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.
Highlights
Plumbagin, an anti-cancer agent, is toxic to cells of multiple species
OVCAR3, SKOV3, MCF7, and ECC1 are human ovarian, breast and endometrial cancer cells. 4T1 is a cell line derived from a spontaneous mammary tumor from BALB/c mouse and the MYC-HRAS MOSE are murine ovarian surface epithelial cells transformed by the introduction of mutant MYC and HRAS
Irrespective of their mutational status or tissue and species of origin, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays conducted with all of these cells showed that plumbagin was effective in inhibiting their proliferation at IC50 between 1.5–3.5 μM (Fig. 1A and B; Supplementary Files 1 and 2)
Summary
An anti-cancer agent, is toxic to cells of multiple species. We investigated if plumbagin targets conserved biochemical processes. Plumbagin produces its anti-cancer effects by inducing apoptosis and G1 cell cycle arrest[3] Evidence supporting these observations includes an increase in Annexin V staining, cleaved caspase-3 activity, and expression of Bax and a corresponding decrease in the anti-apoptotic protein, Bcl-2. Several reports have indicated that exposure to plumbagin causes an increase in intracellular oxygen radicals[6,7] This spike in reactive oxygen species (ROS) causes double strand DNA breaks and likely contributes to cell death[6,8]. Based on this evidence, plumbagin, its chemical analogs and its complexes in nanoparticles and chitosan microspheres are being considered as potential chemotherapeutic and chemopreventive agents
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