Phytoestrogens Induce Apoptosis via Extrinsic Pathway, Inhibiting Nuclear Factor-κB Signaling

Background: Phytoestrogens are known to prevent tumor induction. But their molecular mechanisms of action are largely unknown. This study aimed to examine the effect of genistein and quercetin on proliferation and apoptosis in HER2-expressing breast cancer cells. Materials and Methods: The antiproliferative effects of phytoestrogens were tested by proliferation assays. Flow cytometry was performed to analyze the cell cycle. The effect of phytoestrogens on cellsignaling molecules was determined by Western blotting. Results: Genistein and quercetin inhibited the proliferation of MCF-7 vec and MCF-7 HER2 cells. This growth inhibition was accompanied with an increase of subG0/G1 apoptotic fractions. Genistein and quercetin induced extrinsic apoptosis pathway, up-regulating p53. Genistein and quercetin reduced the phosphorylation level of IκBα, and abrogated the nuclear translocation of p65 and its phosphorylation within the nucleus. Conclusion: Genistein and quercetin exert their antiproliferative activity by inhibiting NFκB signaling. Phytoestrogens could be potential useful compounds to prevent or treat HER2-overexpressing breast cancer. Apoptosis, or programmed cell death (PCD), plays an important role in various biological systems, such as embryonic development, cell turnover, and immune response against tumorigenic or virus-infected cells (1). When apoptosis occurs, different cell morphological changes including the shrinkage of cell and nuclei, membrane blebbing, loss of cell membrane asymmetry and attachment, nuclear fragmentation, and chromatin condensation appear (2). Apoptosis differs from necrosis in its morphological and biochemical features. In most tumor cells, apoptosis occurs via two different signaling pathways: the extrinsic and the intrinsic apoptosis pathways. The extrinsic pathway is related to the activation of the death receptors, such as Fas and tumor necrosis factor receptors (TNFR). Death domains (DD) of Fas are oligomerized and recruit Fas-associated death domain (FADD) and procaspase-8 to form deathinducing signaling complex (DISC). Procaspase-8 is cleaved and activated and released from the DISC into the cytoplasm where it activates caspase-3 to induce apoptosis (1, 3, 4). The intrinsic pathway is related to changes in mitochondrial membrane potential (Δψm) and mitochondrial permeability transition, resulting in mitochondrial release of apoptogenic factors such as cytochrome c and apoptosis-inducing factor (AIF) into the cytoplasm (1, 5). Cytochrosome c binds to APAF1 and recruits procaspase-9 to form an apoptosome; caspase-9 activates effector caspases such as caspase-3 to induce apoptosis (4). Caspase-3 from both extrinsic and intrinsic pathways is responsible for the cleavage of Poly (ADP-ribose) polymerase (PARP) during cell death (6). Extracellular signal-regulated kinases (ERK) pathway and Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling pathway can inhibit this apoptotic signaling (4). The cross-talk between the extrinsic apoptosis pathway and the intrinsic apoptosis pathway exists; caspase8 can cleave BID, a death-inducing member of the BCL2 3301 Correspondence to: Dr. Seong-Gyu Ko, Laboratory of Clinical Biology and Pharmacogenomics and Center for Clinical Research and Genomics, Institute of Oriental Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea. Tel: +82 29610329, Fax: +82 29661165, e-mail: [email protected]