FISETIN, A FLAVONOID, INDUCES CELL CYCLE ARREST AND APOPTOSIS IN HUMAN BREAST CANCER CELLS

Abstract

Significant morbidity and mortality continues to be associated with breast cancer and its treatments. Fisetin, a phytochemical that is present in many fruits and vegetables, has demonstrated anticancer activity. My research explores fisetin as a possible novel therapeutic modality for breast cancer. Breast cancer cell lines (MDA-MB-468, MDA- MB-231, MCF-7, T47-D, SKBR-3; mitoxantrone-resistant (MITX) and paclitaxel- resistant (Tx400) cell lines) were exposed to fisetin and cell survival was assessed by MTT, crystal violet, acid phosphatase, and colony-forming assays. Normal cells (human mammary epithelial cells, fibroblasts, human umbilical vein endothelial cells) were used as negative controls. The mechanism of action of fisetin was explored using cell cycle analysis and assays for apoptosis/necrosis, including Annexin V-propidium iodide staining and LDH-release. Apoptosis induction pathways were studied using Western blotting, as well as caspase inhibitors and cell viability assays. Flow cytometry was used to assess mitochondrial membrane stability (DiOC6 staining) and reactive oxygen species (ROS) production (dihydroethidium staining). Fisetin had a dose- and time-dependent cytotoxic effect on breast cancer cell lines (e.g., 100 ?M fisetin decreased MDA-MB-468 cell number by 70% at 72h in both crystal violet and acid phosphatase assays). In contrast, the viability of normal cells was not substantially affected by concentrations of fisetin that killed breast cancer cells. Fisetin-treated breast cancer cells showed cell cycle arrest (MDA-MB-468 cells arrested at G2/M phase; MDA-MB-231 cells arrested in S- phase) and death by apoptosis (e.g., MDA-MB-468 cells showed up to 50% apoptosis and 8% late apoptosis/necrosis by Annexin V-staining; cell cycle analysis and LDH- release assays supported these results). Fisetin-induced apoptosis was associated with mitochondrial membrane permeabilization, as well as activation of the caspase cascade since the pro-apoptotic effect of fisetin was reduced in the presence of a pan-caspase inhibitor. In addition, fisetin did not cause ROS production in MDA-MB-468 or 231 cells, ruling out a role for ROS in fisetin-mediated cytotoxicity. My findings suggest that fisetin may be useful in the treatment of breast cancer.