Genistein induces receptor and mitochondrial pathways and increases apoptosis during BCL-2 knockdown in human malignant neuroblastoma SK-N-DZ cells

J Neurosci Res. 2010 Mar;88(4):877-86. doi: 10.1002/jnr.22244.


The potent antiapoptotic molecule Bcl-2 is markedly up-regulated in a majority of cancers, including neuroblastoma. Genistein is an isoflavone with antitumor properties. The present study sought to elucidate the molecular mechanism of genistein-induced apoptosis and also to examine the effect of genistein in increasing apoptosis during Bcl-2 knockdown in human malignant neuroblastoma SK-N-DZ cells. The cells were transfected with Bcl-2 siRNA plasmid vector, treated with 10 microM genistein, or the combination, and subjected to TUNEL staining and FACS analysis. Semiquantitative and real-time RT-PCR experiments were performed for examining expression of Fas ligand (FasL), tumor necrosis factor-alpha (TNF-alpha), Fas-associated death domain (FADD), and TNFR-1-associated death domain (TRADD). The cell lysates were analyzed by Western blotting for levels of molecules involved in both receptor- and mitochondria-mediated apoptotic pathways. Treatment with the combination of Bcl-2 siRNA and genistein resulted in more than 80% inhibition of cell proliferation. TUNEL staining and FACS analysis demonstrated apoptosis in 70% of cells after treatment with the combination of both agents. Apoptosis was associated with increases in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and activation of caspases through the mitochondria-mediated apoptotic pathway. Genistein triggered the receptor-mediated apoptotic pathway through upregulation of TNF-alpha, FasL, TRADD, and FADD and activation of caspase-8. Combination of Bcl-2 siRNA and genistein triggered a marked increase in cleavage of DFF45 and PARP that resulted in enhanced apoptosis. Our study demonstrates that Bcl-2 knockdown during genistein treatment effectively induced apoptosis in neuroblastoma cells. Therefore, this strategy could serve as a potential therapeutic regimen to inhibit the growth of human malignant neuroblastoma.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Analysis of Variance
  • Anticarcinogenic Agents / pharmacology*
  • Apoptosis / drug effects*
  • Caspase 8 / metabolism
  • Cell Line, Tumor
  • Cyclin D1 / antagonists & inhibitors
  • Cyclin D1 / metabolism*
  • Fas Ligand Protein / metabolism
  • Fas-Associated Death Domain Protein / metabolism
  • Genistein / pharmacology*
  • Humans
  • In Situ Nick-End Labeling / methods
  • Mitochondria / drug effects*
  • Neuroblastoma / pathology
  • RNA, Small Interfering / pharmacology
  • Signal Transduction / drug effects*
  • TNF Receptor-Associated Death Domain Protein / metabolism
  • Transfection / methods
  • Tumor Necrosis Factor-alpha / metabolism
  • Up-Regulation / drug effects


  • Anticarcinogenic Agents
  • CCND1 protein, human
  • FADD protein, human
  • Fas Ligand Protein
  • Fas-Associated Death Domain Protein
  • RNA, Small Interfering
  • TNF Receptor-Associated Death Domain Protein
  • Tumor Necrosis Factor-alpha
  • Cyclin D1
  • Genistein
  • Caspase 8