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.