Heat Shock Protein (HSP)-90 Is Overexpressed in Gallbladder Carcinoma (GBC)
Jose R Valbuena, Juan C Roa, Pamela Leal, Patricia Garcia, Sergio Gonzalez, David Oddo, Katty Schnettler, Gonzalo Carrasco, Alejandro H Corvalan. Pontificia Universidad Catolica de Chile, Santiago, Region Metropolitana, Chile; Universidad de la Frontera, Temuco, Chile
Background: GBC is one of the most common malignancies in Chile with the highest mortality rate in the world. It is the principal cause of oncologic death in women. Usually it presents with advanced stage with no effective treatment. Heat shock proteins (HSPs) are molecular chaperones that are known to have tumoral activity. Of these, HSP90 had been shown to interact with different molecules related to differentiation, proliferation and oncogenesis in cancer cells. Two molecules have shown to inhibit the action of HSP90 (geldanamycin (GA) and 17-allylamino-17-des-methoxygeldanamycin (17-AAG)) and hence a potential target for adjuvant therapy. HSP90 expression and antitumoral activity of GA and 17-AAG have not been studied in GBC.
Design: Forty-seven GBC cases were selected for this study. Immunohistochemical studies in paraffin-embedded tissue were done using a monoclonal HSP90 antibody (clone JPB24, dilution 1:200, Novocastra, Newcastle upon Tyne, UK). A positive case was interpreted if at least 50% of the tumor showed cytoplasmic staining. Cell lysates (15mg) were prepared from gallbladder cancer cell line (GB-d1), human epithelial gallbladder cells (HGBEC) and normal human embryonic kidney cells (HEK293) for Western Blot (WB) analysis using anti-HSP90 (1:250). For viability assay, GB-d1 cells were seeded in 96-well plates at a density of 5000 cells per well. After an overnight attachment period, cells were exposed to various concentrations of GA and 17-AAG for 24 h, 48 h and 72 h. Control cells received DMSO only. The percentage of viable cells was assessed with a colorimetric MTS cell proliferation assay (Promega, Madison, WI).
Results: HSP90 protein expression was seen in 45 cases by immunohistochemical analysis (45/47). Average expression was 78%. The staining intensity ranged from weak to strong with 35 cases (74%) showing moderate intensity. HSP90 expression was higher in GB-d1 cell line compared to normal gallbladder cells. The MTS cell proliferation assay demonstrated that both GA and 17-AAG similarly elicited cytostatic and cytotoxic effects on GB-d1 cells. After 48 h treatment at concentrations >10 uM, the GB-d1 cell viability was reduced by GA and 17-AAG. Treatment with 0.1% of DMSO as equimolar solvent control does not affect the GB-d1 cell viability.
Conclusions: GBC is a heterogeneous group of neoplasms with overexpression of proteins related to proliferation and survival. Our results showed that HSP90 is highly expressed in GBC making it a good candidate for targeted therapy.
Tuesday, March 20, 2012 9:30 AM
Poster Session III # 141, Tuesday Morning