Combination of N-(4-hydroxyphenyl) retinamide and genistein induced differentiation and multiple molecular mechanisms for apoptosis in human malignant neuroblastoma SK-N-BE(2) xenografts in nude mice

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Neuroblastoma is the second most common extracranial infantile malignancy, mostly affecting adrenal gland. The conventional therapy for this tumor includes surgery, radiation, and chemotherapy but these modalities in most instances show inefficacy. So, there is an urgent need for development of new therapeutic strategies. Here, we examined the efficacy of a retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) and a flavonoid genistein (GST) alone and also in combination for controlling the growth of human malignant (N-type) neuroblastoma SK-N-BE(2) 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-BE(2) 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 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. Further, 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. Our results revealed that treatment of human neuroblastoma SH-N-BE(2) xenografts with combination of 4-HPR and GST induced differentiation and multiple mechanisms for apoptosis in controlling malignant growth. This work was supported in part by the R01 grants CA-91460 and NS-57811.