sdz-psc-833 and Leukemia--T-Cell

Among the mechanisms that induce multidrug resistance (MDR), one of those most frequent is over-expression of a phosphoglycoprotein (Pgp) encoded in the mouse by the mdr-1 and mdr-3 genes. We have demonstrated that cyclosporin-A (CsA) as well as its analogue PSC 833 were able to revert the MDR phenotype in murine cell lines resistant to vincristine (LBR-V160) or doxorubicin (LBR-D160). The aim of this work was to evaluate the ability of PSC 833 and CsA to modulate mdr-1, mdr-3 and mrp-1 genes as well as to induce apoptosis analyzing the mechanism involved in the above tumor cell lines. By semi-quantitative RT-PCR, we demonstrated that mdr-3 was over-expressed in both resistant lines while mdr-1 was over-expressed only in LBR-V160; in contrast, mrp-1 expression was not evidenced in any of the cell lines. After treatment with 0.1 microg ml(-1) of either PSC 833 or CsA, LBR-V160 showed no changes in mdr-1 but decreased mdr-3 expression, while LBR-D160 failed to display any modification in the expression of these genes. Apoptosis was evidenced by fluorescence microscopy, S minuscule accumulation and agarose gel electrophoresis. Our results demonstrated that CsA (1 microg ml(-1)) was able to induce apoptosis in all cell lines: 18.31% (+/-4.46) for LBR-, 25.96% (+/-5.24) for LBR-V160 and 27.36% (+/-4.12) for LBR-D160, while PSC 833 (1 microg ml(-1)) only induced apoptosis 21.51% (+/-5.73) in LBR-V160 cell line. The expression of Bcl-2 family proteins (Bcl-2, Bax and Bcl-x(L)) was analyzed by flow cytometry showing high expression of the three proteins which was not significantly modified after treatment with either PSC 833 or CsA on the sensitive as well as on the resistant cell lines. Single stranded conformation polymorphisms analysis of p53 (Trp53) gene in the cell lines showed no mutation in exons 5-8 of the tumor suppressor gene. We conclude that depending on the concentration used, PSC 833 and CsA may act either by modulating the mdr-3 gene (0.1 microg ml(-1)) or by direct impact on the cells through induction of apoptosis (1 microg ml(-1)), in the latter case through a mechanism that might act independent of the Bcl-2 family proteins.

Topics: Animals; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Transporters; bcl-2-Associated X Protein; bcl-X Protein; Cell Cycle; Cyclosporine; Cyclosporins; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Exons; Gene Expression Regulation, Leukemic; Genes, bcl-2; Genes, MDR; Genes, p53; Leukemia, T-Cell; Mice; Mice, Inbred BALB C; Neoplasm Proteins; Phenotype; Polymorphism, Single-Stranded Conformational; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Vincristine

Multidrug resistance (MDR) lines from a murine T-cell leukemia were selected in increasing vincristine (VCR) or doxorubicin (DOX) concentrations. Daunorubicin (DNR) efflux was evidenced after 25 additional passages with constant 160 ng ml(-1) of either VCR or DOX, an effect that was inhibited by verapamil, cyclosporin-A (CsA) and PSC 833. The expression of Pgp was not evidenced in the resistant cell lines using anti-human Pgp antibodies. Cell proliferation assay showed that cell lines resistant to VCR (LBR-V160) or DOX (LBR-D160) required higher doses of either drug to produce GI50 compared with control cell line obtained after culture in the absence of VCR or DOX. When resistant cell lines were maintained during 60 days in the absence of either VCR or DOX, MDR phenotype reversal was obtained in LBR-D160 while LBR-V160 remained resistant to the drug, as shown by cell proliferation assays and by drug efflux pump functionality. When VCR or DOX were used together with either CsA or PSC 833, the latter was more effective to produce reversal of resistance than the former, whereas CsA presented greater cytotoxic effect than PSC 833 for sensitive and resistant cells. Cross-resistance was found between VCR, DOX and other antineoplasic agents on murine leukemic cell line. VCR was more effective to induce MDR since the resistant cell lines were more stable to the MDR phenotype.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Cyclosporine; Cyclosporins; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Leukemia, T-Cell; Mice; Phenotype; Tumor Cells, Cultured; Vincristine

P-glycoprotein is a cellular efflux pump. The P-glycoprotein inhibitor PSC 833 causes apoptosis of cancer cells and induces a rise in the intracellular levels of ceramide. Our aims were to determine whether a cause and effect relationship exists between these two actions of PSC 833, and to assess whether the PSC 833-induced apoptosis is restricted to transformed cells. Apoptosis was determined by flow cytometry and radioactive quantitation of DNA fragmentation. PSC 833 induced apoptosis in the human T leukemia cell lines: Molt-4 and Jurkat. Analysis of the apoptosis in Molt-4 and Jurkat cells revealed that PSC 833 induced a rise in the cellular ceramide levels (as measured by the DG kinase assay). PSC 833-induced apoptosis was significantly reduced by specific inhibitors of ceramide de novo synthesis (i.e., fumonisin B1 and L-cycloserine). On the other hand, PSC 833 did not induce apoptosis in normal peripheral blood T cells regardless of whether these cells were quiescent, activated, or proliferating. Our results suggest that PSC 833 induces apoptotic death in human transformed T lymphocytes through an increase in ceramide de novo synthesis. In addition, normal lymphocytes are not susceptible to induction of apoptosis by PSC 833. This difference between normal lymphocytes and leukemia cells presents a potential target for chemotherapy.

Topics: Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Transformed; Ceramides; Cycloserine; Cyclosporins; Diacylglycerol Kinase; DNA Fragmentation; DNA, Neoplasm; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Flow Cytometry; Humans; Jurkat Cells; Leukemia, T-Cell; T-Lymphocytes; Tumor Cells, Cultured

P-glycoprotein(P-gp)- related resistance is one of the major obstacles in treating leukemia patients. Therefore, it is of clinical interest to find new potential modulators and compare their P-gp-modulating efficacy. The present analysis investigated the influence of P-gp modulators, such as verapamil, tamoxifen, droloxifene E, droloxifene Z, SDZ PSC 833 (PSC 833) and dexniguldipine in a leukemic T-cell line (CCRF-CEM) and its P-gp-resistant counterparts (CCRF-CEM/ACT400 and CCRF-CEM/VCR1000). P-gp expression was assessed with an immunocytological technique using the monoclonal antibody 4E3.16. It was characterized as the percentage of P-gp positive cells and also expressed as a D value by using the Kolmogorov Smirnov statistic. The efficacy of P-gp modulators was determined with the rhodamine-123 accumulation test and the MTT test. An in vitro modulator concentration between 0.1 microM and 3 microM was determined, where no genuine antiproliferative effect was apparent. The modulators PSC 833 and dexniguldipine were the significant (p

Topics: Antibodies, Monoclonal; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Cell Division; Cyclosporins; Dihydropyridines; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Humans; Leukemia, T-Cell; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Tamoxifen; Tumor Cells, Cultured; Vault Ribonucleoprotein Particles; Verapamil

P-glycoprotein- and multidrug resistance-associated protein (MRP)-mediated multidrug resistance is associated with decreased drug accumulation. The P-glycoprotein-expressing CCRF-CEM/VLB100 subline and the MRP-expressing CCRF-CEM/E1000 subline are both 50-fold resistant to daunorubicin. However, accumulation of daunorubicin and rhodamine 123 was > 85% reduced in the P-glycoprotein-expressing subline compared to 40-50% in the MRP-expressing subline. Further, the CCRF-CEM/E1000 cells were 30-fold resistant to idarubicin, without reduced accumulation. Verapamil and SDZ PSC 833 restored daunorubicin and rhodamine 123 accumulation, while buthionine sulphoximine affected only the CCRF-CEM/ E1000 subline. We conclude that the verapamil associated change in rhodamine 123 accumulation provides a sensitive functional assay for both P-glycoprotein- and MRP-mediated MDR.

Topics: Antibiotics, Antineoplastic; Antimetabolites, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Buthionine Sulfoximine; Calcium Channel Blockers; Cyclosporins; Daunorubicin; Humans; Idarubicin; Leukemia, T-Cell; Multidrug Resistance-Associated Proteins; Rhodamine 123; Rhodamines; Tumor Cells, Cultured; Verapamil

Cyclosporin A (CSA) exhibits greater multidrug resistance (MDR) modulating activity in vitro than other MDR modulators such as verapamil and quinidine. However, the immunosuppressive and nephrotoxic effects of CSA may limit its clinical use. PSC 833, a new cyclosporin D derivative, exerts a higher MDR reversal activity but lacks toxic or immunosuppressive effects. The drug-resistant sublines K/DAU100, K/DAU200, K/DAU300, K/DAU400, K/DAU500 and K/DAU600 have been derived from the drug-sensitive parental cell line, K562 cl.6 and CEM/VLB100 is a drug-resistant derivative of CCRF-CEM. We report a comparison of the effects of PSC 833 and CSA on daunorubicin (DAU) transport kinetics and chemosensitivity in these cell lines. Both CEM/VBL100 and K562 cl.6 DAU-resistant cells displayed high levels of P-glycoprotein (PGP), decreased DAU accumulation and increased DAU efflux when compared to their parental cells. PSC 833 was 1.6-, 3.4-, 4.9- and 4.6-fold more effective than CSA in reversing DAU resistance in higher resistance CEM/VLB100, K/DAU400, K/DAU500 and K/DAU600 cells respectively. DAU transport kinetics showed that PSC 833 was more effective than CSA in increasing cellular DAU accumulation and decreasing DAU efflux in higher resistant leukaemia subclones. PSC 833 could restore DAU retention at lower doses and was more active than CSA in all the resistant cells. A 89-100% restoration of intracellular DAU retention were gained by PSC 833 at 1.0 microM in K562 cl.6 DAU-resistant sublines, whereas a 73-100% restoration of DAU retention was obtained by CSA only at 30.0 microM in the same resistant sublines. PSC 833 at 3.0 microM is sufficient to restore full DAU retention in all resistant cells. CSA, however, even at 30.0 microM, cannot confer full restoration of DAU retention in higher resistance K562 cl.6/DAU sublines. By measuring MDR modulator-mediated short-term inhibition of PGP function, PSC 833 was found to be at least 10-30 times more active than CSA. As no effect on DAU retention and sensitivity has been found in sensitive parental cells with PSC 833, it is suggested that PSC 833 may act by blocking the effluxing function of PGP in the resistant leukaemia cells.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Survival; Cyclosporine; Cyclosporins; Daunorubicin; Drug Resistance, Multiple; Fluorescent Antibody Technique; Humans; Leukemia, Erythroblastic, Acute; Leukemia, T-Cell; Tumor Cells, Cultured

The P-glycoprotein (Pgp) molecules which are expressed on multidrug resistant (MDR) tumor cells can efflux a variety of cytostatics. In both normal and tumoral epitheliums, Pgp molecules are selectively expressed on the apical surface of the epithelial cells. Such a distribution seems to be responsible for the transcellular transport of Pgp substrates, including cyclosporin A (CsA), from the basal to the apical side. Some normal lymphoid cells also express small amounts of Pgp molecules, for as yet unknown functions. Nevertheless, the sensitivity of their mitogen-induced proliferation to cytostatics, including doxorubicin and CsA, could be increased by the Pgp blockers. Using isotopically-labeled CsA and tumoral lymphoid cell lines, we now show a higher CsA retention in Pgp-lacking parental ('Par') cells than in Pgp-expressing MDR cells. The Pgp blockers can restore the CsA retention in the MDR cells to its level in the Par cells.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cyclosporine; Cyclosporins; Daunorubicin; Doxorubicin; Drug Resistance, Multiple; Flow Cytometry; Humans; Immunosuppressive Agents; Leukemia, Monocytic, Acute; Leukemia, T-Cell; Mice; Peptides, Cyclic; Rhodamines; Tumor Cells, Cultured; Verapamil; Vinblastine