Bcl-2 inhibitor HA14-1 and genistein together adeptly down regulated survival factors and activated cysteine proteases for apoptosis in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells

• Published in: Brain research 2009-08, Vol.1283, p.155-166
• Main Authors: Mohan, Nishant, Karmakar, Surajit, Choudhury, Subhasree Roy, Banik, Naren L, Ray, Swapan K
• Format: Article
• Language: English
• Subjects: Annexin A5 - drug effects; Annexin A5 - metabolism; Antineoplastic Combined Chemotherapy Protocols - pharmacology; Antineoplastic Combined Chemotherapy Protocols - therapeutic use; Apoptosis; Apoptosis - drug effects; Apoptosis - physiology; Apoptosis Regulatory Proteins - antagonists & inhibitors; Apoptosis Regulatory Proteins - metabolism; Bcl-2; Benzopyrans - pharmacology; Benzopyrans - therapeutic use; Biological and medical sciences; Caspase; Cell Cycle - drug effects; Cell Cycle - physiology; Cysteine Endopeptidases - drug effects; Cysteine Endopeptidases - metabolism; Dose-Response Relationship, Drug; Enzyme Activation - drug effects; Enzyme Activation - physiology; Enzyme Inhibitors - pharmacology; Enzyme Inhibitors - therapeutic use; Genistein; Genistein - pharmacology; Genistein - therapeutic use; HA14-1; Humans; Medical sciences; Neuroblastoma - drug therapy; Neuroblastoma - metabolism; Neuroblastoma - physiopathology; Neurology; Nitriles - pharmacology; Nitriles - therapeutic use; Protein Kinase Inhibitors - pharmacology; Protein Kinase Inhibitors - therapeutic use; Tumors of the nervous system. Phacomatoses
• ISSN: 0006-8993; 1872-6240
• DOI: 10.1016/j.brainres.2009.05.097

Abstract Neuroblastoma is a pediatric extracranial tumor and a major cause of death in children under age 2. Conventional therapy shows inefficacy in most cases and thus development of new therapeutic strategies is urgently needed. We explored the efficacy of combination of the small molecule Bcl-2 inhibitor HA14-1 (HA) and the isoflavonoid genistein (GST) in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells. Combination of 10 μM HA and 250 μM GST was optimal for SK-N-BE2 cells and combination of 5 μM HA and 100 μM GST was optimal for SH-SY5Y cells for induction of apoptosis. Phase-contrast microscopy and Wright staining showed morphological features of apoptosis. Cell cycle analysis and Annexin V-FITC/PI binding assay showed that combination of HA and GST was more effective in inducing apoptosis in both cell lines than either HA or GST alone. Western blotting showed that combination of HA and GST caused upregulation of Bax and down regulation of Bcl-2 resulting in increased Bax:Bcl-2 ratio and mitochondrial release of cytochrome c , Smac, and AIF. Down regulation of survival factors such as NF-κB, N-Myc, and survivin promoted apoptosis. Activation of caspase-8, calpain, and caspase-3 occurred in course of apoptosis. Increased calpain and caspase-3 activities were confirmed in the degradation of α-spectrin to 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Thus, combination of HA and GST could serve as a promising therapeutic strategy for increasing apoptosis in different human malignant neuroblastoma cells.