Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Feb 21;289(8):5340-7.
doi: 10.1074/jbc.M113.531020. Epub 2014 Jan 3.

A low-dose arsenic-induced p53 protein-mediated metabolic mechanism of radiotherapy protection

Suthakar Ganapathy  1 , Shaowen Xiao, Mei Yang, Min Qi, Doo Eun Choi, Chul S Ha, John B Little, Zhi-Min Yuan
Affiliations

A low-dose arsenic-induced p53 protein-mediated metabolic mechanism of radiotherapy protection

Suthakar Ganapathy et al. J Biol Chem. .

Abstract

Radiotherapy is the current frontline cancer treatment, but the resulting severe side effects often pose a significant threat to cancer patients, raising a pressing need for the development of effective strategies for radiotherapy protection. We exploited the distinct metabolic characteristics between normal and malignant cells for a metabolic mechanism of normal tissue protection. We showed that low doses of arsenic induce HIF-1α, which activates a metabolic shift from oxidative phosphorylation to glycolysis, resulting in increased cellular resistance to radiation. Of importance is that low-dose arsenic-induced HIF-1α requires functional p53, limiting the glycolytic shift to normal cells. Using tumor-bearing mice, we provide proof of principle for selective normal tissue protection against radiation injury.

Keywords: DNA Damage; Glycolysis; Metabolism; Signal Transduction; p53.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Low doses of arsenic induce HIF-1α in an NFκB-dependent manner. Human fibroblasts were treated with sodium arsenite (As) (100 nm) for 12 h. The cells were harvested for HIF-1α immunostaining (A) or analysis of the HIF-1α transcript (B) (Ctl, control). Human fibroblasts were treated with the indicated doses of sodium arsenite. The cells were harvested 12 h later and analyzed for the levels of HIF-1α transcript (C) or GLUT-1 transcript (D). Data are mean ± S.D. from three independent experiments. F, human fibroblasts were pretreated with dimethyl sulfoxide (DMSO), LY294002 (25 μm) (LY294), PD98049 (25 μm) (PD980), or capsaicin (3 μm) (Capsa) for 1 h. The cells were then treated with either solvent or 100 nm arsenic and harvested 12 h later for Western blot analysis using the indicated antibodies. G, human fibroblasts were transfected with either control RNAi (siGL2) or sip65. The cells were subjected to treatment with arsenic (100 nm) 48 h post-transfection. The cells were harvested 12 h later for HIF-1α immunostaining. H, human fibroblasts were transfected with either control RNAi or sip65 for 48 h or pretreated with dimethyl sulfoxide or capsaicin (3 μm) for 1 h. The cells were treated with either solvent (Cntl) or 100 nm arsenic (low dose arsenic, LDA). The cells were harvested 12 h later and analyzed for the levels of HIF-1α, GLUT-1, or GLUT-3 transcript. Data are mean ± S.D. from three independent experiments. E, human fibroblasts were treated with sodium arsenite for the indicated time periods, and the cells were immunostained for HIF-1α.
FIGURE 2.
FIGURE 2.
Low-dose arsenic induces a metabolic shift. A, human fibroblasts were treated with arsenic (As) (100 nm) for 12 h. mRNA was isolated, and the transcripts of the indicated genes were determined by quantitative RT-PCR. Data are mean ± S.D. from three independent experiments. B, arsenic-treated (100 nm) fibroblasts were subjected to Western blot analysis using the indicated antibodies. C, human fibroblasts were transfected with either control RNAi (siGL2) or siHIF-1α. The cells were subjected to treatment with arsenic (100 nm) 48 h post-transfection. The cells were harvested at 12 h later for analysis of the indicated transcripts. Con, control. D and E, fibroblasts were treated with arsenic (100 nm) for 12 h and incubated with d-[1,2-13C]glucose for 15 min prior to metabolite extraction and targeted LC-MS/MS analysis. The ratio of 13C-labeled to unlabeled (12C) metabolites was measured by LC-MS/MS and is presented as mean ± S.D. over three independent samples (*, p < 0.05).
FIGURE 3.
FIGURE 3.
Low-dose arsenic induces radiation resistance via induction of HIF-1α-dependent glycolysis. A, human fibroblasts were pretreated with or without arsenic (As) (100 nm) for 12 h, followed by 4-Gy irradiation. The cells were also subjected to colony survival assays. Data are mean ± S.D. from three independent experiments. **, p < 0.01. Ctl, control. B, fibroblasts were treated as indicated. The cells were harvested 1 h after irradiation and subjected to γH2AX immunostaining. C, quantification of γH2AX-positive cells shown in B. Error bars represent mean ± S.D. of three independent experiments (∼100 cells/sample). **, p < 0.01. D, human fibroblasts were transfected with either control RNAi (siGL2) or siHIF-1α. 48 h post-transfection, the cells were pretreated with arsenic (100 nm) for 12 h and then irradiated at 4 Gy. The cells were analyzed via colony survival assay. Data are mean ± S.D. from three independent experiments. E, siGL2 or siHIF-1α-expressing cells were pretreated with arsenic (100 nm), followed by 4-Gy irradiation as in E. The cells were analyzed for γH2AX 1 h after IR treatment. The numbers of γH2AX-positive cells are presented as mean ± S.D. of three independent experiments (∼100 cells/sample). **, p < 0.01. F, human fibroblasts cultured in normal medium or low-glucose (2 mm) medium were pretreated with arsenic (100 nm) or left untreated, followed after 12 h by 0- or 4.0-Gy treatment. Fibroblasts cultured in normal medium were treated with 2-DG (5 mm) 2 h before 4-Gy treatment. The cells were fixed 1 h post-4-Gy treatment and analyzed as in E. G, siGL2- or siLDH-A-expressing cells were treated and analyzed as in E. H, human lymphocytes were treated as in F and analyzed for the percentage of apoptosis. Bars represent mean ± S.D. of three independent experiments, **, p < 0.01.
FIGURE 4.
FIGURE 4.
Functional p53 is required for mediating low-dose arsenic-induced HIF-1α-dependent radiation resistance. A, fibroblasts were pretreated with or without Nutlin 3A (10 μm) for 2 h prior to being treated with arsenic (As). The cells were harvested 12 h after arsenic treatment and subjected to HIF-1α immunostaining. Ctl, control. B, fibroblasts were treated with or without Nutlin 3A 2 h before being treated with arsenic, followed by irradiation. The cells were analyzed by γH2AX immunostaining and quantified as described in Fig. 3C. DMSO, dimethyl sulfoxide. Bars represent mean ± S.D. of three independent experiments (∼100 cells/sample), **, p < 0.01. C, siGL2- or sip53-expressing cells were pretreated with arsenic (100 nm) or left untreated, followed after 12 h by 0- or 4.0-Gy treatment. The cells were fixed 1 h post-4-Gy treatment and analyzed as in Fig. 3C. Bars represent mean ± S.D. of three independent experiments (100 cells/sample), **, p < 0.01. D, human non-transformed lung epithelial cells (TrBEp-1) or lung carcinoma cells (A549) were treated with arsenic (100 nm) or CoCl2 (100 μm) for 12 h. The cells were analyzed by Western blot analysis using the indicated antibodies. MCF-10A/MCF-7 cells were included as control.
FIGURE 5.
FIGURE 5.
Low-dose arsenic pretreatment alleviates normal tissue toxicity caused by total body irradiation of tumor-bearing mice. Athymic nude mice (BALB/cnu/nu, 4- to 6-week-old, sex-matched) were from Harlan Laboratories. The human lung carcinoma cell line A549 (cells as a 50% suspension in Matrigel) as 3 million cells/mouse in a final volume of 100 μl was injected subcutaneously into the right flank of BALB/c nude mice. When the average tumor volume reached about 100 mm3, mice were randomized into the following groups: control (Ctl), arsenic (As) only, IR only, and arsenic and IR. For arsenic pretreatment, mice were treated with sodium arsenite (0.4 mg/kg body weight) for 3 days (days 0–3). Mice were then treated with total body irradiation at 2 Gy/day for 5 days. A, tumor volumes were measured every 5 days. The tumor volume was calculated using the equation volume = length × width × depth × 0.5236 mm3. Two independent experiments were performed, and the tumor volumes are mean ± S.E. from a total of 10 mice/group. B, the body weight of the mice was monitored throughout the experiment as described in A. The numbers are mean ± S.D. from two independent experiments with a total of 10 mice/group. At the completion of the experiments, the mice were sacrificed by cervical decapitation. Tissue and tumor samples were harvested for histology experiments. C, the small intestine and tumor sections were stained for HIF-1α. H&E staining was performed. Representative images of H&E staining of the small intestine (D) and bone marrow (E) are shown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hall E., Giaccia A. J. (2006) Radiobiology for the Radiobiologist. pp. 5–29, Lippincott Williams & Wilkins, Philadelphia, PA
    1. Koppenol W. H., Bounds P. L., Dang C. V. (2011) Otto Warburg's contributions to current concepts of cancer metabolism. Nat. Rev. Cancer 11, 325–337 - PubMed
    1. Lunt S. Y., Vander Heiden M. G. (2011) Aerobic glycolysis. Meeting the metabolic requirement of cell proliferation. Annu. Rev. Cell Dev. Biol. 27, 441–464 - PubMed
    1. Gordan J. D., Simon M. C. (2007) Hypoxia-inducible factors, central regulators of the tumor phenotype. Curr. Opin. Genet. Dev. 17, 71–77 - PMC - PubMed
    1. Semenza G. L. (2012) Hypoxia-inducible factors in physiology and medicine. Cell 148, 399–408 - PMC - PubMed

Publication types

MeSH terms

-