Cancer Letters

Cancer Letters

Volume 353, Issue 2, 28 October 2014, Pages 248-257
Cancer Letters

Original articles
Systemic DNA damage accumulation under in vivo tumor growth can be inhibited by the antioxidant Tempol

https://doi.org/10.1016/j.canlet.2014.07.030Get rights and content

Highlights

  • Mice bearing tumors exhibited elevated complex DNA damage in normal tissues throughout the body.

  • ROS are thought to cause for this systemic effect.

  • Feeding mice antioxidant tempol reduced this DNA damage.

  • Tempol effects were independent of the functioning adaptive immunity.

Abstract

Recently we found that mice bearing subcutaneous non-metastatic tumors exhibited elevated levels of two types of complex DNA damage, i.e., double-strand breaks and oxidatively-induced clustered DNA lesions in various tissues throughout the body, both adjacent to and distant from the tumor site. This DNA damage was dependent on CCL2, a cytokine involved in the recruitment and activation of macrophages, suggesting that this systemic DNA damage was mediated via tumor-induced chronic inflammatory responses involving cytokines, activation of macrophages, and consequent free radical production. If free radicals are involved, then a diet containing an antioxidant may decrease the distant DNA damage. Here we repeated our standard protocol in cohorts of two syngeneic tumor-bearing C57BL/6NCr mice that were on a Tempol-supplemented diet. We show that double-strand break and oxidatively-induced clustered DNA lesion levels were considerably decreased, about two- to three fold, in the majority of tissues studied from the tumor-bearing mice fed the antioxidant Tempol compared to the control tumor-bearing mice. Similar results were also observed in nude mice suggesting that the Tempol effects are independent of functioning adaptive immunity. This is the first in vivo study demonstrating the effect of a dietary antioxidant on abscopal DNA damage in tissues distant from a localized source of genotoxic stress. These findings may be important for understanding the mechanisms of genomic instability and carcinogenesis caused by chronic stress-induced systemic DNA damage and for developing preventative strategies.

Introduction

Intercellular communication is mediated by substances released by damaged cells which then affect healthy cells. The radiation-induced bystander effect is one example of this phenomenon, where the released factors from irradiated cells may activate pathways in healthy ‘bystander’ cells leading to the induction of DNA damage [1], [2], increased genomic instability and decreased viability [3], [4]. The signal transduction from irradiated to bystander cells in vitro can occur through both cell media and gap junctions [1] and is reminiscent of the inflammatory response mediated by COX-2 related pathways, involving cytokines, growth factors, and membrane-permeable reactive oxygen and nitrogen species (ROS and RNS) [5], [6]. In addition to radiation-damaged cells, recent studies have reported that genetically unstable, senescent, and cancerous cells also can adversely affect their normal neighbors [7], [8], [9], [10], suggesting that the radiation-induced bystander effect is a specific instance of a much more general phenomenon of intercellular communication from damaged or abnormal cells to normal cells.

While these bystander-like phenomena have been well-documented in vitro, as have in vivo counterparts of the radiation-induced bystander (abscopal) effects [11], [12], [13], reports of other extensions of the more general phenomenon in vivo are not so abundant. An interesting example is that of animal tumors in a chronic inflammatory environment [14], with elevated levels of endogenous stress factors and ROS [15], [16], produced either directly by tumors, or indirectly via inflammatory responses, which can induce DNA damage in healthy neighboring cells [17].

While there are several methods for detecting ROS in vitro, they are difficult to monitor in vivo. All ROS detection methodologies have to overcome various limitations such as time, dye specificity, species specificity, and others [18], [19]. In our study with tumor-bearing mice, we employed two endpoints to monitor the effects of oxidative stress, the presence of two potentially lethal DNA lesions, bistranded oxidatively-induced clustered DNA lesions (OCDLs) [20], [21] and foci of phosphorylated histone H2AX (γ-H2AX), a surrogate marker of DNA double strand breaks (DSBs) [22], [23], [24]. Both biomarkers have been used to detect and monitor radiation- and cancer-related DNA damage in mouse and human tissues [25], [26], [27], [28]. While induction of γ-H2AX foci has been reported at non-DSB sites, such as dysfunctional telomeres [29] or in the absence of DNA damage [30], numerous studies related to the bystander effect, have shown a direct link between DSBs and γ-H2AX foci [1], [2], [9], [31], [32]. In our recent study with mice implanted with localized tumors, we showed that the levels of these two types of complex DNA lesions were elevated in several distant tissues [26]. We also showed that the elevated levels of these lesions in distant tissues were mediated by inflammatory macrophages in a CCL2-dependent manner. The elevation of OCDLs and the participation of macrophages both point to ROS involvement in this distant DNA damage.

While ROS homeostasis can be maintained in unstressed healthy cells by a balance of the pathways that produce and destroy ROS, excessive ROS levels may be beyond the capacity of these endogenous systems to regulate. However, they can often be lowered by exogenous antioxidants such as Tempol, a cell-permeable superoxide dismutase mimetic and a free radical scavenger [33]. Belonging to nitroxide stable free radical family, Tempol is a promising agent for clinical use as an antioxidant and radioprotector [34]. It significantly reduces superoxide anion and peroxynitrite-associated inflammation, lowers blood pressure in a variety of animal models and also displays neuroprotective effects [35], [36], [37], [38], [39]. It has been found to be efficient in restoring mitochondrial and cardiac functions in tumour necrosis factor (TNF)α-induced oxidative stress and reducing cardiac hypertrophy in chronically hypoxic rats [40]. It reduces the incidence of hematopoietic neoplasms, increases the survival of irradiated mice [41] and topically protects mice against radiation-induced mucositis [42]. Preclinical studies in guinea pigs, and a Phase I clinical trial in patients receiving whole-brain radiotherapy, suggest that Tempol is effective in suppressing radiation-induced alopecia [43], [44], [45].

To test the hypothesis for an oxidative mechanism fueling these non-targeted effects in the organism, we examined whether an exogenous antioxidant treatment could lower systemic or abscopal oxidative DNA damage levels in tumor-bearing mice. For this reason we incorporated a well-known antioxidant Tempol, into the diets of several tumor-bearing mouse cohorts. Here we report that the local tumor-induced DSB and OCDL accumulation in normal tissues of tumor-bearing mice can be suppressed by feeding the mice a Tempol-supplemented diet. These findings show that oxidative stress pathways leading to elevated DSB and OCDL levels can be interrupted with exogenous antioxidants. Since these two lesions are often precursors to genomic instability and carcinogenesis, and it is estimated that as many as 20% of cancers may be due to chronic inflammatory conditions [14], these findings may have important implications for development of clinical strategies to mitigate chronic stress-induced systemic DNA damage.

Section snippets

Mice and tumors

All necessary permits were obtained for the described study. The protocols were approved by the National Cancer Institute Animal Care and Use Committee. Six-week-old C57BL/6NCr (B6) and nude female mice were obtained from the Animal Production Area, National Cancer Institute (NCI) – Frederick. Cryopreserved murine B16 melanoma (MEL, host strain: B6) and Lewis lung carcinoma (LLC, host strain: B6) were obtained from the Division of Cancer Treatment and Diagnosis tumor repository, NCI –

Tempol effect on animals

Since ROS-induced DNA damage may occur in neighboring or distant normal tissues in tumor-bearing mice, we hypothesized that treatment with an antioxidant such as Tempol may ameliorate the level of the consequential oxidatively-induced DNA damage. The protocol used previously [26] was followed closely to enable direct comparison of the results (Fig. 1). Six cohorts of five B6 mice each were used, two cohorts were implanted with syngeneic B16 melanoma (MEL) cells, two with syngeneic Lewis lung

Discussion

High oxidative stress and inflammation have been connected with transformation of normal cells and tissues to a malignant phenotype [14]. Sustained oxidative stress is a hallmark of cancer, driving DNA damage and genetic instability and shaping the tumor microenvironment by promoting angiogenesis and immune evasion [59], [60], [61]. However, many questions still remain regarding the impact of tumor on neighboring or distant tissues. We have shown previously that the presence of a tumor affects

Conflict of interest

All authors declare no conflicts of interest.

Acknowledgments

We are grateful to the Laboratory Animal Sciences Program and Pathology Histotechnology Laboratory staff (National Cancer Institute – Frederick) for the help with animal maintenance and histological analysis. We thank Roger Martin, Peter MacCallum Cancer Centre, for his advice and comments on the manuscript. This study was partly supported by the NIH Intramural Program, by A.G.'s funding from East Carolina University, EU grant MC-CIG-303514, COST Action CM1201 ‘Biomimetic Radical Chemistry’,

References (91)

  • T. Goffman et al.

    Topical application of nitroxide protects radiation-induced alopecia in guinea pigs

    Int. J. Radiat. Oncol. Biol. Phys

    (1992)
  • B.M. Sutherland et al.

    Quantifying clustered DNA damage induction and repair by gel electrophoresis, electronic imaging and number average length analysis

    Mutat. Res

    (2003)
  • J.B. Mitchell et al.

    A low molecular weight antioxidant decreases weight and lowers tumor incidence

    Free Rad. Biol. Med

    (2003)
  • S. Toyokuni et al.

    Persistent oxidative stress in cancer

    FEBS Lett

    (1995)
  • T.B. Kryston et al.

    Role of oxidative stress and DNA damage in human carcinogenesis

    Mutat. Res

    (2011)
  • R.M. Davis et al.

    Magnetic resonance imaging of organic contrast agents in mice: capturing the whole-body redox landscape

    Free Rad. Biol. Med

    (2011)
  • R.M. Davis et al.

    A novel nitroxide is an effective brain redox imaging contrast agent and in vivo radioprotector

    Free Rad. Biol. Med

    (2011)
  • F. Pinaud et al.

    In vitro protection of vascular function from oxidative stress and inflammation by pulsatility in resistance arteries

    J. Thorac. Cardiovasc. Surg

    (2011)
  • M.H. Tsuhako et al.

    Tempol ameliorates murine viral encephalomyelitis by preserving the blood-brain barrier, reducing viral load, and lessening inflammation

    Free Rad. Biol. Med

    (2010)
  • J. Yamada et al.

    Cell permeable ROS scavengers, Tiron and Tempol, rescue PC12 cell death caused by pyrogallol or hypoxia/reoxygenation

    Neurosci. Res

    (2003)
  • J.M. Hair et al.

    BRCA1 role in the mitigation of radiotoxicity and chromosomal instability through repair of clustered DNA lesions

    Chem. Biol. Interact

    (2010)
  • O.A. Sedelnikova et al.

    Role of oxidatively induced DNA lesions in human pathogenesis

    Mutat. Res

    (2010)
  • E. Shacter et al.

    Oxidative stress interferes with cancer chemotherapy: inhibition of lymphoma cell apoptosis and phagocytosis

    Blood

    (2000)
  • R. Reliene et al.

    Antioxidants suppress lymphoma and increase longevity in Atm-deficient mice

    J. Nutr

    (2007)
  • M.V. Sokolov et al.

    Ionizing radiation induces DNA double-strand breaks in bystander primary human fibroblasts

    Oncogene

    (2005)
  • O.A. Sedelnikova et al.

    DNA double-strand breaks form in bystander cells after microbeam irradiation of three-dimensional human tissue models

    Cancer Res

    (2007)
  • K.M. Prise et al.

    Radiation-induced bystander signalling in cancer therapy

    Nat. Rev. Cancer

    (2009)
  • H. Zhou et al.

    Mechanism of radiation-induced bystander effect: role of the cyclooxygenase-2 signaling pathway

    Proc. Natl Acad. Sci. U.S.A.

    (2005)
  • C. Shao et al.

    Role of TGF-beta1 and nitric oxide in the bystander response of irradiated glioma cells

    Oncogene

    (2008)
  • S. Nagar et al.

    The death-inducing effect and genomic instability

    Radiat. Res

    (2005)
  • J.P. Coppe et al.

    Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor

    PLoS Biol

    (2008)
  • J.S. Dickey et al.

    Intercellular communication of cellular stress monitored by gamma-H2AX induction

    Carcinogenesis

    (2009)
  • M.V. Sokolov et al.

    Gamma-H2AX in bystander cells: not just a radiation-triggered event, a cellular response to stress mediated by intercellular communication

    Cell Cycle

    (2007)
  • I. Koturbash et al.

    Irradiation induces DNA damage and modulates epigenetic effectors in distant bystander tissue in vivo

    Oncogene

    (2006)
  • M. Mancuso et al.

    Oncogenic bystander radiation effects in Patched heterozygous mouse cerebellum

    Proc. Natl Acad. Sci. U.S.A.

    (2008)
  • A. Mantovani et al.

    Cancer-related inflammation

    Nature

    (2008)
  • S.S. Brar et al.

    NOX5 NAD(P)H oxidase regulates growth and apoptosis in DU 145 prostate cancer cells

    Am. J. Physiol. Cell Physiol

    (2003)
  • R. De Bont et al.

    Endogenous DNA damage in humans: a review of quantitative data

    Mutagenesis

    (2004)
  • S.P. Hussain et al.

    Radical causes of cancer

    Nat. Rev. Cancer

    (2003)
  • Y. Wei et al.

    Quantitative measurement of reactive oxygen species in vivo by utilizing a novel method: chemiluminescence with an internal fluorescence as reference

    J. Biomed. Opt

    (2010)
  • E. Owusu-Ansah et al.

    Distinct mitochondrial retrograde signals control the G1-S cell cycle checkpoint

    Nat. Genet

    (2008)
  • A.G. Georgakilas

    Processing of DNA damage clusters in human cells: current status of knowledge

    Mol. Biosyst

    (2008)
  • A. Georgakilas

    Detection of clustered DNA lesions: biological and clinical applications

    World J. Biol. Chem

    (2011)
  • O.A. Sedelnikova et al.

    Histone H2AX in DNA damage and repair

    Cancer Biol. Ther

    (2003)
  • W.M. Bonner et al.

    GammaH2AX and cancer

    Nat. Rev. Cancer

    (2008)
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