HSP70: a therapeutic biomarker for treatment of glioma

Harshada, Shashikant, Patil (2015) HSP70: a therapeutic biomarker for treatment of glioma. Doctoral thesis, University of Central Lancashire.

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Gliomas are amongst the most malignant, invasive and recurrent forms of brain tumour with very short survival rate due to high chemoresistance. Recently, highly inducible molecular chaperones HSP70 and HSP90 are emerging as important anti-cancer targets. Previously, proteomic analysis had demonstrated that post-induction of HSP70 on HSP90 inhibition undermines the efficacy of treatment. The present study has quantified transcriptional levels and Akt/PKB activity of Hsp70 and Hsp90α in glioma cell lines. In order to evaluate the therapeutic value of both chaperones, HSP70 and HSP90 were targeted in glioma cells U87-MG using VER-155008 and 17-AAG, respectively. Improved efficacy of HSP70 and HSP90 inhibitors was evaluated using a chemosensitivity assay. MicroRNAs (miRNAs) are highly conserved small non-coding RNA molecules (21-24 nucleotides) that regulate simultaneously the expression of hundreds of mRNA targets, and are reported to be aberrantly expressed in glioma. Therefore, miRNA microarray technology was used to evaluate the efficacy of these inhibitory drugs compared with Temozolomide (TMZ) which is used as a standard treatment for glioma. Microarray data identified 154 miRNAs using either stringent or non-stringent parameters. 16 miRNAs were overlapped with treatments, 15 were upregulated, while 13 were overlapped between Temozolomide and VER-155008. In Temozolomide and VER-155008 treatment, Hsa-miR-194p was upregulated by 139 and 63 fold, respectively, Hsa-miR-215 was upregulated 165 and 61 fold, respectively, Hsa-miR-449a was upregulated by 62 and 77 fold, respectively and Hsa-miR-744-5p was upregulated by 63 and 43 fold, respectively. 17-AAG and VER-155008 treatment shown only one miRNA overlapping with 29 and 2 fold change, respectively. Hsa-miR-4636 was the only downregulated miRNA in TMZ and VER treatment with a 32 and 33 fold change, respectively. The miRNA target prediction software was used for the highly upregulated miRNAs: hsa-miR-194-5p, hsa-miR-215, hsa-miR-449a, hsa-miR-744-5p and hsa-miR-3161 correlating to Dnmt3a, Alcam, Cdk4, Dnajc16 (Hsp40) and R-Ras2 genes, respectively. Gene validation using qRT-PCR suggested no correlation between miRNA-mRNA levels, and thus, challenges the suitability of miRNAs technology as treatment predictors. In conclusion, the result for the protein data showed that HSP70 was inhibited on treatment with Temozolomide, 17-AAG and VER-155008 to 13, 0 and 20 %, respectively, while HSP90 inhibition was 84, 43 and 65 %, respectively, reflecting the affinities of these three compounds towards HSP90 compared to HSP70, and therefore infers that HSP70 could be a stronger therapeutic approach. In conclusion the result of the study has clearly demonstrated that HSP70 can be better therapeutic biomarker for treatment of glioma.

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