Epolones induce erythropoietin expression via hypoxia-inducible factor-1α activation

Abstract

Induction of erythropoietin (Epo) expression under hypoxic conditions is mediated by the heterodimeric hypoxia-inducible factor (HIF)-1. Following binding to the 3′ hypoxia-response element (HRE) of the Epo gene, HIF-1 markedly enhances Epo transcription. To facilitate the search for HIF-1 (ant)agonists, a hypoxia-reporter cell line (termed HRCHO5) was constructed containing a stably integrated luciferase gene under the control of triplicated heterologous HREs. Among various agents tested, we identified a class of substances called epolones, which induced HRE-dependent reporter gene activity in HRCHO5 cells. Epolones are fungal products known to induce Epo expression in hepatoma cells. We found that epolones (optimal concentration 4-8 μmol/L) potently induce HIF-1α protein accumulation and nuclear translocation as well as HIF-1 DNA binding and reporter gene transactivation. Interestingly, the activity of a compound related to the fungal epolones, ciclopirox olamine (CPX), was blocked after addition of ferrous iron. This suggests that CPX might interfere with the putative heme oxygen sensor, as has been proposed for the iron chelator deferoxamine mesylate (DFX). However, about 10-fold higher concentrations of DFX (50-100 μmol/L) than CPX were required to maximally induce reporter gene activity in HRCHO5 cells. Moreover, structural, functional, and spectrophotometric data imply a chelator:iron stoichiometry of 1:1 for DFX but 3:1 for CPX. Because the iron concentration in the cell culture medium was determined to be 16 μmol/L, DFX but not CPX function can be explained by complete chelation of medium iron. These results suggest that the lipophilic epolones might induce HIF-1α by intracellular iron chelation.

An atypical active cell death process underlies the fungicidal activity of ciclopirox olamine against the yeastSaccharomyces cerevisiae

“…Azoles are the best example, because in addition to their action at the plasma membrane, they can indirectly affect mitochondria (Kontoyiannis & Murray, 2003). Although CPO acts as a chelator of Fe 31 (Wanner et al, 2000;Linden et al, 2003;Niewerth et al, 2003), it seems to be important in the maintenance of mitochondrial membrane potential (Lee et al, 2005b). We therefore assessed yeast mitochondrial function/integrity on the basis of Rh123 staining (Ludovico et al, 2001a).…”

The antimycotic ciclopirox olamine induces HIF‐1α stability, VEGF expression, and angiogenesis

“…This might be explained by the higher lipophilicity of CPX compared with DFX, suggesting that CPX chelates intracellular iron. Indeed, the intracellular chelatable iron concentration has been reported to be ~3 µM in hepatocytes and 6 µM in endothelial cells (50), equivalent to 9 µM and 18 µM of CPX, respectively, which forms a 3:1 complex with iron (31). However, the difference in lipophilicity is probably not sufficient to explain the potency of CPX function because the lipophilic DP was also only active at ~10-fold higher concentrations than CPX.…”

“…The HRCHO5 cells containing a stably transfected, HIF-1-dependent reporter gene have been described previously (31). The human HepG2 hepatoma cell line was obtained from American Type Culture Collection (ATCC numbers HB-8065).…”

“…We first addressed the effects on cell proliferation and on HIF-1-induction mediated by CPX when compared with two other iron chelators, the hydrophilic DFX and the lipophilic DP, both well-known for their HIF-1-inducing capabilities (23,46,47). Therefore, HRCHO5 cells were treated with increasing amounts of CPX, DFX, and DP for 24 h. HRCHO5 cells were derived from the Chinese hamster ovary (CHO) cell line stably transfected with a luciferase reporter gene under the control of the SV40 promoter and six transferrin-derived HIF-1 DNA binding sites, allowing for the rapid determination of HIF-1 activity (31,37). The functional dependence of this plasmid construct on HIF-1 was confirmed by the complete absence of induction by hypoxia or iron chelation following transient transfections into mouse embryonic fibroblasts genetically deficient for HIF-1α (K. Eckhardt and R. H. Wenger, unpublished observations).…”

“…As determined by functional and spectroscopic studies, we previously reported that CPX acts as a bidentate iron chelator, capable of inducing HIF-1α protein accumulation in the nucleus, HIF-1 DNA binding, and subsequent activation of exogenous reporter genes (31). Here, we analyzed CPX function in comparison with other iron chelators, and we explored the possibility that CPX activates endogenous HIF-1 target genes, including VEGF, in both in vitro cell culture and in vivo organ models.…”