azidopine and Breast-Neoplasms

Modulators of P-glycoprotein (P-gp) are often themselves transported out of cells, thereby limiting their effectiveness. It may be possible to develop more effective modulators of multidrug resistance by designing drugs that irreversibly block the function of P-gp. Therefore, we studied the effect of the mustard derivatives of fluphenazine (FPN) and trans-flupenthixol (FPT) on P-gp function. Both fluphenazine-mustard (FPN-M) and trans-flupenthixol-mustard (FPT-M) possessed alkylating activity, as assayed using 4-(p-nitrobenzyl) pyridine. Multidrug-resistant MCF-7/AdrR cells were incubated with FPN or FPN-M, or FPT or FPT-M for 1 h, washed for varying number of times in phosphate-buffered saline (PBS), then resuspended in medium containing [3H]vinblastine (VBL), and assayed for steady-state accumulation of the drug. Washing had far less of an effect on the ability of FPN-M and FPT-M to increase VBL accumulation compared to their parent compounds. After eight washes in excess PBS, the cells initially exposed to FPN or FPT accumulated only 30% and 50% of the initially accumulated drug, whereas the FPN-M- or FPT-M-treated cells accumulated approximately 75% and 90% of the control, respectively. FPN-M and FPT-M also increased the uptake and decreased the efflux of VBL from MDR cells despite repeated washing. We also examined the effects of these modulators on sensitivity of MDR cells to cytotoxic agents. FPN-M and FPT-M sensitized MCF-7/AdrR cells to VBL and doxorubicin to a greater extent than their parent compounds. These studies point out the potential of "irreversible" P-gp modulators to produce prolonged chemosensitization.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Breast Neoplasms; Dihydropyridines; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Flupenthixol; Fluphenazine; Humans; Tumor Cells, Cultured; Vinblastine

The predominant characteristics of multidrug resistant (MDR) cancer cells are broad spectrum resistance to chemotherapeutic agents and a pronounced defect in intracellular accumulation of the drugs, in association with overexpression of the drug efflux pump P-glycoprotein. Protein kinase C (PKC) phosphorylates the linker region of P-glycoprotein. Evidence has been presented that the isozyme PKC-alpha may contribute to the drug resistance phenotype of human breast cancer MCF7-MDR cells, PKC-alpha is markedly overexpressed in MCF7-MDR cells, and artificial overexpression of PKC-alpha in MCF7 constructs that overexpress P-glycoprotein significantly enhances the MDR phenotype of the cells in association with increased P-glycoprotein phosphorylation. Verapamil, cyclosporin A, and a number of other agents that compete with cytotoxic drugs for binding sites on P-glycoprotein can potently reverse MDR, but this is accompanied by severe toxicity in vivo. In this report, we demonstrate that an N-myristoylated peptide that contains a sequence corresponding to the pseudosubstrate region of PKC-alpha (P1) partially reverses multidrug resistance in MCF7-MDR cells by a novel mechanism that involves inhibition of PKC-alpha. P1 and two related PKC inhibitory N-myristoylated peptides restored intracellular accumulation of chemotherapeutic drugs in association with inhibition of the phosphorylation of three PKC-alpha substrates in MCF7-MDR cells: PKC-alpha, Raf-1 kinase, and P-glycoprotein. A fourth N-myristoylated peptide substrate analog of PKC, P7, did not affect drug accumulation in the MCF7-MDR cells and failed to inhibit the phosphorylation of the PKC-alpha substrates. The effects of P1 and verapamil on drug accumulation in MCF7-MDR cells were additive. P1 did not affect P-glycoprotein expression. MCF7-MDR cells were not cross-resistant to P1, which suggest that the peptide was not transported by P-glycoprotein. Furthermore, P1 was distinguished from MDR reversal agents such as verapamil and cyclosporin A by its inability to inhibit [3H]azidopine photoaffinity labeling of P-glycoprotein. P1 actually increased [3H] azidopine photoaffinity labeling of P-glycoprotein in MCF7-MDR cells, providing evidence that the effects of P1 on P-glycoprotein in MCF7-MDR cells are not restricted to inhibition of the phosphorylation of the pump. P1 may provide a basis for developing a new generation of MDR reversal agents that function by a novel mechanism that involves inhibition of PKC

Topics: Affinity Labels; Amino Acid Sequence; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Breast Neoplasms; Dihydropyridines; Drug Resistance, Multiple; Female; Humans; Isoenzymes; Molecular Sequence Data; Myristic Acid; Myristic Acids; Oligopeptides; Phosphorylation; Protein Kinase C; Protein Kinase C-alpha; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Tumor Cells, Cultured

We have performed isobologram analyses of the ability of tamoxifen (TAM) to alter the response to Adriamycin (ADR) and vinblastine (VBL) in human breast cancer cells. MCF-7 cells express functional receptors for estrogen and progesterone but do not express detectable levels of M(r) 170,000 glycoprotein (gp170). CL 10.3 and MCF-7ADR cells are MCF-7 variants which express gp170. CL 10.3 but not MCF-7ADR cells express functional steroid hormone receptors. Tamoxifen (1-2.5 microM) interacts synergistically with ADR and VBL in CL 10.3 and MCF-7ADR cells. TAM increases the cytotoxicity of VBL and ADR and the intracellular levels of [3H]VBL by approximately 2-3-fold. TAM also prevents the binding of [3H]azidopine to gp170. The ability of TAM to concurrently increase the cytotoxic effects of ADR and VBL, increase VBL accumulation, and inhibit the binding of azidopine to gp170 strongly implies that the synergistic effects of TAM are mediated through its effects on gp170. TAM produces an antagonistic to additive interaction with ADR and VBL in MCF-7 cells, and at high concentrations (5 microM) the synergy apparent in CL 10.3 and MCF-7ADR cells is lost. While TAM clearly has a significant potential for use as a chemosensitizing agent, the design of clinical trials may require careful consideration.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Breast Neoplasms; Carrier Proteins; Dihydropyridines; Dose-Response Relationship, Drug; Doxorubicin; Drug Interactions; Drug Resistance; Female; Humans; Membrane Glycoproteins; Phenotype; Tamoxifen; Tumor Cells, Cultured; Vinblastine