Regulation of genistein-induced differentiation in human acute myeloid leukaemia cells (HL60, NB4) Protein kinase modulation and reactive oxygen species generation.
While it has been reported that genistein induces differentiation in multiple tumour cell models, the signalling and regulation of isoflavone-provoked differentiation are poorly known. We here demonstrate that genistein causes G(2)/M cycle arrest and expression of differentiation markers in human acute myeloid leukaemia cells (HL60, NB4), and cooperates with all-trans retinoic acid (ATRA) in inducing differentiation, while ATRA attenuates the isoflavone-provoked toxicity. Genistein rapidly stimulates Raf-1, MEK1/2 and ERK1/2 phosphorylation/activation, but does not stimulate and instead causes a late decrease in Akt phosphorylation/activation which is attenuated by ATRA. Both differentiation and G(2)/M arrest are attenuated by MEK/ERK inhibitors (PD98059, U0126) and ERK1-/ERK2-directed small interfering RNAs (siRNAs), and by the PI3K inhibitor LY294002, but not by the p38-MAPK inhibitor SB203580. Genistein stimulates p21(waf1/cip1) and cyclin B1 expression, phosphorylation/activation of ATM and Chk2 kinases, and Tyr15-phosphorylation/inactivation of Cdc2 (Cdk1) kinase, and these effects are attenuated by MEK/ERK inhibitors, while LY294002 also attenuates ERK and ATM phosphorylation. Caffeine abrogates the genistein-provoked G(2)/M blockade and alterations in cell cycle regulatory proteins, and also suppresses differentiation. Finally, genistein causes reactive oxygen species (ROS) over-accumulation, but the antioxidant N-acetyl-L-cysteine fails to prevent ERK activation, G(2)/M arrest, and differentiation induction. By contrast, N-acetyl-L-cysteine and p38-MAPK inhibitor attenuate the apoptosis-sensitizing (pro-apoptotic) action of genistein when combined with the antileukaemic agent arsenic trioxide. In summary, genistein-induced differentiation in acute myeloid leukaemia cells is a ROS-independent, Raf-1/MEK/ERK-mediated and PI3K-dependent response, which is coupled and co-regulated with G(2)/M arrest, but uncoupled to the pro-apoptotic action of the drug.
Sánchez
Yolanda
Y
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Amrán
Donna
D
de Blas
Elena
E
Aller
Patricio
P
D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D005434:Flow Cytometry; D019833:Genistein / Q000494:pharmacology*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology / Q000473:pathology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*
D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D019833:Genistein; D011494:Protein Kinases