Expression of multidrug resistance proteins in retinoblastoma

Abstract

Aim: To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance.

Methods: Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells.

Results: Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC.

Conclusion: Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

Keywords: chemotherapy; multidrug resistance; multidrug resistance associated proteins; retinoblastoma.

Figure 1. Cell culture of Y79/vincristine cells (B), Y79/etoposide cells (C), Y79/carboplatin cells (D) and compared with Y79 cells (A)

Cells were observed under a phase-contrast microscope (Magnification ×200).

Figure 2. Effect of vincristine (A), etoposide (B) and carboplatin (C) on cell viability

Primary cultures of enucleated eyes after chemotherapy were treated with vincristine etoposide and carboplatin respectively in a dose and time dependent manner. Cell viability was determined by MTT assay. Experiments were done twice in triplicate. Values represent mean±SD cell viability as percentage of untreated control samples.

Figure 3. Western blot analysis of p53 (A) and Bcl-2 (B) in Y79 cells (lane 1) and Y79/vincristine cells (lane 2), Y79/etoposide cells (lane 3), and Y79/carboplatin cells (lane 4); Western blot analysis of p53 (C) and Bcl-2 (D) in primary cultures of primarily enucleated eyes (lanes 1&2) and in primary cultures of enucleated eyes after chemotherapy (lane 3&4)

Cell lysates were resolved by SDS-PAGE, and proteins were immunoblotted and detected using specific antibodies.

Figure 4. Western blot analysis of P-gp (A), Mrp-1 (B) and Lrp (C) in Y79 cells (lane 1) and Y79/vincristine cells (lane 2), Y79/etoposide cells (lane 3), and Y79/carboplatin cells (lane 4)

Cell lysates were resolved by SDS-PAGE, and proteins were immunoblotted and detected using specific antibodies.

Figure 5. Western blot analysis of P-gp (A), Mrp-1(B) and Lrp (C) in primary cultures of primarily enucleated eyes (lanes 1&2) and in primary cultures of enucleated eyes after chemotherapy (lanes 3&4)

Cell lysates were resolved by SDS-PAGE, and proteins were immunoblotted and detected using specific antibodies.