Neuronal apoptosis resulting from high doses of the isoflavone genistein: Role for calcium and p42/44 mitogen-activated protein kinase

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.