Radiation-Generated ROS Induce Apoptosis via Mitochondrial

*Sandra Claro, Alice Teixeira Ferreira and Maria Etsuko Miyamoto Oshiro*

## **Abstract**

Ionizing radiation (IR) causes an increase in intracellular calcium, alters contractility, and triggers apoptosis via the activation of PKCα 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 H2O2 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 KΨmito. In contrast, IR increased the nuclear expression of BAG-1, TNFα, PKCα, and -ε and cyclins A and E. In conclusion, 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.

**Keywords:** ROS, apoptosis, mitochondria, cyclins, smooth muscle

## **1. 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

of the mitochondrial transmembrane electric potential (KΨmito) [9]. Several studies have used tumor cells to investigate the molecular pathways involved in the regulation and triggering of apoptosis by ionizing radiation (IR) [10, 11], but IR is more effective in normal than neoplasic tissue; so it is important to minimize the exposure in it and to clarify the mechanisms involved in the cellular damage [12]. In addition, damage to healthy tissues due to IR used in cancer treatment is frequently associated with the appearance of a second cancer occurring in the radiated field or in its vicinity [13]. This event could be explained by remodeling of the molecular and cellular processes triggering a number of inter- and intracellular signaling cascades that regulate the progression of the cell cycle and cell survival [14–16].

The apoptotic pathway activated by IR is different from the extrinsic pathway activated by ligands and involves the generation of reactive oxygen species (ROS) and H2O2 [10, 17]. According to Orrenius [18], the enhanced ROS production regulates cellular metabolism, for the execution of the suicide program, by proteins released from the mitochondria. One of the factors involved in ROS-induced cell death is tumor necrosis factor alpha (TNFα) [15, 19], and mitochondria appear to participate in the production of this mediator. A number of hypotheses have been put forward to explain the mechanism by which TNFα cytotoxicity induces the intrinsic pathway [11]. Nevertheless, the mechanisms regulated by ROS is not totally clear, but our previous results described an increase in [Ca2+]i [20] and the activation of protein kinase C (PKCα and -ε) [21]. IR has not been directly demonstrated to affect proteins, including cyclins, cyclin-dependent kinases (CDKs), retinoblastoma protein (Rb), and E2F complex proteins [22–24], involved in the orchestration of the cell cycle. The goals of this study were to examine the extrinsic and intrinsic mechanisms involved in the apoptosis, and to investigate ROS and H2O2 generation and the mitochondria role under IR of intestinal smooth muscle cells from the guinea pig ileum.
