Rat C6 glioma model as a tool for discovering new ttherapeutic strategies: characterization of the resistant phenotype in vitro and in vivo

Uspešnost terapije glioblastoma, najĉešćeg i najagresivnijeg malignog tumora centralnog nervnog sistema, je osujećena usled visokog stepena invazivnosti, kao i pojave rezistencije na hemioterapiju. Cilj ove doktorske disertacije je bio da se uspostavi i okarakteriše rezistentni gliomski model koji bi imao primenu u ispitivanju novih terapeutskih strategija u leĉenju ove teške bolesti. Kao poĉetni materijal korišćena je pacovska C6 ćelijska linija glioma, fenotipski sliĉna humanim glioblastomima. Za uspostavljanje rezistencije korišćen je prvi odobreni antigliomski lek 1,3-bis(2-hloroetil)-1-nitrozoureom (BCNU, karmustin), alkilirajući agens koji se koristi u terapiji primarnih i rekurentnih glioblastoma. Kontinuirana primena BCNU je dovela do razvoja rezistencije C6 ćelija ne samo na ovaj hemioterapeutik, već i na druge DNK oštećujuće agense, cisplatin (CPt) i temozolomid (TMZ). Novouspostavljena rezistentna ćelijska linija je oznaĉena kao RC6. Ispitivanje mehanizama rezistencije je obuhvatilo analizu promena na nivou apoptotske mašinerije i oštećenja DNK lanaca, kao i analizu promena u antioksidativnom kapacitetu izmeĊu C6 i RC6 ćelija. Rezistentna RC6 linija pokazuje smanjenu osetljivost na indukciju ćelijske smrti koja je praćena smanjenjem ekspresije antiapoptotskog faktora Bcl-2, proapoptotskog faktora Bad i efektorske kaspaze 3 na nivou iRNK i proteina. TakoĊe je pokazano smanjenje ekspresije iRNK antiapoptoskog Bcl-Xl i proapoptotskog Bax-a. Razvoj rezistencije je doveo i do znaĉajnog povećanja produkcije reaktivnih kiseoniĉnih vrsta (ROS) i smanjenja odnosa koncentracije oksidovanog i redukovanog glutationa. Zapaţeno je povećanje ekspresije iRNK komponenti antioksidativnog sistema (MnSOD, iNOS, GPx i MRP1) i smanjenje ekspresije iRNK za HIF-1α kod RC6 ćelija u odnosu na C6 ćelije. RC6 ćelije pokazuju povećanu osetljivost na delovanje doksorubicina (DOX). UtvrĊeno je da RC6 ćelije akumuliraju više DOX-a od C6 ćelija ĉemu doprinosi znaĉajno smanjenje ekspresije iRNK za ABCB1 membranski transporter, kao i smanjenje unutarćelijskog pH. Pored toga, RC6 ćelije proliferišu znatno sporije u odnosu na C6 ćelije što je karakteristiĉno za rezistentne ćelije, ali i za ćelije sa većim invazivnim sposobnostima. Rezultati potvrĊuju da invazivnosti RC6 ćelija doprinosi povišena ekspresija iRNK za metaloproteinazu MMP9...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..