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. 2020 Oct 30;19(22):3195–3207. doi: 10.1080/15384101.2020.1838793

Astragalus polysaccharide inhibits radiation-induced bystander effects by regulating apoptosis in Bone Mesenchymal Stem Cells (BMSCs)

Yi-Ming Zhang a,*, Li-Ying Zhang a,*, Heng Zhou b, Yang-Yang Li a, Kong-Xi Wei a, Cheng-Hao Li a, Ting Zhou a, Ju-Fang Wang b, Wen-Jun Wei b, Jun-Rui Hua b, Yun He a, Tao Hong a, Yong-Qi Liu a,✉
PMCID: PMC7714477  PMID: 33121344

ABSTRACT

The purpose of this study was to investigate the effects of astragalus polysaccharides (APS) on the proliferation and apoptosis of bone marrow mesenchymal stem cells (BMSCs) induced by X-ray radiation-induced A549 cells bystander effect (RIBE), and to explore their mechanisms. In this study, APS increased the reduced cell proliferation rate induced by RIBE and inhibiting the apoptosis of bystander cells. In terms of mechanism, APS up-regulates the proteins Bcl-2, Bcl-xl, and down-regulates the proteins Bax and Bak, which induces a decrease in mitochondrial membrane potential, which induces the release of Cyt-c and AIF, which leads to caspase-dependent and caspase-independent pathway to cause apoptosis. In addition, we believe that ROS may be the main cause of these protein changes. APS can inhibit the generation of ROS in bystander cells and thus inhibit the activation of the mitochondrial pathway, further preventing cellular damage caused by RIBE.

KEYWORDS: APS, RIBE, BMSCs, Astragalus Polysaccharide, Bystander Effect, Apoptosis

Introduction

Currently, radiotherapy is still a common treatment for many cancers [1]. Among all kinds of cancers, non-small cell lung cancer is most sensitive to ionizing radiation [2,3]. X-rays are most commonly used in clinical radiotherapy, which have short wavelengths but have a strong penetrating power. While killing tumor cells, it also damages normal tissues and cells [4]. However, the radiation-induced bystander effects (RIBE) is one of the major problems of radiotherapy [5]. RIBE is a phenomenon in which unirradiated cells exhibit irradiated effects as a result of signals received from nearby irradiated cells. Studies have shown that soluble cytokines induced by radiation can promote the release of ROS [6]. Thus, the neighboring cells or distant tissues would present the same biological endpoints of radiation such as DNA damage [7], chromosome aberrations [8], cell death [9], and gene mutation [10].

BMSCs are multipotent cells with self-replicative, regenerative, and immunomodulatory properties [11,12] and they can migrate to the inflammation site, injury tissue and even tumors, including lung cancer [13]. It has been reported that the expression of oncogenes and tumor suppressor genes and the chromosomal abnormalities were observed in BMSCs co-cultured with tumor cells [14,15]. It is also shown that RIBE could cause DNA damage, and when the damage could not be repaired, cells were induced to apoptosis [16,17]. ROS is the culprit involved in RIBE. High levels of ROS can cause apoptosis [18]. ROS can cause a decrease in mitochondrial membrane potential and thus induce apoptosis induced by the mitochondrial pathway [19,20]. However, the mechanism of apoptosis of BMSCs caused by the RIBE is still unclear so far.

Astragalus polysaccharide (APS) is a major active ingredient of Astragalus membranaceus [21], APS has many effects, such as immunoregulatory [22], anti-oxidant [23] and anti-inflammatory properties [24]. More evidence shows that APS has a variety of protective effects on BMSCs. APS can promote the proliferation and osteogenic differentiation of BMSCs [25], maintain the normal morphology of the cells [26], and can effectively inhibit the inflammatory response caused by RIBE [27]. Yang has proved that APS suppresses the release of mitochondrial ROS to reduce inflammation in the Mesenchymal Stem Cell line [28]. However, it has not been reported that APS protects BMSCs from apoptosis caused by RIBE. In this experiment, we used the co-culture model to study the difference in apoptosis of BMSCs in the co-culture system and the radiation co-culture system, and the intrinsic apoptotic mechanisms were analyzed and discussed. A549 cells were irradiated by 2 GY X-rays and then co-cultured with BMSCs in Transwell to study the apoptosis on BMSCs. The effects of APS on apoptosis of BMSCs in RIBE were investigated and understand the mechanisms of the mitochondrial pathway involved. This is the first time we have confirmed this finding.

Materials and methods

Cell culture

BMSCs (ScienCell Research Laboratories, USA) were cultured in a mesenchymal stem cell medium (ScienCell Research Laboratories, USA) and maintained in a 5% CO2 humidified incubator (Thermo Fisher Scientific, USA) at 37°C. Human non-small cell lung cancer cell line, A549 (Shanghai Institute for Biological Sciences, China), was cultured in DMEM/F-12 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Hyclone, USA), 100units/mL penicillin and 100 mg/mL streptomycin and maintained in a 5% CO2 humidified incubator at 37°C.

Irradiation

X-ray radiation was generated by X-RAD-225 X-ray source (Precision, North branford, USA). The dose rate was about 200 cGy/min (225.0 kV, 13.3 mA). The total absorbed dose was 6 Gy. All irradiations were performed at room temperature.

Preparation of APS

The purity of APS (Shanghai Yuanye Biological Technology Company, China) was more than 98%. Based on our previous studies (Zhang et al., 2019), APS can be dissolved in the culture medium and the final concentration of APS was 50 μg/mL.

Co-culture model

This experiment was carried out using Transwell. A549 cells were plated at 2.4 × 104 cells per well in the upper compartment and BMSCs were plated at 1 × 105 cells per well in the lower compartment. The next day, the upper compartment was taken to radiation. After irradiation, the two-chamber cells were Co-cultured in an incubator for 3 days. The control group was also cultured in the Transwell, but the upper compartment was not seeded with A549 cells, only the lower compartment was seeded with BMSCs cells.

Groups

In this study, BMSCs were divided into five groups. BMSCs cultured with mesenchymal stem cell medium served as a blank control group (Ctrl).BMSCs cultured with A549 cells in Transwell for 3 days served as a Co-culture group (Co). BMSCs cultured with A549 cells in Transwell with mesenchymal stem cell medium rich in 50 µg/mL APS for 3 days served as a Co-culture and APS group (Co+APS). BMSCs cultured with irradiated A549 cells in Transwell for 3 days served as a Co-culture and Irradiation group (Co+IR). BMSCs cultured with irradiated A549 cells in Transwell with mesenchymal stem cell medium rich in 50 µg/mL APS for 3 d served as a Co-culture and Irradiation and APS group (Co+IR+APS).

Cell counting experiment

1 × 105 BMSCs cells were plated into the lower compartment of the transwells in each group. The number of BMSCs was counted after 3 days using a Coulter Counter (Beckman, Brea, USA) and BMSCs were photographed by microscope in 3 days (Nikon, Tokyo, Japan).

CCK-8 assay

The CCK-8 assay kit (Dojindo, Japan) was used to detect cell viability according to the manufacturer’s instructions. After treatment for 3 days with different media, cells were seeded onto 96-well plates at a density of 2000 cells per well. Then, cell viability was detected at 1, 2, 3, 4 and 5 days. Then, 20 μL of CCK-8 (10% in culture medium) were added to cells at each time point. Cells were then incubated for 4 h at 37°C. After agitation for 10 min on a shaker, absorbance at 450 nm was read using a microplate reader (Tecan Infinite M200; Salzburg, Austria).

Hoechst staining

BMSCs were seeded onto coverslips in lower compartment and then co-cultured with irradiated A549 cells for 3 days. After removal of culture medium, cells were exposed to staining solution containing Hoechst 33,258 (1 mg/ml) at 37°C for 10–30 min. Cells with chromatin condensation were visualized and photographed using a confocal microscope (Olympus) 30 min after addition of the staining solution. Chromatin condensation is the most characteristic feature of apoptosis. Apoptotic cells were countd the number of apoptosis in 1000 BMSCs in 6-8 fieldsrandomly selected areas.

Flow cytometric analysis

Apoptosis was detected using the Annexin V-Fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis assay kit (Sungene Biotech, Tianjin, China). In brief, treated cells were collected by centrifugation, washed twice with ice-cold PBS, and resuspended in 500 μL 1× Annexin V binding buffer containing 5 μL Annexin V-FITC and 5 μL of PI. After incubation for 10 min at room temperature, apoptosis was analyzed by flow cytometry. The levels of ROS (Shanghai, Xiangsheng, Cat #50101ES01, China) in bystander cell were detected by flow cytometric following the manufacturer’s instructions.

ELISA

The levels of ROS (human ROS ELISA Kit; Westang Bio-tech Co Ltd, Shanghai, China; Cat # F02471) in the conditioned medium and the culture supernatant of each group were detected by ELISA kits following the manufacturer’s instructions.

Assays for mitochondrial membrane potential

The mitochondrial membrane potential of BMSCs was detected using a mitochondrial membrane potential assay kit (JC-1; Beyotime). In accordance with the manufacturer's instructions, different groups BMSCs were grown on a glass coverslip in transwell culture plates, pretreated with APS for 72 h, and incubated with JC-1 at 37°C for 20 min. Images were captured using a confocal microscope. Mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control. Images were captured using a confocal microscope.

Western blot analysis

Cells were lysed in RIPA buffer (P0013 C, Beyotime, Shanghai, China). Proteins were separated by 10% SDS-PAGE and transferred to a methanol activated PVDF membrane (GVPPEAC12, Millipore, Billerica, USA). The membrane was blocked for 1 h in PBST containing 5% milk and subsequently probed with Bcl-2 antibody (GTX128736, GeneTex, Texas, USA), Bcl-xl antibody (GTX115960, GeneTex, Texas, USA), Bax antibody (GTX103726, GeneTex, Texas, USA), Bak antibody (GTX105983, GeneTex, Texas, USA), Caspase 3 antibody (GTX135674, GeneTex, Texas, USA), Cyt-c antibody (GTX105983, GeneTex, Texas, USA), AIF antibody (SC-13,116, SantaCruz, USA) and GAPDH antibody (ab9485, Abcam, Cambridge, UK) for 2 h. After a 1 h incubation with a goat-anti-rabbit HRP-conjugated secondary antibody (ab97051, Abcam, Cambridge, UK), the protein bands were detected with luminal reagent (Millipore, Pittsburgh, USA), and their relative intensities were quantified using Image J.

Immunofluorescence staining

The cells of each group were fixed in 4% polyformaldehyde for 15 min and permeabilized in PBS with 0.5% Triton-X 100. After blockage with 5% nonfat milk for 1 h, cells were incubated with Caspase 3 antibody (GTX135674, GeneTex, Texas, USA) for 2 h at room temperature. After incubation with anti-rabbit antibody (G1101, Abcam, USA) for 1 h at room temperature, cells were counter-stained with DAPI (Invitrogen, Cat #P36941) and observed with a confocal microscope. At least 100 cells were calculated in each group.

Statistical analysis

Each in vitro experiment was repeated at least 3 times, and the data were analyzed by SPSS (v13.0). The statistical significance was calculated by one-way ANOVA or by the t test. P < 0.05 was considered significant.

Result

Inhibitory effect of Co-culture after irradiation on bystander cell viability and the protective effects of APS

To determine the effect of APS on cell viability of bystander cell BMSCs, cell count assay and CCK8 assay were performed. There were no differences between the control group, the Co-culture group, and the Co-culture and APS group (P > 0.05). The cell viability (Figure 1a, c) and cellular count rate (Figure 1b) of bystander cell BMSCs were distinctly inhibited in Radiation co-culture system (P < 0.05) compared with control group and Co-culture group. Additionally, cell viability (Figure 1c) and cellular count rate (Figure 1b) of the Co+IR+APS group was significantly higher than in the Co+IR group (P < 0.05). These data show that APS has protective effects on cell survival of bystander cells against Co-culture after irradiation.

Figure 1.

Figure 1.

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APS promote proliferation of bystander cells. (a)The morphology of Co-cultured after radiation and APS-treated BMSCs for 72 h. Images were taken using a microscope. (b) The number of BMSCs counted for the third day using a Coulter Counter. (c) The proliferation rate of BMSCs in each group in 5 days with CCK-8 assay. All data are expressed as the mean±SD (n = 3) and statistical significance (*p < 0.05, or **p < 0.01) is shown for comparison between the two groups as shown in the figures

Radiation-induced bystander effect promotes the apoptosis of BMSCs and APS reduce them

To determine the effect of APS on apoptosis of bystander cell BMSCs, Hoechst staining assay and Flow cytometric assay were performed. The morphological changes of BMSCs were observed by confocal microscope after being cultured for 3 days (Figure 2a) and the number of cell apoptosis was counted. The apoptosis rate of BMSCs was observed by flow cytometer (Figure 2b) and statistic the differences between each group. In terms of the number of apoptosis, there was no difference between the control group, the co-culture group, the co-culture and the APS group (P > 0.05) (Figure 2c); in terms of the apoptosis rate, the co-culture group increased the apoptosis rate (P < 0.05) (Figure 2d). The number of cell apoptosis and apoptosis rate of bystander cell BMSCs was distinctly promoted in radiation co-culture system (P < 0.05) compared with the control group and the co-culture group. Additionally, the number of cell apoptosis (Figure 2c) and apoptosis rate (Figure 2d) of the Co+IR+APS group was significantly lower than in the Co+IR group (P < 0.05). These data show that APS has protective effects on cell apoptosis of bystander cells against radiation-induced bystander effect.

Figure 2.

Figure 2.

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APS inhibit apoptosis of bystander cells. (a) The image of apoptosis in BMSCs in each group with Hoechst staining assay (200× magnification). (b)Flow cytometric analysis of cell apoptosis was conducted using Annexin V-FITC+PI staining. (c) The number of apoptosis in each group of BMSCs that counted 1000 cells. (d)Percentage of apoptotic cells in each group. All data are expressed as the mean±SD (n = 3) and statistical significance (*p < 0.05, or **p < 0.01) is shown for comparison between the two groups as shown in the figures

Radiation-induced bystander effect promotes the production of ROS in BMSCs and APS reduce them

To detect the change of ROS in bystander cells, we tested the relative concentration of ROS in the conditioned medium of each experimental group. In the control group, the ROS expression was maintained at a baseline level. In other groups, the expression of ROS increased first and then decreased, reaching the peak at 24 h. Compared with the Ctrl group, the ROS content of the Co+IR group increased at various times (P < 0.05); compared with the Co+IR group, the ROS content of the Co+IR+APS group decreased at all time points (P < 0.05) (Figure 3a).

Figure 3.

Figure 3.

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APS reduces ROS generation of bystander cells. (a)Relative concentration of ROS in CM for 72 h by ELISA. (b) The ROS rate of BMSCs in each group in 24 h with Flow cytometry (c) Percentage of ROS positive cell rate in each group. All data are expressed as the mean±SD (n = 3) and statistical significance (*p < 0.05, or **p < 0.01) is shown for comparison between the two groups as shown in the figures

After that, we tested the ROS content in the 24 h BMSCs of each experimental group. Similarly, compared with the Ctrl group, the BMSCs ROS content in the Co group and the Co+IR group increased (P < 0.05), compared with the Co group, the ROS content of the Co+IR group increased (P < 0.05), and the ROS content of the Co+APS group decreased (P < 0.05). Compared with the Co+IR group, the BMSCs ROS content in the Co +IR+APS group decreased (P < 0.01) (Figure 3b, c). The above data shows that the ROS content increases in both the co-culture system and the radiation co-culture system, and the increase is more obvious in the radiation co-culture system. APS can reduce the ROS content in bystander cells.

Radiation-Induced bystander effect can induce mitochondrial membrane potential loss and APS regulates it

In order to examine the mitochondrial damage of bystander cells treated by Co-culture after irradiation, we used the fluorescent probe JC-1 to detect changes in mitochondrial membrane potential of BMSCs and observed the expression of Cyt-c and AIF by Western blot. The experimental results found that a lot of green fluorescence (membrane potential loss) was found in the CCCP group and a large amount of red fluorescence (membrane potential active) was found in the Ctrl group. The expression of Cyt-c and AIF was lower in the Ctrl group. Compared with the Ctrl group, a large amount of green fluorescence (membrane potential loss) was found in the Co+IR group (P < 0.01) (Figure 4a), the expression of Cyt-c and AIF was higher (P < 0.05), green fluorescence was also appeared in Co group (P < 0.01), AIF release increased, but Cyt-c expression was not obvious (Figure 4c, e); When cells were treated with 50 μg/mL APS before Co-culture after irradiation, the ratio of red fluorescence to green fluorescence in mitochondrial membrane potential increased (P < 0.01), the level of Cyt-c and AIF decreased (P < 0.05). These results indicate that APS can protect mitochondrial membrane potential damage and inhibit Cyt-c and AIF release.

Figure 4.

Figure 4.

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APS protects bystander cells from apoptosis through the mitochondrial pathway (a) The image of mitochondrial membrane in bystander cells by fluorescent microscope (600× magnification). Positive control group was treated with 10 μM CCCP for 1 h. (b)The JC-1 red/green fluorescence ratio reflects the mitochondrial membrane potential. Fluorescence intensities from five randomly selected microscopic fields per group were measured and analyzed. (c) The western blotting bands of Cyt-c. (d) The protein statistical result of Cyt-c. (e) The western blotting bands of AIF. (f) The protein statistical result of AIF. All data are expressed as the mean±SD (n = 3) and statistical significance (*p < 0.05, or **p < 0.01) is shown for comparison between the two groups as shown in the figures

Radiation-Induced Bystander effect induced BMSCs apoptosis through activation of the mitochondrial pathway and APS regulates it

To explore the mitochondrial pathway activation in bystander cells treated by Co-culture after irradiation, we observed the expression of Bcl-2, Bcl-xl, Bax, Bak, and Caspase 3 in each group by Western blot.

The results showed that compared with the control group, the protein expression of Bcl-2 and Bcl-xl in Co group and Co+IR group was decreased (P < 0.05), and the decrease in Co+IR group was more obvious. After treating cells with APS, Bcl −2 and Bcl-xl expression increased (P < 0.05). The protein levels of Bax, Bak, and Caspase 3 in the Co and Co+IR groups were significantly higher than those in the control group (P < 0.05), and Caspase 3 increased more significantly in the Co+IR group, and they were all down-regulated by APS (Figure 5a, b). In addition, we measured the expression level of Caspase 3 by immunofluorescence staining (Figure 5c, d). The results of immunofluorescence showed a similar trend to Western blotting of Caspase 3, indicating that both the co-culture system and the radiation co-culture system can cause mitochondrial pathway apoptosis in BMSCs, and the apoptosis caused by the radiation co-culture system is more serious, APS could regulate the mitochondrial pathway to protect BMSCs apoptosis from radiation bystander effect.

Figure 5.

Figure 5.

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APS reduce expression of apoptotic proteins of bystander cells. (a) The western blotting bands of Bcl-2, Bcl-xl, Bax, Bak and Caspase 3. (b) The protein statistical result of Bcl-2, Bcl-xl, Bax, Bak and Caspase 3. (c) Representative immunofluorescence images of Caspase 3 at 72 h by fluorescent microscope (600× magnification). (d) Relative expression of Caspase 3 in bystander cells. All data are expressed as the mean±SD (n = 3) and statistical significance (*p < 0.05, or **p < 0.01) is shown for comparison between the two groups as shown in the figures

Discussion

At present, radiotherapy is one of the main treatments for lung cancer, which can significantly improve the survival rate and local tumor control rate of lung cancer patients [29]. Radiation can not only kill tumor cells but also cause damage to unirradiated cells. RIBE can cause cell DNA breaks, growth inhibition, apoptosis and even carcinogenesis [7–10]. The study found that a large number of BMSCs will appear in lung cancer tissues after radiation [30]. The reason may be that RIBE will affect the tumor microenvironment, and make BMSCs recruit tumor tissues from the circulation and adjacent tissues [31]. Due to the effects of RIBE, the lung cancer cells which were irradiated can cause damage to BMSCs. Therefore, how RIBE affects BMSCs and how to protect them from damage have recently been raised.

In the study of RIBE, the transfer conditioned medium model and the co-culture model are the two most common biological models, both of which can transfer signal molecules such as cytokines produced by irradiated cells to unirradiated cells. In our previous studies, Micronuclei and Foci dots are generated in BMSCs after treatment with conditioned medium, which indicates that RIBE can cause cellular DNA damage [27]. Compared with the transfer conditioned medium, the co-culture model can better simulate the human tumor microenvironment, thus better reflecting the principle of RIBE. In this work, we used the co-culture model to observe the effects of RIBE on the apoptosis of BMSCs, and to study the anti-apoptotic effect of APS in RIBE and its possible mechanism.

In this work, we used Transwell co-cultivation for modeling, inoculating A549 cells in the upper layer and BMSCs cells in the lower layer to explore the effect of cytokines released by A549 cells on the apoptosis of BMSCs in the co-culture system. Our study found that after 3 days of co-culture with A549 cells, there was no significant change in the proliferation of BMSCs cells, but the phenomenon of BMSCs apoptosis could occur. There was a slight increase in ROS in the conditioned medium of the co-cultivation system. There is an upward adjustment, which seems to be similar to many reports in the literature. Yang X research found that the co-culture of A549 cells and HUVECs cells can lead to apoptosis of endothelial cells [32]. Li Z research showed that when endothelial cells co-cultured with A549 cells, it can lead to apoptosis of A549 cells, possibly with NO Regarding the production [33], Alberto research believes that when A549 is co-cultured with fibroblasts, the ROS content of these two types of cells increases on average [34]. What’s more, our research also found that the mitochondrial membrane potential of BMSCs after co-cultivation was also lost to varying degrees, which shows that the co-culture system may also potentially activate the mitochondrial pathway, and interestingly, in the co-culture system, AIF release increased, but Cyt-c did not change significantly, pro-apoptotic proteins Bax and Bak were up-regulated, but anti-apoptotic proteins Bcl-2 and Bcl-xl did not change significantly. Therefore, we cannot determine whether the apoptosis caused by the mitochondrial pathway actually exists in the co-culture system. Even though the end point protein of apoptosis, Caspase 3, is also expressed, it is not possible to determine its apoptosis pathway. We suspect that in addition to the mitochondrial pathway, there may be other apoptotic pathways, and that in addition to ROS influencing factors, there may be interference effects of other cytokines. However, the results of this study are relatively clear that compared with the co-culture system, the apoptosis phenomenon in the radiation co-culture system is more obvious, and it is determined that the possibility of apoptosis can be exerted through the mitochondrial pathway. Below we focus on the mechanism of action in radiation co-cultivation.

When irradiating the upper layer of A549 cells, the irradiated cells can release a large number of soluble cytokines [35]. These signaling molecules can pass through the polycarbonate membrane, causing a series of changes in the underlying BMSCs. The most obvious of these signaling molecules is ROS [36]. ROS can enter the cytoplasm of MSCs through the cell membrane by free diffusion, which damages DNA and mitochondria [37–39], causing a series of biological effects of BMSCs. It can cause cell genome instability, growth inhibition and apoptosis. Some scholars have suggested that the content of the inflammatory pathway is very high in the transfer conditioned medium [40], and some scholars have suggested that ROS can cause the activation of mitochondrial pathway [41], then ROS under the radiation co-culture model Will it cause activation of the mitochondrial pathway of the underlying stem cells? Previous studies have shown that ROS produced by the para-radiation effects can cause mitochondrial damage. When the damage is unable to repair, some cells will initiate the apoptotic pathway, the most common being the mitochondrial apoptotic pathway [42]. Mitochondria-mediated pathway of apoptosis is the intrinsic pathway, which is triggered upon mitochondrial injury including loss of integrity of mitochondrial outer membrane. Then, cytochrome c is released from the mitochondria into the cytosol. The Bcl-2 family plays a crucial role in the mitochondrial apoptotic pathway. Bcl-2 and Bcl-xl are anti-apoptotic proteins that inhibit the release of Cyt-c and prevent Cyt-c from reaching the Caspase cascade downstream, thereby inhibiting cell apoptosis [43,44]. Bax and Bak are pro-apoptotic proteins. Over-expression can release Cyt-c in a large amount in mitochondria, further activate the Caspase cascade, and finally activate the apoptosis end point protein Caspase 3 to induce apoptosis [45]. Here, our study found that the mitochondrial membrane potential of bystander cells co-cultured after irradiation decreased, the expression of Bcl-2 and Bcl-xl decreased, and then the expression of Bax and Bak increased, and a large amount of Cyt-c released into the cytoplasm which resulted in increased expression of Caspase3. After APS treatment, mitochondrial membrane potentials increased in bystander cells, Bcl-2, Bcl-xl expressions increased, Bax, Bak, Cyt-c, and Caspase 3 expressions decreased. These phenomena indicate that the bystander effect can activate the mitochondrial pathway and cause apoptosis, and the addition of APS can reduce cell damage.

When cells are subjected to external stimuli such as ROS, the permeability of the mitochondrial membrane may be changed [46]. Mitochondria will not only release Cyt-c but also release Apoptosis-inducing factor (AIF) into the cytoplasm. AIF transfers to the nucleus, leading to chromosome agglutination and DNA breakage, thereby mediating caspase-independent pathway apoptosis [47,48]. This study found that the expression of AIF protein of bystander cells co-cultured after irradiation increased, and it decreased when APS was added. The above results reflect that the RIBE induced apoptosis not only occurs from the caspase-dependent pathway but also occurs from the caspase-independent pathway. APS can protect mitochondrial damage and reduce apoptosis.

Conclusions

In this study, a co-culture model of decreased-RIBE by APS in BMSCs was summarized (Figure 6). This work can confirm that apoptosis of BMSCs in radiation co-culture is caused by mitochondrial pathway, which may be due to the release of ROS from irradiated A549 cells into the medium. ROS can cause mitochondrial damage, thereby activating caspase-dependent way induced by Cyt-c and caspase-independent way induced by AIF, ultimately leading to cell growth inhibition and apoptosis. APS can inhibit the activation of mitochondrial pathway, and promote cell growth and inhibit apoptosis. Mitochondrial pathway may be one of the potential mechanisms of APS against RIBE. Our findings may prove that APS can protect against “Bad stuff” of lung cancer radiotherapy, but “how” APS works is an interesting topic of further exploration.

Figure 6.

Figure 6.

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Proposed model for the protective function of APS in ROS-mediated RIBE. In summary, the ROS delivered from irradiated A549 cells and the bystander cells BMSCs can cause apoptosis. RIBE induced apoptosis through caspase-dependent and caspase-independent pathways.ROS can activate the mitochondrial pathway and cause cells damage. APS might reduce the generation of ROS and block RIBE through regulating the mitochondrial pathway

Funding Statement

This study was supported by the National Natural Science Foundation of China [Nos. 81973595 and 82004094]; Collaborative Innovation Project of Chinese and Tibetan Medicine [No. XBXT2015-1], Project of Gansu Provincial Education Department [No. 2015A-095] and Project of Lanzhou Municipal Science and Technology Bureau[No. 2015-2-48].

Disclosure statement

No potential conflict of interest was reported by the authors.

References

  • [1].Shipley WU, Seiferheld W, Lukka HR, et al. Radiation with or without antiandrogen therapy in recurrent prostate cancer. N Engl J Med. 2017;376(5):417–428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Videtic GMM, Donington J, Giuliani M, et al. Stereotactic body radiation therapy for early-stage non-small cell lung cancer: executive summary of an ASTRO evidence-based guideline. Pract Radiat Oncol. 2017;7(5):295–301. [DOI] [PubMed] [Google Scholar]
  • [3].Ahmed KA, Kim S, Arrington J, et al. Outcomes targeting the PD-1/PD-L1 axis in conjunction with stereotactic radiation for patients with non-small cell lung cancer brain metastases. J Neurooncol. 2017;133(2):331–338. [DOI] [PubMed] [Google Scholar]
  • [4].Zhang H, Zhai Z, Wang Y, et al. Resveratrol ameliorates ionizing irradiation-induced long-term hematopoietic stem cell injury in mice. Free Radic Biol Med. 2013;54:40–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Bazak J, Korytowski W, Girotti AW.. Bystander effects of nitric oxide in cellular models of anti-tumor photodynamic therapy. Cancers (Basel). 2019;11(11):1674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Schaue D, Micewicz ED, Ratikan JA, et al. Radiation and inflammation. Semin Radiat Oncol. 2015;25(1):4–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Yang H, Asaad N, Held KD.. Medium-mediated intercellular communication is involved in bystander responses of X-ray-irradiated normal human fibroblasts. Oncogene. 2005;24(12):2096–2103. [DOI] [PubMed] [Google Scholar]
  • [8].Kadhim MA, Macdonald DA, Goodhead DT, et al. Transmission of chromosomal instability after plutonium alpha-particle irradiation. Nature. 1992;355(6362):738–740. [DOI] [PubMed] [Google Scholar]
  • [9].Mothersill C, Seymour C. Medium from irradiated human epithelial cells but not human fibroblasts reduces the clonogenic survival of unirradiated cells. Int J Radiat Biol. 1997;71(4):421–427. [DOI] [PubMed] [Google Scholar]
  • [10].Sawant SG, Randers-Pehrson G, Geard CR, et al. The bystander effect in radiation oncogenesis: I. Transformation in C3H 10T1/2 cells in vitro can be initiated in the unirradiated neighbors of irradiated cells. Radiat Res. 2001;155(3):397–401. [DOI] [PubMed] [Google Scholar]
  • [11].Rolandsson Enes S, Andersson Sjöland A, Skog I, et al. MSC from fetal and adult lungs possess lung-specific properties compared to bone marrow-derived MSC. Sci Rep. 2016;6:29160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Wu X, Wang Z, Qian M, et al. Adrenaline stimulates the proliferation and migration of mesenchymal stem cells towards the LPS-induced lung injury. J Cell Mol Med. 2014;18(8):1612–1622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Wong SP, Rowley JE, Redpath AN, et al. Pericytes, mesenchymal stem cells and their contributions to tissue repair. Pharmacol Ther. 2015;151:107–120. [DOI] [PubMed] [Google Scholar]
  • [14].Liu C, Chen Z, Chen Z, et al. Multiple tumor types may originate from bone marrow-derived cells. Neoplasia. 2006;8(9):716–724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Shain KH, Yarde DN, Meads MB, et al. Beta1 integrin adhesion enhances IL-6-mediated STAT3 signaling in myeloma cells: implications for microenvironment influence on tumor survival and proliferation. Cancer Res. 2009;69(3):1009–1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Bill MA, Srivastava K, Breen C, et al. Dual effects of radiation bystander signaling in urothelial cancer: purinergic-activation of apoptosis attenuates survival of urothelial cancer and normal urothelial cells. Oncotarget. 2017;8(57):97331–97343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Bewicke-Copley F, Mulcahy LA, Jacobs LA, et al. Extracellular vesicles released following heat stress induce bystander effect in unstressed populations. J Extracell Vesicles. 2017;6(1):1340746. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Shao C, Folkard M, Michael BD, et al. Targeted cytoplasmic irradiation induces bystander responses. Proc Natl Acad Sci U S A. 2004;101(37):13495–13500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Murphy JE, Nugent S, Seymour C, et al. Mitochondrial DNA point mutations and a novel deletion induced by direct low-LET radiation and by medium from irradiated cells. Mutat Res. 2005;585(1–2):127–136. [DOI] [PubMed] [Google Scholar]
  • [20].Maguire P, Mothersill C, Seymour C, et al. Medium from irradiated cells induces dose-dependent mitochondrial changes and BCL2 responses in unirradiated human keratinocytes. Radiat Res. 2005;163(4):384–390. [DOI] [PubMed] [Google Scholar]
  • [21].Yuan LB, Hua CY, Gao S, et al. Astragalus polysaccharides attenuate monocrotaline-induced pulmonary arterial hypertension in rats. Am J Chin Med. 2017;45(4):773–789. [DOI] [PubMed] [Google Scholar]
  • [22].Zhou X, Liu Z, Long T, et al. Immunomodulatory effects of herbal formula of astragalus polysaccharide (APS) and polysaccharopeptide (PSP) in mice with lung cancer. Int J Biol Macromol. 2018;106:596–601. [DOI] [PubMed] [Google Scholar]
  • [23].Hamid M, Liu D, Abdulrahim Y, et al. Amelioration of CCl4-induced liver injury in rats by selenizing Astragalus polysaccharides: role of proinflammatory cytokines, oxidative stress and hepatic stellate cells. Res Vet Sci. 2017;114:202–211. [DOI] [PubMed] [Google Scholar]
  • [24].Lu J, Chen X, Zhang Y, et al. Astragalus polysaccharide induces anti-inflammatory effects dependent on AMPK activity in palmitate-treated RAW264.7 cells. Int J Mol Med. 2013;31(6):1463–1470. [DOI] [PubMed] [Google Scholar]
  • [25].Li Q, Xing W, Gong X, et al. Astragalus polysaccharide promotes proliferation and osteogenic differentiation of bone mesenchymal stem cells by down-regulation of microRNA-152. Biomed Pharmacother. 2019;115:108927. [DOI] [PubMed] [Google Scholar]
  • [26].Zhang YM, Liu YQ, Liu D, et al. The effects of astragalus polysaccharide on bone marrow-derived mesenchymal stem cell proliferation and morphology induced by A549 lung cancer cells. Med Sci Monit. 2019;25:4110–4121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Zhang L, Luo Y, Lu Z, et al. Astragalus polysaccharide inhibits ionizing radiation-induced bystander effects by regulating MAPK/NF-kB signaling pathway in bone mesenchymal stem cells (BMSCs). Med Sci Monit. 2018;24:4649–4658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Yang F, Yan G, Li Y, et al. Astragalus polysaccharide attenuated iron overload-induced dysfunction of mesenchymal stem cells via suppressing mitochondrial ROS. Cell Physiol Biochem. 2016;39(4):1369–1379. [DOI] [PubMed] [Google Scholar]
  • [29].Pouget JP, Georgakilas AG, Ravanat JL. Targeted and off-target (Bystander and Abscopal) effects of radiation therapy: redox mechanisms and risk/benefit analysis. Antioxid Redox Signal. 2018;29(15):1447–1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Yoshino H, Murakami K, Nawamaki M, et al. Effects of Nrf2 knockdown on the properties of irradiated cell conditioned medium from A549 human lung cancer cells. Biomed Rep. 2018;8(5):461–465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Klopp AH, Spaeth EL, Dembinski JL, et al. Tumor irradiation increases the recruitment of circulating mesenchymal stem cells into the tumor microenvironment. Cancer Res. 2007;67(24):11687–11695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Yang X, Li K, Zhang X, et al. Nanofiber membrane supported lung-on-a-chip microdevice for anti-cancer drug testing. Lab Chip. 2018;18(3):486–495. [DOI] [PubMed] [Google Scholar]
  • [33].Li Z, Hao H, Tian W, et al. Nitric oxide, a communicator between tumor cells and endothelial cells, mediates the anti-tumor effects of Marsdenia Tenacissima Extract (MTE). J Ethnopharmacol. 2020;250:112524. [DOI] [PubMed] [Google Scholar]
  • [34].Cruz-Bermúdez A, Laza-Briviesca R, Vicente-Blanco RJ, et al. Cancer-associated fibroblasts modify lung cancer metabolism involving ROS and TGF-β signaling. Free Radic Biol Med. 2019;130:163–173. [DOI] [PubMed] [Google Scholar]
  • [35].Chou CH, Chen PJ, Lee PH, et al. Radiation-induced hepatitis B virus reactivation in liver mediated by the bystander effect from irradiated endothelial cells. Clin Cancer Res. 2007;13(3):851–857. [DOI] [PubMed] [Google Scholar]
  • [36].Burdak-Rothkamm S, Rothkamm K. Radiation-induced bystander and systemic effects serve as a unifying model system for genotoxic stress responses. Mutat Res. 2018;778:13–22. [DOI] [PubMed] [Google Scholar]
  • [37].Dong C, Tu W, He M, et al. Role of endoplasmic reticulum and mitochondrion in proton microbeam radiation-induced bystander effect. Radiat Res. 2020;193(1):63–72. [DOI] [PubMed] [Google Scholar]
  • [38].Gorbunov NV, Garrison BR, McDaniel DP, et al. Adaptive redox response of mesenchymal stromal cells to stimulation with lipopolysaccharide inflammagen: mechanisms of remodeling of tissue barriers in sepsis. Oxid Med Cell Longev. 2013;2013:186795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [39].Shi T, Dansen TB. Reactive oxygen species induced p53 activation: DNA damage, redox signaling, or both? [published online ahead of print, 2020 Jul 21]. Antioxid Redox Signal. 2020. DOI: 10.1089/ars.2020.8074 [DOI] [PubMed] [Google Scholar]
  • [40].Zhang LY, Yong WX, Wang L, et al. Astragalus polysaccharide eases G1 phase-correlative bystander effects through mediation of TGF-β R/MAPK/ROS signal pathway after carbon ion irradiation in BMSCs. Am J Chin Med. 2019;47(3):595–612. [DOI] [PubMed] [Google Scholar]
  • [41].Kim HS, Kim YJ, Seo YR. An overview of carcinogenic heavy metal: molecular toxicity mechanism and prevention. J Cancer Prev. 2015;20(4):232–240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [42].Olechowska-Jarząb A, Ptak-Belowska A, Brzozowski T. Therapeutic importance of apoptosis pathways in pancreatic cancer. Folia Med Cracov. 2016;56(1):61–70. [PubMed] [Google Scholar]
  • [43].Muchmore SW, Sattler M, Liang H, et al. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature. 1996;381(6580):335–341. [DOI] [PubMed] [Google Scholar]
  • [44].Morgan CW, Julien O, Unger EK, et al. Turning on caspases with genetics and small molecules. Methods Enzymol. 2014;544:179–213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [45].Waziri PM, Abdullah R, Yeap SK, et al. Clausenidin induces caspase-dependent apoptosis in colon cancer. BMC Complement Altern Med. 2016;16(1):256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [46].Greco V, Longone P, Spalloni A, et al. Crosstalk between oxidative stress and mitochondrial damage: focus on amyotrophic lateral sclerosis. Adv Exp Med Biol. 2019;1158:71–82. [DOI] [PubMed] [Google Scholar]
  • [47].Norberg E, Orrenius S, Zhivotovsky B. Mitochondrial regulation of cell death: processing of apoptosis-inducing factor (AIF). Biochem Biophys Res Commun. 2010;396(1):95–100. [DOI] [PubMed] [Google Scholar]
  • [48].Cregan SP, Fortin A, MacLaurin JG, et al. Apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death. J Cell Biol. 2002;158(3):507–517. [DOI] [PMC free article] [PubMed] [Google Scholar]

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