Increase in thiol oxidative stress via glutathione reductase inhibition as a novel approach to enhance cancer sensitivity to X-ray irradiation.

Depletion of the reduced form of glutathione (GSH) has been extensively studied for its effect on sensitizing cancer to radiation. However, little is known about the effects of thiol oxidative stress created through an increase in glutathione disulfide (GSSG) on cancer sensitivity to radiation. In this study, an increase in GSSG was effectively created using 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), an irreversible glutathione reductase (GR) inhibitor. Our results demonstrate that the GSSG increase significantly enhanced cancer sensitivity to X-ray irradiation in four human cancer cell lines (A431, MCF7, NCI-H226, and OVCAR-3). When cells were pretreated with 2-AAPA followed by X-ray irradiation, the IC(50) values for X-ray irradiation of A431, MCF7, NCI-H226, and OVCAR-3 cells were reduced, from 24.2 +/- 2.8, 42.5 +/- 3.0, 43.0 +/- 3.6, and 27.8+/-3.5 Gy to 6.75 +/- 0.9, 8.1 +/- 1.1, 6.75 +/- 1.0, and 12.1 +/- 1.7 Gy, respectively. The synergistic effects observed from the combination of X-rays plus 2-AAPA were comparable to those from the combination of X-rays plus buthionine sulfoximine, a reference compound known to increase cancer sensitivity to radiation. The synergistic effect was correlated with an increase in cell thiol oxidative stress, which was reflected by a five-to sixfold increase in GSSG and 25% increase in total disulfides. No change in GSH or total thiols was observed as a result of GR inhibition.

Characterization of a novel dithiocarbamate glutathione reductase inhibitor and its use as a tool to modulate intracellular glutathione.

Thiol redox state (TRS) is an important parameter to reflect intracellular oxidative stress and is associated with various normal and abnormal biochemical processes. Agents that can be used to increase intracellular TRS will be valuable tools in TRS-related research. Glutathione reductase (GR) is a critical enzyme in the homeostasis of TRS. The enzyme catalyzes the reduction of GSSG to GSH to maintain a high GSH:GSSG ratio. Inhibition of the enzyme can be used to increase TRS. Despite the reports of various GR inhibitors, N,N-bis(2-chloroethyl)-N-nitrosourea, an anticancer drug with IC(50) = 647 microm against yeast GR, remains the most commonly used GR inhibitor in the literature. However, the toxicity caused by nonspecific interactions, as well as inhibition of DNA synthesis, complicates the use of N,N-bis(2-chloroethyl)-N-nitrosourea as a GR inhibitor. We report 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a novel irreversible GR inhibitor. 2-AAPA was prepared by one-step synthesis from commercially available reagents. The K(i) and k(inact) of 2-AAPA against yeast GR were determined to be 56 microm and 0.1 min(-1), respectively. At the concentration that produced >80% yeast GR inhibition, 2-AAPA showed no inhibition against glutamylcysteine synthetase, glutathione synthetase, catalase, and superoxide dismutase, but minimal inhibition against glutathione S-transferase and glutathione peroxidase. In CV-1 cells, 2-AAPA (0.1 mm) produced 97% GR inhibition, 25% GSH reduction, and a 5-fold increase in GSSG in 20 min. The compound can be a useful tool in TRS-related research.