Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells

Abstract

Purpose: Protein levels and activity of the hypoxia-inducible transcription factors HIF-1 and HIF-2 are controlled by hydroxylation of the regulatory alpha chains. Proline hydroxylases (PHDs) target the protein for degradation via the von-Hippel-Lindau (VHL)-ubiquitin-ligase complex, and asparagine hydroxylation by Factor Inhibiting HIF (FIH) leads to transcriptional inactivation. In cell-free systems, these enzymes require ascorbate as a cofactor, and this is also inferred to be an intracellular requirement for effective hydroxylation. However, how intracellular concentrations of ascorbate affect hydroxylase activity is unknown. In this study, we investigated the modulation of the regulatory hydroxylases in cancer cells by intracellular ascorbate. Materials and methods: To facilitate this investigation, we used clear cell renal carcinoma cell lines that were VHL-proficient (Caki-1), with a normal hypoxic response, or VHL-defective (Caki-2 and 786-0), with uncontrolled accumulation of HIF-α chains. We monitored the effect of intracellular ascorbate on the hypoxia-induced accumulation of HIF-1α, HIF-2α and the expression of downstream HIF targets BNIP3, cyclin D1 and GLUT1. Changes in hydroxylation of the HIF-1α protein in response to ascorbate were also investigated in 786-0 cells gene-modified to express full-length HIF-1α (786-HIF1). Results: In VHL-proficient cells, hypoxia induced accumulation of HIF-1α and BNIP3 which was dampened in mild hypoxia by elevated intracellular ascorbate. Increased HIF-2α accumulation occurred only under severe hypoxia and this was not modified by ascorbate availability. In VHL-defective cells, ascorbate supplementation induced additional accumulation of HIF under hypoxic conditions and HIF pathway proteins were unchanged by oxygen supply. In 786-HIF1 cells, levels of hydroxylated HIF-1α were elevated in response to increasing intracellular ascorbate levels. Conclusion: Our data provide evidence that the hypoxic pathway can be modulated by increasing HIF hydroxylase activity via intracellular ascorbate availability. In VHL-defective cells, accumulation of HIF-alpha proteins is independent of hydroxylation and is unaffected by intracellular ascorbate levels.

Keywords: PHD; VHL; ccRCC; hypoxia inducible factor-1; kidney cancer; vitamin C.

Figure 1

Ascorbate uptake of ccRCC cells. The ccRCC cell lines Caki-1 (dotted line), Caki-2 (dark gray) and 786-0 (black) were treated with 0–1000 μM ascorbate for 16 hrs and intracellular ascorbate content was measured in whole cell lysates by HPLC-ECD (A). Cell counts for each condition were relatively stable except for 786-0 cells which showed reduced numbers at a concentration of 1 mM ascorbate (B). Data are mean + SD from three independent experiments. Abbreviations: ccRCC, clear cell renal cell carcinoma; HPLC-ECD, high-performance liquid chromatography with electrochemical detection.

Figure 2

Hypoxic induction of HIF pathway proteins in RCC cell lines. HIF pathway proteins were analysed by Western blotting following incubation of Caki-1, Caki-2 and 786-0 in decreasing oxygen tensions for 8 hrs. Bar charts show protein levels of HIF-1α (A, F, K), HIF-2α (B, G, L), GLUT1 (C, H, M), cyclin D1 (D, I, N) and BNIP3 (E, J, O) relative to a positive control (hypoxia-treated T24 cell lysate) which was loaded on each gel and referenced to β-actin. Data represent mean +SD from three independent experiments. Statistical significance was evaluated by One-way ANOVA with Dunnett’s Multiple Comparison Test; * p<0.05, ** p<0.01 *** p<0.001. Abbreviations: HIF-1α/2α, hypoxia-inducible factor-1α/2α; GLUT1, glucose transporter 1; BNIP3, Bcl2/adenovirus E1B 19 kDa interacting protein 3; ND, not detected.

Figure 3

Effect of ascorbate on HIF pathway protein levels at different oxygen tensions in Caki-1 cells. Cells were pre-loaded with increasing concentrations of ascorbate (0–1000 μM) for 16 hrs and then subjected to reduced oxygenation conditions. Shown are representative Western blots for HIF-1α, HIF-2α, GLUT1, cyclin D1 and BNIP3 from one of three independent experiments. β-actin was used as a loading control. Protein levels of HIF-1α and BNIP3 were decreased by ascorbate treatment under mild hypoxia (1–10% O₂). Other proteins were not affected. Abbreviations: HIF-1α/2α, hypoxia-inducible factor-1α/2α; GLUT1, glucose transporter 1; BNIP3, Bcl2/adenovirus E1B 19 kDa interacting protein 3.

Figure 4

Effect of ascorbate on HIF pathway protein levels at different oxygen tensions in Caki-2 cells. Cells were pre-loaded with increasing concentrations of ascorbate (0–1000 μM) for 16 hrs and then subjected to reduced oxygenation conditions. Shown are representative Western blots for HIF-1α, GLUT1, cyclin D1 and BNIP3 from one of three independent experiments. β-actin was used as a loading control. Protein levels of HIF-1α were increased after ascorbate treatment at 1% and 0.1% O2. Expression of all HIF targets was unchanged. Abbreviations: HIF-1α/2α, hypoxia-inducible factor-1α/2α; GLUT1, glucose transporter 1; BNIP3, Bcl2/adenovirus E1B 19 kDa interacting protein 3.

Figure 5

Effect of ascorbate on HIF pathway protein levels at different oxygen tensions in 786-0 cells. Cells were pre-loaded with increasing concentrations of ascorbate (0–1000 μM) for 16 hrs and then subjected to reduced oxygenation conditions. Shown are representative Western blots for HIF-1α, HIF-2α, GLUT1, cyclin D1 and BNIP3 from one of three independent experiments. β-actin was used as a loading control. There was no consistent difference of protein levels after ascorbate treatment under any condition. Abbreviations: HIF-1α/2α, hypoxia-inducible factor-1α/2α; GLUT1, glucose transporter 1; BNIP3, Bcl2/adenovirus E1B 19 kDa interacting protein 3.

Figure 6

Relative quantity of HIF pathway proteins in response to increasing ascorbate loading at different oxygen levels in Caki-1, Caki-2 and 786-0 cells. Densitometry analysis shows relative protein levels of HIF-1α (A, F), HIF-2α (B, J), GLUT1 (C, G, K), cyclin D1 (D, H, L) and BNIP3 (E, I) in Caki-1 (A-E), Caki-2 (F-I) and 786–0 (J-L) cells. Data after treatment with increasing concentrations of ascorbate (50–1000 μM) at different oxygen tensions (0.1–20% O₂), standardised to β-actin and relative to ascorbate-deficient cells (0 μM, white bar) are shown. In Caki-1 cells, protein levels of HIF-1α and BNIP3 were significantly decreased by ascorbate treatment under mild hypoxia. In Caki-2 cells, HIF-1α protein levels were significantly increased under 0.1% O₂ with increasing ascorbate. In 786-0 cells, no significant changes were observed, and no other proteins were affected. Data are mean + SD from three independent experiments. Statistical significance was determined by One-way ANOVA with Dunnett’s Multiple Comparison Test; ** p<0.01, *** p<0.001. Abbreviations: HIF-1α/2α, hypoxia-inducible factor-1α/2α; GLUT1, glucose transporter 1; BNIP3, Bcl2/adenovirus E1B 19 kDa interacting protein 3.

Figure 7

Proline hydroxylation of HIF-1α in ccRCC cell lines with or without intracellular ascorbate. Western blot analysis confirmed expression of HIF-1α in the 786-HIF1 clones at P4 which decreased with increasing passage number (P5-P6). A hypoxia-treated T24 cell lysate was used as a positive control for HIF-1α (+) and β-actin as a loading control (A). Cell growth was monitored for four days, showing slower growth of 786-HIF1 clones compared to parental cells, increasing with repeated passaging. Cell numbers are per well of a 12-well plate. Data shown as mean +SD of three independent experiments. Statistical significance compared to parental 786-0 cells was assessed using 2-way ANOVA with Bonferroni post test (B). The effect of 500 μM ascorbate on P564 hydroxylation of HIF-1α was measured in parental 786-0 and 786-HIF1 cells, and in Caki-1 cells under ambient oxygen conditions or after hypoxia treatment (8 hrs at 1% O₂) using Western blot. No hydroxy-HIF-1α was detected in parental 786-0 or Caki-1 cells (C). Densitometry analysis shows 1.5 fold increased hydroxylation in 786-HIF1 cells supplied with ascorbate compared to untreated 786-HIF1 cells (normalized to total HIF-1α content). Statistical significance was assessed by paired t-test (D). 786-HIF1 cells were treated with 50 μM–1 mM ascorbate and cell lysates subjected to Western blot for the detection of HIF-1α and hydroxy-HIF-1α (P564) (E). Relative hydroxy-HIF-1α (P564) levels (normalized to total HIF-1α levels) were increased after ascorbate treatment, as determined by densitometry analysis (F). Data shown as mean +SD from three independent experiments. Statistical significance compared to control condition was assessed by 1-way ANOVA with Bonferroni post test.* p<0.05, ** p<0.01, *** p<0.001. Abbreviations: HIF-1α, hypoxia-inducible factor-1α; P, passage number.