γ-Rays-generated ROS induce apoptosis via mitochondrial and cell cycle alteration in smooth muscle cells

Abstract

Purpose: γ-rays (IR) cause an increase in intracellular calcium [Ca²⁺], alters contractility and triggers apoptosis via the activation of protein kinase C in intestinal guinea pig smooth muscle cells. The present study investigated the role of the mitochondria in these processes and characterized proteins involved in IR-induced apoptosis.

Materials and methods: Intestinal smooth muscle cells were exposed to 10–50 Gy from a ⁶⁰Co γ-source. Reactive oxygen species (ROS) levels were measured by colourimetry with a fluorescente probe. Protein expression was analyzed by immunoblotting and immunofluorescence.

Results: Apoptosis was inhibited by glutathione, possible by inhibiting the generation or scavenging ROS. Apoptosis was mediated by the mitochondria releasing cytochrome c leading to caspase 3 activation. 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 transmembrane electric potential. In contrast, IR increased the nuclear expression of cytoplasmic proteins and cyclins A and E.

Conclusion: Smooth muscle cells subjected to IR undergo mitochondrial-mediated apoptosis that involves oncoproteins activation and preserves mitochondrial structure. IR also cause alterations in the expression and localization of both pro- and anti-apoptotic proteins.

Keywords::

• γ-ray
• ROS
• apoptosis
• mitochondria
• cyclins
• smooth muscle cells

Acknowledgements

The authors would like to thank: Gus Schoorlemmer and Maria de Fátima Magalhães Lazari for critical reading of the manuscript; Adolfo Garcia Erustes, Chandler Tahan, Marcia Fujie Araguth Tanaka, Nelson Mora and Patricia Milanez for technical support and Adelino José Pereira and Miguel Miziara from Centro Radioterapia e Oncologia - São Paulo/SP/BR.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This study was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo/BR processes 99/10261-7; 03/14076-7; 10/00106-5.