gp-EGFP cells. These results suggest that Rab5 and RalA regulate P-gp trafficking between the plasma membrane and an intracellular compartment. In contrast, coexpression of constitutively active Rab5 (Rab5-Q79L) or dominant negative RalA (RalA-S28N) had no effect on the localisation of P-gp-EGFP. Furthermore, the intracellular accumulation of daunorubicin, a substrate for P-gp, increased significantly with an increased intracellular localisation of P-gp-EGFP. These results imply that it may be possible to overcome MDR by controlling the plasma membrane localisation of P-gp.

STI571 combined with vincristine greatly suppressed the tumor formation of multidrug-resistant K562 cells in a human-nude mice xenograft model

The development of the targeted signal transduction inhibitor STI571 has prompted us to treat chronic myeloid leukemia in different ways. Since STI571 may reverse multidrug-resistance of K562/MDR cells in vitro, we studied the effect of STI571 on multidrug-resistant K562 cells in vivo. Multidrug-resistant human leukemia cell line K562-n/VCR expresses both bcr/abl fusion gene and multi-drug resistance (mdr1) gene. It is a vincristine resistant cell line subcloned from the vincristine (VCR) sensitive cell line K562-n induced by vincristine in vitro. K562-n and K562-n/VCR cells were inoculated subcutaneously into both sides of nude mice breast (5 x 106 cells/each) to establish a human leukemia xenograft model. The incidence and volume of tumor were observed. In the tumor-bearing nude mice, anti-tumor drugs vincristine, daunorubicin (DNR), STI571, and STI571 plus VCR for the treatment of mdr1 and bcr/abl double positive leukemia were studied respectively. The tumor incidence was 100% in the nude mice inoculated with either K562-n or K562-n/VCR. The transcription of the mdr1 gene and expression of P-gp were negative in K562-n cells but positive in K562-n/VCR cells. The intracellular accumulation of DNR in K562-n cells was higher than that in K562-n/VCR cells (P < 0.05). The tumor incidence of K562-n/VCR cells in nude mice was much higher than that of K562-n cells in chemotherapy groups, and the mean volume of the tumors was also larger (P < 0.05). STI571 combined with VCR significantly suppressed the proliferation of K562-n/VCR cells. The MDR characteristics of K562-n/VCR in vivo were the same as in vitro. STI571 had a significant tumor-suppressing effect on VCR-sensitive leukemia cells and a moderate effect on MDR leukemia cells. VCR combined with STI571 had an excellent tumor-suppressing effect on both K562-n/VCR and K562-n in the human-nude mice xenograft model.

Dibenzocyclooctadiene lingnans: a class of novel inhibitors of P-glycoprotein

To determine if five dibenzocyclooctadiene lingnans, a class of naturally occurring compounds from Schisandra chinensis (Turcz.) Baill, have the activities to reverse P-glycoprotein (P-gp) mediated multidrug resistance (MDR). The IC(50)s of four MDR cell lines (K562/Adr, MCF-7/Adr, KBv200, and Bcap37/Adr) toward daunorubicin, vincristine, and paclitaxel in the presence or absence of one of the dibenzocyclooctadiene lingnans were determined by a FACscan assay. The intracellular daunorubicin accumulation in the four MDR cell lines was determined by incubation of cells with daunorubicin (2 microg/ml) in the presence or absence of one of the dibenzocyclooctadiene lingnans by a FACscan assay. The interaction of the five dibenzocyclooctadiene lingnans with P-gp was assayed by their inhibition of (3)H-azidopine photoaffinity labeling of P-gp. Among the five lingnans, while schisandrin A and B, and schisantherin A demonstrated strong and comparable activities to reverse the drug resistance and the intracellular drug accumulation in four MDR cell lines, schisandrol A and B showed very limited activities. The poor activities of schisandrol A and B are possibly caused by the hydroxyl groups on the cyclooctadiene ring, because the activities of the molecules resumed when the hydroxyl group was esterified to form a benzoate. Further studies demonstrated that these compounds physically interacted with P-gp. Schisandrin A and B, and schisantherin A are potent P-gp inhibitor and is of potential for future clinical application.

Reversal of the multidrug resistance in human hepatocellular carcinoma cells by recombinant adenoviral delivery of the MRP antisense RNA in vitro

To explore the effect of multidrug resistance-associated protein (MRP) antisense RNA mediated by recombinant adenoviruses on reversal of the multidrug resistance in human drug-resistant hepatocellular carcinoma (HCC) cells. The HCC cells were first treated in vitro by the recombinant adenoviruses containing antisense MRP. Chemosensitivity of all of the cells to ADM and DNR was determined by MTT assay. The levels of the MRP mRNA and its product P190 were measured by RT-PCR and flow cytometry (FCM) respectively according to the time after transfection. The accumulation of the DNR in these cells was analyzed by FCM simultaneously. IC50 of the cells infected by Ad-Asmrp to ADM and DNR was 0.487 microg/ml and 0.328 microg/ml respectively, with the factors of resistance (RF) decreased by 36.8 and 35.4 respectively (P < 0.05). Continuous decrease in levels of MRP mRNA and P190 was observed 24 h and 48 h after infection respectively (P < 0.01). The intracellular DNR accumulation was increased simultaneously in the Ad-Asmrp transfectants (P < 0.001). MRP antisense RNA mediated by recombinant adenoviruses could increase chemosensitivity and partially reverse the MDR in HCC cells in vitro by inhibit expression of MRP gene. The study would provide experimental basis for the reversal of MDR in HCC by antisense RNA technology mediated by recombinant adenoviruses.

Clinical Significance of Prognostic Factors Related to Multidrug Resistance and Apoptosis for Prediction of Chemotherapy Outcomes in Acute Leukemia.

OBJECTIVE: To study the prognostic value of cd34, P-gp and Bcl-2 expression and MDR functional assays together with patients' characteristics in patients with de novo acute leukemia.METHODS: The expression levels of P-gp, CD34, Bcl-2 and the mean fluorescence intensity ratio of rhodamine 123 efflux assay (MIR (RE)) and intracellular daunorubicin accumulation assay (MIR (IDA)) of patients' leukemia blast samples were prospectively determined with flow cytometry. The results of remission (CR) and non-remission (NR) patients were compared. Patients were given standard chemotherapy for remission induction.RESULTS: CR patients were comparable to NR patients in age (39.2±23.0: 44.4±22.8, p=0.568, n=30). But NR patients had significantly higher peripheral white blood cell count than CR patients (72.84±78.74: 21.38±17.08, p=0.022, n=30). On the other hand, leukemia blasts samples of CR patients tend to express lower levels of CD34 (22.35±29.20: 46.18±39.5, p=0.084, n=30), P-gp (2.23±2.06: 11.95±22.90, p=0.111, n=30) and Bcl-2 (46.90±35.11: 57.02±31.97, p=0.434, n=30), but not significantly. However, the MIR (RE) (1.16±0.38 vs. 1.43±0.26, P=0.045, n=24) and MIR (IDA) (1.02±0.05 vs. 1.47±0.44, P =0.005, n=24) were both significantly lower in CR patients than in NR patients.CONCLUSION: Leukemia patients with much higher peripheral WBC count at diagnosis were more difficult to reach remission after induction chemotherapy than patients with lower WBC counts. Age was not influential to chemotherapy results. The determination of MIR value in MDR functional assays is more valuable than expression levels of CD34, P-gp and Bcl-2 in the prediction of chemotherapy outcomes.

Dynamic and intracellular trafficking of P-glycoprotein-EGFP fusion protein: Implications in multidrug resistance in cancer.

In our present study, a P-glycoprotein-EGFP (P-gp-EGFP) fusion plasmid was constructed and functionally expressed in HeLa cells to investigate the intracellular localization and trafficking of P-glycoprotein (P-gp). Using immunocytochemistry and fluorescent confocal microscopy techniques, colocalization studies showed that after transfection, P-gp-EGFP was progressively transported from the endoplasmic reticulum (ER) to the Golgi and finally to the plasma membrane within 12-48 hr. The degree of intracellular accumulation of daunorubicin was related to the particular localization of P-gp-EGFP. Significant daunorubicin accumulation occurred in transfected cells when P-gp-EGFP was localized predominantly within the ER, and accumulation remained high when P-gp-EGFP was mainly localized in the Golgi. However, there was little or no intracellular accumulation of daunorubicin when P-gp-EGFP was localized predominantly on the plasma membrane. Blocking the intracellular trafficking of P-gp-EGFP with brefeldin A (BFA) and monensin resulted in inhibition of traffic of P-gp-EGFP and retention of P-gp-EGFP intracellularly. Intracellular accumulation of daunorubicin also increased in the presence of BFA or monensin. Our study shows that P-gp-EGFP can be used to define the dynamics of P-gp traffic in a transient expression system, and demonstrates that localization of P-gp on the plasma membrane is associated with the highest level of resistance to daunorubicin accumulation in cells. Modulation of intracellular localization of P-gp with agents designed to selectively modify its traffic may provide a new strategy for overcoming multidrug resistance in cancer cells.

P-glycoprotein, lung resistance-related protein and multidrug resistance-associated protein in de novo adult acute lymphoblastic leukaemia.

P-glycoprotein (P-gp), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP) expression, and blast cell intracellular daunorubicin accumulation (IDA) were evaluated in 95 previously untreated cases of adult acute lymphoblastic leukaemia (ALL) using flow cytometry. Forty-five out of 95 (47%) patients were P-gp positive (+), 12/66 (18%) were LRP+ and 11/66 (17%) were MRP+. Eighteen out of 66 (28%) patients showed a simultaneous multidrug resistance (MDR)-related protein expression higher than controls for more than one protein, while 24/66 (36%) cases did not overexpress any protein. Twenty-one out of 24 (87%) cases overexpressing at least one MDR-related protein had a defect in accumulating daunorubicin into their blast cells, while only 4/24 (16%) cases who did not overexpress any protein had similar features. The complete remission rates were similar in MDR-positive and -negative (-) patients but relapses within 6 months were more frequent in P-gp+ cases, and therefore the disease-free survival duration was shorter in P-gp+ than in P-gp- patients (P = 0.01). The number of MRP+ and/or LRP+ cases was too small to be able to draw any conclusion on their role in affecting or predicting therapy outcome. In conclusion, P-gp overexpression associated with a defect in daunorubicin accumulation is a frequent feature in adult ALL at onset and seems to be related to poorer therapy outcome and, consequently, a shorter disease-free survival. LRP and MRP overexpression seems to be a rare event and no conclusion can be drawn on its prognostic role.

P-glycoprotein and multidrug resistance-associated protein, but not lung resistance protein, lower the intracellular daunorubicin accumulation in acute myeloid leukaemic cells.

The in vitro intracellular daunorubicin accumulation (IDA) of blast cells from 69 patients with newly diagnosed acute myeloid leukaemia (AML) was correlated with the expression and functional activity of the multidrug resistance (MDR) proteins, P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP) and lung-resistance protein (LRP). An inverse and significant association was found between IDA and Pgp-related efflux activity (r = -0.31, P = 0.01) and also MRP (r = -0.25, P = 0.04) but not with LRP (r = -0.13, P = 0.28). Coexpression of the MDR proteins had an additive effect in further lowering of IDA levels, suggesting that the clinical MDR phenotype is dependent on the sum of multiple MDR factors available to the leukaemic cell. Thus, the median IDA of leukaemic cells without any MDR proteins was significantly higher than that of blasts carrying two MDR proteins (0.466 vs. 0.296, P = 0.046). Seven patients with no expression of Pgp, MRP and LRP still had low IDA levels, suggesting the presence of efflux MDR mechanisms other than those studied. The relation of IDA to clinical parameters known to be associated with poor prognosis, such as age, secondary AML, karyotype, peripheral blood blast and CD34 counts, was also studied, but no significance was found on multifactorial analysis. There was a non-significant trend for earlier relapse in patients with low IDA levels (leukaemia-free survival of 16.3 months compared with 21.1 months in patients with high IDA levels). Our data suggest that, while the IDA assay is a quick and relatively easy test for the combined efflux MDR phenotype, it is unable to detect other MDR mechanisms, such as LRP, which may be important to the clinical outcome of patients with AML.

Liposome-encapsulated daunorubicin for PGP-related multidrug resistance.

The possibility that Daunoxome (DNX), a combination of daunorubicin (DNR) with a liposomal targeting system, escapes PGP was tested. Two pairs of leukaemic cell lines, each consisting of the parental non-multidrug resistance (MDR) line and of a MDR variant, were studied for cytotoxicity (MTT test) and for cellular DNR kinetic and accumulation (flow cytometry). DNX and free DNR were equally toxic against non-MDR cells, whereas the liposomal anthracycline was more toxic than the free drug against the MDR variant. Non-MDR cells accumulated DNR more rapidly when they were exposed to free DNR than to DNX, but MDR cells accumulated more DNR when they were exposed to DNX. The kinetics of DNX and free DNR were also studied in the blast cells of 41 cases of acute leukaemia and they were found to be related to blast cell PGP expression. In 15 cases with a low PGP expression intracellular DNR accumulation was faster and higher with free DNR than with DNX. In 26 cases with a high PGP expression the area under the curve was similar with DNX and free DNR, but the kinetics of intracellular DNR accumulation showed an early low plateau with free DNR and a slow and continuous increase with DNX. In MDR cell lines the ratio was more favourable to DNX than to free DNR. We conclude that liposome encapsulated DNR is partially protected from PGP and that it is worth testing for the treatment of PGP-positive acute leukaemia.

P-glycoprotein, lung resistance-related protein and multidrug resistance associated protein in de novo acute non-lymphocytic leukaemias: biological and clinical implications.

P-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance associated protein (MRP) expression and the blast cells' intracellular daunorubicin accumulation (IDA) were evaluated in 96 previously untreated cases of de novo acute non-lymphocytic leukaemia (ANLL). 47/96 patients (49%) were classified as PGP+ 44/ 96 (46%) as LRP+, and 8/96 (8%) as MRP+. The more frequent MDR clusters were PGP-/LRP-/MRP- (32/96 cases, 33%) and the PGP+/LRP+/MRP- (27/96 cases, 28%) followed by PGP+/LRP-/MRP- (15/96 cases, 16%) and PGP-/LRP+/MRP- (14/96 cases, 14%). A favourable karyotype was observed more frequently in PGP- and LRP-cases. A highly significant correlation was found between either PGP or LRP overexpression and leukaemic blast cell IDA. All the patients received standard induction and consolidation treatments containing MDR-related (idarubicin, mitoxantrone, etoposide) and other (arabinosyl cytosine) drugs. Multivariate analysis showed that PGP overexpression was significantly associated with a poor response to treatment, both in terms of primary resistance or shorter survival. Other independent prognostic factors were age and cytogenetics. LRP overexpression did not reach statistical significance, although for LRP+ cases the trend was unfavourable. Due to small numbers, no conclusion could be made regarding MRP overexpression, but 5/8 cases showed unfavourable karyotypic abnormalities, 8/8 had a defective IDA and 6/8 failed to achieve remission. This study showed that both PGP and LRP overexpression are common features in de novo ANLL at onset whereas MRP overexpression is more rare. It suggested that overexpression of one of the MDR related proteins was associated with a defective IDA, and confirmed that, in addition to age and cytogenetics, PGP retains an independent prognostic value. It also suggested that LRP did not affect clinical outcome when patients were treated with idarubicin or mitoxantrone and arabinosyl cytosine.

Comparison between Anthracyclines and Rhodamine-123 Accumulation in Chronic Lymphoid Leukemia: Effect of Cyclosporin A and Verapamil

Multidrug resistance in leukemic cells is associated with decreased drug accumulation. A resistant cell line and cells from 11 patients with chronic lymphoid leukemia B were used for the evaluation of intracellular accumulation of daunorubicin (DNR), idarubicin (IDA), epirubicin (EPI) and rhodamine-123 (Rh-123). Cyclosporin A (CSA) and verapamil were used to test their modulatory effects on anthracyclines and the fluorescent dye. In leukemic samples there was a tendency for a lower accumulation index in samples tested with Rh-123 as compared to anthracyclines. IDA was a poorer substrate to P-glycoprotein (Pgp) than two of its analogues, e.g. DNR and EPI. A good correlation (80%) was found between Rh-123 accumulation and Pgp expression by phosphatase-anti-alkaline phosphatase. A strict correlation (100%) was found between modulation by CSA of Rh-123 accumulation and immunoreactivity to Pgp. Two discordant results were seen suggesting that other mechanisms of resistance could be present. The Rh-123 accumulation test seems to give a better indication than anthracyclines, however, it is not selective and may allow the detection of other drug-transport pumps.

P-glycoprotein (PGP) and lung resistance-related protein (LRP) expression and function in leukaemic blast cells.

P-glycoprotein (PGP) lung resistance protein (LRP) and multidrug resistance associated protein (MRP) expressions and function were evaluated by flow cytometry in 65 leukaemic patients (38 acute non-lymphocytic leukaemias, eight acute lymphocytic leukaemias, 19 Ph-positive chronic myeloid leukaemias in blastic phase). By using the MRK-16, the LRP-56 and the MRPm6 MoAbs, 34% of the cases did not over-express any proteins (-); 24.5% over-expressed (+) only PGP, 11% only LRP, 1.5% only MRP, 24.5% both PGP and LRP, and 4.5% both PGP and MRP. The mean intracellular daunorubicin accumulation (IDA) and rhodamine 123 (Rh123) retention in the presence or absence of the reversal agent SDZ PSC 833 (PSC) of the PGP-/LRP-/MRP- cases were comparable to the ones observed in normal leucocytes. With respect to the non-over-expressing cases, the PGP-/LRP+/MRP- cases showed only an impaired IDA (mean 204 +/- 29; P < 0.001). The PGP+/ LRP+/MRP- cases had a defect both in IDA (mean 166 +/- 47, P < 0.001) and Rh123 retention (mean 0.42 +/- 0.14: P < 0.001), which were both corrected by PSC. All the PGP+/LRP+/MRP- cases had a defect in IDA (mean daunorubicin (DNR) accumulation 192 +/- 44; P < 0.001). However, only in 8/16 of them an evident defect in Rh123 retention was found. In conclusion, both PGP and LRP over-expression were common in leukaemia. An impaired IDA was found in all cases over-expressing PGP, LRP or both. The study of Rh123 retention could give incorrect information about the blast cells' ability to accumulate cytotoxic drugs in patients over-expressing both PGP and LRP.