Dihydroartemisinin Exerts Its Anticancer Activity through Depleting Cellular Iron via Transferrin Receptor-1

<div><p>Artemisinin and its main active metabolite dihydroartemisinin, clinically used antimalarial agents with low host toxicity, have recently shown potent anticancer activities in a variety of human cancer models. Although iron mediated oxidative damage is involved, the mechanisms underlying these activities remain unclear. In the current study, we found that dihydroartemisinin caused cellular iron depletion in time- and concentration-dependent manners. It decreased iron uptake and disturbed iron homeostasis in cancer cells, which were independent of oxidative damage. Moreover, dihydroartemisinin reduced the level of transferrin receptor-1 associated with cell membrane. The regulation of dihydroartemisinin to transferrin receptor-1 could be reversed by nystatin, a cholesterol-sequestering agent but not the inhibitor of clathrin-dependent endocytosis. Dihydroartemisinin also induced transferrin receptor-1 palmitoylation and colocalization with caveolin-1, suggesting a lipid rafts mediated internalization pathway was involved in the process. Also, nystatin reversed the influences of dihydroartemisinin on cell cycle and apoptosis related genes and the siRNA induced downregulation of transferrin receptor-1 decreased the sensitivity to dihydroartemisinin efficiently in the cells. These results indicate that dihydroartemisinin can counteract cancer through regulating cell-surface transferrin receptor-1 in a non-classical endocytic pathway, which may be a new action mechanism of DHA independently of oxidative damage.</p> </div

DHA induced disturbance of iron homeostasis could not be reversed by NAC.

<p>(A) MCF7 cells were pretreated with 20 mM NAC or left untreated for 30 min and then DHA (25 µM) were added to further treatment. After 24 hours, cell lysates were prepared and immunoblotted. (B) HepG2 cells were pretreated with NAC or not and further incubated with 25 µM DHA for 24 hours. Quantitative RT-PCR was performed to detect the mRNA level. **, <i>P</i><0.01. Data are represented as mean ±SD of three different experiments.</p

DHA induced TfR1 internalization in a lipid rafts/caveolae mediated way.

<p>(A) After pretreatment with CPZ (20 µM) or nystatin (25 µg/ml) or left untreated for 30 min, HepG2 cells were incubated with 25 µM DHA for another 4 hours. Cells were harvested and the membrane-associated TfR1 was determined by flow cytometric analysis. *, <i>P</i><0.05 compared with DMSO-treated cells. Data are represented as mean ±SD of two different experiments. (B) HEK293 cells expressed GFP-TfR1 were treated with DMSO or 25 µM DHA for 24 hours and subjected to confocal microscope analysis. Scale bar, 5 µm. (C) HepG2 cells were treated with DMSO or 25 µM DHA for 24 hours and the endogenous TfR1 protein was immunoprecipitated to perform the palmitoylation assay as described in Materials and Methods.</p

The proposed mechanisms by which DHA disrupts iron homeostasis.

<p>The proposed mechanisms by which DHA disrupts iron homeostasis.</p

DHA decreased cell-surface TfR1 and inhibited Tf uptake.

<p>(A and B) After treated with DHA, HepG2 cells were harvested and incubated with PE-conjugated TfR1 antibody for 30 min and then subjected to flow cytometric analysis to determine membrane-associated TfR1 level. *, <i>P</i><0.05; **, <i>P</i><0.01 compared with control cells. Data are represented as mean ±SD of three different experiments. (C) HepG2 cells were treated with different concentrations of DHA for 24 hours and then incubated with Alexa fluor 633-conjugated transferrin for 2 hours. Harvested cells were subjected to flow cytometric analysis for internalized transferrin. *, <i>P</i><0.05; **, <i>P</i><0.01 compared with control cells. Data are represented as mean ±SD of three different experiments. (D) HepG2 treated with DMSO or 25 µM DHA for 24 hours were incubated with Alexa fluor 633-conjugated transferrin for the time indicated. Cells were harvested and subjected to flow cytometric analysis. *, <i>P</i><0.05; **, <i>P</i><0.01. Data are represented as mean ± SEM of three different experiments.</p