Detection of Glutathione in Oral Squamous Cell Carcinoma Cells With a Fluorescent Probe During the Course of Oxidative Stress and Apoptosis.

PURPOSE: To detect glutathione (GSH) in oral squamous carcinoma cells (OSCCs) with a GSH selective fluorescent probe during the course of oxidative stress and apoptosis.

MATERIALS AND

METHODS

A novel GSH probe was applied to assess GSH in human tongue squamous cell carcinoma cells (cal-27). The cellular GSH and reactive oxygen species (ROS) levels were assessed with a GSH probe and DCF-DA (2,7-dichlorofluorescin diacetate) probe. The mitochondrial GSH and ROS levels were assessed with a GSH probe, DCF-DA probe, and Mitotracker Red CM-H2XRos probe (Invitrogen, Carlsbad, CA). To further study whether oxidative stress would induce apoptosis of OSCCs, we then applied a GSH probe and annexin V-fluorescein isothiocyanate probe to assess cellular GSH levels and eversion of phosphatidylserine, and the cellular GSH levels and mitochondrial membrane potential (ΔΨm) were assessed with a GSH probe and JC-1 probe during the course of oxidative stress and apoptosis induced by hydrogen peroxide and ethacrynic acid. The fluorescence was observed under laser confocal fluorescence microscopy.

RESULTS

The intensity of fluorescence that represented intracellular alteration of GSH levels, cellular ROS formation, mitochondrial ROS formation, and apoptosis occurrence, respectively, could be visualized under laser confocal fluorescence microscopy.

CONCLUSIONS

The GSH selective fluorescent probe can evaluate cellular GSH levels sensitively during the course of oxidative stress and apoptosis of OSCCs induced by exogenous hydrogen peroxide, which could be enhanced by depletion of mitochondrial GSH.