Model ćelija C6 glioma pacova kao oruđe za pronalaženje novih terapeutskih strategija: karakterizacija rezistentnog fenotipa in vitro i in vivo
Rat C6 glioma model as a tool for discovering new therapeutic strategies: characterization of the resistant phenotype in vitro and in vivo
Doctoral thesis (Published version)
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The most common and aggressive malignant tumor of the central nervous system is glioblastoma which successful treatment is compromised due to its high invasiveness, and resistance to chemotherapy. This study aimed to establish and characterize resistant glioma model for testing new therapeutic strategies for treatment of this serious disease. Rat C6 glioma cell line, which is phenotypically similar to human glioblastoma, was chosen as starting material. The first approved antiglioma drug, 1,3-bis (2-chloroethyl) -1-nitrosourea (BCNU, carmustine), an alkylating agent for treatment of primary and recurrent glioblastoma was used for establishing the resistant phenotype. Continuous treatment with BCNU led to the development of resistance not only to this chemotherapeutic, but also to other DNA damaging agents, cisplatin (CPt) and temozolomide (TMZ). The newly established drug resistant cell line was labeled as RC6. Resistance mechanisms analysis adressed the changes in apoptotic machinery and DNA damage, as well as the changes in antioxidant capacity between C6 and RC6 cells. Resistant RC6 line showed reduced sensitivity to cell death induction which was accompanied by a significant decrease in mRNA and protein expression of antiapoptotic factor Bcl-2, proapoptotic factor Bad and effector caspase 3. The mRNA expression of antiapoptotic factor Bcl-Xl and proapoptotic factor Bax was also decreased. The development of resistance was accompanied by significantly increased reactive oxygen species (ROS) production and decreased oxidized to reduced glutathione ratio. mRNA expression of antioxidant system components (MnSOD, iNOS, GPx and MRP1) was significantly increased in RC6 compared to C6 cells, while HIF-1α mRNA expression was decreased.RC6 cells exibited increased sensitivity to doxorubicin (DOX). It was determined that RC6 cells accumulated more DOX than C6 cells as a result of decreased mRNA expression of ABCB1 membrane transporter, as well as lower intracellular pH.In addition, RC6 cells proliferated significantly slower compared to C6 cells which is characteristic for drug resistant cells and cells with greater invasive capacity. The results confirmed that increased mRNA expression of metalloprotease MMP9 contributed to RC6 cells invasiveness...
Keywords:Glioblastoma; Chemoresistance; Carmustine (BCNU); Temozolomide (TMZ); Collateral sensitivity; Invasiveness; Antiglioma therapy; CoQ10
Source:University of Belgrade, Faculty of Biology, 2017
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