Binding of dihydroartemisinin to differentiating neuroblastoma cells and rat cortical homogenate

The binding characteristics of dihydroartemisinin to neural tissues and cells in culture were studied by incubating differentiating C6 and NB2a cells and rat cerebral cortex homogenate with I to 200 μM ¹⁴C-labelled dihydroartemisinin with or without 2 μM haemin for 24 h. The role of protein thiol and amine groups in dihydroartemisinin binding was assessed by pre- incubation of the cortex homogenate with sodium cyanate and/or iodoacetamide. Rosenthal plots of binding data demonstrated that there were two discrete phases of dihydroartemisinin binding. Haemin increased the total binding of dihydroartemisinin to both cortex and cell proteins. In each preparation, haemin increased B(max) of the high affinity binding sites and also increased k(D) of NB2a high affinity binding sites and decreased k(D) of cortex low affinity binding sites. The effects of haemin on the binding parameters of cortex resembled its effects on C6 cells rather than NB2a cells. NB2a high and low affinity binding sites had a greater affinity for dihydroartemisinin than those of either rat cortex or C6 cells. Iodoacetamide and sodium cyanate reduced binding to cortex proteins by approximately 70%. Co-incubation of ¹⁴C-dihydroartemisinin with arteether reduced binding of dihydroartemisinin but co-incubation with desoxyartemisinin did not. These studies demonstrate that the known haemin-induced increase in toxicity of dihydroartemisinin to differentiating neuroblastoma cells is accompanied by a corresponding increase in dihydroartemisinin binding to cell proteins, that protein thiol and amine groups react with the products of the reaction between dihydroartemisinin and haemin and point to a relationship between the toxicity of artemisinin derivatives and protein binding.