Temozolomidin glioma hücrelerindeki eksozomal stres proteinleri üzerine etkileri

Brain tumors are one of the cancer types with lethal consequences worldwide. Gliomas are the most frequent brain tumors originated from glial cells of the central nervous system and most common and aggressive primary brain malignancy is called glioblastoma multiform (GBM grade IV astrocytoma). The median survival time for GBM patients is only 8-12 months. Despite the surgery, radiotherapeutic and chemotherapeutic interventions, the survival rates do not change remarkably. Thus, scientists across the world are still seeking for innovative therapies for gliomas. The presence of the blood-brain barrier (BBB) is an important factor in reducing the efficacy of chemotherapy. Temozolomide (TMZ), a DNA alkylating agent, which can penetrate the BBB due to its structure used as the most promising treatment for recurrent glioma. In recent cancer researches, treatment of brain tumors has been shaped to reduce Hsp levels. The reasons for this are to show that there is a relationship between stress proteins and brain tumor malignancy, and that those proteins are effective in the proliferation and progression of tumor cells and that cancerous cells increasingly show resistance to treatment due to the increment in the expression levels of these proteins. Stress proteins have become an important focal point in developing new therapies due to the contribution to the suppression of these proteins' expression to the treatment. There are many studies in the literature that suppress the expression of these proteins using distinct methods including targeting the TMZ agent and reducing the expression level of heat shock proteins. Recent research shows that cells can communicate through extracellular vesicles (EVs). Exosomes with spherical structures of 30-150 nm in diameter and surrounded by lipid bilayers have become the most studied group of the EV family. Exosomes have been indicated to have important roles in cancer improvement and can alter the tumor microenvironment with materials, which they can cargo (DNA, RNA, and proteins-some Hsps). Therefore, it is thought that cancer-associated exosomes and exosome surface HSPs can be a therapeutic target. The main objective of this study was to evaluate the effects of TMZ on the expression of heat shock proteins in exosomes released from U87MG cells. At the beginning of the study, the effect of TMZ on cell viability was analyzed. According to the cytotoxicity analyzes, the agent was treated to the cells at concentrations that did not show any toxic or proliferative effect on the cells (100% cell viability) or that affected cell viability by less than 20% and 50% cell viability. After the exosome isolation was performed, the effect of TMZ application on both exosomal and cell-derived soluble protein profiles was demonstrated by 1-dimensional electrophoresis technique. The proteins were separated on the gels then transferred to the membrane by Western blotting technique and incubated with specific antibodies for target proteins. The most common exosomal marker proteins demonstrated the yield of the isolation by Western Blot and Scanning Electron Microscopy (SEM) images. Cell migration assay was carried out to explain the role of exosomes in metastasis. According to the results of this study, it has been seen that TMZ at various concentrations in dose dependent manner treated to glioma cells increased the expression level of intracellular Hsp70, while the expression level of Hsp70 that is secreted by extracellular-exosomal pathway was decreased. It was determined that TMZ concentration was 750 μM, which the most increased Hsp70 expression in U87MG glioma cells and decreased expression of exosomal Hsp70. The results could be considered a promising approach in slowing cell migration and consequently slowing angiogenesis by decreasing the amount of extracellular Hsp70 by TMZ treatment. These results indicate that the chemotherapy agent was administered to cells, perhaps becomes an effective therapeutic option for the brain tumors treatments. The data from this study are promising to the development of innovative therapies for the treatment of aggressive brain tumors with high fatality rates.