Homeobox A9 (HOXA9) is a transcription factor that is overexpressed in acute myeloid leukemia (AML). It is associated with the pathogenesis and progression of AML, and is a factor responsible for a poor prognosis. Therefore, the development of HOXA9-targeting molecules may contribute to not only better understanding of the mechanism of HOXA9 regulation, but also the development of therapeutic applications. We constructed a reporter assay system using the promoter region of the KBTBD10 gene, to which HOXA9 directly binds and regulates transcription, in the human acute monocytic leukemia cell line THP-1. Using this luciferase gene assay, we screened 1120 plant extracts and a methanol extract of the unripe fruits of Cerbera manghas was found to suppress the reporter gene expression mediated by the KBTBD10 promoter. From the extract, five steroid-type compounds were identified as the active constituents: 7α-neriifolin (1), 17β-neriifolin (2), 17α-digitoxigenin β-d-glucosyl-(1 → 4)-α-l-thevetoside (3), 17β-digitoxigenin β-d-glucosyl-(1 → 4)-α-l-thevetoside (4), and acetylthevetin B (5). Among the five compounds, 17β-neriifolin most potently inhibited HOXA9-dependent gene expression without affecting the HOXA9 mRNA levels, and suppressed cell proliferation by inducing apoptosis. The findings on the structure–activity relationships of the compounds from C. manghas may contribute to the development of small molecule inhibitors of HOXA9.
