17-AAG and sihsp90α combinational therapy as a novel anti-cancer approach

Mehta, Adi Behram (2012) 17-AAG and sihsp90α combinational therapy as a novel anti-cancer approach. Doctoral thesis, University of Central Lancashire.

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Heat Shock Protein 90 (Hsp90) is a molecular chaperone which plays an active role in maintaining protein homeostasis. Hsp90 is known to be highly expressed in tumour cells where it regulates stability and function of several key oncogenic client proteins including Akt kinase, EGFR, CDK and PDGFR. These client proteins are mutated or overexpressed in tumours and are involved in tumour progression and metastasis due to their roles in signalling pathways, cell cycle and apoptosis.

Hsp90 has two isoforms, namely Hsp90α and Hsp90β and share 85% sequence homology. Hsp90β is the constitutive isoform, however, Hsp90α is highly induced in many cancers and is responsible for tumourigenesis. Previous studies from our laboratory established Hsp90α mRNA and protein to be highly expressed in glioma cell lines and tissues compared to normal tissue and cells. In a follow up study, Hsp90α was silenced using predesigned small interfering RNA (sihsp90α) with high target specificity and it showed a clinical impact on the chemosensitivity to Temozolomide (TMZ). Thus, Hsp90α could be a therapeutic target for the treatment of glioma.

This study utilized a Hsp90-inhibitor 17-AAG and hsp90α-specific siRNA (sihsp90α), either as single agent or in combination, to inhibit Hsp90 function in glioblastoma. Hsp90α mRNA and protein expression levels post treatment were evaluated using qRT-PCR and confocal microscopy. To determine if Hsp90α inhibition influenced Akt kinase activity, a Hsp90 client protein, Akt activity was examined in treated and control cells using a Akt Kinase activity kit. Novel results from the study revealed that Hsp90α was significantly down-regulated at both the mRNA and protein levels which were associated with reduced cell viability. Hsp90α inhibition resulted in loss of Akt kinase activity which validated the role of Hsp90α in chaperoning tumour progression. The use of 17-AAG concomitant with sihsp90α did not demonstrate synergistic anti-tumour effects in glioblastoma in vitro.

Furthermore, the application of siRNA as an alternative to small-molecule inhibitors in the treatment of human disease has shown therapeutic potential. However, a major hurdle to its utility has been the difficulty in delivering these anionic macromolecules in vivo. In this study, the ability of Tat peptide to enhance siRNA-mediated knockdown of hsp90α and siRNA stability in serum was investigated. In glioblastoma, Tat-mediated sihsp90α transfection concomitant with 17-AAG exhibited significant downregulation of Hsp90α mRNA and protein levels with increased peptide concentrations. Hsp90α suppression was associated with reduced Akt kinase activity and cell viability. The sihsp90α/Tat complex significantly improved sihsp90α stability in human serum for up to 36 h post serum exposure.

Finally, the combination therapy induced hsp90α knockdown and reduced Akt kinase activity in vivo. Although these results are preliminary and requires further validation, the anti-cancer activity is promising. These results suggest that the combination treatment with sihsp90α and 17-AAG may have therapeutic potential in GBM.

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