Original articlesSystemic DNA damage accumulation under in vivo tumor growth can be inhibited by the antioxidant Tempol
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’,
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These authors contributed equally.