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 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

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

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

miR-106a-5p Inhibits the Proliferation and Migration of Astrocytoma Cells and Promotes Apoptosis by Targeting FASTK

<div><p>Astrocytomas are common malignant intracranial tumors that comprise the majority of adult primary central nervous system tumors. MicroRNAs (miRNAs) are small, non-coding RNAs (20–24 nucleotides) that post-transcriptionally modulate gene expression by negatively regulating the stability or translational efficiency of their target mRNAs. In our previous studies, we found that the downregulation of miR-106a-5p in astrocytomas is associated with poor prognosis. However, its specific gene target(s) and underlying functional mechanism(s) in astrocytomas remain unclear. In this study, we used mRNA microarray experiments to measure global mRNA expression in the presence of increased or decreased miR-106a-5p levels. We then performed bioinformatics analysis based on multiple target prediction algorithms to obtain candidate target genes that were further validated by computational predictions, western blot analysis, quantitative real-time PCR, and the luciferase reporter assay. Fas-activated serine/threonine kinase (FASTK) was identified as a direct target of miR-106a-5p. In human astrocytomas, miR-106a-5p is downregulated and negatively associated with clinical staging, whereas FASTK is upregulated and positively associated with advanced clinical stages, at both the protein and mRNA levels. Furthermore, Kaplan-Meier analysis revealed that the reduced expression of miR-106a-5p or the increased expression of FASTK is significantly associated with poor survival outcome. These results further supported the finding that FASTK is a direct target gene of miR-106a-5p. Next, we explored the function of miR-106a-5p and FASTK during astrocytoma progression. Through gain-of-function and loss-of-function studies, we demonstrated that miR-106a-5p can significantly inhibit cell proliferation and migration and can promote cell apoptosis <i>in vitro</i>. The knockdown of FASTK induced similar effects on astrocytoma cells as those induced by the overexpression of miR-106a-5p. These observations suggest that miR-106a-5p functions as a tumor suppressor during the development of astrocytomas by targeting FASTK.</p></div

FASTK is a direct target gene of miR-106a-5p.

<p>(A) A schematic description of the hypothesized duplexes formed by interactions between the FASTK 3′-UTR binding sites and miR-106a-5p. The predicted free energy of each hybrid is indicated. The complementary seed sites are marked in red, and all of the nucleotides in these regions are completely conserved across several species. (B) Representative western blots showing FASTK protein levels in U251 cells treated with pre-ncRNA, pre-miR-106a-5p, anti-ncRNA and anti-miR-106a-5p. (C) Statistical analysis of three independent experiments. (D) Quantitative real time-PCR analysis of FASTK mRNA expression levels in U251 cells treated with pre-ncRNA, pre-miR-106a-5p, anti-ncRNA and anti-miR-106a-5p. The results shown represent data from three independent experiments. (E) Direct recognition of the FASTK 3′-UTR by miR-106a-5p. Firefly luciferase reporters containing either wt or mut FASTK 3′-UTRs were co-transfected into U251 cells with pre-miR-106a-5p, anti-miR-106a-5p and their corresponding negative controls. The parental luciferase plasmid was also transfected as a control. At 24 h post-transfection, the cells were assayed using luciferase assay kits. The results are presented as the mean ± SD of three independent experiments (** p<0.01; *** p<0.001). (F) Relative miR-106a-5p expression levels in NAT samples and WHO grade I-IV astrocytomas. (G) Representative western blots showing FASTK protein levels in NAT samples and WHO I-IV astrocytomas. (H) Statistical analysis of three independent experiments. (I) Relative FASTK mRNA expression levels in NAT samples and WHO grade I-IV astrocytomas. (J) The relationship between miR-106a-5p expression and astrocytoma patient survival time. (K) The relationship between FASTK expression and astrocytoma patient survival time.</p

Differentially regulated genes in cells with increased or decreased expression of miR-106a-5p.

<p>(A) Overexpression or knockdown of miR-106a-5p. U251 cells were seeded into 6-well plates and transfected the following day using Lipofectamine 2000. For each well, 100 pmol of pre-ncRNA, pre-miR-106a-5p, anti-ncRNA or anti-miR-106a-5p was transfected. The intercellular levels of miR-106a-5p were evaluated by qRT-PCR at 24 h after transfection. For comparison, the expression levels of miR-106a-5p in pre-ncRNA- or anti-ncRNA-transfected cells were arbitrarily set at 1. The results are presented as the mean ± SD of three independent experiments (*** p<0.001). (B) The scatter plot of altered genes that were inversely expressed with increased or decreased expression of miR-106a-5p. Left: downregulated genes when miR-106a-5p is upregulated; Right: upregulated genes when miR-106a-5p is downregulated. (C) A Venn diagram of the overlap of altered genes with increased or decreased miR-106a-5p expression. The differentially expressed genes are depicted as two overlapping circles. The green circle indicates the number of genes that are downregulated when miR-106a-5p is upregulated, whereas the red circle indicates the number of genes that are upregulated when miR-106a-5p is downregulated. The number in the overlapping area indicates the number of mRNAs that belong to the intersecting sets.</p