The herbal compound miltirone from Salvia miltiorrhiza targets DJ-1 to induce mitophagic dysfunction and ROS-dependent hippo activation in gastric cancer.

[BACKGROUND] The protein DJ-1/PARK7 is highly expressed in various cancers and is involved in oxidative stress and mitophagy, making it a potential anti-cancer target. Salvia miltiorrhiza Bunge, a traditional Chinese herb, exhibits significant anti-tumor activity through its ethanol extract, though its specific active components and molecular mechanisms remain largely unknown.

[PURPOSE] This study aims to screen the active components from the ethanol extract of Salvia miltiorrhiza, elucidate the molecular mechanism underlying its anti-gastric cancer effects.

[METHODS] Liquid chromatography-mass spectrometry was employed to construct a compound library of SME, and limited proteolysis-mass spectrometry was employed for the screening of potential targets. The binding between compounds and DJ-1 was validated through surface plasmon resonance and molecular docking. In vitro experiments, including CCK-8, EdU, flow cytometry, Western blot, immunofluorescence, and transmission electron microscopy, were conducted to assess cell proliferation, apoptosis, autophagy, and mitochondrial function. In vivo anti-tumor efficacy was evaluated by the nude mouse xenograft model. Mechanistic studies involved ROS detection, kinase activity analysis, and gene knockdown.

[RESULTS] A total of 596 compounds were identified from SME, among which Miltirone directly bound to the DJ-1 protein (KD = 3.89 μM) and inhibited its antioxidant function. Miltirone significantly suppressed gastric cancer cell proliferation and induced apoptosis both in vitro and in vivo, while also causing mitochondrial dysfunction, autophagy impairment, and ROS accumulation. Furthermore, Miltirone activated the CBP/MOB1 axis in a ROS-dependent manner, promoting MOB1 phosphorylation and subsequently activating the Hippo pathway, leading to YAP phosphorylation and degradation. Inhibition of the Hippo pathway reversed the anti-tumor effects of Miltirone, indicating that activation of the Hippo signaling pathway is responsible for Miltirone-mediated anti-gastric cancer activity.