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P-glycoprotein overexpression cannot explain the complete doxorubicin-resistance phenotype in rat glioblastoma cell
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.
Insights
Multidrug resistance (MDR) in glioblastoma cells involves P-glycoprotein but also other mechanisms at higher resistance levels. Verapamil reversed drug incorporation but not full cytotoxicity in highly resistant cells.
Area of Science:
- Pharmacology
- Cancer Biology
- Molecular Biology
Background:
- Glioblastoma cells can develop resistance to chemotherapy drugs like doxorubicin.
- Multidrug resistance (MDR) is a major challenge in cancer treatment.
- P-glycoprotein is a key transporter involved in MDR.
Purpose of the Study:
- To investigate if classical multidrug resistance (MDR) mediated by P-glycoprotein explains the doxorubicin resistance in rat glioblastoma cells.
- To characterize the mechanisms of resistance in progressively doxorubicin-resistant glioblastoma cell variants.
Main Methods:
- Development of doxorubicin-resistant rat glioblastoma cell lines (C6 0.001, C6 0.1, C6 0.5) through long-term culture.
- Semi-quantitative evaluation of P-glycoprotein expression.
- Assessment of doxorubicin incorporation and cytotoxicity.
- Pharmacological studies using verapamil to reverse drug resistance.
Main Results:
- Resistant cell variants exhibited cross-resistance to other chemotherapy drugs.
- Doxorubicin incorporation was reduced in resistant cells, but less so in highly resistant variants.
- P-glycoprotein overexpression correlated with resistance factor in lower-resistance lines but not the highest.
- Verapamil reversed drug incorporation but only partially reversed cytotoxicity in highly resistant cells.
Conclusions:
- Classical MDR, involving P-glycoprotein, is the primary mechanism in early-stage doxorubicin resistance in these glioblastoma cells.
- Additional, non-classical MDR mechanisms contribute to the high-level resistance observed in the most resistant cell lines.
- Complete reversal of doxorubicin resistance requires targeting both classical and other resistance mechanisms.