Effects of temozolomide on extracellular vesicle contents released from brain cancer cells

Cancer, is one of the most prominent member of the diseases that causes deaths worldwide and in vitro experiments are of paramount importance to elucidate its molecular mechanisms. Although brain tumors are not commonly observed among all types of cancer, glioblastomas (GBM) are the most malignant and the fastest-progressing primary brain tumor in adults. Despite all the treatments applied to the patients, the average life expectancy is 14.6 months. The main difficulties encountered in GBM treatment are the presence of the blood brain barrier and resistance that occurs after treatment. Temozolomide, a chemotherapy agent, is an important therapeutic used in the treatment of GBM, as it can cross the blood brain barrier due to its structure. Recent studies have indicated that drugs applied to cancer cells can change the expression levels of some genes involved in different processes such as apoptosis, DNA damage response, cell cycle regulation, stress response, tumor formation and suppression. Increased expression of these genes is generally associated with resistance to treatment. Suppression of the expression levels of these genes is one of the main strategies used in order to increase the chance of success of the treatment. Exosomes, which forms the smallest subgroup of extracellular vesicles known so far, play a role in intercellular communication, signal transmission and pathological processes of many diseases, including cancer. Exosomes can involve in cancer progression, metastasis, invasion and resistance formation through their various nucleic acid, miRNA, mRNA, protein (including some stress proteins (Hsp)) contents. Therefore, exosome contents released from cancer cells are considered to be a therapeutical target. Within the scope of the thesis, by applying temozolomide, which is used as a cancer drug, to human glioblastoma cells (U87-MG and LN229), primarily we determined the expression levels of different stress proteins and various RNAs in the cell and then the levels of certain cellular proteins and RNAs were compared with the EV contents released from these cell lines into the medium. In this context, firstly, the cytotoxic effects of TMZ on U87-MG and LN229 human glioma cell lines were evaluated and by applying certain TMZ concentrations to cells, the level of stress proteins and RNA levels of 92 genes in the cells were detected by expression studies. In the next stage, the isolation and characterization studies of exosomes released from GBM cells into the medium were carried out and expression levels of selected proteins and RNAs after drug administration were evaluated. In the experiments, it was determined that RAD51 gene expression increased only in U87-MG cells, while MDM2 gene expression increased both in LN229 cells and in exosomes originating from these cells. Immunological analysis showed that TMZ administration increased Hsp70 expression in both glioma cells, while Hsp27 expression increased only in LN229 cells. When the effects of TMZ application on proteins in exosomes originating from glioma cells were examined, it was determined that Hsp60 and Hsp70 expressions increased. The findings obtained within the scope of this thesis will contribute to the development of innovative approaches in the treatment of glioblastoma patients.