Neuroblastoma, which shows a complex clinical and biological heterogeneity, is a childhood tumor of the sympathetic nervous system. Although many other childhood malignancies are nowadays curable, malignant (N-type) neuroblastoma remains an enigma to both the basic scientists and clinicians as it is not yet amenable to current treatment modalities and the affected children usually die at pre-school age. Therefore, new and innovative therapeutic strategies must be devised for controlling this deadly childhood disease. Malignant growth of neuroblatoma is characterized by poor differentiation and uncontrolled proliferation of the immature neuroblasts in adrenal gland in most cases. Retinoids such as all-trans retinoic acid (ATRA), 13-cis retinoic acid (13-CRA), and N-(4-hydroxyphenyl) retinamide (4-HPR) at low doses are capable of inducing cell differentiation. Plant-derived flavonoids (-)-epigallocatechin-3-gallate (EGCG) and genistein (GST) at relatively high doses can control cancer cell growth by induction of apoptosis. In this investigation, we used human N-type neuroblastoma SH-SY5Y cells to show that treatment with a low dose of a retinoid (1 μM ATRA, 1 μM 13-CRA, or 0.5 μM 4-HPR) for 7 days induced neuronal differentiation with down regulation of telomerase activity and subsequent treatment with a relatively high dose of a flavonoid (25 μM EGCG or 25 μM GST) for 1 day sensitized the differentiated cells for enhancement of apoptosis. Apoptosis in SH-SY5Y cells was associated with an increase in intracellular free [Ca2+], as determined by fura-2 assay. Western blotting showed alterations in the levels of Bax and Bcl-2 proteins resulting in an increase in Bax:Bcl-2 ratio and upregulation of calpain and caspases in course of apoptosis. The reverse transcriptase-polymerase chain reaction (RT-PCR) indicated down regulation of the transcriptional expression (mRNA) of calpastatin (the endogenous inhibitor of calpain) and baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins (the endogenous inhibitors of caspases) in apoptotic cells. Treatment of SH-SY5Y cells with EGCG activated caspase-8, indicating induction of the receptor-mediated pathway of apoptosis. Colorimetric assays using chromogenic synthetic peptide substrates confirmed involvement of different proteolytic mechanisms in apoptotic death. The results of this investigation suggested that retinoid induced neuronal differentiation with down regulation of telomerase activity and enhanced the action of flavonoid for apoptosis in malignant neuroblastoma cells. Therefore, combination of a retinoid and a flavonoid appears to be an effective therapeutic strategy for controlling the malignant growth of neuroblastoma cells. This investigation was supported in part by the R01 grants (CA-91460 and NS-57811) from the National Institutes of Heatlth (Bethesda, MD, USA).
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA