Genotoxic damage to glioblastoma cells treated with 6 MV X-radiation in the presence or absence of methoxy estradiol, IUDR or topotecan

ABSTRACT

To explore the cumulative genotoxic damage to glioblastoma [GBM] cells, grown as multicellular spheroids, following exposure to 6 MV X-rays [2 Gy, 22 Gy] with or without, 2- methoxy estradiol [2ME2], iododeoxyuridine [IUDR] or topotecan [TPT], using the Picogreen assay. The U87MG cells cultured as spheroids were treated with 6 MV X-ray using linear accelerator. Specimens were divided into five groups and irradiated using X-ray giving the dose of 2 Gy after sequentially incubated with one of the following three drug combinations TPT, 2-ME2/TPT, IUDR/TPT or 2ME2/IUDR/TPT. One specimen was used as the irradiated only sample [R]. The last group was also irradiated with total dose of 22 Gy [each time 2 Gy] of 6 MV X-ray in 11 fractions and treated for three times. DNA damage was evaluated using the Picogreen method in the experimental study. R/TPT treated group had more DNA damage [double strand break [DSB]/single strand break [SSB]] compared with the untreated group [P<0.05]. Moreover the R/TPT group treated with 2ME2 followed by IUDR had maximum DNA damage in spheroid GBM indicating an augmented genotoxicity in the cells. The DNA damage was induced after seven fractionated irradiation and two sequential treatments with 2ME2/IUDR/TPT. To ensure accuracy of the slope of dose response curve the fractionated radiation was calculated as 7.36 Gy with respect to alpha/beta ratio based on biologically effective dose [BED] formulae. Cells treated with 2ME2/IUDR showed more sensitivity to radiation and accumulative DNA damage. DNA damage was significantly increased when GBM cells treated with TPT ceased at S phase due to the inhibition of topoisomerase enzyme and phosphorylation of Chk1 enzyme. These results suggest that R/TPT-treated cells increase sensitivity to 2ME2 and IUDR especially when they are used together. Therefore, due to an increase in the level of DNA damage [SSB vs. DSB] and impairment of DNA repair machinery, more cell death will occur. This in turn may improve the treatment of GBM