Nutrient deprivation and reactive oxygen species (ROS) play an important role in breast cancer mitochondrial adaptation. Adaptations to these conditions allow cells to survive in the stressful microenvironment of the tumor bed. This study is directed at defining the consequences of High Nitric Oxide (HNO) exposure to mitochondria in human breast cancer cells. The breast cancer cell line BT-20 (parent) was adapted to HNO as previously reported, resulting in the BT-20-HNO cell line. Both cell lines were analyzed by a variety of methods including MTT, LDH leakage assay, DNA sequencing, and Western blot analysis. The LDH assay and the gene chip data showed that BT-20-HNO was more prone to use the glycolytic pathway than the parent cell line. The BT-20-HNO cells were also more resistant to the apoptotic inducing agent salinomycin, which suggests that p53 may be mutated in these cells. Polymerase chain reaction (PCR) followed by DNA sequencing of the p53 gene showed that it was, in fact, mutated at the DNA-binding site (L194F). Western blot analysis showed that p53 was significantly upregulated in these cells. These results suggest that free radicals, such as nitric oxide (NO), pressure human breast tumor cells to acquire an aggressive phenotype and resistance to apoptosis. These data collectively provide a mechanism by which the dysregulation of ROS in the mitochondria of breast cancer cells can result in DNA damage.
Breast cancer Nitric oxide p53 Reactive oxygen species (ROS) Glycolysis and apoptosis Aerobic and anaerobic metabolisms
This is a preview of subscription content, log in to check access.
Siegel R, et al. Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin. 2011;61(4):212–36.Google Scholar
Semenza GL. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene. 2010;29(5):625–34.PubMedCrossRefGoogle Scholar
Paradise WA, et al. Nitric oxide: perspectives and emerging studies of a well known cytotoxin. Int J Mol Sci. 2010;11(7):2715–45.PubMedCrossRefGoogle Scholar
Ralph SJ, et al. The causes of cancer revisited: “mitochondrial malignancy” and ROS-induced oncogenic transformation—why mitochondria are targets for cancer therapy. Mol Aspects Med. 2010;31(2):145–70.PubMedCrossRefGoogle Scholar
Bentz BG, et al. The yin and yang of nitric oxide: reflections on the physiology and pathophysiology of NO. Head Neck. 2000;22(1):71–83.PubMedCrossRefGoogle Scholar
De Vitto H, Mendonça BS, Elseth KM, et. al. Part II. Mitochondrial mutational status of high nitric oxide adapted cell line BT-20 (BT-20-HNO) as it relates to human primary breast tumors (submitted in conjunction with current manuscript). Tumor Biol. 2012. doi:10.1007/s13277-012-0555-4.
Bentz BG, et al. Nitric oxide synthase type 3 is increased in squamous hyperplasia, dysplasia, and squamous cell carcinoma of the head and neck. Ann Otol Rhinol Laryngol. 1999;108(8):781–7.PubMedGoogle Scholar
Radosevich JA, Elseth KM, Vesper BJ, et al. Long-term adaptation of lung tumor cell lines with increasing concentrations of nitric oxide donor. Open Lung Canc J. 2009;2:35–44.CrossRefGoogle Scholar
Vesper BJ, et al. Long-term adaptation of breast tumor cell lines to high concentrations of nitric oxide. Tumor Biol. 2010;31(4):267–75.CrossRefGoogle Scholar
Yarmolyuk YR, et al. Part I. Development of a model system for studying nitric oxide in tumors: high nitric oxide-adapted head and neck squamous cell carcinoma cell lines. Tumor Biol. 2011;32(1):77–85.CrossRefGoogle Scholar
Onul A, et al. Long-term adaptation of the human lung tumor cell line A549 to increasing concentrations of hydrogen peroxide. Tumor Biol. 2012; 33(3):739–48.Google Scholar
Scudellari M. Does mitochondrial dysfunction lie at the heart of common, complex diseases like cancer and autism? The Scientist. 2011;25:30–5.Google Scholar
Nelson DA, et al. Hypoxia and defective apoptosis drive genomic instability and tumorigenesis. Genes Dev. 2004;18(17):2095–107.PubMedCrossRefGoogle Scholar
Samper E, et al. Increase in mitochondrial biogenesis, oxidative stress, and glycolysis in murine lymphomas. Free Radic Biol Med. 2009;46(3):387–96.PubMedCrossRefGoogle Scholar
Wong TS, et al. Physical and functional interactions between human mitochondrial single-stranded DNA-binding protein and tumour suppressor p53. Nucleic Acids Res. 2009;37(2):568–81.PubMedCrossRefGoogle Scholar
Lasfargues EY, Ozzello L. Cultivation of human breast carcinomas. J Natl Cancer Inst. 1958;21(6):1131–47.PubMedGoogle Scholar
Mossmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth. 1983;65:55–63.CrossRefGoogle Scholar
Decker T, Lohmann-Matthes ML. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods. 1988;115(1):61–9.PubMedCrossRefGoogle Scholar
Petitjean A, et al. Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum Mutat. 2007;28(6):622–9.PubMedCrossRefGoogle Scholar
Blin N, Stafford DW. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 1976;3(9):2303–8.PubMedCrossRefGoogle Scholar
Cubillos-Rojas M, et al. Simultaneous electrophoretic analysis of proteins of very high and low molecular mass using tris-acetate polyacrylamide gels. Electrophoresis. 2010;31(8):1318–21.PubMedCrossRefGoogle Scholar
Vesper BJ, et al. Part I. Molecular and cellular characterization of high nitric oxide-adapted human breast adenocarcinoma cell line. Tumor Biol. doi:10.1007/s13277-012-0530-0.
Warburg O. On respiratory impairment in cancer cells. Science. 1956;124(3215):269–70.PubMedGoogle Scholar
Kim KY, et al. Salinomycin-induced apoptosis of human prostate cancer cells due to accumulated reactive oxygen species and mitochondrial membrane depolarization. Biochem Biophys Res Commun. 2011;413(1):80–6.PubMedCrossRefGoogle Scholar
Lu J, Sharma LK, Bai Y. Implications of mitochondrial DNA mutations and mitochondrial dysfunction in tumorigenesis. Cell Res. 2009;19(7):802–15.PubMedCrossRefGoogle Scholar
Ishikawa K, Takenaga K, Akimoto M, et al. ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis. Science. 2008;320:661–4.PubMedCrossRefGoogle Scholar
Kulawiec M, Owens KM, Singh KK. mtDNA G10398A variant in African-American women with breast cancer provides resistance to apoptosis and promotes metastasis in mice. J Hum Genet. 2009;54(11):647–54.PubMedCrossRefGoogle Scholar
Gupta PB, et al. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell. 2009;138(4):645–59.PubMedCrossRefGoogle Scholar
Switzer CH, et al. Nitric oxide and protein phosphatase 2A provide novel therapeutic opportunities in ER-negative breast cancer. Trends Pharmacol Sci. 2011;32(11):644–51.PubMedCrossRefGoogle Scholar