Abstract
Purpose::
Heat shock protein 90 (Hsp90) is a chaperone for many key regulators involved in signal transduction, apoptosis and cell cycle control during malignant transformation. Hsp90 is abundantly expressed by a variety of tumors and has been recently targeted for cancer therapy. Previous data showed that expression of Hsp90 in uveal melanoma correlated with largest tumor dimension, a marker of poor prognosis. Furthermore, the inhibition of HSP90 function by the small-molecule 17-allylamino-17-demethoxy-geldanamycin (17-AAG) resulted in a reduction in proliferation of uveal melanoma cell lines. The objective of this study was to further investigate the role of HSP90 in promoting the survival, migration and invasion abilities in uveal melanoma cells. We then evaluated the effects of 17-AAG on cell motility, invasion, cell cycle and apoptosis of uveal melanoma cell lines.
Methods::
Changes in cell migration and invasion were evaluated in in vitro assays, in the presence or absence of 17-AAG. A QCMTM 24-Well Colorimetric Cell Migration Assay (Chemicon) was used for this experiment. Cell cycle fractions were determined by flow cytometry using an Epics XL flow cytometer (Beckman Coulter, Miami, Florida). The caspase 3 protease activity was detected using the BD ApoAlertTM Caspase Colorimetric Assay (BD Biosciences Clontech) using the manufacturer protocol. In addition expression of intracellular proteins was determined by Western blot analysis.
Results::
Exposure to 17-AAG significantly reduced the migratory and invasive capabilities of all 5 uveal melanoma cell lines. Cell cycle analysis showed that exposure to 17-AAG induced accumulations of cells in G1 and loss of cells in S phase. Caspase 3 protease activity analysis, a marker for apoptosis, showed a significant increase in expression after drug exposure. The cytotoxic effect of 17-AAG was associated with decreased levels of phospho-AKT after 24 h exposure to the drug.
Conclusions::
Inhibition of HSP90 function by 17-AAG had an effect on cell motility and invasive potential, inducing cell cycle arrest and promoting apoptosis in all uveal melanoma cell lines. These results showed that downregulation of p-AKT in response to 17-AAG could indicate a potent modulation of the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway in uveal melanoma. Inhibitors of Hsp90 should be further tested as a local and systemic antineoplastic therapy for the treatment of uveal melanoma.