Steroidal alkaloids isolated from Veratrum grandiflorum Loes. as novel Smoothened inhibitors with anti-proliferation effects on DAOY medulloblastoma cells

Abstract

Constitutive activation of Hedgehog (Hh) pathway is intimately related with the occurrence and development of several malignancies, such as medulloblastoma (MB) and other tumors. Therefore, small molecular inhibitors of Hh pathway are urgently needed. In this study, three new steroidal alkaloids, ⊿⁵ (20R, 24R) 23-oxo-24-methylsolacongetidine, ⊿⁵ (20S, 24R) 23-oxo-24-methylsolacongetidine and veralinine 3-O-α-l-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside, together with six known alkaloids, 20-epi-verazine, verazine, protoverine 15-(l)-2'-methylbutyrate, jervine, veramarine and β1-chaconine, were isolated and determined from Veratrum grandiflorum Loes. The dual-luciferase bioassay indicated that all compounds exhibited significant inhibitions of Hh pathway with IC₅₀ values of 0.72-14.31 μM against Shh-LIGHT 2 cells. To determine whether these Hh pathway inhibitors act with the Smoothened (Smo) protein, which is an important oncoprotein and target for this pathway, BODIPY-cyclopamine (BC) competitive binding assay was preferentially performed. Compared with BC alone, all compounds obviously reduced the fluorescence intensities of BC binding with Smo in Smo-overexpression HEK293T cells through fluorescence microscope and flow cytometer. By directly interacting with Smo, it revealed that they were actually novel natural Smo inhibitors. Then, their anti-tumor effects were investigated against the human MB cell line DAOY, which is a typical pediatric brain tumor cells line with highly expressed Hh pathway. Interestingly, most of compounds had slight proliferation inhibitions on DAOY cells after treatment for 24 h same as vismodegib, while β1-chaconine showed the strongest inhibitory effect on the growth of DAOY with IC₅₀ value of 5.35 μM. In conclusion, our studies valuably provide several novel natural Smo inhibitors for potential targeting treatment of Hh-dependent tumors.

Keywords: Alkaloids; Anti-proliferation; Hedgehog pathway; Medulloblastoma; Smo inhibitor; Veratrum grandiglorum Loes..