Dihydroartemisinin Exhibits Biologic Activity Against Canine Osteosarcoma Cell Lines
Artemisinin-derived trioxanes are potent antimalarials whose antineoplastic property has been discovered recently. Their parasiticidal and tumoricidal effects are thought to be due to iron-dependant free radical generation. Artemisinins have been anecdotally used for dogs with osteosarcoma; however, no study has been done to evaluate biologic activity of artemisinins against canine tumors.
Cell viability assays were performed on four canine osteosarcoma cell lines (D17, OSCA2, OSCA16, OSCA50) after 48 hours of exposure to dihydroartemisinin, an active metabolite of most artemisinins, at concentrations of 0.1 to 100 µM. Apoptosis was assessed by ELISA for free nucleosomal DNA fragmentation and by Western blot for caspase 3 cleavage. Cell cycle analysis was performed using propidium iodine staining and flow cytometry. Detection of reactive oxygen species (ROS) was undertaken in the D17 cell line using 6-carboxy-2V,7V-dihydrofluorescein-diacetate and flow cytometry.
The concentration of dihydroartemisinin required for 50% inhibition of cell viability (IC50) was reached in all four canine osteosarcoma cell lines and ranged from 8.7 to 43.6 M. Induction of apoptosis was demonstrated by increased nucleosomal DNA fragmentation, cleavage of caspase 3, and an increase in the subG0/G1 population by flow cytometry. Exposure to dihydroartemisinin also resulted in decreased G0/G1 population. Lastly, iron-dependent ROS generation was detected in dihydroartemisinin treated D17 cells in a dose-dependent manner.
Dihydroartemisinin exhibits biologic activity against canine osteosarcoma cell lines, inducing cell cycle arrest and apoptosis. These studies lay the groundwork for future clinical trials of dihydroartemisinin in dogs with osteosarcoma.