Role of transferrin receptor and the ABC transporters ABCB6 and ABCB7 for resistance and differentiation of tumor cells towards artesunate

Abstract

The anti-malarial artesunate also exerts profound anti-cancer activity. The susceptibility of tumor cells to artesunate can be enhanced by ferrous iron. The transferrin receptor (TfR) is involved in iron uptake by internalization of transferrin and is over-expressed in rapidly growing tumors. The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are also involved in iron homeostasis. To investigate whether these proteins play a role for sensitivity towards artesunate, Oncotest's 36 cell line panel was treated with artesunate or artesunate plus iron(II) glycine sulfate (Ferrosanol). The majority of cell lines showed increased inhibition rates, for the combination of artesunate plus iron(II) glycine sulfate compared to artesunate alone. However, in 11 out of the 36 cell lines the combination treatment was not superior. Cell lines with high TfR expression significantly correlated with high degrees of modulation indicating that high TfR expressing tumor cells would be more efficiently inhibited by this combination treatment than low TfR expressing ones. Furthermore, we found a significant relationship between cellular response to artesunate and TfR expression in 55 cell lines of the National Cancer Institute (NCI), USA. A significant correlation was also found for ABCB6, but not for ABCB7 in the NCI panel. Artesunate treatment of human CCRF-CEM leukemia and MCF7 breast cancer cells induced ABCB6 expression but repressed ABCB7 expression. Finally, artesunate inhibited proliferation and differentiation of mouse erythroleukemia (MEL) cells. Down-regulation of ABCB6 by antisense oligonucleotides inhibited differentiation of MEL cells indicating that artesunate and ABCB6 may cooperate. In conclusion, our results indicate that ferrous iron improves the activity of artesunate in some but not all tumor cell lines. Several factors involved in iron homeostasis such as TfR and ABCB6 may contribute to this effect.

Figure 1. Expression of ABCB6 and ABCB7 in MCF7 (A.) and CCRF-CEM cells (B.) after treatment with artesunate.

The expression after 24 hours is given in relation to the expression before treatment.

Figure 2. Effect of artesunate on MEL cell proliferation, differentiation, and ABCB6/ABCB7 expression.

A. Effect on cell growth. MEL cells were cultured without or with 1.0 µg/ml artesunate for the indicated time. The cell numbers were measured by trypan blue exclusion. B. Protein expression of ABCB6 and ABCB7 after treatment with 2% DMSO for different time points. Actin expression served as loading control. C. Effect on differentiation. MEL cells (100,000 cells/ml) were cultured in the presence of various concentrations of artesunate for 48 h. The content of heme was estimated, by the method with oxalic acid. D. The cells transfected with sense- or antisense oligonucleotides for ABCB6 were induced with 2% DMSO without or with 0.18 µg/ml artesunate for 48 h.