Development of Resistance towards Artesunate in MDA-MB-231 Human Breast Cancer Cells
Figure 4
Effects of artesunate on NFκB (A) and AP-1 (B).
Binding of 32P-labeled NFκB and AP-1 specific oligonucleotides to the cognate transcription factor present in nuclear extracts of MDA-MB-231 cells was monitored by EMSA. Artesunate treatment of MDA-MB-231 cells for different time intervals (lane 2, 2 h; lane 3, 4 h; lane 4, 6 h) led to a induced binding of NFκB (A) and AP-1 (B) to its response element as compared to carrier-treated cells (lane 1). The specificity of the binding was assessed for all three oligos by addition of a 50× molar excess of cold oligonucleotides (lanes 5). Furthermore, addition of appropriate specific antibodies(supershift) against p65 or c-jun respectively resulted in the formation of very faint or rather diffuse double bands (lanes 6–9). Experiments were repeated at least three times. Fig. 4 C-K illustrates the expression of p65 and c-jun in MDA-MB-231 and -468 cells. mRNA expression of p65 was induced in MDA-MB-231 cells upon artesunate treatment only in non-pretreated cells (C), while p65 expression did not alter upon artesunate treatment in pre-treated cells (D). MDA-MB-468 cells p65 expression was down-regulated statistically significantly after 18 h and 24 h upon artesunate treatment, no matter whether the cells were pretreated with artesunate (F) or not (E). The effect of acquired resistance as seen for p65 expression could also be found concerning the expression of c-jun. While c-jun was statistically significantly up-regulated in MDA-MB-231 cells after 18 h and 24 h, no significant change could be observed for cells pretreated 24 h with artesunate. (* P<0.05; ** P<0.01; *** P<0.001; one way Anova with Bonferroni's post test). Experiments were performed in triplicates.