Cytotoxicity of Artemisinin, a Dimer of Dihydroartemisinin, Artemisitene and Eupatoriopicrin as Evaluated by the MTT and Clonogenic Assay

Artemisinin and its derivatives possess an endoperoxide bridge, which is thought to lead to the production of free-radical species. The cytotoxicity of some of these agents to a murine Ehrlich ascites (EN19) and a human HeLa S3 cancer cell line was determined using the MTT and the clonogenic assay. The MTT assay cannot distinguish between growth inhibition and cell killing, while the clonogenic assay detects actual cell death. The use of both assays to test a certain drug may give information on the mode of its cytotoxicity (i.e. growth inhibition versus cell killing). The endoperoxides artemisinin and the dimer of dihydroartemisinin showed much higher cytotoxicity in the MTT assay compared with the clonogenic assay. Thus these drugs mainly induced growth inhibition. For artemisitene and eupatoriopicrin, which possess an exocyclic methylene with alkylating properties, both tests yielded comparable results. For these compounds the MTT assay merely determined cell killing. For the reference drugs cisplatin and doxorubicin the MTT assay showed lower cytotoxicity than the clonogenic assay. This may be explained by the metabolic activity of cells that were clonogenically dead. Moreover, our experiments have shown that the MTT assay may lead to misinterpretations concerning the mode of action of certain drugs, when it is used as a substitute for the clonogenic assay.