Cu(II)-coumestrol interaction leads to ROS-mediated DNA damage and cell death: a putative mechanism for anticancer activity.

Phytoestrogens have attracted considerable interest as natural alternatives to hormone replacement therapy and their potential as cancer therapeutic agents. Among phytoestrogens, coumestrol has shown multipharmacological properties such as antiinflammatory, neuroprotective, osteoblastic differentiation and anticancer. Though several studies have described anticancer effects of coumestrol, a clear underlying molecular mechanism has not been elucidated. Unlike normal cells, cancer cells contain elevated copper levels that play an integral role in angiogenesis. Copper is an important metal ion associated with the chromatin DNA, particularly with guanine. Thus, targeting copper in cancer cells can serve as effective anticancer strategy. Using human peripheral lymphocytes, we assessed lipid peroxidation, protein carbonylation, reactive oxygen species (ROS) generation, DNA damage and apoptosis by coumestrol in the presence of exogenously added Cu(II) in cells to simulate malignancy-like condition. Results showed that Cu(II)-coumestrol interaction leads to lipid peroxidation and protein carbonylation (markers of oxidative stress), DNA fragmentation and apoptosis in treated lymphocytes. Further, incubation of lymphocytes with ROS scavengers and membrane-permeant copper chelator, neocuproine, resulted in inhibition of DNA damage and apoptosis. This suggests that coumestrol engages in redox cycling of Cu(II) to generate ROS that leads to DNA fragmentation and apoptosis. In conclusion, this is the first report showing that coumestrol targets cellular copper to induce prooxidant death in malignant cells. We believe that such a prooxidant cytotoxic mechanism better explains the anticancer activity of coumestrol. These findings will provide significant insights into the development of new chemical molecules with better copper-chelating and prooxidant properties against cancer cells.

Effect of phytoestrogens on basal and GnRH-induced gonadotropin secretion.

Plant-derived estrogens (phytoestrogens, PEs), like endogenous estrogens, affect a diverse array of tissues, including the bone, uterus, mammary gland, and components of the neural and cardiovascular systems. We hypothesized that PEs act directly at pituitary loci to attenuate basal FSH secretion and increase gonadotrope sensitivity to GnRH. To examine the effect of PEs on basal secretion and total production of FSH, ovine pituitary cells were incubated with PEs for 48 h. Conditioned media and cell extract were collected and assayed for FSH. Estradiol (E2) and some PEs significantly decreased basal secretion of FSH. The most potent PEs in this regard were coumestrol (CM), zearalenone (ZR), and genistein (GN). The specificity of PE-induced suppression of basal FSH was indicated by the absence of suppression in cells coincubated with PEs and an estrogen receptor (ER) blocker (ICI 182 780; ICI). Secretion of LH during stimulation by a GnRH agonist (GnRH-A) was used as a measure of gonadotrope responsiveness. Incubation of cells for 12 h with E2, CM, ZR, GN, or daidzein (DZ) enhanced the magnitude and sensitivity of LH secretion during subsequent exposure to graded levels of a GnRH-A. The E2- and PE-dependent augmentation of gonadotrope responsiveness was nearly fully blocked during coincubation with ICI. Collectively, these data demonstrate that selected PEs (CM, ZR, and GN), like E2, decrease basal secretion of FSH, reduce total FSH production, and enhance GnRH-A-induced LH secretion in a manner that is dependent on the ER.