Abstract
Purpose: :
The resistance of tumors to chemotherapeutics has been described as being a function of their multidrug resistant protein pumps. Two of these pumps are P–glycoprotein (P–gp) and the multi–drug resistance associated protein–1 (MRP1). The aim of this project was to determine the proportion of multidrug resistance in retinoblastoma cell lines that is due to each pump. Recent studies have shown that treatment with 17–allylamino–17–demethoxygeldanamycin (17AAG), an inhibitor of heat shock protein 90, inhibited the function of P–gp. We investigated the ability of 17AAG to inhibit the function of P–gp in these retinoblastoma cell lines.
Methods: :
Three separate studies were performed. In the first, the MultiDrugQuant Assay (Chemicon) was used on the retinoblastoma cell lines (WERY–1 and Y79). This assay uses an inhibitor to block both MDR–1 and MRP–1 and one that blocks only MRP1. Flow cytometry was then used to determine the fluorescence remaining in the cell after efflux. The second part of the study involved using the Multidrug resistance direct dye efflux assay (Chemicon) with the Retinoblastoma cell lines. A time course study using DiCO3 at half hour, one hour and a three hour time points was done to establish the optimal time for efflux. DiCO3, is a dye which is specifically effluxed by P–gp. Vinblastine, a competitive inhibitor of P–gp was added to one set of samples and another set was incubated at 4°C, which inactivates the pump and is used as a control. In the third portion, the Multi–drug Resistance Direct Dye Efflux Assay was used in combination with 17AAG
Results: :
Using the MultiDrugQuant Assay we found that P–gp accounted for approximately 100% of the dye efflux. During the time course experiment the cells incubated for 3 hours showed the most efflux. Vinblastine inhibited efflux of DiCO3 at levels comparable to control at thirty minutes. Although, at the one hour and three hour time points there was a significant drop in the inhibitory effects of Vinblastine. 17AAG inhibited P–gp activity at higher levels than control after three hours of incubation.
Conclusions: :
P–gp is the major cause of multi–drug resistance in these retinoblastoma cell lines. Resistance to Vinblastine gradually increased until inhibition of P–gp was completely lost at the three–hour time point. In comparison, 17AAG not only maintained inhibition of P–gp at three hours, but exceeded control levels. This indicates that 17AAG may be used as a potential inhibitor of P–gp to increase the effectiveness of chemotherapeutic agents in retinoblastoma patients.
Keywords: retinoblastoma • drug toxicity/drug effects