Abstract
We have associated pharmacological studies to a semi-quantitative evaluation of P-glycoprotein(s) expression, to establish if classical multidrug resistance (MDR) could account for the complete resistance phenotype exhibited by progressively doxorubicin-resistant rat glioblastoma cells. Three resistant variants (C6 0.001, C6 0.1 and C6 0.5) of the C6 glioblastoma cell line (C6 S) were selected by long-term culture in the presence of three concentrations of doxorubicin (0.001, 0.1 and 0.5 microgram.ml-1 respectively). The degree of doxorubicin resistance was respectively 7, 33 and 400, and all the cell variants were cross-resistant to m-AMSA, etoposide and vincristine. Doxorubicin incorporation was reduced similarly in all resistant cells, irrespective of the level of resistance. When exposed to their respective doxorubicin IC50, the 7-fold resistant cells had the same intracellular drug incorporation as the sensitive cells, whereas the 33-fold and 400-fold resistant cells could incorporate respectively 3.7 and 17 times more drug. The ratio of doxorubicin exposures required for 50% DNA synthesis inhibition and 50% growth inhibition was dependent on the degree of resistance; this ratio was 12.8 in C6 S, 11.6 in C6 0.001, 6.3 in C6 0.1 and 1.8 in C6 0.5. P-glycoprotein(s) overexpression was of the same magnitude as the resistance factor in variants C6 0.001 and C6 0.1, but was lower than resistance factor in variant C6 0.5. Reversal of drug incorporation by verapamil was complete in all resistant cell lines; however, reversal of doxorubicin cytotoxicity was complete only in the 7-fold resistant line and was only partial in the most resistant lines, which remained 10-fold and 20-fold resistant to doxorubicin. These results suggest that classical MDR was the first phenotype selected by doxorubicin in C6 0.001, whereas mechanism(s) of doxorubicin resistance other than classical MDR are added in the most resistant lines.
Highlights
Multidrug-resistance can be reversed to various extents by treatment of cells with the calcium channel blocker verapamil (Tsuruo et al, 1981)
Cross resistance patterns of the cell lines Multidrug resistance character of the C6 variants used in this study was evaluated by the measure of the cross-resistance between doxorubicin, vincristine, m-AMSA and etoposide (Table I)
Cross resistance to the other drugs of the classical multidrug resistance (MDR) profile was of the same order of magnitude as resistance to doxorubicin in C6 0.001 cells and in C6 0.1 cells; resistance to doxorubicin was much higher than resistance to other drugs in C6 0.5 cells
Summary
Multidrug-resistance can be reversed to various extents by treatment of cells with the calcium channel blocker verapamil (Tsuruo et al, 1981). It is clear that verapamil exerts its action by a mechanism that does not involve voltage-gated calcium channels (Huet & Robert, 1988). The circumvention of resistance is associated with increased drug accumulation, probably through interaction of verapamil with the outward drug transporter of resistant cells, P-glycoprotein. Data demonstrating that verapamil inhibits the vinblastine photoaffinity labelling of P-glycoprotein suggest that interaction of this agent with P-glycoprotein could be the biochemical basis for its pharmacological effects in MDR cells (Cornwell et al, 1986). Using photoaffinity analogs of verapamil, Safa (1988) has shown that Pglycoprotein in MDR cells is a specific acceptor for verapamil
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