sdz-psc-833 and Leukemia

This study was conducted to define the maximum tolerated dose (MTD), dose limiting toxicity (DLT), and pharmacokinetics of idarubicin when administered with and without the P-glycoprotein inhibitor PSC-833 in combination with cytarabine, and etoposide. Fifteen patients with relapsed and refractory acute leukemia were enrolled and received cytarabine as a 7-day continuous infusion, with etoposide and idarubicin administered for any three consecutive days during the cytarabine infusion. Two hours prior to the second dose of idarubicin, PSC-833 administration was initiated. The pharmacokinetics of idarubicin alone and with PSC-833 was assessed at three idarubicin dose levels (6, 8 and 10 mg/m(2)). The MTD of idarubicin in this combination was 8 mg/(m(2) day) with a DLT of oral mucositis. The complete remission rate (on an intent-to-treat basis) for this regimen was 33%, with a median duration of 6 months. The clearance of idarubicin was 140 +/- 200 and 181 +/- 94.3 l/h for idarubicin alone and with PSC-833, respectively. The volume of distribution of the central compartment was 423 +/- 443 and 337 +/- 394 l for idarubicin alone and in combination with PSC-833, respectively. This combination including PSC-833 was well tolerated. Although a pharmacokinetic interaction might have been expected, PSC-833 did not significantly alter the disposition of idarubicin.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Cyclosporins; Cytarabine; Drug Interactions; Drug Resistance, Neoplasm; Etoposide; Female; Humans; Idarubicin; Leukemia; Male; Maximum Tolerated Dose; Middle Aged; Treatment Outcome

Cancer cells that escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics, and stable isotope labeling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared with sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid β-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies.

Topics: 3-Hydroxyacyl CoA Dehydrogenases; Acetyl-CoA C-Acyltransferase; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carbon-Carbon Double Bond Isomerases; Cell Line, Tumor; Cyclosporins; Daunorubicin; Drug Resistance, Neoplasm; Drug Synergism; Enoyl-CoA Hydratase; Fatty Acids; Glutamine; Glycolysis; Humans; Leukemia; Metabolome; Oxidation-Reduction; Pantothenic Acid; Perhexiline; Racemases and Epimerases; RNA, Messenger; Transcriptome

A vinblastine resistant cell line, KCVB2, was established by co-selecting the parental erythroleukemic cell line K562 with step-wise increased concentrations of vinblastine (Velban) in the presence of the cyclosporin D analogue PSC 833 (2 microM), a potent modulator of the multidrug resistance phenotype. KCVB2 cells are 8-fold resistant to the selecting agent, vinblastine, but also exhibit significant resistance to other vinca alkaloids, including 14-fold resistance to vinorelbine, as well as 6-fold cross-resistance to paclitaxel. Doubling time and morphology were similar to the parental K562 cells. Rt-PCR analysis revealed no alterations in the expression of the mdr1 and MRP genes. Intracellular vinblastine accumulation was unchanged. Disruption of the mitotic spindles and multiple mitotic asters occurred in both cell lines but required higher concentrations of vinblastine in KCVB2 cells than in K562 cells. Significant differences were observed in the tubulin content of KCVB2 cells: reduction of total tubulin content, increased polymerized fraction of total tubulin, and overexpression of class III beta-tubulin which is expressed at very low levels in the parental K562 cells. K562 cells transfected with murine class III beta-tubulin did not display the resistance pattern observed in KCVB2 cells. Revertants of KCVB2 manifested reversion to parental drug sensitivity, an increase in total tubulin level, and a decrease in polymerized tubulin. In conclusion, the KCVB2 cell line displays a novel mechanism of resistance to both depolymerizing and stabilizing microtubule-targeted cytotoxins which does not involve altered cellular drug accumulation, but corresponds to alterations in the total tubulin content and polymerization status, and may involve an effect on microtubule dynamics.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclosporins; Drug Resistance, Neoplasm; Genes, MDR; Humans; Leukemia; Microtubules; Mitosis; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, Protein; Spindle Apparatus; Transfection; Tubulin; Vinblastine

Inappropriate expression of the multidrug resistance (MDR1) gene encoding the P-glycoprotein (Pgp) has been frequently implicated in resistance to different chemotherapeutic drugs. We have previously generated chronic myeloid leukemia (CML) cell lines resistant to the tyrosine kinase inhibitor imatinib mesylate (STI571), and one line (LAMA84-r) showed overexpression not only of the Bcr-Abl protein but also of Pgp. In the present study, we investigated this phenomenon in other cell lines overexpressing exclusively Pgp. Thus, cells from the K562/DOX line, described as resistant to doxorubicin due to MDR1 gene overexpression, grew continuously in the presence of 1 microM imatinib, but died in 4 to 5 days if the Pgp pump modulators verapamil or PSC833 were added to the imatinib-treated culture. Analysis of cell proliferation by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay confirmed the differential sensitivity of K562/DOX to imatinib, which was also reversed by verapamil or PSC833. Flow cytometric analysis of the total phosphotyrosine content by intracytoplasmic staining after a 2-hour incubation with escalating doses of imatinib showed that the inhibitory concentrations of 50% (IC(50)) for inhibition of cellular protein tyrosine phosphorylation were 15, 10, and 5 microM for K562/DOX, K562/DOX plus verapamil, and K562, respectively. Retroviral-mediated transfection of the BCR-ABL(+) AR230 cell line with the MDR1 gene decreased its sensitivity to imatinib, an effect that was also reversed by verapamil. The possible role of MDR overexpression in clinical resistance to imatinib remains to be defined. We therefore confirm that imatinib should be added to the extensive list of drugs that can be affected by the MDR phenomenon.

Topics: Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzamides; Cell Death; Cell Division; Colony-Forming Units Assay; Cyclosporins; Doxorubicin; Drug Resistance, Neoplasm; Flow Cytometry; Gene Expression; Genes, MDR; Humans; Imatinib Mesylate; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Phosphotyrosine; Piperazines; Pyrimidines; Transfection; Tumor Cells, Cultured; Verapamil

Overexpression of a 170kD membrane glycoprotein, P-glycoprotein (Pgp), which acts as an energy dependent efflux pump for cytotoxic drugs is believed to be one of the factors that is responsible for clinical drug resistance. Recent studies suggest that Pgp is also responsible for the intracellular transport of cholesterol from the plasma membrane to the endoplasmic reticulum. Leukemic cells from patients with acute myelogenous leukemia have an elevated uptake of low density lipoprotein (LDL) when compared with white blood cells from healthy individuals. Since elevated LDL receptor expression and multidrug resistance are both common events in leukemic cells, we investigated LDL receptor expression in sensitive and drug resistant human leukemic cell lines. We found a 2- to 10-fold higher uptake of LDL in five out of five drug resistant K562 cell lines. All three drug resistant HL60 cell lines studied also had higher uptake than the parental cells. The LDL receptor expression in vincristine resistant Pgp positive K562 cells was less sensitive to downregulation by sterols than in parental cells. There was no selective effect of the Pgp inhibitor PSC-833 or other Pgp modulators on LDL receptor activity in Pgp positive cells. Since also resistant Pgp, multidrug resistance protein 1, and breast cancer resistance protein negative cells exhibited an elevated LDL receptor activity, we conclude that overexpression of these proteins is not the mechanism behind the elevated LDL uptake in the drug resistant leukemic cell lines. The findings are of interest for the concept of using lipoproteins as carriers of cytotoxic drugs in cancer treatment.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Cell Line; Cholesterol; Cyclosporins; Drug Resistance, Neoplasm; Humans; Hydroxymethylglutaryl CoA Reductases; Leukemia; Lipoproteins, LDL; Membrane Fluidity; Receptors, LDL; Sterols; Tumor Cells, Cultured; Verapamil

Circumvention of chemoresistance in cancer may involve several modulator drugs with high affinity for the multidrug transporter P-glycoprotein (Pgp), which is expressed in a number of multi-resistant malignancies. Pgp acts as a membrane efflux pump with broad substrate specificity including antineoplastic drugs and endogenous substances such as certain cytokines and sphingolipids. Therefore, the consequence of Pgp blockade could be far more complex than intracellular drug retention. In the present study exposure of the Pgp inhibitor, PSC 833 (1200 ng/ml), to Pgp expressing KG1a/200 human leukemia cells provoked cell cycle arrest and apoptosis in vitro. This finding was put to test in vivo using a xenotransplant model of KG1a/200 human cells intravenously inoculated into non-obese diabetic severe combined immunodeficient (NOD-SCID) mice. The animals were randomly allocated to receive treatment with PSC 833 (n = 32) or placebo (n = 24). PSC 833 (30 mg/kg) was subcutaneously injected six or 12 times separated by 48-96 h. The overall mean whole blood concentration of PSC 833 was 1191 +/- 60 ng/ml (s.e.m.) at 20 h after administration. Tumor engraftment was significantly reduced in the treatment group (P = 0.037), which also had prolonged survival compared to control animals (P = 0.0016). This is the first study that demonstrates antileukemic effects of a Pgp inhibitor as single agent therapy in vivo, and the present data raise the possibility of alternative exploitation of modulators in cancer chemotherapy.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Survival; Cyclosporins; Drug Evaluation, Preclinical; Drug Resistance, Multiple; Graft Survival; Humans; Leukemia; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Survival Rate; Transplantation, Heterologous

A convenient functional assay of the multidrug resistance (MDR) pump is useful for the diagnosis of MDR-1 cancers and the quantitative determination of the potency of inhibitors of the pump. Calcein-AM, a substrate of the MDR pump, was used to determine the concentration of SDZ PSC833 needed to completely inhibit the pump in CEM/VLB100 drug-resistant cells. The initial rates (in percent) for calcein retention by these MDR-1 cells were used to calculate values for the percent initial efflux of calcein-AM through the MDR pump in the presence of the inhibitors PSC833, cyclosporinA, and dexniguldipine. The percent efflux values at 250 and 60 nM calcein-AM were used to calculate the required concentration of each inhibitor to produce half-inhibition (I50) of initial efflux through the pump. These results are consistent with a noncompetitive inhibition of the MDR pump by each of the three inhibitors.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Cyclosporine; Cyclosporins; Dihydropyridines; Drug Resistance, Neoplasm; Fluoresceins; Half-Life; Humans; Inhibitory Concentration 50; Kinetics; Leukemia; Lymphocytes; Molecular Weight; Tumor Cells, Cultured

The multidrug transporter P-glycoprotein (Pgp), which is frequently overexpressed in multidrug resistant leukemia, has many proposed physiological functions including involvement in transmembraneous transport of certain growth-regulating cytokines. Therefore, we studied cell growth of three pairs of drug resistant and sensitive leukemia cell lines (KG1a, K562 and HL60) exposed to three different inhibitors of Pgp. The resistant KG1a and K562 sublines, which expressed high levels of Pgp, responded to low doses of the cyclosporin SDZ PSC 833, the cyclopeptolide SDZ 280-446, and the cyclopropyldibenzosuberane LY335979 with a dose-dependent growth inhibition. In the resistant variants of KG1a and K562 cells the mean half-maximal growth inhibitory doses (GI50) of SDZ PSC 833 were 312 (SE 41) and 414 (SE 50) nM, those of SDZ 280-446 were 685 (SE 51) and 578 (SE 54) nM, and those of LY335979 were 66 (SE 1) and 48 (SE 8) nM, respectively. Exposure to 1 microM SDZ PSC 833 resulted in tetraploidization, cytokinesis failure and apoptosis of the KG1a and K562 MDR variants. Conversely, parental cells with no or low levels of Pgp and the non-Pgp resistant variant of HL60 cells were not receptive to these cytotoxic effects. We conclude that inhibition of Pgp may exercise selective cytotoxicity in Pgp-rich leukemia cells indicating a possible therapeutic target in multiresistant leukemia.

Topics: Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cyclosporins; Daunorubicin; Dibenzocycloheptenes; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Leukemia; Peptides, Cyclic; Phenotype; Quinolines; Tumor Cells, Cultured

Multidrug resistance (MDR) to anti-cancer agents is frequently associated with overexpression of the drug efflux transporter P-glycoprotein (Pgp) in cancer cells, ensuing drug expulsion and maintenance of tolerable intracellular levels of certain cytotoxic drugs. Pgp may also be present in normal tissue, providing protection against toxic substances, but the physiological role of Pgp is not fully understood. Recently, it was shown that Pgp also takes part in the transport of certain growth-regulating cytokines (Drach et al, 1996; Raghu et al, 1996). Therefore, we studied the effect of the highly potent Pgp inhibitor PSC 833 on proliferation of three pairs of MDR and parental human cell lines (HB8065 hepatoma cells, KG1a and K562 leukaemia cells). The MDR phenotypes were characterized by Pgp overexpression, which was demonstrated by flow cytometry using the anti-Pgp antibody MRK16. Electronic cell counting of 72-96 h cultures revealed a dose-dependent antiproliferative effect of PSC 833 in the resistant KG1a/200 and K562/150 cells. The half-maximal growth inhibitory concentrations (GI50) were 0.2 microM and 0.7 microM respectively. Exposure to PSC 833 induced cell death by apoptosis in both cell types, as revealed by flow cytometry and detection of 3'-hydroxy ends of DNA (the result of DNA fragmentation associated with apoptosis), by terminal transferase-mediated dUTP-biotin nick end-labelling (TUNEL). Similar effects were not found in the hepatoma cell lines or the parental leukaemia lines. These results demonstrated a discriminating cytotoxicity of PSC 833 in two human leukaemia MDR variants, representing a possible therapeutic indication which warrants consideration during the ongoing clinical evaluation of this drug.

Topics: Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Hepatocellular; Cell Division; Cyclosporins; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Flow Cytometry; Fluorescent Antibody Technique; Humans; Leukemia; Tumor Cells, Cultured

In the present study it was investigated whether and by which mechanisms the co-administration of interleukin-3 (IL-3) and the P-glycoprotein blocker PSC 833 can augment mitoxantrone (MX) and daunorubicin (DAU) cytotoxicity in two human growth factor dependent P-glycoprotein (P-gp) positive myeloid leukemic cell lines, Mo-7 and GF-D8. Cytotoxicity was determined in MTT assay. Increased cytotoxicity occurred in Mo-7 cells preincubated with 24h IL-3 followed by 1 h MX (cell survival: 85% +/- 6 vs 68% +/- 2, at 0.05 microM MX, P < 0.005) or DAU (79% +/- 8 vs 62% +/- 9 at 0.8 microg/ml DAU, P < 0.05). Similar results were obtained for the GF-D8 cell line. In this cell line, at 0.5 microM MX the cell survival decreased from 84% +/- 13 to 61% +/- 19 (P < 0.05) and at 5.0 microg/ml DAU from 102% +/- 8 to 69% +/- 5, (P < 0.002). The IL-3 administration did not affect the P-gp and bcl-2 protein expression, cellular MX concentration or MX efflux but coincided with an increased percentage of cells in S-phase and topoisomerase II (topo II)-alpha mRNA and topo II activity especially in the Mo-7 cell line. PSC 833 enhanced DAU cytotoxicity in both cell lines. The administration of IL-3 plus PSC 833 in the Mo-7 cell line resulted in an additive effect on DAU cytotoxicity. At 0.8 microg/ml DAU and 2 microg/ml PSC 833, the percentage surviving cells decreased from 62% +/- 9 in the absence of IL-3 to 37% +/- 3 in the presence of IL-3 (P < 0.01). The additive effect of combined treatment was most pronounced in GF-D8 cells which also had the highest P-gp expression. In contrast, PSC 833 did not modulate the MX effects, irrespective of the presence of IL-3. In summary, the results demonstrate that the combined administration of IL-3 and PSC 833 can enhance the cytotoxic effects of DAU but not MX in these P-gp positive cell lines whereas the effects of MX could be modulated by factors which influence topo II activity.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Survival; Cyclosporins; Daunorubicin; Dose-Response Relationship, Drug; Drug Interactions; Drug Resistance, Multiple; Drug Synergism; Humans; Interleukin-3; Kinetics; Leukemia; Mitoxantrone; Tumor Cells, Cultured

Pharmacologically active in vivo doses of P-glycoprotein (Pgp) blockers, specifically verapamil, Cremophor EL and PSC833 cause toxicity in addition to that from the concomitantly used cancer chemotherapeutic drugs. It was shown before that these blockers cause different types of toxicities in vivo. We found that these 3 chemically distinct Pgp blockers exert different biophysical effects on the membranes of L1210 MDR cells. They also affect the general metabolism of these cells differently, but all block affinity labeling of Pgp. We could also show that the combination of suboptimal doses of these blockers can restore the uptake of the Pgp substrate rhodamine 123 into L1210MDR, 3T3MDR and KB-VI cells and can reduce the survival rate of these cells when treated in combination with daunorubicin. Our results suggest that the combination of suboptimal doses of these Pgp blockers may be advantageous in clinical practice.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Calcium Channel Blockers; Cell Division; Cell Membrane; Cyclosporins; Glycerol; Humans; Leukemia; Transfection; Tumor Cells, Cultured; Verapamil

Recent studies have shown that cyclosporin A (CsA) may affect ricin A-chain immunotoxin (RTA-IT) therapy. In this study, we evaluated the ability of CsA and its nonimmunosuppressive analog, SDZ PSC 833, to enhance anti-CD5 T101 RTA-ITs in vitro. Both 4 mumol/L CsA and 4 mumol/L SDZ PSC 833 significantly and specifically enhanced the cytotoxic activity of T101 RTA-IT on the human lymphoblastic T-cell line, CEM III (101-fold and 105-fold, respectively). Furthermore, these Cs also enhanced the cytotoxicity of the more potent T101 F(ab')2 RTA-IT (ninefold and eightfold, respectively). The effect of human plasma, originating from four patients enrolled in a phase I high-dose CsA regimen, was examined on T101 RTA-IT cytotoxicity on CEM III cells. In each case, with plasma CsA levels between 3,090 and 4,860 ng/mL (2.5 to 4 mumol/L), a significant increase in T101 RTA-IT-mediated cytotoxicity was observed ranging from 31% to 60%. Neither CsA nor SDZ PSC 833 affected the rate of RTA-IT binding, internalization, intracellular trafficking, or degradation. Analysis of internalized T101 RTA-IT molecules showed that these were essentially intact, which suggests that these enhancers may act only on a small population of RTA-ITs that escapes present investigational techniques. In conclusion, because the concentrations used are clinically achievable, Cs appear to be promising agents for in vivo enhancement of RTA-ITs.

Topics: Antigens, CD; CD5 Antigens; Cell Line; Cyclosporine; Cyclosporins; Drug Synergism; Humans; Immunotoxins; Leukemia; Protein Biosynthesis; Ricin; T-Lymphocytes

In the clinical therapy of cancer, resistance to many cytostatic drugs is a major cause of treatment failure. Among other mechanisms, the expression and pumping activity of P-glycoprotein (PGP) in the membrane of resistant cancer cells is responsible for the reduced uptake of cytostatics. The blockade or inhibition of PGP activity by chemosensitisers seems to be a tenable way to restore sensitivity to antineoplastic drugs and therapeutic efficacy. In the present work the influence of the new chemosensitiser dexniguldipine on rhodamine-123 accumulation in multidrug-resistant leukaemia cells was investigated. Dexniguldipine increases cellular rhodamine-123 accumulation dose-dependently.pEC50 values (-log concentration of drug showing a half maximal effect) in accumulation studies are dependent on pH of the test system and are in the range of 6.5 (pH 7.2) to 7.2 (pH 8.0) for dexniguldipine. In comparison with other chemosensitisers such as SDZ PSC 833, cyclosporin A, verapamil, dipyridamole, quinidine and amiodarone, dexniguldipine is the most potent drug in this test system. In addition to equilibrium measurements of rhodamine-123 accumulation, efflux of rhodamine-123 was analysed in the absence and presence of chemosensitisers. A clear dose-dependency was seen and, moreover, a dramatic decrease in efflux rates was achieved in the presence of chemosensitisers. The described system can be used to investigate PGP-mediated drug transport on a pharmacological and biochemical basis.

Topics: Antineoplastic Agents; Cyclosporins; Dihydropyridines; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Humans; Hydrogen-Ion Concentration; Leukemia; Rhodamine 123; Rhodamines; Tumor Cells, Cultured