Abstract

Background Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with high lethality, asymptomatic in early stages and prone to metastasis, with poor response to current treatments. β,β-Dimethylacrylshikonin (DMAS) is a naphthoquinone derivative with potent anticancer activity. Nevertheless, The anti-tumor mechanisms of DMAS in HCC have not yet been fully elucidated. Purpose This study was designed to investigate the effects of DMAS on HCC and to clarify the associated molecular mechanisms. Study design To investigate the effects of DMAS, we conducted transcriptomic sequencing, network pharmacology analyses and functional experiments in HCC cells. An in vivo xenograft model was also employed to further confirm these observations. Methods CCK-8, Transwell, EdU, colony formation, flow cytometry, wound healing, immunofluorescence, and Western blot analysis were conducted to assess the effects of DMAS on HCC cells. The interaction between DMAS and TGM2 was investigated using DARTS, molecular docking, and CETSA. Adenoviral transfection, JC-1, mitochondrial-lysosomal colocalization, and autophagy inducers were applied to investigate interactions with autophagy/mitophagy, and TGM2 overexpression in HuH-7 cells clarified the underlying antitumor mechanism. The antiproliferative activity of DMAS against HCC cells was demonstrated through in vitro studies, and its in vivo therapeutic performance and safety profile were corroborated in a xenograft mouse model. Results In vitro analyses demonstrated that DMAS effectively inhibits HCC cell proliferation. Moreover, DMAS suppresses cell migration by inducing mitochondria-dependent apoptosis and antagonizing epithelial-mesenchymal transition (EMT). Mechanistically, DMAS induces mitochondrial dysfunction and activates the PINK1/Parkin signaling axis, initiating mitophagy-related responses. However, concomitant binding to and inhibition of TGM2 suppresses Beclin1 and ATG5 expression, impairing autophagosome biogenesis and