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Increased vinblastine binding to membrane vesicles from multidrug-resistant KB cells
Abstract:
Human KB carcinoma cells resistant to high levels of colchicine, vinblastine, vincristine, adriamycin, and actinomycin D exhibit reduced accumulation of these structurally unrelated chemotherapeutic agents (Akiyama, S.-I., Fojo, A., Hanover, J. A., Pastan, I., and Gottesman, M. M. (1985) Somatic Cell Mol. Genet. 11, 117-126; Fojo, A., Akiyama, S.-I., Gottesman, M. M., and Pastan, I. (1985) Cancer Res. 45, 3002-3007). To examine the mechanism of reduced drug accumulation in these cells, we measured [3H]vinblastine ([3H]VBL) binding to membrane vesicles made from drug-sensitive (KB-3-1), drug-resistant (KB-C4), and revertant (KB-R1) cells. Membrane vesicles from KB-C4 cells bound up to 8-fold more [3H]VBL than vesicles from the parental KB-3-1 or revertant KB-R1 cell lines. No difference in binding of [3H]dexamethasone, to which the cells are equally sensitive, was observed. The difference in [3H]VBL binding by vesicles from resistant and sensitive cells was eliminated by the addition of 10 micrograms/ml verapamil, which is known to reverse the multidrug-resistance phenotype. Drug binding by KB-C4 vesicles was osmotically insensitive, temperature-dependent, and trypsin-sensitive. Binding of [3H]VBL by KB-C4 vesicles was inhibited by vinblastine, vincristine, and daunomycin (in decreasing order). Dexamethasone at 100 microM, colchicine at 100 microM, and actinomycin D at 100 microM did not significantly inhibit [3H]VBL accumulation. No significant differences in tubulin content were detected among vesicles from sensitive and resistant cells. These data demonstrate that membrane vesicles from multiply drug-resistant cells bind increased amounts of vinblastine.
Insights
Multidrug-resistant cancer cells show reduced accumulation of chemotherapy drugs. Membrane vesicles from these resistant cells bind significantly more vinblastine, indicating a mechanism for drug resistance.
Area of Science:
- Molecular Biology
- Cancer Research
- Pharmacology
Background:
- Multidrug resistance (MDR) in cancer cells involves reduced intracellular accumulation of various chemotherapeutic agents.
- Structurally unrelated drugs like colchicine, vinblastine, and adriamycin are often effluxed by resistant cells.
- Understanding the molecular mechanisms of MDR is crucial for improving cancer treatment efficacy.
Purpose of the Study:
- To investigate the mechanism behind reduced drug accumulation in multidrug-resistant human KB carcinoma cells.
- To quantify the binding of [3H]vinblastine ([3H]VBL) to membrane vesicles from drug-sensitive and drug-resistant cell lines.
- To explore the characteristics of [3H]VBL binding in resistant cells and its modulation by verapamil.
Main Methods:
- Preparation of membrane vesicles from drug-sensitive (KB-3-1), drug-resistant (KB-C4), and revertant (KB-R1) human KB carcinoma cell lines.
- Measurement of [3H]vinblastine ([3H]VBL) binding to these membrane vesicles.
- Assessment of [3H]dexamethasone binding to rule out non-specific uptake differences.
- Evaluation of the effect of verapamil on [3H]VBL binding.
- Analysis of drug binding characteristics (osmotic sensitivity, temperature dependence, trypsin sensitivity) and inhibition by other drugs and tubulin content.
Main Results:
- Membrane vesicles from multidrug-resistant KB-C4 cells exhibited up to an 8-fold increase in [3H]VBL binding compared to sensitive KB-3-1 or revertant KB-R1 cells.
- No difference in [3H]dexamethasone binding was observed between sensitive and resistant cells.
- The increased [3H]VBL binding in resistant cells was reversed by verapamil, a known modulator of MDR.
- [3H]VBL binding was osmotically insensitive, temperature-dependent, and trypsin-sensitive, and inhibited by vinblastine, vincristine, and daunomycin.
- No significant differences in tubulin content were detected between cell lines.
Conclusions:
- The data demonstrate that membrane vesicles from multidrug-resistant cells bind increased amounts of vinblastine.
- This increased binding suggests a specific mechanism involving the drug-binding site within the cell membrane, potentially related to drug efflux pumps.
- The findings contribute to understanding the molecular basis of vinblastine resistance in cancer cells.