Identification of acetylshikonin as a novel tubulin polymerization inhibitor with antitumor activity in human hepatocellular carcinoma cells.

Abstract

Microtubules are attractive targets for anticancer drugs. However, the microtubule-targeting agents (MTAs) currently in clinical use exhibit inevitable drug resistance. Therefore, there is an urgent need to discover novel MTAs for the clinical treatment of cancer. Bioactive compounds extracted from Lithospermum erythrorhizon were assessed for in vitro anti-proliferative activities against a panel of human cancer cell lines using cell counting kit-8 (CCK-8) assay. Tubulin polymerization inhibition assay, colchicine competitive binding site assay, and immunofluorescence were used to validate the tubulin inhibition effect of acetylshikonin. Flow cytometry, Hoechst staining, and caspase-3 activity evaluation were performed to assess cell cycle arrest and cell apoptosis. 5,5',6,6'-tetrachloro-1,1',3,3'-tetramethylbenzimidazolylcarbocyanine iodide (JC-1) staining and dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining were used to evaluate mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), respectively. Acetylshikonin exhibited potent anti-proliferative activities against a panel of human cancer cell lines (IC50 values: 1.09-7.26 µM) and displayed comparable cytotoxicity against several drug-resistant cell lines. Further mechanism studies revealed that acetylshikonin induced cell cycle arrest of MHCC-97H cells at G2/M phase, and significantly promoted apoptosis marked by a collapse of MMP and abnormal ROS accumulation. In this study, acetylshikonin was identified as MTA against hepatocellular carcinoma and can serve as a promising lead compound for further development of anti-cancer drug, underscoring its potential clinical significance.