Interactions of the major metabolite of the cancer chemopreventive drug oltipraz with cytochrome c: A novel pathway for cancer chemoprevention

Abstract

The major metabolite of the cancer chemopreventive agent oltipraz, a pyrrolopyrazine thione (PPD), has been shown to be a phase 2 enzyme inducer, an activity thought to be key to the cancer chemopreventive action of the parent compound. In cells, mitochondria are the major source of reactive oxygen species (ROS) and cytochrome c (cyt c) is known to participate in mitochondrial electron transport and confer antioxidant and peroxidase activities. To understand possible mechanisms by which PPD acts as a phase 2 enzyme inducer, a study of its interaction with cyt c was undertaken. UV-visible spectroscopic results demonstrate that PPD is capable of reducing oxidized cyt c. The reduced cyt c is stable for a long period of time in the absence of an oxidizing agent. In the presence of ferricyanide, the reduced cyt c is rapidly oxidized back to its oxidized form. Further, UV-visible spectroscopic studies show that during the reduction process the coordination environment and redox state of iron in cyt c are changed. Low-temperature EPR studies show that during the reduction process, the heme iron changes from a low-spin state of s = 1/2 to a low-spin state of s = 0. Room-temperature EPR studies demonstrate that PPD inhibits the peroxidase activity of cyt c. EPR spin trapping experiments using DMPO show that PPD inhibits the superoxide radical scavenging activity of oxidized cyt c. From these results, we propose that PPD interacts with cyt c, binding to and then reducing the heme, and this may enhance ROS levels in mitochondria. This in turn could contribute to the mechanism by which the parent compound, oltipraz, might trigger the cancer chemopreventive increase in transcription of phase 2 enzymes. The modifications of cyt c function by the oltipraz metabolite may have implications for the regulation of apoptotic cell death.

Oxidized Metabolites of Oltipraz Exert Cytoprotective Effects against Arachidonic Acid through AMP-Activated Protein Kinase-Dependent Cellular Antioxidant Effect and Mitochondrial Protection

“…PPD, a metabolic intermediate of the conversion to M2 by oltipraz, has the ability to induce the activity of phase 2 enzymes, react with GSH and thereby generate oxygen free radicals (Velayutham et al, 2005b). More recently, it was found that PPD inhibits the antioxidant activity of cyt c and thus enhances ROS levels in mitochondria (Velayutham et al, 2007). In the present study, we observed that oltipraz treatment promoted superoxide production in mitochondria (Fig.…”

“…In contrast, oltipraz showed no inhibitory effect. Rather, oltipraz treatment alone increased the mitochondrial MitoSOX fluorescence, which presumably results from the production of PPD (Velayutham et al, 2007). Either M1 or M2 treatment alone did not do so.…”

“…A previous study has shown that an intermediate metabolite of oltipraz, pyrrolopyrazine thione (PPD), interacts with cytochrome c (cyt c) by binding to and thereby reducing the heme iron (Velayutham et al, 2007). By this interaction, PPD inhibits the peroxidase activity of tight mitochondrial inner membrane-bound cyt c as well as the superoxide radical scavenging activity of oxidized cyt c. Consequently, PPD enhances ROS levels in mitochondria.…”

Nitric Oxide Signaling in Biology

“…Cu, Zn-superoxide dismutase (SOD1) in the mitochondrial intermembrane space dismutates O − 2 to hydrogen peroxide (H 2 O 2 (Fukai and Ushio-Fukai, 2011). ROS (O − 2 and H 2 O 2 in turn may act as a second messenger (Velayutham et al, 2007). The increased production of ROS from mitochondria thus initiates downstream signaling events (Erusalimsky and Moncada, 2007).…”

“…It has been proposed that under physiological conditions the interaction between NO and CcO increases the mitochondrial ROS, which would act as a second messenger to maintain high constitutive levels of cytoprotective (antioxidant or phase II) enzymes (see Fig. 3) (Erusalimsky and Moncada, 2007;Velayutham et al, 2007).…”