ABSTRACT
Glioblastoma multiforme [GBM], one of the most common and aggressive malignant brain tumors, is highly resistant to radiotherapy. Numerous approaches have been pursued to find new radiosensitizers. We used a picogreen and colonogenic assay to appraise the DNA damage and cell death in a spheroid culture of GBM cells caused by iodine-131 [I-131] beta radiation in the presence of topotecan [TPT]. U87MG cells were cultured as spheroids with approximate diameters of 300 microm. Cells were treated with beta radiation of I-131 [at a dose of 2 Gy] and/ or TPT [1 microg/ml for 2 hours]. The numbers of cells that survived were compared with untreated cells using a colonogenic assay. In addition, we evaluated possible DNA damages by the picogreen method. The relation between DNA damage and cell death was assessed in the experimental study of groups. The findings showed that survival fraction [SF] in the I-131+TPT group [39%] was considerably less than the I-131 group [58.92%; p<0.05]. The number of single strand breaks [SSB] and double strand breaks [DSB], in the DNA of U87MG cells treated with beta radiation of I-131 and TPT [I-131+TPT] significantly increased compared to cells treated with only I-131 or TPT [p<0.05]. The amount of SSB repair was more than DSB repair [p<0.05]. The relationship between cell death and DNA damage was close [r>/=0.6] and significant [p<0.05] in the irradiated and treated groups. Also the maximum rate of DNA repair occurred 24 hours after the treatments. A significant difference was not observed on other days of the restoration. The findings in the present study indicated that TPT can sensitize U87MG cells to radiation and increase DNA damages. Potentially, TPT can cause an increase in damage from DSB and SSB by its inhibitory effects on topoisomerase enzyme and the cell cycle. The increased complex damages following the use of a genotoxic agent and beta I-131 radiation, causes a significant increase the cell death because of the difficult repair process. By assessing the relationship between DNA damage and cell death, the picogreen method can be useful in predicting colonogenic assay. Consequently, it is suggested that co-treatment with I-131 beta radiation and TPT can improve GBM treatment
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