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. 2015 Sep;56(5):777-83.
doi: 10.1093/jrr/rrv039. Epub 2015 Jul 9.

Radiation-induced bystander effect in non-irradiated glioblastoma spheroid cells

Fahime Faqihi  1 , Ali Neshastehriz  2 , Shokouhozaman Soleymanifard  3 , Robabeh Shabani  4 , Nazila Eivazzadeh  5
Affiliations

Radiation-induced bystander effect in non-irradiated glioblastoma spheroid cells

Fahime Faqihi et al. J Radiat Res. 2015 Sep.

Abstract

Radiation-induced bystander effects (RIBEs) are detected in cells that are not irradiated but receive signals from treated cells. The present study explored these bystander effects in a U87MG multicellular tumour spheroid model. A medium transfer technique was employed to induce the bystander effect, and colony formation assay was used to evaluate the effect. Relative changes in expression of BAX, BCL2, JNK and ERK genes were analysed using RT-PCR to investigate the RIBE mechanism. A significant decrease in plating efficiency was observed for both bystander and irradiated cells. The survival fraction was calculated for bystander cells to be 69.48% and for irradiated cells to be 34.68%. There was no change in pro-apoptotic BAX relative expression, but anti-apoptotic BCL2 showed downregulation in both irradiated and bystander cells. Pro-apoptotic JNK in bystander samples and ERK in irradiated samples were upregulated. The clonogenic survival data suggests that there was a classic RIBE in U87MG spheroids exposed to 4 Gy of X-rays, using a medium transfer technique. Changes in the expression of pro- and anti-apoptotic genes indicate involvement of both intrinsic apoptotic and MAPK pathways in inducing these effects.

Keywords: U87MG spheroid; apoptosis; radiation-induced bystander effect.

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Figures

Fig. 1.
Fig. 1.
Viability percent using Trypan Blue dye 24 h after irradiation and medium transfer. Each bar represents the mean ± SD of the results for three independent experiments.
Fig. 2.
Fig. 2.
Plating efficiency using U87MG of various concentrations of cells of spheroids cultured in MEM supplemented with 10% FBS. Each bar represents the mean ± SD of the results for three independent experiments.
Fig. 3.
Fig. 3.
Relative fold-changes in gene expression levels of BAX in U87MG spheroid following 4-h and 24-h exposure to 4 Gy direct and bystander X-ray irradiation. Each bar represents the mean ± SD of the results of three independent experiments. Asterisk represents significant changes.
Fig. 4.
Fig. 4.
Relative fold-changes in gene expression levels of BCL2 in U87MG spheroid following 4-h and 24-h exposure to 4 Gy direct and bystander X-ray irradiation. Each bar represents the mean ± SD of the results of three independent experiments. Asterisk represents significant changes.
Fig. 5.
Fig. 5.
Relative fold-changes in gene expression levels of JNK in U87MG spheroid following 4-h and 24-h exposure to 4 Gy direct and bystander X-ray irradiation. Each bar represents the mean ± SD of the results of three independent experiments. Asterisk represents significant changes.
Fig. 6.
Fig. 6.
Relative fold-changes in gene expression levels of ERK in U87MG spheroid following 4-h and 24-h exposure to 4 Gy direct and bystander X-ray irradiation. Each bar represents the mean ± SD of the results of three independent experiments. Asterisk represents significant changes.
Fig. 7.
Fig. 7.
BAX/BCL2 relative fold expression ratio in U87MG spheroid following 4-h and 24-h exposures to 4 Gy direct and bystander X-ray irradiation Each bar represents the mean ± SD of the results of three independent experiments.
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