The role of dna repair mechanisms in trabectedin mediated enhanced cytotoxic effect in radiation treated human glioblastoma multiforme cell cultures (U-87 MG AND T98G)

Glioblastoma multiforme (GBM) is one of the most common and lethal brain tumors. One of the most important problems in the treatment is drug and radiation resistance observed in glioblastoma cells. Thus, current research strategies include determining molecular targets, developing new chemotherapeutics and sensitization of tumor cells to radiation. Trabectedin (ET-743) is a molecule that was originally isolated from the Caribbean sea squirt, Ecteinascidia turbinata and is currently prepared semi-synthetically. It is known that it binds to minor groove of the DNA and leads to cell death. The main objective of the study is to investigate whether the cytotoxic and apoptotic effect of sequential treatment of the low doses of radiation and Trabectedin on human glioblastoma multiforme (U-87 MG ve T98G) cells would enhance. Moreover it is to investigate the role of DNA repair mechanisms on the enhanced cytotoxic/apoptotic effect of sequential treatment of the low doses of radiation and Trabectedin. xCELLigence system was used for investigating the effects of Trabectedin on the cell viability. The effects of ionizing radiation and sequential treatments on the cell viability were determined by using Trypan blue dye exclusion assay. Apoptosis was shown by measuring the level of DNA fragmentation and caspase 3/7 activation. Changes in mRNA levels of DNA repair related genes which are affected by sequential treatment of efficient Trabectedin and radiation doses were determined by Human DNA Repair Array PCR Array. The IC50 values of Trabectedin in U-87 MG and T98G cell lines were 8.4 nM and 17 nM, respectively, at 24h; 5 and 13 nM, respectively, at 48h. The IC50 values of ionizing radiation in U-87 MG and T98G cell lines were 22 Gy and 25 Gy, respectively, at 24h; 8 Gy and 9.8 Gy, respectively, at 48h. Enhanced cytotoxicity was observed in both tested cell lines by sequential treatment of Trabectedin and ionizing radiation. Increased DNA fragmentation and caspase 3/7 activation was observed in T98G cell lines treated with increasing concentrations (0.01-100 nM) of Trabectedin in a time and dose dependent manner. Increased levels of DNA fragmentation and caspase 3/7 activation were observed in sequentially treated T98G cell lines. Sequential treatment was also caused significant changes in mRNA levels of DNA repair mechanisms related genes such as Rad51B, XRCC4, ATM, ATR, MGMT in T98G cell lines; OGG1, Rad51B, XRCC4 in U-87 MG cell lines. This is the first study in the GBM cell cultures that both cytotoxic/ apoptotic effects of the sequential treatment of Trabectedin and ionizing radiation and the role of DNA repair mechanisms in these effects were investigated. The preliminary data indicates that Trabectedin may be a promising chemotherapeutic for sensitizing cells to ionizing radiation in the treatment of GBM.