The role of HSF1 on stress proteins expressions in glioma cells

The incidence of primary malignant brain tumors has increased rapidly over the last 30 years, with changing living conditions. Among these tumors, gliomas are considered the most common and most malignant tumor type. The prognosis for high-grade glioblastoma multiforme (GBM), the most common form of glial tumors, is very poor. Despite the development of surgical techniques and temozolomide treatment along with radiotherapy as a standard therapy, survival is limited to an average of 15-16 months. Comprehension the normal developmental pathways and gliomagenesis in the central nervous system will help to better understanding the biology of these tumors and to provide more effective treatment methods for patients with glioma. A large molecular chaperone family, known as Heat Shock Proteins (HSPs) or stress proteins, is highly expressed in gliomas. There is a positive correlation between the increased expression of HSPs and the invasive capacity of glial tumors. Overexpression of HSPs leads to mutational changes in glioma and ultimately drives the cells to malignancy and invasion. Overexpression of HSPs that provide cytoprotection to glioma cells also contributes to radio- and chemo-resistance. The role of HSPs in both normal and stressed cells requires the existence of complex regulator processes that allow the production of the correct expression pattern. The induction of HSPs depends on the activation of certain members of the transcription factors family. The main mechanism of this regulation in mammalian cells depends on a protein called Heat Shock Factor 1 (HSF1) and its interactions. In several studies, down-regulation of HSF1 is aimed by various factors such as changes in post-translational modifications, proteasomal degradation, use of small molecule inhibitors and mechanisms of gene regulation. Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a natural flavonoid found in various fruits and vegetables, is an HSF1 inhibitor and has been reported to facilitate apoptosis of tumor cells. In addition, it is thought that knock-out of HSF1 at gene level may use as an effective mechanism. In this study, knock-out of the HSF1 gene with the highest yield by the CRISPR/Cas9 method in glioma cells was determined as the main target. Depending on the purpose of the study, both quercetin application and CRISPR / Cas9 mediated HSF1 knock-out were performed in U87-MG human glioma cells and expression of various stress proteins was examined by Western Blot technique. As a result of 2, 10 and 30 μM quercetin applications performed for the purpose of HSF1 knock-out, a significant reduction in HSF1 expression was detected for 10 and 30 μM concentrations. For two different sgRNAs used in CRISPR / Cas9 application for the same purpose, HSF1 expression was found to be decreased by more than 50%. When both applications were compared, it was determined that the application of quercetin in doses causing significant changes had a disadvantage over CRISPR / Cas9 application due to the high toxicity on cell viability. In addition, CRISPR / Cas9 has been shown to be an effective pathway in HSF1 knock-out with low cytotoxicity and high efficiency. The results of the analyzes performed to determine the changes in the expression levels of Hsp27, Hsp70, Hsp90, pHsp27 proteins in the groups with applied quercetin and CRISPR / Cas9, showed that both applications have different effects on the expression levels of these proteins. In conclusion, HSF1 is not an unattended factor that regulates the expression of stress proteins in glioma cells. It will be possible to prevent the advantages of HSPs to cancer cells after fully understanding of the pathways that cause high expressions of these proteins in cancer cells with further studies.