title: Neuronal apoptosis resulting from high doses of the isoflavone genistein: role for calcium and p42/44 mitogen-activated protein kinase.
abstract: 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 microM). 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 microM, 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 microM) partially blocks genistein-induced apoptosis, whereas the p38 MAPK inhibitor SB202190 (10 microM) has no effect. Genistein elevates intracellular calcium and both 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (1 microM) and dantrolene (10 microM) 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.
authors.affiliation: Department of Pharmacology, University of Washington, Seattle, Washington 98195, USA. nantzee@u.washington.edu
authors.forename: N J
authors.initials: NJ
authors.lastname: Linford
authors.forename: Y
authors.initials: Y
authors.lastname: Yang
authors.forename: D G
authors.initials: DG
authors.lastname: Cook
authors.forename: D M
authors.initials: DM
authors.lastname: Dorsa
mesh_terms: D000818:Animals; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology; D053938:DNA Fragmentation / Q000187:drug effects; D019254:Genes, bcl-2 / Q000235:genetics; D019833:Genistein / Q000494:pharmacology*; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000378:metabolism
chemical_list: D016588:Anticarcinogenic Agents; D007202:Indicators and Reagents; D011960:Receptors, Estrogen; D019833:Genistein; D007770:L-Lactate Dehydrogenase; D019950:Mitogen-Activated Protein Kinase 1; D020169:Caspases; D002118:Calcium