Dihydroartemisinin induces caspase-8-dependent apoptosis in murine GT1-7 hypothalamic neurons

The cytotoxicity of dihydroartemisinin (DHART; an active metabolite of artemisinin or ART) was investigated using murine GT1-7 hypothalamic neurons. A decrease in neuronal cell viability was observed in DHART-treated cells typically after 6 h of incubation. When neuronal cells were exposed to DHART for 24 h, the value of IC₅₀ was found to be 24 ± 3.2 µM (n = 6). Based on acridine orange/ethidium bromide (dual) staining and increases in oligonucleosomal fragmentation, the cell death at lower concentrations of DHART (≤ 20 µM) was suggestive of apoptotic in nature. On the other hand, at higher concentrations of DHART (≥ 50 µM), the cell death appeared to be predominantly necrotic. A potentiation of cytotoxic effects was seen in Fe(II)-containing medium whereas inclusion of deferoxamine (chelator of Fe) attenuated such effects. This would imply that the cleavage of the endoperoxide bridge of DHART by Fe(II) and the subsequent formation of O- and C-centered radical(s) are important determinants of the cytotoxicity that was observed. The activities of caspase-3/7, caspase-8 and caspase-9 were maximally seen at 12-h after exposure to DHART. Inhibitors of caspase-8 (C8I) but not caspase-9 (C9I) reduced the DHART-induced increase in caspase-3/7 activity. A relatively higher activity of caspase-8 to that of caspase-9 and the inhibition of caspsase-3/7 activity by C8I suggest that DHART induces caspase-8-mediated apoptosis involving the extrinsic pathway. Taken together, this study demonstrates that DHART and, possibly, other ART derivatives have considerable neurotoxic potential and facilitate our understanding of these agents.