Immune Expression and Inhibition of Hsp90 in Retinoblastoma

Abstract

Purpose:: The up-regulation of heat shock protein 90 (Hsp90), a molecular chaperone, has been described in different neoplasms and is currently being investigated in clinical trials as a potential anticancer target. We have previously shown that an Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), inhibited the function of P-glycoprotein (P-gp) in two human retinoblastoma cell lines (WERY-1 and Y79). P-gp was shown to be the major cause of multi-drug resistance in these cell lines and the observed efficacy of 17-AAG suggests that Hsp90 may stabilize P-gp. Moreover, previous studies have demonstrated that P-gp had higher expression in well-differentiated retinoblastomas, especially those treated by chemotherapy before enucleation. The goal of this study is to examine the immunohistochemical profile of Hsp90 in retinoblastoma and to evaluate the sensitivity of retinoblastoma cells to the cytotoxic effects of 17-AAG.

Methods:: Forty-eight paraffin-embedded sections of retinoblastoma were obtained from the Henry C. Witelson Ocular Pathology Registry. Hsp90 expression was determined by avidin-biotin complex (ABC) immunohistochemistry method, using a monoclonal anti-Hsp90 and scored semi quantitatively. Two human Rb cell lines were used for this study (WERY-1 and Y79). A MTT based proliferation assay was used to compare retinoblastoma cell growth within in a range of concentrations of 17-AAG. The averaged results per condition were recorded. The Student's t-test was used to compare results from cells cultured with and without 17- AAG.

Results:: High immunohistochemical expression of Hsp90 was identified in all 48 retinoblastomas analyzed. When 17- AAG was added to the cell lines, a statistically significant reduction in proliferation of retinoblastoma cell lines in a dose dependent manner was observed. A significant decrease in proliferation was observed at concentrations of 100 µM to 1 µM for Y79. In comparison a significant decrease in proliferation for WERY-1 was seen for all concentrations tested up to 0.0001µm.

Conclusions:: To the best of our knowledge, this is the first report showing the immunoexpression of Hsp90 in retinoblastoma as well as the inhibitory effect on proliferation of retinoblastoma cells using 17-AAG to target Hsp90. The possible modulation of P-gp by altering the Hsp90 function may indicate that Hsp90 represents a potential adjuvant treatment for patients with retinoblastoma.

Keywords: retinoblastoma • immunohistochemistry • chaperones