Radiation-Generated ROS Induce Apoptosis via Mitochondrial

Ionizing radiation (IR) causes an increase in intracellular calcium, alters con-tractility, and triggers apoptosis via the activation of PKC α and - ε in irradiated smooth muscle cells. The present study investigated the role of the mitochondria in these processes and characterized the proteins involved in IR-induced apoptosis. Intestinal smooth muscle cells were exposed to 10–50 Gy from a γ -source. ROS and H 2 O 2 levels were measured with colourimetry and a DCFH-DA probe, and protein expression was analyzed by immunoblotting and immunofluorescence. The IR-induced generation of ROS was inhibited by glutathione, and apoptosis was mediated by the mitochondria via BAX, cytochrome c, and caspase 3. IR increased the expression of the cyclins A, B2, and E, and led to unbalanced cellular growth in an absorption dose-dependent manner. However, radiation did not induce alterations in the mitochondrial ultrastructure or in K Ψ mito . In contrast, IR increased the nuclear expression of BAG-1, TNF α , PKC α , and - ε and cyclins A and E. In conclu-sion, IR triggers the activation of antiapoptotic proteins and enhances the risk of a second type of cancer in patients undergoing radiotherapy. In addition to increasing the radioresistance of cells, antiapoptotic proteins can also stimulate uncontrolled cell proliferation that culminates in mutagenesis.


Introduction
The molecular pathways that induce and regulate apoptosis have been extensively studied [1,2]. Apoptosis is characterized by the condensation of nuclear chromatin and blebbing of nuclear and cytoplasmic membranes, a process that leads to the formation of membrane-bound apoptotic bodies [3]. The proteolytic caspase cascade plays a central role in the apoptotic response, and proteins of the BCL-2 family are key checkpoints in the regulation of apoptosis [4,5]. In healthy cells, the BCL-2 family is kept in an inactive form, with a complex distribution in the mitochondrial outer membrane (MOM), sarco/endoplasmic reticulum (SER), cytosol, and nuclear envelope [6].
The mitochondria also play a key role in Ca2+ homeostasis and oxidative stress [7]. Elevated intracellular calcium concentrations ([Ca2+] i ) do not seem to inhibit mitochondrial motility [8] but can lead to the opening of the mitochondrial transition pore (MTP) complex during the process of swelling, which is responsible in turn for the permeability of the MOM to large molecules and the collapse Radiation-Generated ROS Induce Apoptosis via Mitochondrial DOI: http://dx.doi.org /10.5772/intechopen.86747 c. To test if the generation of ROS contributes to apoptosis, some cultured cells were incubated with glutathione (GSH), 10 −3 M reduced glutathione, and yeast glutathione reductase type II (0.08 units/mg protein) and then fixed and stained as described in section b [29].
d. The generation of H 2 O 2 was measured with 2′7′-dichlorofluorescein diacetate (DCFH-DA, as described by Hasui [30]) in live cultured cells. The cells were suspended in PBS, mixed with 0.3 mM DCFH-DA at 37°C to allow the conversion to DCF, and analyzed at 570/530 nm in the FL-1 channel.
e. To measure the degree of unbalanced growth, cultured cells were detached and stained with acridine orange (AO) for the evaluation of the ratio of RNA content, according to Traganos [31].
g. The cyclins A, B2, and E, and the DNA content were analyzed by MODFIT 3.0 software as described by Gong [33].

Western blot analysis
The experimental procedure was performed as previously described [21] using LSMLGPI homogenates. The following antibodies were used, anti-: caspase 3, cyclin A and cyclin B2, cyclin E, BCL-xL, BAX, cytochrome c, and BCL-2.

Confocal microscopy
LSMLGPI cells were seeded onto glass coverslips and exposed to IR. The mitochondria were stained with a probe as described by Claro [20] in living cells.
For analysis of the KΨ mito , 0.5 μM DiOC 6 (3) was used in DMEM, in vivo. The fluorescence was measured between 546/500 nm. To confirm the mitochondrial accumulation of DiOC 6 (3), the cells were incubated with KΨ mito inhibitors [34] for different periods of time.

Electron micrography
The cells were seeded as described by Claro [21], and were then radiated and fixed before being analyzed with a transmission electron microscope (1200 EXII, JEOL, Tokyo, Japan).

Fluorescence microscopy
Living cells were incubated with 2 μg/ml bisbenzimides diluted in DMEM and were analyzed between 461 and 350 nm, for DNA labeling.

Statistical analysis
Differences between irradiated and nonradiated groups were identified using the analysis of variance (ANOVA) of the unpaired Newman-Keuls tests (GraphPad Prism 5 software). Statistical significance was set at P < 0.05.

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 2 correlates the changes in cyclin expression and the alteration of the cell cycle caused by IR. The α r 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 H 2 O 2 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 KΨ mito were incubated with DiOC 6 (3), in living cells.   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).

Discussion
IR generates ROS and H 2 O 2 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

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 Radiation-Generated ROS Induce Apoptosis via Mitochondrial DOI: http://dx.doi.org /10.5772/intechopen.86747 protein breakage and the degradation of membrane phospholipids. However, ROS and H 2 O 2 also cause DNA fragmentation and prevent the repair mechanisms elicited by sublethal damage [20,21,37]. ROS and H 2 O 2 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 H 2 O 2 , 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]. H 2 O 2 is a weak oxidizing agent but can form hydroxyl radicals. These findings suggest that IR-generated ROS or H 2 O 2 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 H 2 O 2 through its transformation in water ( [38], pp. [10][11][12][13][14][15][16][17][18][19][20][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 H 2 O 2 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], KΨ mito 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 H 2 O 2 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 H 2 O 2 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][49][50][51][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 [Ca 2 +] 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 H 2 O 2 . 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 H 2 O 2 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 H 2 O 2 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