Abstract. The nuclear factor erythroid 2-related factor 2 (Nrf2) plays an important role in cellular defense against oxidative stress. Recent studies have demonstrated that Nrf2 is a useful target for cancer treatment, including radiation therapy. Ionizing radiation affects, not only the irradiated cells, but also the non-irradiated neighboring cells, and this effect is known as radiation-induced bystander effect. Upon exposure to radiation, the irradiated cells transmit signals to the non-irradiated cells via gap junctions or soluble factors. These signals in turn cause biological effects, such as a decrease in the clonogenic potential and cell death, in the non-irradiated neighboring cells. Nrf2 inhibition enhances cellular radiosensitivity. However, whether this modification of radiosensitivity by Nrf2 inhibition affects the radiation-induced bystander effects is unknown. In this study, we prepared an Nrf2 knockdown human lung cancer cell A549 and investigated whether the effects of irradiated cell conditioned medium (ICCM) on cell growth and cell death induction of non-irradiated cells vary depending on the Nrf2 knockdown. We found that Nrf2 knockdown resulted in a decrease in the cell growth and an increase in the radiosensitivity of A549 cells. When non-irradiated A549 cells were transfected with control siRNA and treated with ICCM, no significant difference was observed in the cell growth and proportion of Annexin V + dead cells between ICCM from non-irradiated cells and that from 2 or 8 Gy-irradiated cells. Similarly, no significant difference was observed in the cell growth and cell death induction upon treatment with ICCM in the Nrf2 knockdown A549 cells. Taken together, these results suggest that Nrf2 knockdown decreases cell growth and enhances the radiosensitivity of A549 cells; however, it does not alter the effect of ICCM on cell growth. IntroductionIonizing radiation causes biological effects, such as cell death and chromosomal aberrations, on cells. There are many evidences that ionizing radiation affects, not only the irradiated cells, but also the non-irradiated neighboring cells (1-3). Such response is known as radiation-induced non-targeted effects, which includes genomic instability and radiation-induced bystander effects. Genomic instability is characterized by effects such as delayed gene mutation and chromosomal aberrations that occur in the progeny of irradiated cells (3). In radiation-induced bystander effects, it has been suggested that the irradiated cells transmit signals to the non-irradiated cells via gap junctions or soluble factors (such as cytokines and growth factors) (1,2). To investigate the soluble factor-mediated bystander effects in vitro, non-irradiated cells were co-cultured with irradiated cells or cultured in irradiated cell conditioned medium (ICCM). It has been reported that non-irradiated cells co-cultured with irradiated cells or treated with ICCM undergo various biological responses, such as DNA double-strand breaks, decrease in clonogenic cell survival, and cell...
Ionizing radiation induces a cellular response not only in the irradiated cells, but also in the neighboring non-irradiated cells, and this effect is known as the radiation-induced bystander effect. The irradiated cells transmit signals to the non-irradiated cells via gap junctions or soluble factors, exerting biological effects on the neighboring non-irradiated cells. In this study, we investigated the effects of irradiated cell conditioned medium (ICCM) on the response of human lung cancer cells (A549 and H1299) to anticancer treatment. First, we analyzed the effects of ICCM on the induction of apoptosis by anticancer treatment (ionizing radiation or gefitinib). Human lung cancer cells were cultured with ICCM from non-irradiated and 8 Gy-irradiated cells and, subsequently, exposed to 8 Gy X-ray. In the A549 cells, the proportion of Annexin V + apoptotic cells was significantly lower in the cells treated with ICCM from 8 Gy-irradiated cells when compared with that in the cells treated with ICCM from non-irradiated cells (P<0.05), whereas this effect was not observed in the H1299 cells. Furthermore, no significant difference in the proportion of Annexin V + cells was noted following treatment with gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, between the cells treated with ICCM from non-irradiated cells and those treated with ICCM from the 8 Gy-irradiated cells. We then examined the effects of ICCM on the migration of A549 cells by scratch assay. ICCM from the 8 Gy-irradiated cells enhanced the migration of the 8 Gy-irradiated cells, but not that of the non-irradiated cells. Taken together, these results suggest that cancer cells treated with ICCM exhibit resistance to ionizing radiation in terms of apoptosis and cellular migration, although this phenomenon depends on the cell type.
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