Bir pro-apoptotik Bcl-2 ailesi üyesi olan Harakiri'nin (HRK) Glioblastoma Multiforme tümörlerindeki rolünün ve düzenlenmesinin incelenmesi

Glioblastoma Multiforme (GBM) is the most common and aggressive type of brain tumors and the patient survival rate is approximately 12 months after diagnosis. Unfortunately, there is not any effective therapy because of poor delivery of therapeutics due to the blood brain barrier and tumor cells' resistance to the therapy. Tumor-necrosis-factor related apoptosis-inducing ligand (TRAIL) is a promising factor that induces apoptosis specifically on tumor cells; and its efficiency is preclinically tested. However, approximately %50 of GBM cell lines are resistant to TRAIL. Evidence shows that aberrant expressions of apoptotic machinery might be responsible for TRAIL resistance, but which mechanisms are particularly responsible for this innate difference is not fully understood. We have recently observed that expression of Harakiri (HRK) that is a BH3-only Bcl-2 family protein is highly increased in TRAIL-sensitive subpopulation of a human GBM cell line; and it is significantly repressed in the TRAIL-resistant subpopulation. This difference in HRK expression is remarkably higher compared to differences in other members of apoptosis. HRK is a sensitizer BH3-only protein and regulates apoptosis by interfering with anti-apoptotic Bcl-2 and Bcl-xL proteins and blocking their function. While its function is characterized in the context of the nervous system, its implications in tumorigenesis are not well studied. Few studies show the suppressed expression levels of HRK in tumors and exogenous expression of HRK attenuates the tumor growth in some cancers. However the functional role of HRK and the relations with TRAIL have not been studied in GBM before. In this study, we investigated the role of HRK in GBMs. We found that HRK is differentially expressed among established GBM cell lines. In order to assess the role of HRK by gain-of- and loss-of-function approaches, we generated HRK overexpression and knockdown vectors and performed experiments that tested the effects of HRK on cell viability and apoptosis. As a result, we observed that HRK overexpression itself induced apoptosis in different GBM cells at different levels. Also, we showed that this phenotype could be blocked by forced expression of Bcl2- and Bcl-xL, suggesting the functional interaction of Bcl-2/Bcl-xL and HRK in tumor cells. Moreover, HRK overexpression showed additive activity with TRAIL in GBM cells. In addition, we identified that the agents that cooperate with TRAIL to induce apoptosis, such as the histone deacetylase inhibitor MS-275, significantly increased HRK expression levels. Also, knockdown of HRK blocked TRAIL-sensitizing effect of MS-275. This suggests that HRK induction might be one mechanism during TRAIL sensitization and apoptosis. In order to better understand how HRK gene expression is regulated and to identify novel agents with the ability to induce GBM cell apoptosis, we also developed a reporter system that monitors HRK regulation in GBM cells. Taken together, our results suggest that HRK upregulation is associated with GBM cell apoptosis and has significant role for TRAIL-induced apoptosis. Thus, novel small compounds that can increase HRK levels in cancer cells might offer new therapeutic approaches in the future.