Dihydroartemisinin suppresses COX-2-mediated apoptosis resistance in hepatocellular carcinoma under endoplasmic reticulum stress

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, and its treatment still faces numerous challenges. Dihydroartemisinin (DHA), a derivative of artemisinin, has shown significant antitumor activity in preclinical research. Our study seeks to uncover the molecular mechanisms of DHA in HCC, potentially providing scientific evidence for its use as a supportive therapy in clinical settings. This study was conducted using various experimental approaches to systematically analyze the effects of DHA in HCC. Cell viability was evaluated by CCK-8 to determine the IC₅₀ of DHA in HCC cells. Flow cytometry was used to measure the rates of apoptosis and reactive oxygen species (ROS) levels. Colony formation assays were performed to examine the inhibitory effects of DHA on HCC cell proliferation. The toxicity of DHA on HCC cells was evaluated through the lactate dehydrogenase release assay. Western blot was conducted to examine expression levels of proteins related to endoplasmic reticulum (ER) and apoptosis. Fluo-3 AM was utilized to label calcium ions (Ca²⁺), allowing for the detection of intracellular Ca²⁺ level changes. Additionally, ER tracker was employed to label the ER, with its morphological changes observed via immunofluorescence. DHA notably inhibited the vitality and proliferation of HCC cells and promoted cell apoptosis. Following DHA exposure, there were notable increases in ER stress markers, ROS, and Ca²⁺ levels. The morphology of the ER exhibited a loose and expanded state. The use of ER stress inhibitors attenuated these effects. Additionally, ER stress inducers facilitated the upregulation of COX-2, mediating apoptosis in HCC cells. Upon COX-2 knockdown, the apoptotic effect of DHA was markedly amplified. In HCC, DHA induces apoptosis in tumor cells by curbing the COX-2-mediated apoptotic resistance that arises during ER stress. This breakthrough reveals the molecular pathways through which DHA can aid in HCC treatment, offering valuable experimental data to support its clinical use as an adjuvant drug.