Abstract 5342: Xanthohumol induces apoptosis and reduces notch in neuroblastoma

Abstract

Abstract Background: Neuroblastoma (NB) is a common neuroendocrine tumor with a high incidence of malignancy and recurrence. It signifies the most common extracranial solid cancer in childhood and accounts for 15% of childhood cancer-related deaths. More than 40% of patients present at diagnosis with aggressive disease and distant metastases which poses numerous therapeutic challenges. Despite intensive treatment options, the 5-year disease free survival remains less than 50%. Therefore, effective treatment approaches are required. Although recent studies revealed deregulated expression of Notch signaling in NB, therapeutic targeting of Notch represents a significant challenge. Xanthohumol (XN) (3′-[3,3-dimethyl allyl]-2′,4′,4-trihydroxy-6′-methoxychalcone), prenylated flavonoid from the Humulus lupulus L. (hops) exerts anti-proliferative activity against various cancers but the molecular mechanism of action remains unclear. In addition, the effect of XN on NB is not known. Based on our findings, we hypothesize that the XN treatment will reduce NB growth and the growth suppression may be mediated by Notch reduction. Therefore, the present study is designed to elicit, for the first time, anti-proliferative effect of XN on NB cell lines as well as understanding the molecular mechanism of action of XN. Methods: The proliferation effects of XN on a panel of human NB cell lines (SK-NA-S, NGP and SH-5Y-SY) were assessed by MTT assay as well as in real-time cellular proliferation assay using IncuCyte Live-Cell Imaging system. The expression levels of pro-apoptotic (c-PARP and cleaved caspase-3) and anti-apoptotic proteins (Bcl2, and survivin) were analyzed by Western blotting after XN treatment. Effects of XN on Notch pathway proteins (Notch3, Hes1, and ASCL1) were studied by Western blotting in addition to quantitative RT-PCR. Short hairpin RNA (shRNAs) targeting Notch3 was used to determine the XN sensitivity in NB. Results: NB cells treated with increasing concentrations of XN (0-30 μM) had a dose-dependent reduction in growth. A significant reduction in growth was noted at or above 10 μM of XN (40% or above; p<0.01) in all three cell lines tested. This was confirmed by real-time imaging of the cells after treatment with XN. The growth suppression effect is due to apoptosis as evidenced by increased expression of c-PARP and cleaved caspase-3 and decreased levels of Bcl2 and survivin proteins. Furthermore, XN inhibited Notch signaling by reducing Notch3 and associated downstream targets; Hes1 and ASCL1. Concomitant treatment with XN and Notch3 shRNA augmented growth suppression. Conclusions: We report for the first time that XN targets Notch3 signaling in NB cells. In summary, the similarity of XN metabolism between animals and humans, the antiproliferative effects on various cancers, and safety profile, we anticipate that XN could potentially be used in future clinical studies on patients with disease. Citation Format: Mariappan Balamurugan, Selvi Kunnimalaiyaan, T.Clark Gamblin, Muthusamy Kunnimalaiyaan. Xanthohumol induces apoptosis and reduces notch in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5342. doi:10.1158/1538-7445.AM2015-5342