Induction of apoptosis in Caco-2 cells by exogenously added O₂⁻ produced by a nanodevice

Significant statement

The effects of reactive oxygen species (ROS) on cells have attracted great attention from both physiological and pathological aspects. The primary source of ROS in vivo is mostly superoxide (O2−). The formed O2− is readily converted to H2O2 either spontaneously or enzymatically. However, as O2− is a highly reactive anion radical, high levels of O2− can damage cells and tissues. To examine how O2− affects cells, a tool is required that can constantly produce O2−. Xanthine oxidase has sometimes been used for this purpose, but the enzyme has several demerits for cell experiments. We have recently established a new nanodevice based on an O2–generating enzyme Nox2 that can produce O2− constantly at a high rate being free from above demerits. Intestinal epithelial cells sometimes suffer from high concentrations of ROS during host defense, inflammation, and autoimmunity. ROS are thought to cause damage to the intestinal mucosal barrier, a symptom of inflammatory bowel disease. Using the newly developed nanodevice we investigated the effects of O2− on Caco-2 cells, which are derived from colon epithelial cells. The data indicated that exogenously added O2− efficiently induced apoptosis in Caco-2 cells. The new nanodevice can be applied to other cell lines to mimic in vivo situations in which they are exposed to high levels of O2−.

Figure legend

Schematic illustration of O₂^–-generating nanodevice (upper) and its application for oxidative stress studies in cultured cells (lower). [c.f., Tamura M. (2015) Biotech. Reports, 6, 45-50]

Journal Reference

Exp Cell Res. 2015 Feb 15;331(2):408-15. Yoshioka Y¹, Fujibayashi H¹, Kameda K², Kan D¹, Tone S³, Tamura M⁴.

[expand title=”Show Affiliations”]

¹Department of Applied Chemistry, Graduate School of Science and Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan.

²INCS Shigenobu Station, Ehime University, Toon, Ehime 791-0295, Japan.

³Department of Biochemistry, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan.

⁴Department of Applied Chemistry, Graduate School of Science and Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan. Electronic address: [email protected].[/expand]

Abstract

The effects of reactive oxygen species on cells have attracted considerable attention in relation to oxidative stress and related disorders. Superoxide (O2(-)) is the primary reactive oxygen species formed in animals as a byproduct or purposeful product of enzymes. We recently established an O2(-)-generating nanodevice that produces O2(-) continuously even in culture medium, by improving an original nanodevice. The new nanodevice, named Device II, efficiently induced cell death in Caco-2 cells in a time- and dose-dependent manner. Catalase largely recovered the cell viability, while superoxide dismutase rather lowered the viability. Flow cytometric and fluorescence microscopic analyses revealed that phosphatidylserine was exposed on the cells and that caspase-3 was activated in the cells after treatment with Device II. These findings indicated that exogenously added O2(-) caused apoptosis in Caco-2 cells through its derivative H2O2.

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