DNA damage of glioblastoma multiform cells induced by beta radiation of iodine-131 in the presence or absence of topotecan: a picogreen and colonogenic assay

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

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

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

Evaluation of the combined effects of hyperthermia, cobalt-60 gamma rays and IUdR on cultured glioblastoma spheroid cells and dosimetry using TLD-100

In radiation treatment, the irradiation which is effective enough to control the tumors far exceeds normal-tissues tolerance. Thus to avoid such unfavourable outcomes, some methods sensitizing the tumor cells to radiation are used. Iododeoxyuridine [IUdR] is a halogenated thymidine analogue that known to be effective as a radiosensitizer in human cancer therapy. Improving the potential efficacy of radiation therapy after combining to hyperthermia depends on the magnitude of the differential sensitization of the hyperthermic effects or on the differential cytotoxicity of the radiation effects on the tumor cells. In this study, we evaluated the combined effects of IUdR, hyperthermia and gamma rays of 60Co on human glioblastoma spheroids culture. In this experimental study,the cultured spheroids with 100microm diameter were treated by 1 microM IUdR, 43°C hyperthermia for an hour and 2 Gy gamma rays, respectively. The DNA damages induced in cells were compared using alkaline comet assay method, and dosimetry was then performed by TLD-100. Comet scores were calculated as mean +/- standard error of mean [SEM] using one-way ANOVA. Comparison of DNA damages induced by IUdR and hyperthermia + gamma treatment showed 2.67- and 1.92-fold enhancement, respectively, as compared to the damages induced by radiation alone or radiation combined IUdR. Dosimetry results showed the accurate dose delivered to cells. Analysis of the comet tail moments of spheroids showed that the radiation treatments combined with hyperthermia and IUdR caused significant radiosensitization when compared to related results of irradiation alone or of irradiation with IUdR. These results suggest a potential clinical advantage of combining radiation with hyperthermia and indicate effectiveness of hyperthermia treatment in inducing cytotoxicity of tumor cells

Relative biological effectiveness [RBE] of [131]I radiation relative to [60]Co gamma rays

To assess relative biological effectiveness [RBE] of [131]I radiation relative to [60]Co gamma rays in glioblastoma spheroid cells. In this experimental study, glioblastoma spheroid cells were exposed to [131]I radiation and [60]Co gamma rays. Radiation induced DNA damage was evaluated by alkaline comet assay. Samples of spheroid cells were treated by radiation from [131]I for four different periods of time to find the dose-response equation. Spheroid cells were also exposed by 200 cGy of [60]Co gamma rays as reference radiation to induce DNA damage as endpoint. Resulted RBE of [131]I radiation relative to [60]Co gamma rays in 100 microm giloblastoma spheroid cells was equal to 1.16. The finding of this study suggests that [131]I photons and electrons can be more effective than [60]Co gamma rays to produce DNA damage in glioblastoma spheroid cells

[Evaluation of diagnostic reference dose levels in ct-scan examinations of adolescence in Tehran: a brief report]

Today, a CT scan examination play an important role in disease diagnosis and thus allocates a significant dose of medical X-ray examinations. One of the important principles in the use of ionizing radiations is to observe radiation protection principles. Evaluating patient's absorbed dose and implementing the strategies for reducing dose are prior in safety issues. To achieve this purpose, sufficient studies should be performed in this regard. Therefore, patient's absorbed dose and the factors affecting have been studied in this research. This study aims to present and develop an acceptable level of absorbed dose in CT scan examinations. Diagnostic reference dose level in the adult age group [older than 15 years] has been studied in eleven CT scan centers in Tehran. For this purpose, CT scan examinations prevalent in above-mentioned centers were chosen and Weighted Computed Tomography dose index [CTDIw] and dose length product [DLP] parameters were studied. Standard phantoms with 16 and 32 diameters of Polymethylmethacrylate [PMMA] origin have been used in the centers for CT scan calibration process. CTDIw third quartile has been considered as the diagnostic reference dose level [DRL]. Rate of diagnostic reference dose in the adult age group [older than 15 years] for the head, sinus, lungs, abdomen and pelvis are 50.87, 38.27, 8.05 and 9.11 mg, respectively. Dose measurements made in this survey have been used to set up local DRLs and can be used as a template for national DRLs. Value of diagnostic reference dose for the head examinations is more than the other parts and diagnostic reference dose in Tehran city in the Adult age group and the protocols compared is less than national reference dose.

Evaluation of the combined effect of 2ME2 and 60Co on the inducement of DNA damage by IUdR in a spheroid model of the U87MG glioblastoma cancer cell line using alkaline comet assay

In this study, we investigated the combined effect of 2-Methoxyestradiol [2ME2] and 60Co on the cytogenetic damage of iododeoxyuridine [IUdR] in the spheroid model of U87MG glioblastoma cancer cell lines by alkaline comet assay. U87MG cells were cultured as spheroids with diameters of 350 micro m. As control, the spheroids of one plate were not treated. Other cultures were pretreated with 2ME2 [250 micro M] for one volume doubling time [1 VDT]. After this time, the subsequent treatments were performed according to the following groups: 1. Vehicle [this sample was not treated in the 2nd VDT] 2. Treated with 2ME2 [250 micro M] for 1 VDT 3. Treated simultaneously with 2ME2 [250 micro M] and IUdR [1 micro M] for 1 VDT 4. Treated with 2ME2 [250 micro M] for 1 VDT then irradiated with 60Co [2 Gy] 5. Treated simultaneously with 2ME2 [250 micro M] and IUdR [1 micro M] for 1 VDT then irradiated with 60Co [2 Gy] Then the DNA damage was evaluated using the alkaline comet assay method. The results showed that 2ME2 in combination with gamma irradiation of 60Co significantly [p<0.001] increased the DNA damage by IUdR as compared to the control group. Thus the combination of these two agents increased the cytogenetic effects of IUdR in the spheroid culture model of U87MG glioblastoma cell lines. By inhibiting the HIF-1 protein and preventing the G0 phase arrest, 2ME2 causes an increased progression into S phase and increases the IUdR absorption. Then the DNA damage in the spheroid cells increases as the uptake of IUdR is increased. These results suggest that the combined use of 2ME2 and 60Co can increase the radiosensitization effect of IUdR

Cytogenetic damages from iododeoxyuridine-induced radiosensitivity with and without methoxyamine in human glioblastoma spheroids

lododeoxyuridine-induced Radiosensitivityi [IUdR] is a halogenated thymidine analogue recognized to be effective in vitro and in vivo radioserisitizer in human cancers. It is reported that Methoxyamine [MX] potentiates DNA damages in cancer cells with blocking the repair pathway of lUdR damages. But studies, entirely, are restricted on monolayer culture cells from human colon cancer cells. Spheroids are 3D form of cells that aggregate and grow together which resemble in vivo tumor models in several aspects and the results of such studies can be extended to tumor in vivo. The aim of the current study was to evaluate DNA damages from IUdR and gamma rays with and without Methoxyamine in human Glioblastoma spheroids. The DNA induced damages in U87MG cell line were compared using alkaline comet assay method. Experiments were performed with two different sizes of spheroids [100omicrom and 300microm]. Evaluation of the effects of IUdR with and without MX pretreatment on spheroids following ionizing radiation showed that MX increased the cell damages of lUdR with and without irradiation in both diameters spheroids. The damages were further increased in 100microm compared with 300microm diameter. Comparisons of tail moments in spheroids with 100 and 300microm diameter showed that cell damages in larger spheroids, 300microm, are lesser than smaller one, 100microm. This could be due to existence of G[0] cells and cells with longer cycle which lUdR was less incorporated into them. Thus, decrease in lUdR radiosensitization and base wxcision repair [BER], results in reduction of MX activities. Using agents for Inhibiting the activities of proteins which are responsible for carrying the cells to G[0] may be beneficial in solving such problems

Comparsion between intracerebral biodistribution of [125I] IUdR in a rat glioma model and [125I] IUdR uptake in spheroid multicellular culture: effect of proliferative heterogencity

Radioiodinated iodo deoxyuridine [lUdR] is a novel, cycle-specific agent that has potential for the treatment of residual malignant glioma after surgery. Because this thymidine analogue kills only roliferating cells. However, maligment cells which are not synthesizing DNA during exposure to the radio pharmaceutical will be spared. To determine whether tumour incorporation [125 I]IUdR could be enhanced by protracted administration we employed three different delivery methods such as single injection, polymer implant and using osmotic pump in an in vivo study in compare with an in vitro study using multicellular glioma spheroids of a range of sizes and incubation times. We used a C6 cell line, growing in the brains of Wistar rats, as a glioma model and compared biodistribution of radiopharmaceutical in glioma cells by using autoradiography method. Au toradiogarphy technique also used to describe means of [125 I] lUdR incorporation at different times and depths within UVW multicellular glioma spheroids of a range of sizes. Twenty-four hours after administration of drug, autoradiography of brain section demonstrated nuclear uptake of the radiopharmaceutical in cells throughout tumour while normal brain cells remained free of radioactivity. The [125 I] IUdR labeling indices [percent +/- s.e.m.] achieved were 6.2 [0.4] by single injection, 22.5 [4.1] using a sustained release polymer implant [poly lactide-co-glycolide] and 34.3 [2.0] mini-osmotic pump. The results of the spheroids study confirm that incorporation of [125 I] lUdR decreased markedly with increasing size of spheroid. The distribution of lUdR was uniform thought small spheroids [<200 /micro], while the concentration of lUdR occurred predominantly in the peripheral cells of larger spheroids. The lUdR uptake enhancement occurred by increasing the incubation time from 52 hours to 104 hours i.e. one or two multiples of the initial volume doubling time. The results obtained from in vitro and in vivo studies emphasize the need for a sustained delivery system as a prerequisite for effective treatment. These findings are also encouraging for the development of a sustained release for radiolabel led IUdR for use in the treatment of intracranial tumours, particularly in the immediate postoperative setting