BAY 87-2243, a highly potent and selective inhibitor of hypoxia-induced gene activation has antitumor activities by inhibition of mitochondrial complex I

Abstract

The activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression, and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high-throughput screening led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87-2243 was found to inhibit HIF-1α and HIF-2α protein accumulation under hypoxic conditions in non-small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF-1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87-2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel-Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase-2. Antitumor activity of BAY 87-2243, suppression of HIF-1α protein levels, and reduction of HIF-1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors.

Keywords: Antitumor activity; hypoxia; hypoxia-inducible factor-1; mitochondrial complex 1.

Figure 1

(A) Chemical structure of BAY 87-2243. (B) BAY 87-2243 suppresses hypoxia-induced reporter gene activity (left), and CA9 protein levels in vitro in HCT-116 cell lysates (right). HCT-116 cells were stably transfected with 4×VEGF promoter-derived HIF response element in front of a luciferase reporter gene and incubated for 24 h under hypoxia (1% pO₂). IC₅₀ values were calculated by GraphPad Prism. VEGF, vascular endothelial growth factor; HIF, hypoxia-inducible factor.

Figure 2

BAY 87-2243 inhibits HIF target gene expression in vitro. (A) H460 cells were cultured for 16 h under normoxia or hypoxia (1% pO₂) in the absence or presence of various concentrations of BAY 87-2243. MRNA expression levels of the HIF target genes CA9, ADM, ANGPTL4 and the negative control gene EGLN2 were quantified by RT-PCR. (B) RNA from vehicle-treated normoxic H460 cells and from hypoxic H460 cells incubated with 1, 10, and 100 nmol/L BAY 87-2243, respectively, was subjected to microarray hybridization. A heatmap shows the mRNA signal intensity of those 30 genes that were most strongly suppressed by 100 nmol/L BAY 87-2243 in hypoxic H460 cells compared to DMSO-treated hypoxic H460 cells. HIF, hypoxia-inducible factor; RT-PCR, real-time polymerase chain reaction.

Figure 3

BAY 87-2243 inhibits hypoxia-inducible factor (HIF-1α) and HIF-2α protein accumulation in hypoxic H460 cells H460 under hypoxia but has no effect on HIF-1α protein levels induced by hypoxia mimetics and has no effect on prolyl hydroxylase 2 (PHD2) activity. (A, B) H460 cells were cultured for 16 h under normoxia or hypoxia (1% pO₂) in the absence or presence of various concentrations of BAY 87-2243. HIF-1α (A) and HIF-2α (B) protein levels were assessed by Western Blot in whole cell extracts. β-actin was used as a loading control. (C) H460 cells were cultured for 16 h under normoxia with the PHDs desferrioxamine (DFO) and CoCl₂ plus/minus BAY 87-2243 before the HIF1α protein levels in cellular extracts were quantified by Western Blot. β-actin was used to as a loading control. (D) Effect of BAY 87-2243 on the recombinant PHD2-mediated hydroxylation of HIF-1α peptide over time was measured in a biochemical assay. Hydoxylated peptide was quantified after incubation with purified VBC complex labeled with europium using fluorescence as a readout. The known PHD inhibitor N-oxalylglycine served as a positive control.

Figure 4

BAY 87-2243 is inactive in RCC4 cells lacking functional VHL protein or in H460 cells silenced for EGLN1. (A) RCC4 cells were incubated under normoxia or hypoxia for 16 h with the indicated concentrations of BAY 87-2243. Expression levels of HIF-1 target genes CA9, ANGPTL4, EGLN3, and of negative control EGLN2 were quantified by real-time PCR. (B) HIF-1α protein levels in hypoxic RCC4 cells treated for 16 h with 100 nmol/L BAY 87-2243 were assessed by Western Blot in cytosolic and nuclear extracts. β-actin was used to as a loading control for cytosolic extracts and Lamin B1 as a loading control for nuclear extracts. (C) H460 cells were transfected with either an EGLN1-silencing RNA or a scrambled control siRNA for 48 h before incubation with 100 nmol/L BAY 87-2243 for additional 24 h under normoxia. RNA was isolated and the expression levels of EGLN1 (to confirm silencing), and the HIF-1 target genes CA9, EGLN3, and VEGF were quantified by real-time PCR. VHL, Von Hippel–Lindau; HIF, hypoxia-inducible factor; VEGF, vascular endothelial growth factor.

Figure 5

BAY 87-2243 reduces tumor weight, hypoxia-inducible factor (HIF)-1α protein levels, and HIF-1 target gene expression in H460 xenograft tumors. (A) Nude mice bearing established H460 human tumor xenografts were treated orally, once daily with BAY 87-2243 (0.5, 1.0, 2.0, and 4.0 mg/kg for 21 days and tumor weight was analyzed. (B) Total RNA from H460 xenograft tumors was isolated 16 h after last oral dosage of BAY 87-2243 at day 22 and the mRNA expression levels of HIF-1 target genes CA9, ANGPTL4, EGLN3, and of negative controls EGLN2 and HIF-1A was quantified by real-time PCR. (C) Nuclear extracts were isolated from four animals of the 4 mg/kg BAY 87-2243-treated group and from four animals from the vehicle-treated group. HIF-1α protein levels were assessed by Western blot analysis. A cell lysate from H460 cells incubated with 250 μmol/L CoCl₂ for 16 h served as a positive control for the HIF-1α antibody. Lamin B1 served as a loading control.

Figure 6

BAY 87-2243 inhibits mitochondrial complex I activity. (A) H460 cells were cultured in medium containing either glucose, galactose, or lactate as carbohydrate source and cell proliferation was measured by cell titer glow assay after 48 h of incubation with BAY 87-2243. (B) Inhibition of complex-I-dependent oxygen consumption in isolated mitochondria from PC3 cells by BAY 87-2243 was measured using LUX-MitoXpress system. (C) Luciferase activity was measured in H1299luc cells 1 h after incubation with either BAY 87-2243, rotenone, or antimycin A and a second measurement was performed after addition of 25 mmol/L succinate and 1 h further incubation. (D) Cellular ATP-dependent luciferase activity was measured in H1299luc cells and in H1299luc cells transfected with NDI1 after 1 h of incubation with either BAY 87-2243, rotenone, or antimycin A.