Radiation quality effects alteration in COX-2 pathway to trigger radiation-induced bystander response in A549 lung carcinoma cells

Abstract

This study aimed to determine whether the radiation-induced bystander effect (RIBE) is affected by radiation quality. To mimic the different radiation qualities of the direct action (D)/indirect action (ID) ratio, A549 cells were exposed to X-rays, with either 100 mM of the radical scavenger, thio-urea (TU+), or null (TU-). Biological responses in irradiated and bystander cells were compared at equal lethal effects of a 6% survival dose, which was estimated from the survival curves to be 8 Gy and 5 Gy for TU+ and TU-, respectively. Cyclooxygenase-2 (COX-2) expression in TU- irradiated cells increased up to 8 h post-irradiation, before decreasing towards 24 h. The concentration of prostaglandin E2 (PGE2), a primary product of COX-2 and known as a secreted inducible factor in RIBE, increased over 3-fold compared with that in the control at 8 h post-irradiation. Conversely, COX-2 expression and PGE2 production of TU+ irradiated cells were drastically suppressed. These results show that the larger D/ID suppressed COX-2 expression and PGE2 production in irradiated cells. However, in contrast to the case in the irradiated cells, COX-2 expression was equally observed in the TU- and TU+ co-cultured bystander cells, which showed the highest expression levels at 24 h post-irradiation. Taken together, these findings demonstrate that radiation quality, such as the D/ID ratio, may be an important factor in the alteration of signalling pathways involved in RIBE.

Fig. 1.

Measurement of ROS production by X-ray exposure. Plots were fitted by linear regression after background subtraction. The slopes (rfu/Gy) of the graphs for TU^– and TU⁺ were 5931 and 2059, respectively. DCF = dichlorofluorescein, rfu = relative fluorescence units.

Fig. 2.

Plots were fitted according to the linear quadratic model, S = exp (–α × D – β × D²), where S is the survival fraction, D is the dose in Gy, and α [Gy⁻¹] and β [Gy⁻²] are the fitting parameters. The α, β, 10% survival dose (D₁₀) and 1% survival dose (D₁) for TU^– and TU⁺ were calculated to be TU^–: 0.38, 0.037, 4.27 Gy and 7.14 Gy and TU⁺: 0.175, 0.022, 6.96 Gy and 10.9 Gy, respectively.

Fig. 3.

Panel A shows the PGE₂ level of irradiated cells measured at 8 h post-irradiation with various X-ray doses. PGE₂ concentrations of 5 Gy–irradiated TU^– cells and TU⁺ cells were 93.4 pg/ml and 38.1 pg/ml, respectively. PGE₂ concentration for 8 Gy TU⁺ irradiated cells was 59.5 pg/ml, which was lower than 5 Gy TU^– irradiated cells (*P < 0.05). Panel B shows COX-2 expression after the addition of 0.1 ng/ml PGE₂ (#P = 0.1).

Fig. 4.

Comparison of COX-2 expression in TU^– and TU⁺ irradiated and bystander cells. Panels A-1 and B-1 are representative images of western blot analyses of irradiated cells and bystander cells, respectively. Panels A-2 and B-2 show the COX-2 expression for each hour post-irradiation of irradiated cells and bystander cells, respectively. *P < 0.05.