Emetine dihydrochloride inhibits invasiveness and motility of hepatocellular carcinoma cells by blocking the MAPK pathway and inducing destabilization of Twist1

Background Hepatocellular carcinoma (HCC) is a malignant tumor that causes both extrahepatic and intrahepatic metastases. Epithelial to mesenchymal transition (EMT) is a crucial step in the development and metastasis of cancer. Emetine dihydrochloride (EDH) has been previously used as an anti-emetic and is now proposed as a replication inhibitor of SARS-CoV-2, but its effect against cancer metastasis has not been evaluated. Therefore, this study sought to investigate the regulatory mechanisms of cell migration from an EMT perspective using EDH in HCC cell lines. Methods HCC cell lines (Huh7, Hep3B, SNU449, SNU886, and PLC-PRF-5) were used to measure cell viability against EDH. The effect of EDH on migration was verified by wound healing analysis and migration analysis using Transwell. The effect of EDH on invasion was determined using an invasion assay in Matrigel-coated Transwell chambers. Spheroid invasion and soft agar colony formation assays were performed to verify the effect of EDH on anchorage-independent growth. Gelatin zymography was used to determine the activities of matrix metalloproteinase − 2 and − 9. The protein expression levels of Twist1, downstream target genes, and the mitogen-activated protein kinases (MAPKs) and AKT signaling pathways were determined through immunoblotting. The RNA expression levels of each gene were analyzed through RT-PCR and quantitative RT-PCR. Results EDH inhibited the motility of various HCC cell lines at non-toxic concentrations. The inhibitory effect of EDH on cancer cell motility resulted from a decrease in protein levels of Twist1, a key transcription factor involved in EMT. Depletion of Twist1 due to EDH treatment suppressed the expression of mesenchymal markers (N-cadherin and Vimentin) while increasing the expression of epithelial markers (E-cadherin). The regulatory pathway for the destabilization of Twist1 by EDH was mediated through the inactivation of the MAPK pathway. EDH specifically inactivated JNK and p38, thereby destabilizing the Twist1 protein, which is dependent on the S68 phosphorylation of Twist1. Conclusion EDH induces MAPK inactivation, which decreases Twist1 protein levels and ultimately suppresses mesenchymal properties. These results provide the first report on EDH from an EMT perspective and suggest its potential as an anticancer agent for HCC.