β,β-Dimethylacryloyl Alkannin from <i>Arnebia euchroma</i> Roots Suppresses Triple-Negative Breast Cancer Growth via AKT/Gli1 Signaling

After confirmation of <i>Arnebia euchroma</i> by genetic analysis, we identified the key compounds from the root: alkannin (<b>1</b>), acetylalkannin (<b>2</b>), β-acetoxyisovaleryl alkannin (<b>3</b>), isobutyryl alkannin (<b>4</b>), and β,β-dimethylacryloyl alkannin (DMA) (<b>5</b>). Among these, DMA most effectively inhibited the proliferation of triple-negative breast cancer (TNBC) cells, with IC₅₀ values of 5.1 μM (MDA-MB-231) and 8.7 μM (MCF10DCIS.com). DMA and the Hedgehog (Hh) inhibitor Gant61 targeting Gli1 significantly induced apoptosis, as indicated by increased Bax and cleaved PARP, and decreased Bcl-2 levels (<i>p</i> < 0.01) in both cell lines. We also identified AKT as a potential target of DMA, as treatment reduced phosphorylated AKT (Ser473) protein levels by 66.3% ± 0.7% and 30.1% ± 5.7% in MDA-MB-231 and MCF10DCIS.com cells, respectively (<i>p</i> < 0.01). In vivo, DMA (25 mg/kg) suppressed MDA-MB-231 xenograft tumor growth by approximately 78% (<i>p</i> < 0.01) and induced apoptosis through regulating AKT/Hh/Gli1 axis. Interestingly, the reduced form of DMA (<b>5'</b>) lost its efficacy in inhibiting proliferation, p-AKT expression, and Gli1 transcriptional activity and nuclear localization, indicating that the Michael acceptor in DMA is critical for inhibiting TNBC growth. Overall, DMA suppressed TNBC in vitro and in vivo through AKT/Gli1 pathway and shows potential as a potent agent against TNBC.