Hypoxia upregulates the expression of the NDRG1 gene leading to its overexpression in various human cancers

Abstract

Background: The expression of NDRG1 gene is induced by nickel, a transition metal sharing similar physical properties to cobalt. Nickel may create hypoxia-like conditions in cells and induce hypoxia-responsive genes, as does cobalt. Therefore NDRG1 is likely to be another gene induced by hypoxia. HIF-1 is a transcription factor which has a major role in the regulation of hypoxia-responsive genes, and thus it could be involved in the transcriptional regulation of NDRG1 gene. Hypoxia is such a common feature of solid tumours that it is of interest to investigate the expression of Ndrg1 protein in human cancers.

Results: Hypoxia and its mimetics induce in vitro expression of NDRG1 gene and cause the accumulation of Ndrg1 protein. Protein levels remain high even after cells revert to normoxia. Although HIF-1 is involved in the regulation of NDRG1, long term hypoxia induces the gene to some extent in HIF-1 knock-out cells. In the majority of human tissues studied, Ndrg1 protein is overexpressed in cancers compared to normal tissues and also reflects tumour hypoxia better than HIF-1 protein.

Conclusions: Hypoxia is an inducer of the NDRG1 gene, and nickel probably causes the induction of the gene by interacting with the oxygen sensory pathway. Hypoxic induction of NDRG1 is mostly dependent on the HIF-1 transcription factor, but HIF-1 independent pathways are also involved in the regulation of the gene during chronic hypoxia. The determination of Ndrg1 protein levels in cancers may aid the diagnosis of the disease.

Figure 1

The induction of NDRG1 gene expression by hypoxia and its mimetics. A) A549 cells were exposed to normoxia (20% O₂, control) for 20 hours or to hypoxia (0.5% O₂) for the time periods indicated in the figure. 15 μg of total RNA was isolated and subjected to a Northern blot analysis as described in 'Methods' section. The blot first hybridized with NDRG1 probe (top panel), and then the membrane was stripped and rehybridized with actin probe (bottom panel) to show loading. B) A549 cells were exposed to 0.5 mM NiCl₂ (Nickel), 200 μM CoCl₂ (Cobalt), 200 μM desferrioxamine (DFX), or hypoxia (0.5% O₂) for 20 hrs. 40 μg of whole cell protein extracts were loaded into each lane and subjected to Western blot analysis as described in 'Methods' section, using antibody against Ndrg1. Bottom panel (actin) shows loading. C) A549 cells were first exposed to hypoxia (0.5% O₂) for 20 hrs, then taken out of hypoxic chamber and incubated additionally for the time periods indicated under normoxic (20% O₂) conditions. Western blot analysis with antibody against Ndrg1 was carried out in whole cell protein extracts. Bottom panel (actin) shows loading.

Figure 2

The confirmation of Ndrg1 protein induction in different cell lines. (A) HTE cells were incubated with different concentrations of NiCl₂ (Ni), hypoxia, and 300 μM of CoCl₂ (Co) for 20 hrs. (B) HOS and MCF-7, (C) PW and DU-145 cells were incubated with 500 μM of NiCl₂ (nickel), hypoxia, and 300 μM of CoCl₂ (cobalt) for 20 hrs. Western blot analyses were done as described in 'Methods' section. The membranes were first incubated with anti-Ndrg1 antibody (top panels), then stripped, and rehybridized with anti-actin antibody (bottom panels) to show loading.

Figure 3

The role of HIF-1 in the regulation of NDRG1 gene expression. A) HIF-1 proficient (HIF-1α^+/+) and deficient (HIF-1α^-/-) cells were exposed to 0.5 mM NiCl₂, 300 μM CoCl₂, and hypoxia (0.5% O₂) for 20 hrs. 15 μg of total RNA was isolated and subjected to a Northern blot analysis using NDRG1 probe (top panel). Ethidium bromide staining was used to adjust loading (bottom panel). B) HIF-1α^+/+ and HIF-1α^-/- cells were exposed to 0.5 mM NiCl₂, 300 μM CoCl₂, and hypoxia (0.5% O₂) for 20 hrs. 40 μg of protein extracts were subjected to Western blot analysis using antibody against Ndrg1 protein (top panel). Actin bands in the bottom panel show loading. C) Cells were first incubated for 24 hours under normoxic conditions for attachment (Day 0) and then exposed to hypoxia for up to three days. Western blot analysis with antibody against Ndrg1 was carried out in 25 μg of whole cell protein extracts (top panel). Actin bands in the bottom panel show loading.

Figure 4

Immunohistochemical detection of Ndrg1 protein in human tissues. The nature of the tissue is indicated on top of each picture. Original magnifications are as follows: A, ×400; B, ×400; C, ×100; D, ×100; E, ×400; F, ×400; G, ×400; H, ×400; I, ×100; J, ×100; K, ×400; L, ×400; M, 100; N, ×100.

Figure 5

Immunohistochemical detection of HIF-1α protein in human tissues. The nature of the tissue is indicated on top of each figure. Original magnifications are as follows: A, ×400; B, ×400; C, ×100; D, ×100; E, ×100; F, ×100; G, ×40; H, ×40.

Figure 6

The illustration of the hypothetical mechanism by which nickel and cobalt upregulate the expression of the NDRG1 gene