Shikonin supresses hepatocellular carcinoma by inhibiting JAG1/Notch1/uPA Signaling

BackgroundShikonin, a bioactive naphthoquinone from Arnebiae Radix, exhibits hepatoprotective properties and anti-coagulation effects via inhibiting urokinase-type plasminogen activator (uPA). In hepatocellular carcinoma (HCC), high uPA activity drives invasion and metastasis, and which expression is reported to be boosted by ligand-activated Notch1 signaling. Despite these associations, the specific therapeutic mechanisms of Shikonin against HCC remain unclear.PurposeThis study aims to systematically evaluate the anti-HCC efficacy of Shikonin and to elucidate its underlying molecular mechanism, using an integrated investigation combining network pharmacology, bioinformatics analysis, and in vitro as well as in vivo validation specifically focusing on its role in modulating the JAG1/Notch1/uPA pathway.MethodsThe pharmacological efficacy and potential targets of Shikonin were first identified through bioinformatics analysis. The intersection between predicted Shikonin targets and HCC-related genes was further examined using public databases. Subsequent in vitro experiments compared the expression of the identified key target, Notch1, between HCC cell lines and normal hepatocytes. The effects of Shikonin on cell proliferation, colony formation, and EMT in HepG2 and H22 cells were then evaluated, together with its influence on the JAG1/Notch1/uPA signaling axis. In vivo studies using H22 tumor-bearing mice were conducted to confirm the anti-HCC efficacy of Shikonin and its suppressive effect on the JAG1/Notch1/uPA pathway. Finally, recombinant JAG1 (rhJAG1, a Notch1 agonist) and DAPT (a Notch1 inhibitor) were applied to verify that Shikonin exerts its anti-HCC action through modulation of the JAG1/Notch1/uPA signaling axis.ResultsBioinformatic analysis identified 57 overlapping targets of Shikonin in HCC, with significant enrichment in Notch pathway. Clinically, Notch1 is upregulated in HCC tissues, and correlated with advanced stage, proliferation and invasion. Consistently, elevated Notch1 expression was validated in HCC cell lines compared to normal hepatocytes. Shikonin treatment dose-dependently suppressed proliferation, colony-forming and EMT in both HepG2 and H22 cells, while downregulating JAG1/Notch1/uPA signaling. In H22 tumor-bearing mice, Shikonin effectively inhibited tumor growth and JAG1/Notch1/uPA axis. Mechanistically, the Notch1 agonist rhJAG1 substantially promoted proliferation, invasion and upregulated Notch1 signaling proteins. Whereas, co-treatment with Shikonin effectively counteracted rhJAG1-induced activation of Notch1 signaling, and suppressed cell proliferation as well as invasion. Conversely, the Notch 1 inhibitor DAPT mitigated Shikonin-induced suppression on HCC cell proliferation and invasiveness, and abrogated its inhibitory effect on Notch1 signaling in HepG2 cells. All these collectively suggest that Shikonin inhibits HCC progression by intervening the JAG1/Notch1/uPA axis.ConclusionShikonin suppresses HCC proliferation, invasion, and tumor growth by inhibiting the JAG1/Notch1/uPA signaling axis, highlighting its potential as a therapeutic agent targeting Notch1-driven oncogenic pathways.