Genistein-induced neuronal apoptosis and G2/M cell cycle arrest is associated with MDC1 up-regulation and PLK1 down-regulation.
The aim of the present study is to investigate the effect of genistein on human neuroblastoma SK-N-MC cells. MTT proliferation assay, LDH cytotoxicity assay, flow cytometric analysis, real-time quantitative RT-PCR and western blotting were used to investigate the effect of genistein on cell survival, cellular toxicity, cell cycle progression, and mRNA and protein alterations of selected DNA damage-, cell cycle- and apoptosis-related genes in SK-N-MC cells. Genistein suppressed cell proliferation, increased LDH release and modulated cell cycle distribution through accumulation of cells at G2/M- and S-phase and sub-G0 (cell death) with a concurrent decrease of cells at G0/G1 phase. Genistein increased the MDC1 (Mediator of DNA damage Checkpoint protein 1), p53, p21(waf1/cip1), Cdc2 and Bax mRNA levels in a dose-dependent manner. However, PLK1 (Polo-Like Kinase 1) and Cyclin B1 mRNAs were down-regulated after genistein treatment. Furthermore, Genistein did not alter Chk2 (Checkpoint Kinase 2), Bcl-2 and Cdc25C mRNA levels. On western blotting analyses; genistein increased the protein level of MDC1, p53, p21(waf1/cip1), and Bax in a dose-dependent manner. Genistein also increased the phosphorylation of Chk2 and Cdc25C at Thr-68 and Ser-216, respectively. In addition, consistently with PLK1 down-regulation, the phosphorylation of Cdc25C at Ser-198 was markedly decreased after genistein treatment. Additionally, Chk2, Cdc25C, Cyclin B1, p-Cyclin B1 (Ser-147), and Cdc2 as well as Bcl-2 proteins were down-regulated after genistein treatment. Altogether, these results suggest for the first time the involvement of MDC1 up-regulation after genistein treatment in DNA damage-induced Chk2 activation- and PLK1 down-regulation-mediated apoptosis and cell cycle checkpoint pathways.
Ismail
Ismail Ahmed
IA
Department of Pathology, Kyungpook National University School of Medicine, Daegu, Republic of Korea.
Kang
Ku-Seong
KS
Lee
Hae Ahm
HA
Kim
Jung-Wan
JW
Sohn
Yoon-Kyung
YK
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D019926:Cyclin B / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects* / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D016195:G2 Phase / Q000187:drug effects; D019833:Genistein / Q000494:pharmacology*; D006801:Humans; D009447:Neuroblastoma / Q000139:chemically induced*; D009687:Nuclear Proteins / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016196:S Phase / Q000187:drug effects; D015534:Trans-Activators / Q000378:metabolism*; D015854:Up-Regulation; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D020687:cdc25 Phosphatases / Q000378:metabolism; D000097869:Polo-Like Kinase 1
D048868:Adaptor Proteins, Signal Transducing; D016203:CDC2 Protein Kinase; D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D019926:Cyclin B; D004268:DNA-Binding Proteins; D019833:Genistein; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D051028:bcl-2-Associated X Protein; D020687:cdc25 Phosphatases; D000097869:Polo-Like Kinase 1; C413163:MDC1 protein, human; C578634:CHEK2 protein, human; C517009:CDC25C protein, human