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The clinical hallmark of neuroblastoma is heterogeneity, with the likelihood of tumor progression varying widely according to age at diagnosis and extent of disease. A number of prognostic parameters for the disease have been identified, including age, loss of heterozygosity at chromosome 1p36, DNA ploidy, histopathologic stage, and amplification of the N-myc proto-oncogene. The latter condition is strongly correlated with advanced disease, insensitivity to chemotherapy, and poor outcome. The long-term survival rate of such high-risk patients has remained very low over the last decades. This situation has called for development of innovative therapeutic approaches. The concept of ‘differentiation therapy’ has gained considerable interest in recent years. 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. The plant-derived flavonoids such as (-)-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 neuroblastoma IMR-32 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 N-myc and thereafter treatment with a relatively high dose of a flavonoid (25 μM EGCG or 25 μM GST) for 1 day sensitized the differentiated cells for increase in apoptosis. Apoptosis in IMR-32 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 transcription-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 IMR-32 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 retinoids induced neuronal differentiation with down regulation of telomerase activity and enhancement of the action of flavonoids 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 work was supported by the R01 NS-57811 grant from the NINDS.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA