Neuroblastoma is a malignant cancer, especially, in children older than 1 year . These patients do not respond to any currently available therapeutic regimen and eventually die. Thus, there is an urgent need for development of new therapeutic approaches for treatment of malignant neuroblastoma. We examined the efficacy of retinoids and flavonoids alone and in combination setting for controlling the growth of human malignant neuroblastoma SH-SY5Y cells in culture as well as in xenografted athymic nude mice. Treatment of SH-SY5Y cells with a retinoid such as 1 μM all-trans retinoic acid (ATRA), 1 μM 13-cis retinoic acid (13-CRA), or 0.5 μM N-(4-hydroxyphenyl) retinamide (4-HPR) for 8 days caused astrocytic differentiation, as evidenced by in situ methylene blue staining. Trypan blue dye exclusion test showed a decrease in cell viability with an increasing dose of a flavonoid such as apigenin (APG), (−)-epigallocatechin (EGC), (−)-epigallocatechin-3-gallate (EGCG), or genistein (GST). Wright staining identified morphological features of apoptosis in SH-SY5Y cells following exposure to 50 μM APG, 50 μM EGC, 50 μM EGCG, or 100 μM GST for 24 h. A combination treatment strategy involving 0.5 μM 4-HPR for 8 days and then 50 μM GST for 24 h produced significant amounts of apoptosis in SH-SY5Y cells in culture. The efficacy of this combination of 4-HPR and GST was subsequently evaluated in nude mice with SH-SY5Y xenografts. For xenotransplantation of neuroblastoma, 6 weeks-old nude mice (about 20 g each) were subcutaneously (sc) injected with a (1:1) mixture of exponentially growing SH-SY5Y (6 million cells/mouse) and Matrigel and allowed to develop the tumors. Animals with the 3 to 4 weeks-old xenografts were randomly assigned to 4 different groups: control, 4-HPR alone, GST alone, and 4-HPR plus GST . Animals in control group did not receive any therapy. Each animal in other group received intraperitoneal (ip) injection of a daily dose of 4-HPR (1 μg/kg), GST (75 μg/kg), or 4-HPR (1 μg/kg) plus 4 h later GST (75 μg/kg) for 8 days before tumors from all groups were collected for evaluation of therapeutic outcomes. Histopathological examination of tumor sections following H&E staining showed that tumors of control group maintained characteristic growth of neuroblastoma, treatment with 4-HPR alone caused differentiation of tumor cells, GST alone induced apoptosis, and treatment with 4-HPR plus GST produced significant amounts of apoptosis in tumors. In situ TUNEL and double immunofluorescent labeling of tumor sections demonstrated an increase in calpain expression in apoptotic cells. Taken together, these results suggested that treatment with a combination of the retinoid 4-HPR and the flavonoid GST could control the growth of malignant neuroblastoma. This investigation was supported in part by the R01 grants from the NCI and NINDS, and also a grant from the State of SC.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]