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Relationship of VP-16 to the classical multidrug resistance phenotype
M Sehested1, E Friche, P B Jensen
1Department of Pathology, Sundby Hospital, Copenhagen, Denmark.
Abstract:
The classical multidrug resistance (MDR) phenotype is characterized by cross-resistance between a number of chemically unrelated drugs due to an increased efflux across the plasma membrane via a P-glycoprotein-mediated mechanism. The epipodophyllotoxin derivatives etoposide (VP-16) and teniposide (VM-26) are usually included among the drugs recognized by this MDR phenotype, and the MDR EHR2/DNR cell line is greater than 50-fold cross-resistant to VP-16. The steady-state accumulation of VP-16 in EHR2/DNR cells is only half that of wild-type EHR2 cells, and deprivation of energy by sodium azide surprisingly increased accumulation to a similar extent in both sublines. Efflux was rapid (halflife of 32-35 s) and similar in both sublines, while initial influx was markedly lower in the resistant cells. The temperature coefficients over 10 degrees C for VP-16 in- and efflux indicated passive transport in both sublines. In agreement with this finding, up to 10-fold molar excess (50 microM) VM-26 had no effect on VP-16 accumulation in MDR cells. VP-16 at a 100-fold molar excess inhibited azidopine photoaffinity labeling of P-glycoprotein by only 30% and vincristine binding to plasma membrane vesicles from EHR/DNR cells by 45%. However, VP-16 itself did not differentially bind to plasma membrane vesicles from EHR2 and EHR2/DNR cells. Finally, neither VP-16 accumulation nor cytotoxicity in EHR2/DNR cells were increased to the same degree as for daunorubicin and vincristine by verapamil, and the modulation was similar in wild-type and resistant cells. Thus, although VP-16 may be a substrate for P-glycoprotein, its other transport characteristics such as rapid diffusion and sensitivity to membrane perturbation in wild-type cells lessen any effect of P-glycoprotein-mediated efflux, resulting in a lack of differential modulation by verapamil. These results may be considered when planning clinical trials involving MDR modulators and epipodophyllotoxin derivatives.
Insights
Multidrug resistance (MDR) involves P-glycoprotein efflux, but etoposide (VP-16) resistance is complex. VP-16 transport is primarily passive, limiting P-glycoprotein
Area of Science:
- Pharmacology
- Cell Biology
- Biochemistry
Background:
- Classical multidrug resistance (MDR) is mediated by P-glycoprotein, leading to cross-resistance against various drugs.
- Epipodophyllotoxin derivatives, etoposide (VP-16) and teniposide (VM-26), are typically substrates for P-glycoprotein.
- The EHR2/DNR cell line exhibits significant cross-resistance to VP-16, exceeding 50-fold.
Purpose of the Study:
- To investigate the mechanisms underlying etoposide (VP-16) resistance in the MDR EHR2/DNR cell line.
- To determine the role of P-glycoprotein in VP-16 transport and accumulation.
- To assess the interaction of VP-16 with P-glycoprotein and its modulation by MDR modulators like verapamil.
Main Methods:
- Comparative analysis of VP-16 accumulation and efflux in wild-type EHR2 and MDR EHR2/DNR cells.
- Assessment of VP-16 transport kinetics, including influx and efflux rates, and temperature dependence.
- Evaluation of VP-16 interaction with P-glycoprotein using photoaffinity labeling and drug binding assays; assessment of verapamil's effect on VP-16 accumulation and cytotoxicity.
Main Results:
- VP-16 accumulation was reduced in resistant cells, but energy deprivation affected both cell types similarly.
- VP-16 efflux was rapid and similar in both cell lines, while influx was lower in resistant cells, suggesting passive transport.
- VP-16 showed limited inhibition of P-glycoprotein labeling and vincristine binding; verapamil had minimal differential effect on VP-16 accumulation or cytotoxicity in resistant cells.
Conclusions:
- Etoposide (VP-16) resistance is not solely mediated by P-glycoprotein-dependent efflux.
- VP-16's transport characteristics, including rapid diffusion and sensitivity to membrane changes, diminish the impact of P-glycoprotein.
- These findings have implications for clinical strategies involving MDR modulators and epipodophyllotoxin derivatives.