Neuroblastoma is an extracranial solid tumor that mostly occurs in adrenal glands in children. The conventional therapeutic strategies are ineffective in most cases. So, new therapies are urgently warranted for treatment of malignant neuroblastoma. Overexpression of N-Myc occurs in high-risk neuroblastoma. We examined the efficacy of sorafenib (an anti-angiogenic agent) and genistein (a pro-apoptotic agent) for inhibiting angiogenesis and inducing apoptosis, respectively, in human malignant neuroblastoma SK-N-DZ (N-Myc amplified) and SH-SY5Y (N-Myc non-amplified) cell lines. Sorafenib and genistein dose-dependently decreased cell viability in both neuroblastoma cell lines. Based on the dose-response studies, we decided to treat both cell lines with 2.5 µM sorafenib and 25 µM genistein alone and in combination for 24 h. Combination therapy was more effective in inhibiting angiogenic factors and cell proliferation in both SK-N-DZ and SH-SY5Y cell lines. Wright staining confirmed induction of morphological features of apoptosis. Cell cycle analysis and Annexin-V staining showed that treatment with combination of sorafenib and genistein caused the most significant amounts of apoptosis in both cell lines. Western blotting showed that combination therapy most effectively inhibited expression of angiogenic factors (e.g., VEGF, bFGF) and down regulated expression of cell survival factors (e.g., hTERT, p-Akt, NF-\#954;B, Mcl-1, c-IAP2, survivin) in both cell lines. Moreover, combination therapy suppressed expression of N-Myc and multidrug resistance protein (MDR) while dramatically increased expression of p53 and p21 leading to growth arrest and apoptosis in both cell lines. Following combination therapy, apoptosis occurred with activation of caspase-8, cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and Smac overexpression indicating activation of both extrinsic and intrinsic apoptotic pathways in both SK-N-DZ and SH-SY5Y cell lines. In course of apoptosis, increased activities of the cysteine proteases calpain and caspase-3 were confirmed in the cleavage of 270 kD \#945;-spectrin at specific sites that generated 145 kD spectrin breakdown product (SBDP) and 120 kD SBDP, respectively. Further increased activity of caspase-3 cleaved inhibitor of caspase-3-activated DNase (ICAD) in both cell lines. The activity of lysosomal cysteine protease cathepsin B was also markedly elevated for apoptosis following combination therapy. These data strongly suggest that combination of sorafenib and genistein can inhibit angiogenesis and trigger multiple molecular mechanisms for inducing apoptosis in human malignant neuroblastoma cells. This investigation was supported in part by the R01 grants (NS-57811 and CA-91460) from the National Institutes of Health (Bethesda, MD).
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5511.
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO