**2. Results**

We tested if LSMLGPI cells die by apoptosis in response to IR and observed that the maximum number of apoptotic bodies appeared 72 h following radiation with 10–50 Gy [21, 28]. The first step was to evaluate the effects of IR on the expression of cell-cycle proteins in LSMLGPI cells (**Figure 1**). In contrast to the cyclins B2 and E, the expression of cyclin A was unchanged at 24, 48, and 72 h postradiation. Subsequently, all proteins were analyzed at 24 h postradiation.

#### **Figure 1.**

*Effects of IR on the expression and localisation of cell cycle proteins. Cell cultures from LSMLGPI were fixed and labeled with specific primary and secondary antibodies. (A) Representative time-course histograms of activation of cyclins by IR. (B) Quantification of cells resuspended in PBS 24 h postradiation; besides, representative histograms of the acquisition data of relative cell size and analysis of fluorescence intensity distribution are shown. \* P < 0.01 compared to control, # P < 0.01 compared to 10 Gy, Newman-Keuls test. Error bars indicate SEM. (C) Western blot analysis in whole-cell lysates demonstrating expression of cyclin a, B2 and E detected with appropriate antibodies. (D) Images of irradiated cells are representative of three independent experiments. Cyclin a and E co-localized with nucleus are light blue. Nuclear staining was done using DAPI (blue). Scale bar indicates 20 μm.*

**57**

*and §*

**Figure 2.**

*Effects of IR the on synthesis of RNA and DNA and on the cyclins in the cell cycle. (A) DNA, RNA and αr RNA/ (DNA + RNA) distribution 24 h postradiation; besides, representative histograms are shown. (B) Quantification of cell cycle phases by DNA content and analysis of G1, S, and G2 phases of cell cycle at different times of postradiation. (C) Scheme illustrating the analysis performed to estimate the cells expressing cyclins versus cell-cycle phases in measurements of cellular DNA content (PI) and the intensity of cyclins* 

*P < 0.01 compared to control, #*

*P < 0.01 compared to 10 and 30 Gy, respectively. Newman-Keuls test. Error bars indicate SEM.*

*associated Alexa Fluor immunofluorescence analyzed by MODFIT 3.0 software. \**

*Radiation-Generated ROS Induce Apoptosis via Mitochondrial*

*DOI: http://dx.doi.org/10.5772/intechopen.86747*

*Radiation-Generated ROS Induce Apoptosis via Mitochondrial DOI: http://dx.doi.org/10.5772/intechopen.86747*

*Free Radical Medicine and Biology*

We tested if LSMLGPI cells die by apoptosis in response to IR and observed that the maximum number of apoptotic bodies appeared 72 h following radiation with 10–50 Gy [21, 28]. The first step was to evaluate the effects of IR on the expression of cell-cycle proteins in LSMLGPI cells (**Figure 1**). In contrast to the cyclins B2 and E, the expression of cyclin A was unchanged at 24, 48, and 72 h postradiation.

*Effects of IR on the expression and localisation of cell cycle proteins. Cell cultures from LSMLGPI were fixed and labeled with specific primary and secondary antibodies. (A) Representative time-course histograms of activation of cyclins by IR. (B) Quantification of cells resuspended in PBS 24 h postradiation; besides, representative histograms of the acquisition data of relative cell size and analysis of fluorescence intensity* 

*bars indicate SEM. (C) Western blot analysis in whole-cell lysates demonstrating expression of cyclin a, B2 and E detected with appropriate antibodies. (D) Images of irradiated cells are representative of three independent experiments. Cyclin a and E co-localized with nucleus are light blue. Nuclear staining was done using DAPI* 

*P < 0.01 compared to 10 Gy, Newman-Keuls test. Error* 

*P < 0.01 compared to control, #*

Subsequently, all proteins were analyzed at 24 h postradiation.

**2. Results**

**56**

**Figure 1.**

*distribution are shown. \**

*(blue). Scale bar indicates 20 μm.*

#### **Figure 2.**

*Effects of IR the on synthesis of RNA and DNA and on the cyclins in the cell cycle. (A) DNA, RNA and αr RNA/ (DNA + RNA) distribution 24 h postradiation; besides, representative histograms are shown. (B) Quantification of cell cycle phases by DNA content and analysis of G1, S, and G2 phases of cell cycle at different times of postradiation. (C) Scheme illustrating the analysis performed to estimate the cells expressing cyclins versus cell-cycle phases in measurements of cellular DNA content (PI) and the intensity of cyclins associated Alexa Fluor immunofluorescence analyzed by MODFIT 3.0 software. \* P < 0.01 compared to control, # and § P < 0.01 compared to 10 and 30 Gy, respectively. Newman-Keuls test. Error bars indicate SEM.*

#### **Figure 3.**

*Measurements of IR-generated-ROS and H2O2. (A) TBARS and lipid peroxidation measured in homogenate of LSMLGPI via colourimetric assays. (B) Detection of intracellular H2O2 using DCFH-DA probe analyzed at flow cytometer, and (C) the representative histograms. \* P < 0.01 compared to 0 Gy, # and § P < 0.01 compared to 10 and 30 Gy respectively. (D) Effect of glutathione on irradiated cells and fixed in 50% ethanol, and loaded with PI in the presence (+) or absence (−) of GSH, measured 72 h postradiation using flow cytometry and (E) representative histograms. \* P < 0.01 indicates statistical difference between GSH-treated and untreated cells, # P < 0.01 compared to GSH-untreated control. \* , § and <sup>ζ</sup> P < 0.01 indicate statistical difference between untreated cells compared to control, 10 and 30 Gy, respectively. Newman-Keuls test. Error bars indicate SEM.*

**59**

**Figure 4.**

*Radiation-Generated ROS Induce Apoptosis via Mitochondrial*

**Figure 2** correlates the changes in cyclin expression and the alteration of the cell cycle caused by IR. The αr ratio of RNA to total nucleic acid content decreased in an absorption dose-dependent manner, and it visualizes nuclear content. The radiated population of cells did not divide because the G2 phase was arrested despite a significant increase in the accumulation of RNA and DNA during the S phase. Cyclins were continuously expressed during the cell cycle, however it was observed

**Figure 3** indicates that IR caused dose-dependent increases in the generation of thiobarbituric acid reactive substances (TBARS) and H2O2 with maximal ROS generation and a decrease in ROS levels. IR effects were suppressed by GSH, with a reduction in the number of cells in the M2 region. GSH reduced cell death independent on the dose of radiation, resulting in levels similar to those in control cells. Apoptosis was assessed 24 h later by the binding of antibodies specific for BAX, cytochrome c, and caspase 3 (**Figure 4**). IR also increased the expression levels of BCL-xL and BCL-2, suggesting that these oncoproteins attempted to promote cell

**Figure 5** shows the stained apoptotic bodies and the localization of Bax, caspase 3,

**Figure 6** proves that mitochondria presented no evidence of damage other

*Effects of IR on (A) pro- and (C) antiapoptotic proteins of LSMLGPI cells measured in the flow cytometer 24 h postradiation. Cells were fixed, permeabilised and incubated with specific primary and secondary* 

*respectively, Newman-Keuls test. Error bars indicate SEM. (B) and (D) western blot analyses demonstrating* 

*and §*

*P < 0.01 compared to 10 and 30 Gy,* 

*antibodies and resuspended in PBS. \*P < 0.001 compared to control, #*

*BAX, cytochrome c, caspase 3, BCL-xL, and BCL-2 expression, respectively.*

than the appearance of several lysosomes. To prove that the mitochondria were healthy, various agents known to reduce the KΨmito were incubated with

*DOI: http://dx.doi.org/10.5772/intechopen.86747*

the G2 phase.

proliferation.

cytochrome c, Bcl-2, and BCL-xL.

DiOC6(3), in living cells.

*Radiation-Generated ROS Induce Apoptosis via Mitochondrial DOI: http://dx.doi.org/10.5772/intechopen.86747*

*Free Radical Medicine and Biology*

**58**

*cells, #*

**Figure 3.**

*flow cytometer, and (C) the representative histograms. \**

*P < 0.01 compared to GSH-untreated control. \**

*(E) representative histograms. \**

*Measurements of IR-generated-ROS and H2O2. (A) TBARS and lipid peroxidation measured in homogenate of LSMLGPI via colourimetric assays. (B) Detection of intracellular H2O2 using DCFH-DA probe analyzed at* 

*10 and 30 Gy respectively. (D) Effect of glutathione on irradiated cells and fixed in 50% ethanol, and loaded with PI in the presence (+) or absence (−) of GSH, measured 72 h postradiation using flow cytometry and* 

> *, § and <sup>ζ</sup>*

*untreated cells compared to control, 10 and 30 Gy, respectively. Newman-Keuls test. Error bars indicate SEM.*

*P < 0.01 compared to 0 Gy, #*

*P < 0.01 indicates statistical difference between GSH-treated and untreated* 

*and §*

*P < 0.01 indicate statistical difference between* 

*P < 0.01 compared to* 

**Figure 2** correlates the changes in cyclin expression and the alteration of the cell cycle caused by IR. The αr ratio of RNA to total nucleic acid content decreased in an absorption dose-dependent manner, and it visualizes nuclear content. The radiated population of cells did not divide because the G2 phase was arrested despite a significant increase in the accumulation of RNA and DNA during the S phase. Cyclins were continuously expressed during the cell cycle, however it was observed the G2 phase.

**Figure 3** indicates that IR caused dose-dependent increases in the generation of thiobarbituric acid reactive substances (TBARS) and H2O2 with maximal ROS generation and a decrease in ROS levels. IR effects were suppressed by GSH, with a reduction in the number of cells in the M2 region. GSH reduced cell death independent on the dose of radiation, resulting in levels similar to those in control cells.

Apoptosis was assessed 24 h later by the binding of antibodies specific for BAX, cytochrome c, and caspase 3 (**Figure 4**). IR also increased the expression levels of BCL-xL and BCL-2, suggesting that these oncoproteins attempted to promote cell proliferation.

**Figure 5** shows the stained apoptotic bodies and the localization of Bax, caspase 3, cytochrome c, Bcl-2, and BCL-xL.

**Figure 6** proves that mitochondria presented no evidence of damage other than the appearance of several lysosomes. To prove that the mitochondria were healthy, various agents known to reduce the KΨmito were incubated with DiOC6(3), in living cells.

#### **Figure 4.**

*Effects of IR on (A) pro- and (C) antiapoptotic proteins of LSMLGPI cells measured in the flow cytometer 24 h postradiation. Cells were fixed, permeabilised and incubated with specific primary and secondary antibodies and resuspended in PBS. \*P < 0.001 compared to control, # and § P < 0.01 compared to 10 and 30 Gy, respectively, Newman-Keuls test. Error bars indicate SEM. (B) and (D) western blot analyses demonstrating BAX, cytochrome c, caspase 3, BCL-xL, and BCL-2 expression, respectively.*

#### **Figure 5.**

*Effects of IR on apoptotic proteins localisation 24 h postradiation. (A) Cell death by apoptosis is shown by apoptotic bodies formation in irradiated living cells labeled with 2 μg/ml Hoechst 33342 resuspended in cultured medium DMEM maintained at 37°C. Control cells exhibit low blue fluorescence, while irradiated cells exhibit high blue fluorescence and some apoptotic bodies (arrows). Images of irradiated cells present (B) proapoptotic and (C) antiapoptotic proteins with mitochondria stained with Mitotracker (red), and cells incubated with specific primary and secondary antibodies. Nuclear staining was done using DAPI (blue). Proteins co-localized with mitochondria are yellow. Arrows indicate apoptotic bodies. Images are representative of three independent experiments observed in confocal microscope. Scale bar indicates 20 μm.*

**61**

**3. Discussion**

**Figure 6.**

*Radiation-Generated ROS Induce Apoptosis via Mitochondrial*

The increased levels and activation/translocation of PKCα and -ε to the nucleus induced IR. Similarly, a large part of the TNFα was internalized and BAG-1 immu-

*Effect of IR on mitochondria in LSMLGPI cells cultures 72 h postradiation. (A) Electron microscopic analysis showing the mitochondria (Mito) with normal morphology scattered in the cytosol of control and lysosomes (Lyso); scale bar indicates 0.5 μm. (B) Confocal microscopy images in living cells loaded with DiOC6(3) and kept at 37°C. Cells were photographed before administration of ionophores, and after exposure to 4.5 nM valinomycin, 1 μM gramicidin, 1 mM DNP, 10 mM sodium azide, and 6.5 μM oligomycin; scale bar indicates* 

IR generates ROS and H2O2 and promotes changes related to the expression and localization of cyclins, and in the cellular cycle phase distributions in a dosedependent manner in LSMLGPI. Cyclins were continuously expressed during the cell cycle after treatment with IR; however, an arrest of the G2 phase and enhanced DNA replication at the initiation of the S phase occurred. The G2 phase is known

nofluorescence appears next to the nucleus (**Figure 7**).

*50 Zm. The figures are representative of three independent experiments.*

*DOI: http://dx.doi.org/10.5772/intechopen.86747*

*Radiation-Generated ROS Induce Apoptosis via Mitochondrial DOI: http://dx.doi.org/10.5772/intechopen.86747*

#### **Figure 6.**

*Free Radical Medicine and Biology*

**60**

**Figure 5.**

*indicates 20 μm.*

*Effects of IR on apoptotic proteins localisation 24 h postradiation. (A) Cell death by apoptosis is shown by apoptotic bodies formation in irradiated living cells labeled with 2 μg/ml Hoechst 33342 resuspended in cultured medium DMEM maintained at 37°C. Control cells exhibit low blue fluorescence, while irradiated cells exhibit high blue fluorescence and some apoptotic bodies (arrows). Images of irradiated cells present (B) proapoptotic and (C) antiapoptotic proteins with mitochondria stained with Mitotracker (red), and cells incubated with specific primary and secondary antibodies. Nuclear staining was done using DAPI (blue). Proteins co-localized with mitochondria are yellow. Arrows indicate apoptotic bodies. Images are representative of three independent experiments observed in confocal microscope. Scale bar* 

*Effect of IR on mitochondria in LSMLGPI cells cultures 72 h postradiation. (A) Electron microscopic analysis showing the mitochondria (Mito) with normal morphology scattered in the cytosol of control and lysosomes (Lyso); scale bar indicates 0.5 μm. (B) Confocal microscopy images in living cells loaded with DiOC6(3) and kept at 37°C. Cells were photographed before administration of ionophores, and after exposure to 4.5 nM valinomycin, 1 μM gramicidin, 1 mM DNP, 10 mM sodium azide, and 6.5 μM oligomycin; scale bar indicates 50 Zm. The figures are representative of three independent experiments.*

The increased levels and activation/translocation of PKCα and -ε to the nucleus induced IR. Similarly, a large part of the TNFα was internalized and BAG-1 immunofluorescence appears next to the nucleus (**Figure 7**).

#### **3. Discussion**

IR generates ROS and H2O2 and promotes changes related to the expression and localization of cyclins, and in the cellular cycle phase distributions in a dosedependent manner in LSMLGPI. Cyclins were continuously expressed during the cell cycle after treatment with IR; however, an arrest of the G2 phase and enhanced DNA replication at the initiation of the S phase occurred. The G2 phase is known

#### **Figure 7.**

*Effects of IR on the expression and localisation of TNFα and BAG1, PKCα, and -ε, of LSMLGPI cell cultures 24 h postradiation. Cells were fixed and incubated with specific primary and secondary antibodies. (A) Quantification using flow cytometry in cells resuspended in PBS.\*P < 0.01 compared to control, # and § P < 0.01 compared to 10 and 30 Gy, respectively, Newman-Keuls test. Error bars indicate SEM. Figures are representative of three independent experiments and present enhanced green fluorescence of (B) PKCα, PKCε, TNFα, and (C) BAG-1 co-localized with mitochondria that are yellow and with nucleus that are light blue (arrow shows apoptotic bodies). Nuclear staining was done using DAPI (blue). Scale bar indicates 20 μm.*

to be the most radiosensitive phase of the cell cycle, followed by the G1 phase [35]; thus, cells in the G2 phase did not continue to synthesize RNA or DNA. IR induced an excess of DNA in relation to RNA content. These results demonstrate that IR interferes in the cell-cycle distribution, but it does not cause cyclins degradation.

Cell death was effectively triggered by the activation and translocation of BAX to the mitochondria, resulting in cytochrome c release into the cytosol in an absorption dose-dependent manner. Ultrastructural changes and DNA fragmentation characteristics of apoptosis were also identified in vitro [21] and it was confirmed by Hoechst which stained the apoptotic bodies in living cells.

The BAX fluorescence intensity was increased next to the perinuclear region, with some co-localization with the MOM (yellow). Caspase 3 was overexpressed in the nucleus and co-localized with the mitochondria (yellow), and possible retention in the intermembrane space. We also observed caspase 3 localization in the nucleolus which is an atypical form. As cytochrome c mediates the activation of caspases via BAX disruption, we hypothesized that it might also induce the activation of antiapoptotic proteins. According to Edlich [36], activation of BCL-xL and BCL-2 increased the cellular resistance to death and could also cause the retrotranslocation of BAX to the cytosol, confirming our results. Our results demonstrated that there is more than one type of cellular response to IR, namely death or survival. The mitochondrial ultrastructure and function appeared normal in IR-induced apoptosis.

We have shown that IR causes apoptosis which is preceded by the activation of PKCα and -ε and suggests a role for the PKC-mediated pathway [21] and caspase 12 translocation to the cytosol [20]. We and other authors have shown that single absorption doses induce early reactions in normal smooth muscle cells, including

**63**

*Radiation-Generated ROS Induce Apoptosis via Mitochondrial*

protein breakage and the degradation of membrane phospholipids. However, ROS and H2O2 also cause DNA fragmentation and prevent the repair mechanisms elicited by sublethal damage [20, 21, 37]. ROS and H2O2 have been implicated in several mechanisms of cellular injury, including peroxidation of membrane phospholipids, which increases membrane permeability and leads to apoptosis ([38], pp. 196–208). In the present study, however, we observed that up to 50 Gy of IR led to cell death by apoptosis, despite the preservation of the plasma membrane. It is possible that H2O2, rather than ROS, can cross cell membranes rapidly and cause LP in small, discrete sites on smooth muscle membranes ([38], pp. 79–80). In contrast, ROS can mediate necrosis in neurons by the MTP pathway [18]. H2O2 is a weak oxidizing agent but can form hydroxyl radicals. These findings suggest that IR-generated ROS or H2O2 favors the internalization of TNFα. Several mechanisms may have protected the cells against injury in the presence of GSH, including the prevention of protein oxidation, the accumulation of H2O2 through its transformation in water ([38], pp. 10–21), the provision of a substrate for glutathione peroxidase, and the scavenging of hydroxyl radicals. Nevertheless, the most remarkable effect of GSH appears to be protection against alterations in the cell cycle ([38], pp. 247–251). In fact, here, we show that high concentrations of ROS or H2O2 generated by IR were followed by the release of cytochrome c from the mitochondria into the cytosol. Several models of cytochrome c release have been proposed [2, 5], such as

The mechanisms involving BAX, which is inserted into the MOM, may include the formation of channels, by oligomerization, and the preservation of mitochondrial membrane integrity [40]. Although we cannot discount the possible involvement of heterodimers among activated BCL-2, BCL-xL and BAG-1 proteins, there is

The mitochondrial membranes were maintained intact in radiated cells, with similar fluorescence as the control cells, in which the electronegativity of the probe allowed its retention in the mitochondrial interior [34], KΨmito was maintained. Our data indicate an intrinsic mechanism of IR-induced apoptosis. Moreover,

Another potential repair mechanism is the decrease in the cellular ROS or H2O2 levels induced by BCL-2 [42]. This mechanism may also be activated by increased levels of antiapoptotic proteins BCL-xL and BAG-1. However, it has been suggested

In addition to this survival pathways, that prevented cell death, we observed that BCL-2, BCL-xL, and BAG-1 were activated by direct IR and/or indirect via ROS or H2O2 action [44, 45]. Besides, the mitochondrial pattern can vary on different cells and it causes apoptosis that could be independent on the mitochondrial pathway [15, 37]. The radioresistance of mitochondria may be due to the action of natural

Increases in [Ca2+]i can potentiate the effects of ROS by enhancing LP [8, 14, 47]. ROS and increased [Ca2+]i have been shown to induce opening of the MTP, which triggers the mitochondrial of cell death [47]. It is noteworthy that mitochondria are located close to the SER, which sequestrates part of the Ca2+ released by these organelles, and this may affect the release of apoptotic and antiapoptotic factors from the SER [48–52]. The mitochondrial morphology may be altered by Ca2+ overload, with an increase in the MOM permeability culminating in the release of proapoptotic factors [8, 11]. However, our data demonstrated that the mitochondrial motility was maintained even in elevated [Ca2+]i after IR [20]. Increases of [Ca2+]i can also inhibit DNA and protein synthesis as well as nuclear transport, resulting in an accumulation of cells in the quiescent state (G0) [23]. In addition, [Ca2+]i up to 500 nM has been implicated in the regulation of the mammalian cell cycle during the

this mechanism may be different in different types of mitochondria [15, 37].

that BCL-2 survival factors are characteristic of cancer cell metabolism [43].

antioxidants ([37, 38], pp. 97–98) and/or other compounds [46].

no clear evidence that any of these have pore-forming activity [41].

*DOI: http://dx.doi.org/10.5772/intechopen.86747*

release through the MTP mega channel [39].

*Free Radical Medicine and Biology*

**62**

in IR-induced apoptosis.

**Figure 7.**

*§*

*Effects of IR on the expression and localisation of TNFα and BAG1, PKCα, and -ε, of LSMLGPI cell cultures 24 h postradiation. Cells were fixed and incubated with specific primary and secondary antibodies. (A) Quantification using flow cytometry in cells resuspended in PBS.\*P < 0.01 compared to control, #*

*P < 0.01 compared to 10 and 30 Gy, respectively, Newman-Keuls test. Error bars indicate SEM. Figures are representative of three independent experiments and present enhanced green fluorescence of (B) PKCα, PKCε, TNFα, and (C) BAG-1 co-localized with mitochondria that are yellow and with nucleus that are light blue (arrow shows apoptotic bodies). Nuclear staining was done using DAPI (blue). Scale bar indicates 20 μm.*

to be the most radiosensitive phase of the cell cycle, followed by the G1 phase [35]; thus, cells in the G2 phase did not continue to synthesize RNA or DNA. IR induced an excess of DNA in relation to RNA content. These results demonstrate that IR interferes in the cell-cycle distribution, but it does not cause cyclins degradation. Cell death was effectively triggered by the activation and translocation of BAX to the mitochondria, resulting in cytochrome c release into the cytosol in an absorption dose-dependent manner. Ultrastructural changes and DNA fragmentation characteristics of apoptosis were also identified in vitro [21] and it was confirmed

The BAX fluorescence intensity was increased next to the perinuclear region, with some co-localization with the MOM (yellow). Caspase 3 was overexpressed in the nucleus and co-localized with the mitochondria (yellow), and possible retention in the intermembrane space. We also observed caspase 3 localization in the nucleolus which is an atypical form. As cytochrome c mediates the activation of caspases via BAX disruption, we hypothesized that it might also induce the activation of antiapoptotic proteins. According to Edlich [36], activation of BCL-xL and BCL-2 increased the cellular resistance to death and could also cause the retrotranslocation of BAX to the cytosol, confirming our results. Our results demonstrated that there is more than one type of cellular response to IR, namely death or survival. The mitochondrial ultrastructure and function appeared normal

We have shown that IR causes apoptosis which is preceded by the activation of PKCα and -ε and suggests a role for the PKC-mediated pathway [21] and caspase 12 translocation to the cytosol [20]. We and other authors have shown that single absorption doses induce early reactions in normal smooth muscle cells, including

by Hoechst which stained the apoptotic bodies in living cells.

*and* 

protein breakage and the degradation of membrane phospholipids. However, ROS and H2O2 also cause DNA fragmentation and prevent the repair mechanisms elicited by sublethal damage [20, 21, 37]. ROS and H2O2 have been implicated in several mechanisms of cellular injury, including peroxidation of membrane phospholipids, which increases membrane permeability and leads to apoptosis ([38], pp. 196–208). In the present study, however, we observed that up to 50 Gy of IR led to cell death by apoptosis, despite the preservation of the plasma membrane. It is possible that H2O2, rather than ROS, can cross cell membranes rapidly and cause LP in small, discrete sites on smooth muscle membranes ([38], pp. 79–80). In contrast, ROS can mediate necrosis in neurons by the MTP pathway [18]. H2O2 is a weak oxidizing agent but can form hydroxyl radicals. These findings suggest that IR-generated ROS or H2O2 favors the internalization of TNFα. Several mechanisms may have protected the cells against injury in the presence of GSH, including the prevention of protein oxidation, the accumulation of H2O2 through its transformation in water ([38], pp. 10–21), the provision of a substrate for glutathione peroxidase, and the scavenging of hydroxyl radicals. Nevertheless, the most remarkable effect of GSH appears to be protection against alterations in the cell cycle ([38], pp. 247–251).

In fact, here, we show that high concentrations of ROS or H2O2 generated by IR were followed by the release of cytochrome c from the mitochondria into the cytosol. Several models of cytochrome c release have been proposed [2, 5], such as release through the MTP mega channel [39].

The mechanisms involving BAX, which is inserted into the MOM, may include the formation of channels, by oligomerization, and the preservation of mitochondrial membrane integrity [40]. Although we cannot discount the possible involvement of heterodimers among activated BCL-2, BCL-xL and BAG-1 proteins, there is no clear evidence that any of these have pore-forming activity [41].

The mitochondrial membranes were maintained intact in radiated cells, with similar fluorescence as the control cells, in which the electronegativity of the probe allowed its retention in the mitochondrial interior [34], KΨmito was maintained.

Our data indicate an intrinsic mechanism of IR-induced apoptosis. Moreover, this mechanism may be different in different types of mitochondria [15, 37].

Another potential repair mechanism is the decrease in the cellular ROS or H2O2 levels induced by BCL-2 [42]. This mechanism may also be activated by increased levels of antiapoptotic proteins BCL-xL and BAG-1. However, it has been suggested that BCL-2 survival factors are characteristic of cancer cell metabolism [43].

In addition to this survival pathways, that prevented cell death, we observed that BCL-2, BCL-xL, and BAG-1 were activated by direct IR and/or indirect via ROS or H2O2 action [44, 45]. Besides, the mitochondrial pattern can vary on different cells and it causes apoptosis that could be independent on the mitochondrial pathway [15, 37]. The radioresistance of mitochondria may be due to the action of natural antioxidants ([37, 38], pp. 97–98) and/or other compounds [46].

Increases in [Ca2+]i can potentiate the effects of ROS by enhancing LP [8, 14, 47]. ROS and increased [Ca2+]i have been shown to induce opening of the MTP, which triggers the mitochondrial of cell death [47]. It is noteworthy that mitochondria are located close to the SER, which sequestrates part of the Ca2+ released by these organelles, and this may affect the release of apoptotic and antiapoptotic factors from the SER [48–52]. The mitochondrial morphology may be altered by Ca2+ overload, with an increase in the MOM permeability culminating in the release of proapoptotic factors [8, 11]. However, our data demonstrated that the mitochondrial motility was maintained even in elevated [Ca2+]i after IR [20]. Increases of [Ca2+]i can also inhibit DNA and protein synthesis as well as nuclear transport, resulting in an accumulation of cells in the quiescent state (G0) [23]. In addition, [Ca2+]i up to 500 nM has been implicated in the regulation of the mammalian cell cycle during the early G1 phase and in the transition from the G1 to S phase [53]. Ca2+/calmodulin may also modulate the activity of cyclin-dependent kinases (CDK) and/or cyclin E [54]. In previous studies [20], we observed an increase in basal [Ca2 +]i cells was observed and it was suggested that IR causes modifications in the plasma membrane and/or in the sarco/endoplasmic reticulum, but the capacitative Ca2+ entry into radiated cells was reduced [55].

The cyclins A and E are constitutively nuclear proteins when involved in mitosis [14, 16]; nevertheless, in radiated cells, they leaked from the nucleus to the cytosol. The cyclin B2 complex appears to be localized predominantly in the SER [14, 16, 22, 23]. At the start of mitosis, cyclin B2 is rapidly transported into the nucleus [14]. An important fact to consider is that IR induced unbalanced growth [31]. Similar mechanism to Polavarapu [56] could be explained is the penetration of TNFα in the intestinal smooth muscle. According to our results, TNFα may penetrate the intracellular compartment through damage caused by lipid peroxidation in small, discrete sites of plasma membrane, since there is an ability of TNFα to form pores in biomembranes, or through the conventional receptor/lysosome route [46]. Also, activated TNFα can contribute to the apoptosis, as caused by ROS or H2O2. The increased TNFα expression in the cytosol could be explained by the presence of lysosomes in radiated cells, and we can infer that the TNFα was not subject to lysosomal autodigestion, since the mitochondrial membranes were preserved. TNFα can induce cell survival by the polymerization and depolymerization of actin filaments, which prevent the nuclear translocation of proapoptotic molecules and subsequently inhibit caspase 3 [57]. The activation involving ROS or H2O2 has been associated with the triggering of cell death modulated by TNFα [10, 15], through the activation of BAX or the protease cascade [58]. TNFα can also be involved in cell survival similar to IR models with higher doses [41]. In addition, we can infer that caspase 3 may enter into the MOM through membrane openings caused by activated BAX or TNFα [39, 59].

IR induces the formation of apoptotic bodies which will remain in the medium of cultured cells or they will be phagocytosed and digested by adjacent cells in the tissue [60]. Although DNA lesions induced by IR are lethal if not properly repaired, it is clear that membrane events may also contribute to radiation-induced apoptosis [61].

Our experiments demonstrated that radiation induced atypical activation of PKCα and -ε, and there is evidence that this may be related to a conservative regulation of cell cycle events, which act as a molecular link connecting signal transduction pathways and constituents of the cell-cycle machinery [62]. PKC participate in the control of G1 and G2/M, and PKCα and -ε may be regulators of the G1 phase and cause a delay in the G1/S transition, thereby halting DNA synthesis and contributing to cellular differentiation or death. In addition, we suggest that PKCα and -ε trigger cyclin activation and translocation to the nucleus, which occur through the C-terminal region [63]. The mechanism involved in the nuclear localization of PKCα and -ε after IR could be similar to that of PKCγ [63] but still remains to be determined. In contrast, the activation of PKCα and -ε may also have been induced by TNFα, with apoptosis triggered via activation of the TNF-receptor, in addition to elevated calcium, ROS and H2O2 levels [10, 15, 54]. PKCα and -ε may interact with the cyclins A, B2, and E in the mechanism of cellular survival, similar as the CDKs and PKC which have domains that may activate serine/threonine protein kinases [64, 65], in an atypical fashion. The involvement of PKCα and -ε activation in apoptosis has already been suggested [21].

We can speculate that cyclin E modulates PKCα and -ε when involved in the apoptosis. This possible involvement of PKCε would constitute a new finding, as currently it has only been associated with oncogenesis [66, 67]. Similar to TNFα, PKCε also contains an actin binding site, and its direct interaction with actin is

**65**

*Radiation-Generated ROS Induce Apoptosis via Mitochondrial*

essential for the invasion and metastasis of tumors grown in vitro or in vivo in the

An important outcome of the complex network of events triggered by IR is the activation of antiapoptotic proteins in patients with cancer, and radiation therapy may lead to an increased risk of a second cancer [13]. In addition to their maleficent role in increasing radioresistance in normal cells, antiapoptotic proteins can stimulate uncontrolled cellular proliferation that culminates in carcinogenesis and mutagenesis [43]. Takayama et al*.* [69] identified BAG-1 and BCL-2 heterodimers that suppress apoptosis. Furthermore, BAG-1 overexpression is an important prognostic indicator of malignant tumors and may help to identify the metastatic potential of tumoral cells in vivo [70]. BCL-2 can alter the distribution of intracellular BAG-1, thereby changing the cancer risk [70]. Therefore, the overexpression of BCL-2, BCL-xL, and BAG-1 in normal cells may be a predictive indicator of carcinogenesis [69, 70]. In addition, PKCε is an important signaling molecule that influences the levels/activation of antiapoptotic proteins of the BCL-2 family and may regulate mitochondrial integrity, which is associated with cancer [71, 72]. However, the mechanism by which proteins of the BCL-2 family regulate cell death remains controversial. Our data suggest that not only apoptosis but also cellular repair mechanisms are activated in smooth muscle cells subjected to a low absorption dose.

Additionally, the expression level and localization of these proteins may be an important survival indicator in irradiated normal cells and may inform the progno-

The authors would like to thank Fundação de Amparo e Pesquisa do Estado de São Paulo (FAPESP); Federal University of São Paulo (UNIFESP); Edgar Paredes-Gamero, Soraya Smaili, Gustavo José Pareira and Renato de Arruda Mortara.

Sandra Claro\*, Alice Teixeira Ferreira and Maria Etsuko Miyamoto Oshiro Departamento de Biofísica, Federal University of São Paulo, São Paulo, Brazil

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: claro.sandra@unifesp.br

provided the original work is properly cited.

*DOI: http://dx.doi.org/10.5772/intechopen.86747*

sis of cancer patients undergoing radiotherapy.

**Acknowledgements**

**Author details**

regulatory domain [66–68].

*Radiation-Generated ROS Induce Apoptosis via Mitochondrial DOI: http://dx.doi.org/10.5772/intechopen.86747*

essential for the invasion and metastasis of tumors grown in vitro or in vivo in the regulatory domain [66–68].

An important outcome of the complex network of events triggered by IR is the activation of antiapoptotic proteins in patients with cancer, and radiation therapy may lead to an increased risk of a second cancer [13]. In addition to their maleficent role in increasing radioresistance in normal cells, antiapoptotic proteins can stimulate uncontrolled cellular proliferation that culminates in carcinogenesis and mutagenesis [43]. Takayama et al*.* [69] identified BAG-1 and BCL-2 heterodimers that suppress apoptosis. Furthermore, BAG-1 overexpression is an important prognostic indicator of malignant tumors and may help to identify the metastatic potential of tumoral cells in vivo [70]. BCL-2 can alter the distribution of intracellular BAG-1, thereby changing the cancer risk [70]. Therefore, the overexpression of BCL-2, BCL-xL, and BAG-1 in normal cells may be a predictive indicator of carcinogenesis [69, 70]. In addition, PKCε is an important signaling molecule that influences the levels/activation of antiapoptotic proteins of the BCL-2 family and may regulate mitochondrial integrity, which is associated with cancer [71, 72]. However, the mechanism by which proteins of the BCL-2 family regulate cell death remains controversial. Our data suggest that not only apoptosis but also cellular repair mechanisms are activated in smooth muscle cells subjected to a low absorption dose.

Additionally, the expression level and localization of these proteins may be an important survival indicator in irradiated normal cells and may inform the prognosis of cancer patients undergoing radiotherapy.
