Oxidative stress via inhibition of the mitochondrial electron transport and Nrf-2-mediated anti-oxidative response regulate the cytotoxic activity of plumbagin

Abstract

Plumbagin, an anti-cancer agent, is toxic to cells of multiple species. We investigated if plumbagin targets conserved biochemical processes. Plumbagin induced DNA damage and apoptosis in cells of diverse mutational background with comparable potency. A 3–5 fold increase in intracellular oxygen radicals occurred in response to plumbagin. Neutralization of the reactive oxygen species by N-acetylcysteine blocked apoptosis, indicating a central role for oxidative stress in plumbagin-mediated cell death. Plumbagin docks in the ubiquinone binding sites (Q0 and Qi) of mitochondrial complexes I–III, the major sites for oxygen radicals. Plumbagin decreased oxygen consumption rate, ATP production and optical redox ratio (NAD(P)H/FAD) indicating interference with electron transport downstream of mitochondrial Complex II. Oxidative stress induced by plumbagin triggered an anti-oxidative response via activation of Nrf2. Plumbagin and the Nrf2 inhibitor, brusatol, synergized to inhibit cell proliferation. These data indicate that while inhibition of electron transport is the conserved mechanism responsible for plumbagin’s chemotoxicity, activation of Nrf2 is the resulting anti-oxidative response that allows plumbagin to serve as a chemopreventive agent. This study provides the basis for designing potent and selective plumbagin analogs that can be coupled with suitable Nrf2 inhibitors for chemotherapy or administered as single agents to induce Nrf2-mediated chemoprevention.

Plumbagin-induced oxidative stress leads to inhibition of Na+/K+-ATPase (NKA) in canine cancer cells

“…Our experiments with human cancer cell lines showed that plumbagin induces oxidative stress [20]. When the CTAC cells were labeled with the ROS sensing fluorescent dye, H2DCFDA and treated with plumbagin (5 µM), a rapid increase in intracellular oxygen radicals was detected by flow cytometry analysis of the cells ( Fig.…”

“…We recently demonstrated that plumbagin decreased the intracellular ATP levels in cancer cells [20]. We therefore tested if the inhibition of NKA observed in plumbagin-treated CTAC was due to decreased availability of intracellular ATP.…”

“…Oxygen radical formation in the canine cancer cell lines in response to plumbagin was monitored as reported previously [20,24,25]. Briefly, cells were labeled with 10 combination of pipette and bath solution allowed us to isolate NKA activity [26].…”

“…The naphthoquinone unit of plumbagin likely mimics the quinone ring of ubiquinone (CoQ10) ( Fig. 1A) and interferes with electron transport in the oxidative phosphorylation pathway [20]. To obtain support for the use of plumbagin as a chemotherapeutic agent in cancer patients, we are developing pre-clinical data in a canine cancer model.…”

“…We therefore asked if agents that trigger oxidative stress in cancer cells could also negatively influence NKA function. Recently, we have demonstrated that the natural product, plumbagin, increases intracellular oxygen radicals in cancer cells by interfering with mitochondrial electron transport [20]. The oxidative damage caused through treatment with plumbagin results in apoptosis of the cancer cells.…”