Effects of Nrf2 knockdown on the properties of irradiated cell conditioned medium from A549 human lung cancer cells

Abstract

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...

Effects of irradiated cell conditioned medium on the response of human lung cancer cells to anticancer treatment in vitro

“…However, under the present experimental conditions, at 0 Gy irradiation no significant differences in the proportion of Annexin V + apoptotic cells were observed between the cells treated with ICCM from the non-irradiated cells and those treated with ICCM from 8 Gy-irradiated cells ( Fig. 2A), as we have previously reported (11). In the A549 cells, the proportion of Annexin V + apoptotic cells was significantly increased following exposure to 8 Gy X-ray when compared to the cells exposed to 0 Gy X-ray after being cultured with ICCM from irradiated and non-irradiated cells (P<0.01).…”

“…Medium transfer experiments were performed, as previously reported (11). A schematic illustration of the medium transfer experiments is presented in Fig.…”

The Role of Nrf2 Activity in Cancer Development and Progression

“…Throughout our review, we analyzed the protective role of Nrf2 in the case of cancer, which leads to a weaker treatment response. In lung cancer, it was proven in vitro that A549 cells transfected with Nrf2siRNA were more prone to cell death as a response to radiotherapy than the control A549 cells [158]. At the same time, as Tian et al pointed out, it should be considered that Nrf2 also protects healthy cells from radiation-induced inflammation and a rise in oxidative stress [159].…”

The Therapeutic Potential of Kaemferol and Other Naturally Occurring Polyphenols Might Be Modulated by Nrf2-ARE Signaling Pathway: Current Status and Future Direction

“…The relationship between phytochemicals and Nrf2 signaling is shown In human lung cancer, persistent Nrf2 modulation has been shown to result in toxicity attenuation. Conversely, cellular response to drug therapy and ionizing radiations was enhanced upon Nrf2 knockdown [34]. In tumor metastasis, Nrf2 modulation has been shown to result in inhibition of pro-metastatic transcription factor Bach1 degradation, which resulted in promoting lung cancer [35].…”