Vol. 299, Issue 1, 67-75, October 2001

Neuronal Apoptosis Resulting from High Doses of the Isoflavone Genistein: Role for Calcium and P42/44 Mitogen-Activated Protein Kinase

Nancy J. Linford, Yaxiong Yang, David G. Cook and Daniel M. Dorsa

Departments of Pharmacology (N.J.L., Y.Y., D.G.C., D.M.D.) and Psychiatry and Behavioral Neurosciences (D.M.D.), University of Washington, Seattle, Washington

Genistein is a potent plant-derived isoflavone displaying estrogenic activity at low (nanomolar) concentrations and antiproliferative and antiangiogenic properties at higher concentrations (above 10-50 µM). The antiproliferative potential of genistein has made it an interesting candidate for cancer chemotherapy at high concentrations; however, the potential for genistein toxicity in neurons at such concentrations has not been previously addressed. We show that genistein is toxic to rat primary cortical neurons at a concentration of 50 µM, whereas daidzein, a structural analog, shows no toxicity at similar concentrations. The dying cells display an apoptotic morphology that is characterized by fragmented nuclei, appearance of apoptotic bodies, DNA laddering, and caspase-dependent poly(ADP-ribose) polymerase cleavage. This cell death is partially dependent on caspase activity, independent of estrogen receptors, and does not result in a significant loss of Bcl-2 or Bcl-X_L protein. Genistein exposure induces delayed and prolonged activation of p42/44 mitogen-activated protein kinase (MAPK) and p38 MAPK but not c-Jun NH₂-terminal kinase. The specific p42/44 MAPK kinase inhibitor PD98059 (50 µM) partially blocks genistein-induced apoptosis, whereas the p38 MAPK inhibitor SB202190 (10 µM) has no effect. Genistein elevates intracellular calcium and both 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (1 µM) and dantrolene (10 µM) inhibit genistein-induced apoptosis, suggesting a link between genistein-induced intracellular calcium release and apoptosis. The combination of dantrolene and PD98059 block genistein-induced apoptosis in an additive manner compared with either compound alone. These findings provide evidence for a proapoptotic function of p42/44 MAPK and raise caution about potential side effects in the nervous system with genistein use as a high-dose therapeutic agent.