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A series of artemisinin-related endoperoxides was tested for cytotoxicity to Ehrlich ascites tumor (EAT) cells using the microculture tetrazolium (MTT) assay. Artemisinin [1] had an IC50 value of 29.8 microM. Derivatives of dihydroartemisinin [2], being developed as antimalarial drugs (artemether [3], arteether [4], sodium artesunate [5], artelinic acid [6], and sodium artelinate [7]), exhibited a somewhat more potent cytotoxicity. Their IC50 values ranged from 12.2 to 19.9 microM. The presence of an exocyclic methylene fused to the lactone ring, as for artemisitene [9], led to higher cytotoxicity than 1. From the two epimeric 11-hydroxyartemisinin derivatives, the R form 12 showed a considerably higher cytotoxicity than the S form 13. Opening of the lactone ring of 1 dramatically reduced the cytotoxicity. The ether dimer 8 of 2 was the most potent cytotoxic agent, its IC50 being 1.4 microM. The variations in cytotoxicity between the structurally related compounds mostly correlated well with the theoretical capacity of radical formation and stabilization. In some cases lipophilicity or the presence of an electrophilic moiety seemed to have a determinant influence on cytotoxicity. The artemisinin-related endoperoxides showed cytotoxicity to EAT cells at higher concentrations than those needed for in vitro antimalarial activity, as reported in the literature.

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Protein denaturation and aggregation are most likely the cause for the noxious effects of heat shock. There are some indications that the nucleus is onc of the most sensitive cellular compartments. To test the possibility that the intranuclear microenvirc~nment might be detrimental to the heat stability of proteins, we compared the iu sitct thermal stability of a rcporter protein localized in the nucleus 01-in the cytoplasm. A recombinant firefly (PhoD/nus pyrulis) luciferase carrying a point mutation in the C-terminal domitin remains in the cytoplasm (cyt-luciferase). A nuclear localization sequence wns fused to the N-terminal domain of cyt-luciferase; the resulting nuc-luciferase was efficiently targcted to the cell nucleus.In both cases, decreased luciferase activity and solubility were found in lysates from heat-shocked cells. These characteristics were taken as an indication of thermal denaturation in ,vim. The heat-inactivated luciferases were partially reactivated during recovery after stress, indicating the capacity of both the cytoplastnic and nuclear compartments to reassetnble proteins from an aggregated state.Although both the nuc-itnd the cyt-luciferases were heat inactivated at similar rates in v i m , nucluciferase was more susceptible to thermal denaturation in siiu cotnpared to cyt-luciferase. This ohscrvation suggests that the microenvironment of an intracellular compartment may modulate the thermal stability of proteins. The local concentration might be one element of this microcnvironment affecting the heat-stability of proteins.In cells made thertnotolerant by a priming shock, the thermid inactivation of the recombinant luciferases occurred at a slower rate during a second challenging stress. However, this decreased thermal scnsitivity was less pronounced for the nuc-luciferase (threefold) than for the cyt-luciferase (sevcnfold). The nuclear luciferase might become a useful tool to investigate the action of molecular chapcrones in the nucleus.Keywords: protein denaturation; heat shock; luciferase; nuclear localization ; thermotolcrance.In cells exposed to heat stress, some proteins denature and aggregate [ 1-51, Although proteins distributed in the various intracellular conipartnients have been found to denature, there are some indications that the nucleus is onc of the most sensitive compartments. Most protcin aggregates after heating cells are found within the nucleus 161. A decreasc in solubility of many nuclear proteins has been found lo occur during heat shock. They include the proto-oncogene protcins c-Myc, c-Myb and p53, DNA polymerases, RNA polymerase 11, and topoisoinerase I [7-121. This may reflect an overall reduction in nuclear protein solubility that contributes to the increment in protein content of nuclei isolated frotn heat-treated cells [ 13 1. Such protein aggregation may eventually lead to the killing effects of hyperthermia [ 2, 4, 51. A good correlation was found between h e extent and duration of nuclear protein aggregation and heat killing [I 41. At the beginning of...

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