Genistein induces receptor and mitochondrial pathways and increases apoptosis during BCL-2 knockdown in human malignant neuroblastoma SK-N-DZ cells.
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.
George
Joseph
J
Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina, USA.
Banik
Naren L
NL
Ray
Swapan K
SK
D000704:Analysis of Variance; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053181:Caspase 8 / Q000378:metabolism; D045744:Cell Line, Tumor; D019938:Cyclin D1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053222:Fas Ligand Protein / Q000378:metabolism; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D019833:Genistein / Q000494:pharmacology*; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000379:methods; D008928:Mitochondria / Q000187:drug effects*; D009447:Neuroblastoma / Q000473:pathology; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D053199:TNF Receptor-Associated Death Domain Protein / Q000378:metabolism; D014162:Transfection / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D016588:Anticarcinogenic Agents; C530104:CCND1 protein, human; C505533:FADD protein, human; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D034741:RNA, Small Interfering; D053199:TNF Receptor-Associated Death Domain Protein; D014409:Tumor Necrosis Factor-alpha; D019938:Cyclin D1; D019833:Genistein; D053181:Caspase 8