Mobilization of Intracellular Copper by Gossypol and Apogossypolone Leads to Reactive Oxygen Species-Mediated Cell Death: Putative Anticancer Mechanism

Abstract

There is compelling evidence that serum, tissue and intracellular levels of copper are elevated in all types of cancer. Copper has been suggested as an important co-factor for angiogenesis. It is also a major metal ion present inside the nucleus, bound to DNA bases, particularly guanine. We have earlier proposed that the interaction of phenolic-antioxidants with intracellular copper leads to the generation of reactive oxygen species (ROS) that ultimately serve as DNA cleaving agents. To further validate our hypothesis we show here that the antioxidant gossypol and its semi-synthetic derivative apogossypolone induce copper-mediated apoptosis in breast MDA-MB-231, prostate PC3 and pancreatic BxPC-3 cancer cells, through the generation of ROS. MCF10A breast epithelial cells refractory to the cytotoxic property of these compounds become sensitized to treatment against gossypol, as well as apogossypolone, when pre-incubated with copper. Our present results confirm our earlier findings and strengthen our hypothesis that plant-derived antioxidants mobilize intracellular copper instigating ROS-mediated cellular DNA breakage. As cancer cells exist under significant oxidative stress, this increase in ROS-stress to cytotoxic levels could be a successful anticancer approach.

Keywords: anticancer; apogossypolone (ApoG2); copper; gossypol; prooxidant.

Figure 1

(a) The cell proliferation of normal breast epithelial cells (MCF10A) in presence of gossypol and ApoG2 was detected using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. MCF10A cell were incubated with increasing concentration of gossypol/ApoG2, as indicated, for 72 h. Similarly, the cell proliferation of pancreatic cancer cell line, BxPC3; breast cancer cell line, MDA-MB-231; and prostate cancer cell line, PC3; in presence of increasing concentrations of (b) gossypol and (c) apogossypolone, as indicated, was detected using MTT assay. The cells were incubated for 72 h. MTT assay was performed as described in the Materials and Methods. All results are present as percentage of control (±SEM) of three independent experiments.

Figure 2

The Cell Death Detection ELISA Kit (Roche, Palo Alto, CA, USA) was used to detect apoptosis in MDA-MB-231, BxPC3 and PC3 cancer cell lines. Cells were treated with increasing concentrations of gossypol and ApoG2, as indicated, and processed as described in the Materials and Methods. Values reported are ±SEM of three independent experiments.

Figure 3

Cancer cell lines were incubated with 50 μM redox-metal-specific chelators, and treated with 5 μM gossypol/ApoG2 and further processed as described in the Materials and Methods. Neo: neocuproine; DM: desferrioxamine mesylate; His: Histidine. All results presented are mean (±SEM) of three independent experiments.

Figure 4

Cancer cell lines were plated in 24-well plates as described in the Methods. Culture was supplemented with 5 μM gossypol/ApoG2 with or without metal chelator neocuproine (Neo, 50 μM). Colonies (>50 cells), after appropriate culture time (22 days), were counted. Experiments were done in quadruplicate and mean values are reported. p < 0.01 when compared to untreated control cells.

Figure 5

Normal breast epithelial MCF10A cells and MCF10A cells culture media supplemented with 25 μM of copper (designated as MCF10A-Cu) were treated with 5 μM gossypol/ApoG2 and cell growth inhibition assay was done by MTT assay as described in the Methods.

Figure 6

The redox cycling of the ternary complex involving gossypol/ApoG2, copper ions and DNA leading to the generation of various ROS species has been demonstrated in the schematic model along with the chemical structures of gossypol and ApoG2.