Neuroblastoma is childhood malignancy that mostly occurs in the adrenal glands. It is an aggressive metastatic disease, especially, in children older than 1 year of age. Malignant neuroblastoma continues to defy all available chemotherapeutic regimens. So, there is an urgent need for development of new therapeutic strategies for successful treatment of malignant neuroblastoma. In our current investigation, we examined the efficacy of the retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) and the flavonoid genistein (GST) alone and also in combination for controlling the growth of human malignant neuroblastoma SK-N-MC xenografts in nude mice. For development of xenografts, 6-week old nude mice (about 20 g each) were subcutaneously injected with a (1:1) mixture of exponentially growing SK-N-MC cells (6 million cells/mouse) and Matrigel. The mice were allowed to develop the tumors. Animals with the 3-week old xenografts were randomly assigned to four 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 injection of a daily dose of 4-HPR (20 µg/kg/day), GST (2 mg/kg/day), or 4-HPR (20 µg/kg/day) plus 4 h later GST (2 mg/kg/day) for 8 days before the tumors from all groups were collected for evaluation of therapeutic outcomes. Histopathological examination of tumor sections after 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 to some extent, and combination of 4-HPR and GST produced significant amounts of apoptosis in tumors. Western blotting showed that combination of 4-HPR and GST caused changes in expression of Bax and Bcl-2 proteins leading to increase in Bax:Bcl-2 ratio, mitochondrial release of apoptosis-inducing factor (AIF) and Smac/Diablo, and activation of caspase-3 in course of apoptosis. Cytosolic upregulation of Smac/Diablo caused down regulation of the baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins such as BIRC-2 and BIRC-3 to facilitate the apoptotic process. In situ immunofluorescent labeling showed overexpression of calpain, caspase-12, and caspase-3, and also AIF in apoptotic cells, suggesting potential role of these cysteine proteases and AIF in mediation of apoptosis. Also, in situ TUNEL and double immunofluorescent labeling confirmed cell death with increase in levels of calpain, caspase-12, caspase-3, and AIF in tumors treated with combination of 4-HPR and GST. Results revealed that treatment of SK-N-MC xenografts with combination of 4-HPR and GST induced differentiation and multiple mechanisms for apoptosis. This work was supported in part by the R01 grants CA-91460 and NS-57811.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA