2-Dodecyl-6-Methoxycyclohexa-2,5-Diene-1,4-Dione from Averrhoa carambola L. roots: Suppressing hepatocellular carcinoma progression through ROS accumulation and p53 pathway-mediated apoptosis

Abstract

This study explores the anti-tumor effects of 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD), a compound derived from Averrhoa carambola L roots, on hepatocellular carcinoma (HCC) cells and a xenograft mouse model, focusing on its underlying mechanisms. Cell viability following DMDD treatment was assessed using the CCK-8 assay. Flow cytometry determined changes in cell cycle distribution and apoptosis rates, while migration and invasion capabilities were assessed using wound healing and transwell assays, respectively. Transcriptome sequencing (RNA-seq) was conducted to analyze differential gene expression and pathway enrichment. Z-VAD-FMK, a pan-caspase inhibitor, was used to confirm the apoptotic mechanism induced by DMDD. The expression levels of p53, Bax, Bcl-2, and cleaved caspase 3 were quantified via Western blot analysis. A xenograft mouse model was developed to assess the in vivo effects of DMDD on HCC. DMDD suppressed proliferation, migration, and invasion, and induced apoptosis in Huh7 and Hep3b cells. RNA-seq revealed significant enrichment of p53 and apoptosis signaling pathways among differentially expressed genes. DMDD downregulated Bcl-2 expression and upregulated p53, Bax and cleaved caspase 3. In addition, Z-VAD-FMK partially inhibited DMDD-induced apoptosis. DMDD also inhibited tumor growth in mice. DMDD effectively inhibited tumor growth in HCC cell lines and xenograft models, potentially through ROS elevation and p53-mediated activation of the intrinsic apoptotic pathway.