This study is based on investigating the effects of TMZ on Hsp levels and several genes involved in apoptosis, DNA damage response, cell cycle regulation, stress response, tumour formation and suppression in the glioma cancer cells (U87-MG and LN229) and their EV content. Herein, our results indicated that TMZ inhibited cell growth in a dose and time-dependent manner. According to MTT assay results, U87-MG cells were more sensitive to the cytotoxic effects of the TMZ compared to LN229 cells. IC50 values of TMZ in U87-MG cells were lower than IC50 values of LN229 cells for 48 and 72 h treatment periods, and also IC50 values of LN229 were twofold higher than the other cell line. Furthermore, the IC50 value of TMZ administered for 72 hours for U87-MG cells has been found to be 397.2 µM in one study . Also, it has been reported that the IC50 value of TMZ applied to LN229 cells for 72 hours is 954.2 µM . This result indicates that TMZ has a higher cytotoxic effect in the U-87 MG cell line than the LN229 cell line due to the more aggressiveness of LN229 cells than U87-MG cells [20, 21]. Hence, to evaluate the difference in cellular and EV response on TMZ treatment, we use fixed two doses (100 and 200 µM) of TMZ and 72 h treatment time for both the cell lines. In addition, the selected doses are among the most preferred concentrations in TMZ studies in the literature as they do not have much toxic effect [7, 20, 22].
Western blot is the most preferred method in characterisation studies of EVs. The expression of ALIX and TSG101 antibodies, which are considered to be EV markers, were demonstrated by Western blot analysis (Fig. 2a). In addition to immunological analysis, in this study, qualitative analyses of isolated EVs were performed using the PS Capture ™ Exosome ELISA Kit based on the presence of CD63, which is considered a common EV marker. According to the analyses, there was no statistically significant difference in the amount of CD63 in the EVs released from the control and experimental groups of TMZ application in both cell lines (Fig. 2b). However, the results indicated a significant difference between the amounts of CD63 in the EVs released from both cells (Fig. 2c). In a study conducted by Yoshioka et al. (2013) it has been indicated that the amount of exosomal marker proteins can be different in various cell lines. For this reason, different methods are preferred for EV quantification due to the variable protein contents of different EV types. In this study, we chose the NTA method capable of analysing EV parameters such as size and number. According to the results, it was not observed statistically meaningful difference in the concentration of EV released from the untreated and treated groups with TMZ application in both cell lines (Fig. 2e). Moreover, in a study conducted by Simon et al. (2018), it has been shown that an anti-cancer drug applied to U87-MG and LN18 cells cause a negligible change in the EV concentration . The results of this study reveal that TMZ application affects the content of EV without changing the amount. Besides, our results indicated that there was a significant difference between the amounts of EV released from both cells (Fig. 2f). What is more, there are several studies demonstrated that the amount of EV changes depending on the cell type. Simon et al. (2018) has reported that U87-MG and LN18 cells released different concentrations of EV, Tian et al. (2014) has found that immature dendritic cells excreted a limited number of EVs, and Chen et al. (2011) has indicated that mesenchymal stem cells secreted a large number of EVs [24–26].
In order to indicate the effect of successful drug therapy on the target cell, the therapeutic index must be determined. Because HSPs have a capability to be presented as possible therapeutic targets in the treatment of GBM, we investigated the effect of TMZ on HSPs and HSF-1 (which as a transcription factor plays a substantial role in expression of heat shock protein) in cell and EV content. While HSF-1, Hsp90 and Hsp70 expression was triggered by TMZ treatment in the U87-MG cell line, Hsp60 expression did not change (Table 5). In the LN229 cell line, application of TMZ led to increased expression of HSF1, Hsp90, Hsp70 and Hsp27 (Table 5). Studies have shown that TMZ application increases Hsp70 expression in U87-MG , T98G and U251cells . Castro et al. (2015) have revealed that TMZ treatment increases Hsp70 expression in Gli36 cells, decreases it in DBTRG cells and does not change it in U87-MG cells . The same study has determined that TMZ has increased Hsp27 expression in U87-MG, DBTRG and Gli36 glioma cell lines. The results of these studies have been shown that TMZ has different responses in different glioma cells. Furthermore, Hsp70 and Hsp27 with increased expression levels are known to be associated with poor prognosis and treatment resistance in various types of cancer (breast, cervix, hepatocellular carcinoma) [30,31]. Therefore, the results of our study provided evidence that increased HSPs levels may be associated with the TMZ resistance.
In our study, we also showed that TMZ affected HSPs, which are found in EV content originated both cell lines. HSP expression exhibited a similar pattern in EV and cell contents as well as TMZ application led to increased expression of Hsp90, Hsp70, and Hsp60. Moreover, we found that Hsp70, especially, was correlated in EV and parental cells in both cell lines as a result of TMZ treatment (Table 5). However, EV-HSF1 and EV-Hsp27 expression in both cell lines could not be determined. In a study, it has been shown that TMZ affects the content of EV proteins, and secreted EVs after TMZ application play a role in intercellular communication . It has been indicated in different studies that HSP, whose expression levels are increased, especially in EVs released from cancer cells, can be used as biomarkers in cancer diagnosis [33–35]. Moreover, these HSPs may be associated with drug resistance, poor prognosis, response to treatment, migration, and invasion . In a study conducted by Lv et al. (2012), it has been determined that anti-cancer drugs increase the release of Hsp-containing EVs from hepatocellular carcinoma cells. These EVs play a role in cytotoxic responses .
It is widely known that different genes may contribute to increases in expression levels of HSPs. In this context, to figure out which genes are responsible for the rises, we investigated the effects of TMZ applied to U87-MG and LN229 cells on a variety of genes such as cell cycle regulation, DNA damage response, apoptosis, tumour formation and suppression with Real-time PCR analyses. Herein, although there was a change in expression level of several analysed genes at different doses, expression levels in genes without primer dimer formation which may be caused by the use of SYBR-Green Supermix were examined. In the result of the analyses, we found that the expression of the RAD51 gene, which plays a role in the DNA repair mechanism, increased approximately 5 times and the protein level of RAD51 enhanced by 92.7% in U87-MG cells treated with 200 µM TMZ. These results suggest that this increase may be associated with resistance. It has been reported in many studies that the RAD51 expression level was high in numerous tumour types such as prostate cancer and ovarian cancer [38, 39]. The high level of RAD51 also has explained the resistance against DNA damaging reagents such as chemotherapy, as it causes an increase in homologous recombination. In addition, the expression level of the RAD51 gene has increased in various GBM cells treated with TMZ, and this increase was associated with resistance [40, 41]. Therefore, different treatment approaches targeting the RAD51 gene have been tested in order to increase the sensitivity of TMZ in GMB cells . Nevertheless, why and how RAD51 overexpression occurs in GBM cells remains unclear. Moreover, there are studies revealing the relationship between both RAD51 and HSPs. After the creation of single or double-strand breaks, DNA Damage Response Pathway (DDR) occurs, and DDR proteins, such as RAD51, are kept active by Hsp70 and Hsp90 [43, 44]. However, the role of Hsp27 in the DNA repair mechanism is still under investigation . All these results have suggested that the increase in RAD51 gene expression may associated with the rise of expression level in HSPs and this may lead to TMZ resistance.
Besides these consequences, we found that 200 µM TMZ administration leaded to increased expression of the MDM2 oncogene, but it was not caused any changes in the MDM2 protein level in LN229. MDM2, mediating the degradation of the p53 protein under normal conditions, acts as a negative regulator to suppress the activity of the p53 protein. In the studies, overexpression of the MDM2 gene with the p53 mutation has been observed [46,47]. It has been reported that tumours with both the p53 mutation and high MDM2 expression level are associated with a worse prognosis . Moreover, MDM2 whose expression level is increased has also been demonstrated to provide resistance to anticancer drugs such as cisplatin and doxorubicin in breast cancer [49, 50]. In another study conducted by Sato et al. (2011) have shown that MDM2 inhibition increased p53 expression and stem cell-like GBM cells became more sensitive to TMZ . In this study, it was shown for the first time that TMZ application to LN229 cells with mutant p53 activity increased MDM2 expression. Furthermore, there is a study that show a link between MDM2 and HSPs. HSPs also prevent the degradation of mutant p53s by MDM2. Molecular chaperones assist in folding mutant p53 intermediates and stabilise their interaction with p73. When the MDM2 oncogene is over-expressed, HSPs are displaced, and a stable multi-protein complex comprising of mutated p53-TAp73α-MDM2 is formed, additionally amplifying cancer cells chemoresistance . Taking into account all of the data, it's possible that this increase in MDM2 contributes to HSPs expression in LN229 cells and is linked to TMZ resistance.
Herein, we also examined the expression level of RAD51 and MDM2 genes as a result of TMZ application in EVs originating from both glioma cells. Although the RAD51 gene expression level was altered by TMZ treatment in U87-MG cells, when EV-RAD51 was examined, primer dimer formation was determined depending on the SYBR green. Therefore, TaqMan probe was preferred to prevent primary dimer formation caused by SYBR-green to precisely detect expression levels of EV-RAD51. As a result, RAD51 was found to not carry in EV content in this study first time. Additionally, it was found for the first time that TMZ administration increases the MDM2 gene in EV content and MDM2 mRNA levels have an excellent correlation in EV and parental cells (Fig. 5c). TMZ therapy has been known to cause changes in the expression levels of numerous genes in GBM cells; however, little information has been found that it may also affect EV content. In a study, the mRNA expression level of many genes thought to be responsible for TMZ resistance such as GSTp1, MGMT, APNG, ERCC1, ERCC2, MVP, ABCC3, CASP8, and IGFBP2 have correlated in EV and parental cells, and it has suggested that these genes can be potential biomarkers for TMZ resistance . In this scope, according to the results of our study, MDM2 mRNA is thought to be a potential EV-mRNA marker related to TMZ resistance.
In conclusion, TMZ application causes resistance in U87-MG and LN229 cells, and despite the fact that TMZ is utilised as a chemotherapeutic in the treatment of GBM with no alternatives, it may not be the only good anticancer agent in the therapy of GBM. For this purpose, using TMZ in combination with different drugs can be an excellent strategy to increase the effectiveness of treatment. In addition, the fact that EVs correlate with expression levels in the cell at both the mRNA level and the protein level suggests that EVs in the treatment of GBM may have potential biomarkers that can be used to investigate the treatment response. However, different studies are required to determine how some mRNA and Hsp, whose expression levels are increased with TMZ treatment, may affect the recipient cells.