Fig 1.
Chemical structure of Xanthohumol.
Fig 2.
Effects of XN on NGP, SH-SY-5Y, and SK-N-AS cellular proliferation.
MTT assay (A), Colony forming unit (B-C), and IncuCyte Live-Cell imaging (D-F) demonstrate a dose-dependent reduction in cell growth compared to control. * p<0.05; ** p<0.001; *** p<0.0001.
Fig 3.
XN induces apoptosis in NGP, SH-SY-5Y, and SK-N-AS cells.
A. Western blot analysis showed an increase in apoptotic markers, cleaved PARP, cleaved caspase 3 (CC3) and Bax after treatment with XN for 72hrs. This was associated with a reduction in anti-apoptotic protein, Bcl-2. B. Caspase-3/-7 activities increased in a dose-dependent fashion in XN-treated NGP, SK-N-AS, and SH-SY-5Y cells. * p<0.05; ** p<0.001; *** p<0.0001. (C). Annexin-V-FITC flow cytometry analysis showed an increase in apoptosis. (D). Live cell image of cell player with YOYO-1 assay by Incucyte also showed induction of apoptosis.
Fig 4.
XN regulates the Akt pathway and associated with an increase in DR5 expression.
A. Notch signaling is minimally active in NGP, SK-N-AS, and SH-SY-5Y, as evidenced by minimal expression of Notch intracellular Domain1, whereas there is increased expression of achaete-scute complex-like1 (ASCL1), a downstream target of Notch signaling. B. Inhibition of Akt pathway by XN treatment was observed by reduction in phosphorylation of Akt (ser473) as early as 6 hrs. This was associated with a reduction in CyclinD1. C. NB cell line, SK-N-AS was treated with indicated concentrations of XN for 6–24 hours’ time points and lysates were prepared. Western blot analysis of the lysates shows a time-dependent increase in DR5 expression following XN treatment. GAPDH was used as loading control. D. DR5 gene expression increases with XN treatment in a dose-dependent fashion in quantitative RT PCR analysis. GAPDH was used as a control and for normalization. * p<0.05; ** p<0.01; *** p<0.001.
Fig 5.
Effects of XN and TRAIL treatment after 48hrs on SK-N-AS cellular proliferation.
MTT assay (A) and Western blot analysis (B) showed synergism between XN and TRAIL on cell viability and increased apoptotic marker expression (cleaved PARP and CC3). * P-value <0.05; **0.005. C. A proposed mechanism of synergistic effects of XN and TRAIL on NB cells. XN increases DR5 expression, which increases the probability of TRAIL binding to DR5, thereby inducing apoptosis.