Hsp90 as a molecular target.
Doctoral thesis, University of Central Lancashire.
Heat shock protein 90 (Hsp90), a highly conserved molecular chaperone, has been proposed to play a vital role in tumorigenesis. Hsp90 has two isoforms, of which Hsp90α is the major isoform of the Hsp90 complex and has an inducible expression profile. The molecular chaperone Hsp90α has been recognized in different cancers and it is implicated to play a role in cell cycle progression, apoptosis, regulates invasion, angiogenesis and metastasis. It is being recognized as a promising target in cancer treatment. Previous studies in our laboratory have demonstrated hsp90α expression in both primary glioma tissue and cell lines, but not in normal healthy brain tissues and cell lines. Enhanced chemosensitivity was observed upon specific inhibition of hsp90α expression by siRNA, suggesting that inhibiting hsp90α expression could possibly be a favourable therapeutic approach compared to conventional chemotherapies. In this novel study, Hsp90 was inhibited by either treatment with 17AAG or shRNA oligonucleotide targeting hsp90α (shhsp90α) in the U87-MG glioma cell line. The inhibition profile of Hsp90α was observed at the protein levels in control and treated cells by FACS analysis (quantitative) using a flow cytometer and Hsp90α ELISA kit. The results demonstrated a significant reduction of Hsp90α protein levels post treatment with 17AAG and shhsp90α. The activity of Hsp90α was assayed by quantifying the levels of Akt/PKB in the samples. Significant reductions (>50 %) of Akt/PKB levels were observed post hsp90α inhibition. Cell cycle analysis carried out reported S and G2 phase arrest, post Hsp90 inhibition by either 17AAG or shhsp90α. Interestingly, it was reported that 17AAG shows a better silencing profile compared to shhsp90α.
To analyse the downstream effects of Hsp90 inhibition and to determine the client proteins affected, proteomic analysis was performed. Proteomic analysis identified several proteins which were either upregulated/downregulated post Hsp90 inhibition. IPA analysis further identified “cancer” as the top network significantly transformed post Hsp90 inhibition. Upregulated proteins include Hsp70 family members, Hsp27 and gp96, thereby suggesting the role of Hsp90 co-chaperones in compensating for Hsp90 function post Hsp90 inhibition. Moreover, members of the glycolysis/glucogenesis pathway were also upregulated, demonstrating increased dependency on glycolysis for energy supply by the treated glioma cells. Considering Hsp70 and its role in anti-apoptosis, it was postulated that a combination therapy involving a multi-target approach could be carried out. Subsequently, inhibition of both Hsp90 and Hsp70 in U87-MG glioma cell line was carried out resulting in 60 % cell death along with S and G2 phase arrest. Thus, in the effective treatment of glioma, the inhibition of multiple targets needs to be taken into consideration.
Conclusion: It can be thus concluded that, combination therapy involving silencing of Hsp90 and Hsp70 could be of possible significance in glioma therapy.
|Item Type:||Thesis (Doctoral)|
|Additional Information:||1. Cruickshanks, N., Shervington, L., Patel, R., Munje, C., Thakkar, D., Shervington, A. Can hsp90alpha-Targeted siRNA Combined With TMZ Be a Future Therapy for Glioma? Cancer Invest. 2010. DOI: 10.3109/07357901003630967.|
|Uncontrolled Keywords (separate with ;):||Hsp90; Glioma; Proteomics; Hsp70|
|Subjects:||Q Science > Q Science (General)|
Q Science > QD Chemistry
Q Science > QR Microbiology
|Schools:||School of Forensic & Investigative Sciences|
Khalil Ahmed Patel
|Deposited On:||30 Nov 2011 17:27|
|Last Modified:||16 Feb 2012 16:27|
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