Carnosol is an anti-oxidant and anti-inflammatory compound from rosemary. In this paper, we investigated antitumor activity of carnosol against human osteosarcoma cells. We found the viability of human osteosarcoma MG-63 cells was significantly decreased in the presence of carnosol (cell viabilities: 17.2% for 20[Formula: see text]μg/ml of CS vs. 100% for control, [Formula: see text]). Carnosol induced apoptosis and cell cycle arrest in a dose-dependent manner in MG-63 cells. Furthermore, carnosol exposure increased the levels of reactive oxygen species (ROS). The pre-treatment of NAC, the ROS scavenger, blocked the inhibition of cell viability in the carnosol treatment, indicating that ROS is important in the antiproliferation effect. Moreover, we demonstrated that carnosol significantly induced autophagy and co-administration of autophagy inhibitor reduced the antiproliferating effect of carnosol. This result exhibited the cytotoxic effect of autophagy induced by carnosol in MG-63 cells. Interestingly, the treatment of NAC decreased carnosol-induced autophagy. Collectively, these data indicate that carnosol suppresses the viability of human osteosarcoma MG-63 cells by upregulation of apoptosis and autophagy, which are both mediated by ROS. Thus, carnosol might serve as a potential therapeutic agent against osteosarcoma.

Betulin is a lupane type pentacyclic triterpenoid, and commonly found in the bark of birch trees. It displays various pharmacological properties, such as antibacterial, anti‐inflammation, antitumor, and antiviral. In this report, we attempted to investigate the anti‐proliferative and pro‐apoptotic effects of betulin on osteosarcoma cell lines. Our results revealed that betulin significantly decreased cell viability and colony formation in osteosarcoma cell lines. Dose‐dependent induction of Annexin V positive cells, activated caspase 8, activated caspase 9, activated caspase 3, and the cleavage of poly (ADP‐ribose) polymerase were observed after the treatment with betulin, indicating betulin induces apoptosis in osteosarcoma cell lines. mTOR has been identified as a key modulator of autophagy in response to different stresses. In this study, we found that the treatment with betulin suppressed the activation of mTOR, and increased the level of LC 3‐II, the autophagy marker, in osteosarcoma cell lines. Co‐administration of the autophagy inhibitor chloroquine significantly rescued the cell viability and the clonogenic activity in betulin‐treated osteosarcoma cell lines. Our data showed that betulin induced autophagy, and the up‐regulated autophagy positively contributed to the apoptosis. Taken together, our findings suggested that betulin may serve as a promising anti‐proliferative agent for treating osteosarcoma.