Survivin knockdown increased anti-cancer effects of (−)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells

• Published in: Experimental cell research 2012-08, Vol.318 (13), p.1597-1610
• Main Authors: Hossain, Md. Motarab, Banik, Naren L., Ray, Swapan K.
• Format: Article
• Language: English
• Subjects: (−)-Epigallocatechin-3-gallate; 60 APPLIED LIFE SCIENCES; ANGIOGENESIS; Antineoplastic Agents - pharmacology; APOPTOSIS; Apoptosis - drug effects; Base Sequence; BEVERAGES; Caspases - metabolism; Catechin - analogs & derivatives; Catechin - pharmacology; Cell Differentiation; Cell Line, Tumor; Cell Movement - drug effects; CELL PROLIFERATION; Chemotherapy; CHILDREN; Children & youth; CLEAVAGE; Differentiation; Gene Knockdown Techniques; Humans; IN VITRO; Inhibitor of Apoptosis Proteins - antagonists & inhibitors; Inhibitor of Apoptosis Proteins - genetics; Inhibitor of Apoptosis Proteins - metabolism; Medical prognosis; MESSENGER-RNA; METHYLENE BLUE; MICROSCOPY; MITOCHONDRIA; NEOPLASMS; Neovascularization, Pathologic - prevention & control; Neuroblastoma; Neuroblastoma - drug therapy; Neuroblastoma - genetics; Neuroblastoma - metabolism; Neuroblastoma - pathology; NEUTRON SPECTROSCOPY; Plasmids; PROTEINS; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Neoplasm - genetics; RNA, Neoplasm - metabolism; RNA, Small Interfering - genetics; SPIN ECHO; Survivin shRNA; THERAPY; Tumors
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/j.yexcr.2012.03.033

Neuroblastoma is a solid tumor that mostly occurs in children. Malignant neuroblastomas have poor prognosis because conventional chemotherapeutic agents are hardly effective. Survivin, which is highly expressed in some malignant neuroblastomas, plays a significant role in inhibiting differentiation and apoptosis and promoting cell proliferation, invasion, and angiogenesis. We examined consequences of survivin knockdown by survivin short hairpin RNA (shRNA) plasmid and then treatment with (−)-epigallocatechin-3-gallate (EGCG), a green tea flavonoid, in malignant neuroblastoma cells. Our Western blotting and laser scanning confocal immunofluorescence microscopy showed that survivin was highly expressed in malignant neuroblastoma SK-N-BE2 and SH-SY5Y cell lines and slightly in SK-N-DZ cell line. Expression of survivin was very faint in malignant neuroblastoma IMR32 cell line. We transfected SK-N-BE2 and SH-SY-5Y cells with survivin shRNA, treated with EGCG, and confirmed knockdown of survivin at mRNA and protein levels. Survivin knockdown induced morphological features of neuronal differentiation, as we observed following in situ methylene blue staining. Combination of survivin shRNA and EGCG promoted neuronal differentiation biochemically by increases in the expression of NFP, NSE, and e-cadherin and also decreases in the expression of Notch-1, ID2, hTERT, and PCNA. Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing, respectively, morphological and biochemical features of apoptosis. Apoptosis occurred with activation of caspase-8 and cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and increases in the expression and activity of calpain and caspase-3. Combination therapy decreased migration of cells through matrigel and inhibited proliferative (p-Akt and NF-κB), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro network formation ability of cells was significantly inhibited by survivin silencing and completely by combination of survivin silencing and EGCG treatment. Collectively, survivin silencing potentiated anti-cancer effects of EGCG in human malignant neuroblastoma cells having survivin overexpression. ► Survivin shRNA+EGCG controlled growth of human malignant neuroblastoma cells. ► Survivin knockdown induced neuronal differentiation in neuroblastoma cells. ► Survivin shRNA+EGCG induced morphological and b