Hypoxic tumor cell radiosensitization through nitric oxide

Nitric Oxide 19 (2008) 164–169

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Review

Hypoxic tumor cell radiosensitization through nitric oxide Mark De Ridder *, Dirk Verellen, Valeri Verovski, Guy Storme UZ Brussel, Oncologisch Centrum, Dienst Radiotherapie, Laarbeeklaan 101, B-1090 Brussels, Belgium

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Article history: Received 25 January 2008 Revised 15 April 2008 Available online 24 April 2008 Keywords: Nitric oxide Nitric oxide synthase Radiosensitization Nuclear factor-jB Hypoxia Macrophages

a b s t r a c t Hypoxia is a principal signature of the tumor microenvironment and is considered to be the most important cause of clinical radioresistance and local failure. Oxygen is so far the best radiosensitizer, but tumor oxygenation protocols are compromised by its metabolic consumption and therefore limited diffusion inside tumors. Many chemical radiosensitizers can selectively target hypoxic tumor cells, but their systemic toxicity compromises their adequate clinical use. NO is an efficient hypoxic radiosensitizer, as it may mimic the effects of oxygen on fixation of radiation-induced DNA damage, but the required levels cannot be obtained in vivo because of vasoactive complications. Our laboratory explored whether this problem may be overcome by endogenous production of NO inside tumors. We demonstrated that iNOS, activated by pro-inflammatory cytokines, is capable of radiosensitizing tumor cells through endogenous production of NO, at non-toxic extracellular concentrations. We observed that this radiosensitizing effect is transcriptionally controlled by hypoxia and by NF-jB. Tumor-associated immune cells may contribute to the iNOS-mediated radiosensitization by the generation of pro-inflammatory cytokines and NO, which may diffuse towards bystander tumor cells. Our findings indicate a rationale for combining immunostimulatory and radiosensitizing strategies in the future. Ó 2008 Elsevier Inc. All rights reserved.

Tumor hypoxia and radioresistance The response of cells to ionizing radiation is strongly dependent upon oxygen, which is traditionally explained by the ‘‘oxygen fixation hypothesis” (Figs. 1 and 2). To exert its effect on radiosensitivity, oxygen must be present during or within milliseconds after radiation [1,2]. The growth and survival of cells in solid tumors is dependent on the adequate supply of oxygen and nutrients, which diffuse from the blood vessels and are consumed by the tumor cells. In order to meet their increasing oxygen and nutrient demand, tumors develop their own blood supply. However, the neovasculature is morphologically and functionally abnormal, and is generally unable to meet the increasing demands, resulting in a diffusion limited chronic hypoxia. Hence, tumor cells can roughly be divided in two categories with regard to their oxygenation and radiosensitivity. Cells lying near the capillaries, within the diffusion distance of oxygen (±100 lm), are well oxygenated and radiosensitive. Cells lying at the edge and beyond the diffusion distance are hypoxic and radioresistant [3]. In the late 1970s, Brown postulated that another type of hypoxia, being transient in nature, existed in solid tumors as well [4]. This was later confirmed and shown to result from temporary cessations in blood flow [5]. The mechanisms responsible for the intermittent closure of tumor blood vessels

* Corresponding author. Fax: +32 2 477 6212. E-mail address: [email protected] (M. De Ridder). 1089-8603/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2008.04.015

are not entirely understood yet. They may include plugging of blood vessels by blood cells or by circulating tumor cells, collapse of blood vessels in regions with high interstitial pressure, spasms and spontaneous vasomotion in incorporated host arterioles. This temporary closing of blood vessels results in perfusion limited acute hypoxia. The most widely accepted technique for assessing tumor oxygenation in vivo and in the clinic is the measurement of tissue oxygen tension (pO2) by polarographic needle electrodes (Eppendorf electrodes), which are introduced in the tumor and moved forward by an automatic stepping motor [6]. This approach allows measuring the pO2 along several electrode tracks. Generally, more than 50 pO2 readings are performed, to assess the oxygenation of solid tumor. A collective body of evidence demonstrates that cervix carcinomas and head-and-neck cancers are poorly oxygenated, with a median pO2 of about 10 mmHg, and that low pO2 levels before radiotherapy are the most significant adverse prognostic factor [7,8]. Low and highly heterogeneous levels of oxygenation were found in other types of solid tumors as well, and are considered to be a principal signature of the tumor microenvironment [9]. Strategies to overcome hypoxia-induced radioresistance One of the earliest clinical attempts to eliminate hypoxia-induced radioresistance involved patients breathing high oxygen content gas under hyperbaric conditions (3 atmosphere). The British Medical Research Council randomized 1669 patients between

M. De Ridder et al. / Nitric Oxide 19 (2008) 164–169

Surviving fraction

0 1 .1 .01 .001 anoxic aerated

.0001 0

5

10 15 Radiation dose (Gy)

20

Fig. 1. Survival cells of EMT-6 cells in aerated and anoxic conditions. In order to obtain a surviving fraction of 0.1, a radiation dose of 18 Gy is required in anoxic and 6 Gy in aerated conditions, resulting in an oxygen enhancement ratio (OER) of 3.

DNA-H

Reduction in hypoxia irradiation DNA-H

DNA Oxidation in normoxia

DNA-OO

DNA strand breaks Cell death

Fig. 2. The oxygen fixation hypothesis. When radiation is absorbed in a biological material, free radicals are produced. These radicals can be produced directly in the DNA, or indirectly in water molecules and diffuse far enough to damage the DNA. It is the fate of the free radicals produced in the DNA (DNA) that determines the biological damage. In the presence of oxygen, DNA-OO is produced and further processed to DNA-OOH. This results in a changed chemical composition of the DNA and thus fixation of the DNA damage. In the absence of oxygen, DNA can react with H+, restoring the DNA in its original form.

radiotherapy with or without hyperbaric oxygen [10]. Hyperbaric oxygen significantly improved both survival and local control after radiotherapy, but never entered routine clinical use because of logistic problems. Another way to improve the oxygen availability is to optimize the hemoglobin concentration. Generally, anemic patients have a reduced locoregional tumor control and survival [11]. The Danish Head-and-Neck Cancer studies 5, 6 and 7 randomized patients with low hemoglobin level to receive blood transfusions or not [12]. Surprisingly, correction of anemia did not result in a significantly improved local control. This negative result was more recently confirmed by randomized placebo-controlled trials studying the effect of erythropoietin [13]. Another way to improve tumor oxygenation is to prevent perfusion-limited acute hypoxia. Experimental studies showed that nicotinamide, a vitaminB3 analog, is an efficient radiosensitizer in mouse models, primarily by decreasing transient occlusions of blood vessels and thereby perfusion-limited acute hypoxia. Nicotinamide is currently being evaluated in a phase III trial in combination with accelerated radiotherapy and carbogen breathing (ARCON) in laryngeal cancer (CKTO 2000-09, NCT00147732) [14]. In 1969, Adams and Cooke introduced the concept of hypoxic cell radiosensitizers, which are chemicals that mimic oxygen and enhance thereby radiation damage [15]. These compounds are, in contrast to oxygen, not metabolized by tumor cells and can therefore diffuse further away from the capillaries than oxygen. Exper-

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imental studies showed complete reversal of hypoxia-induced tumor cell radioresistance by nitroimidazole compounds, without increasing the response of aerated tissues. However, the results of misonidazole, the lead compound were disappointing in clinical trials because of the low plasma concentrations achievable with this neurotoxic drug [16]. Selective killing of hypoxic cells can also be achieved with bioreductive drugs, which undergo intracellular reduction to form active cytotoxic species under low oxygen tension. Animal studies indicated that mitomycin-c can be used in combination with radiotherapy to kill the hypoxic fraction of a solid tumor, while radiotherapy eradicates the oxygenated fraction. But, mitomycin-c failed in clinical trials, because it has a small differential killing effect between aerobic and hypoxic cells, and can be administered only once or twice during the course of radiotherapy. The most promising group of bioreductive drugs are the organic nitroxides, of which tirapazamine is the lead compound. This molecule is metabolized by intracellular reductases to form a transient oxidizing radical that can be efficiently scavenged by molecular oxygen in aerated tissues to reform the parent compound. In the absence of oxygen, the oxidizing radicals abstract protons from the DNA to form DNA radicals and finally strand breaks [17]. It is currently under evaluation in a phase III Study in Patients With Stage IB-IVA Carcinoma of the Cervix (GOG0219, CAN-NCIC-GOG-0219, NCT00262821). In conclusion, after more than 50 years of extensive research no single hypoxia-selective molecule entered into routine radiotherapeutical use and the search continues. Ideally, a novel hypoxiaselective radiosensitizer should mimic the radiosensitizing effect of oxygen but its diffusion should not be limited by its metabolic consumptions in tumors. Based on biochemical characteristics NO appears such a potential candidate [18]. Radiosensitizing properties of NO and NO-donors As early as 1957, Howard-Flanders showed that the authentic NO-gas is an efficient radiosensitizer of hypoxic bacteria, and postulated fixation of radiation-induced DNA damage, thus mimicking the effects of oxygen on DNA lesions, as primary mechanism [19]. An alternative mechanism might be interaction of NO with ironsulfur containing enzymes, resulting in inhibition of mitochondrial respiration and sparing of the natural radiosensitizer oxygen [20]. In the early 1990s, Mitchell and colleagues evaluated the radiosensitizing potential of the NONOates, which have a X-[N(O)NO]structure. When X is a secondary amine group, 2 molecules of NO per molecule of NONOate are spontaneously generated when dissolved in aqueous media. The NO level produced by the NONOates can easily be controlled, since the generation of NO is constant, predictable and independent from the pO2. Mitchell and colleagues demonstrated that spontaneous release of NO by diethylamine nonoate (DEA/NO) and dipropylamine nonoate (PAPA/NO) radiosensitizes hypoxic Chinese hamster V79 lung fibroblast to a similar extent as oxygen [21]. Griffin and colleagues reported comparable activity for DEA/NO and spermine nonoate (SPER/NO), with enhancement ratios of 2.8–3.0 in hypoxic SCK mammary carcinoma cultures [22]. However, the generation of NO by the NONOates is not tumor selective and their in vivo application at radiosensitizing concentrations would result in high NO levels in the circulation, causing vasoactive complications (septic-like shock). To be able to decrease the concentration of NO in the circulation, our laboratory explored the possibility of exploiting the hypoxic tumor microenvironment for selective generation of NO by bioreduction. We showed that bioreduction of sodium nitroprusside (SNP) and S-niroso-N-acetylpenicillamine (SNAP) results in generation of NO and radiosensitization of hypoxic mouse mammary carcinoma and human pancreatic cancer cells [23,24]. In our

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model of metabolic-induced hypoxia, the radiosensitizing effect of NO was close to that of oxygen, while no radiosensitization was observed in aerobic cells. Radiosensitization through NO-synthase (NOS) In an attempt to further decrease the extracellular concentration of NO, we decided to explore the possibility to endogenously generate NO inside tumor cells. Our laboratory was the first to demonstrate that the inducible isoform of NOS (iNOS), activated by cytokines (IL-1b + IFN-c), is capable of radiosensitizing tumor cells through endogenous production of NO [25]. We found that the iNOS pathway has a serious advantage over NO-donors, since a comparable level of radiosensitization was achieved at a 10–30 times lower extracellular NO background, which is favorable for in vivo applications. The levels of NO required for this radiosensitizing effect are relatively high and can be produced by the inducible isoform of NOS only. The vasodilating effects of NO occur at lower levels, which are produced by the endothelial isoform in blood vessels. Jordan and colleagues reported that NO-dependent modifiers of tumor hemodynamics were able to radiosensitize experimental tumors in vivo. Three strategies were considered for that purpose; administration of isosorbide dinitrate [26], slow infusion of insulin [27], and electrical stimulation of the host tissue [28].The first treatment acted by a direct release of exogenous NO, whereas the others were shown to be mediated by endogenous NO production through activation of the endothelial isoform of NOS (eNOS). All treatments were able to increase the level of oxygenation in rat tumor models. This was caused by an increased tumor blood flow and/or a reduced oxygen consumption. Regulation of the iNOS promoter in murine tumor cells To choose an optimal iNOS induction schedule for radiosensitizing purposes, we studied the transcriptional activation of iNOS in murine EMT-6 mammary carcinoma cells. It is well documented that full activation of the murine iNOS gene requires co-operation of two promoter regions, located from 40 to 300 bp (region I) and 900 to 1100 bp (region II) upstream of the TATA box (Fig. 3). Standard combinations like interferon (IFN)-c + interleukin (IL)-1b, prime iNOS activation via an interferon-responsive element (ISRE) or an interferon-c activated site (GAS) in region II. To maintain a high iNOS transcription rate, a second stimulus toward region I is required and can be provided by IL-1b or lipopolysaccharide (LPS) via the nuclear factor (NF)-jB signaling pathway [29]. This pathway involves kinase IKKa-induced phosphorylation and proteasome-mediated degradation of the inhibitory IjB subunit in the cytoplasm followed by translocation of the p50/p65 NF-jB complex to the nucleus and its binding to the iNOS promoter. In macrophages, the second stimulus may be provided by hypoxia via hypoxia-inducible factor-1 (HIF-1) that has a binding site in region I [30]. We examined the effect of hypoxia on iNOS induction in tumor cells. Our data confirmed that chronic hypoxia up-regulates the mRNA and protein expression of iNOS exposed to IFN-c + IL-1b. Low concentrations of cytokines in 1% but not in 21% induced a remarkable increase in NO production and a 1.8-fold hypoxic tumor cell radiosensitization. Hence, chronic hypoxia may potentially be exploited for selective radiosensitization of hypoxic tumors through the iNOS pathway [31]. The dual role of NF-jB in tumor cell radioresponse We suspected that NF-jB may have conflicting roles of in the radioresponse of tumor cells, keeping in mind that NF-jB signaling is triggered by diverse stimuli and involved in the regulation of

Kinase IKKα IL-1β hypoxia LPS, lipid A P IκB

IκB

proteasome

NF-κB cytoplasm nucleus NF-κB NF-κB

dNF-κB GAS ISRE

-1100

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pNF-κB HIF-1α

-300

-40

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iNOS +132

Fig. 3. NF-jB signaling towards the iNOS promoter. Pro-inflammatory stimuli (e.g. IL-1b, LPS and lipid A) and hypoxia may activate NF-jB signaling by kinase IKKainduced phosphorylation and proteasome-mediated degradation of the inhibitory IjB subunit in the cytoplasm, followed by translocation of the NF-jB complex to the nucleus. Full activation of the murine iNOS gene requires co-operation of two promoter regions, located from 40 to 300 bp (region I) and 900 to 1100 bp (region II) upstream of the TATA box.

multiple downstream genes. Extensive literature strongly suggested that dysregulation or constitutive activation of NF-jB is linked to tumorigenesis, angiogenesis and metastasis, and that it protects tumor cells from radiation damage [32,33]. Consistently, NF-jB inhibition has been used as an approach to radiosensitize tumor cells, aiming at stimulating apoptosis and inhibiting DNA repair. This approach did work, but disruption of NF-jB signaling by indomethacin, by the proteasome inhibitors MG-132 and PS-341 or by transfection with the super-repressor IjBa resulted in a moderate radiosensitization by 1.2–1.4 times only [34–36]. The range of radiosensitization covered by NF-jB inhibition was not impressive but, we considered, it might be amplified under hypoxic conditions, which were missing in the reports mentioned above. Our rationale was: (a) it is difficult to further improve the radioresponse of already radiosensitive aerobic tumor cells, and (b) hypoxic rather than aerobic cells are an obstacle for radiotherapy. Therefore, we treated EMT-6 cells with phenylarsine oxide (PAO) or lactacystin (a proteasome inhibitor) to inhibit NF-jB, and afterwards analyzed their hypoxic cell radioresponse. Once again, the increase in radioresponse was rather moderate (up to 1.4-fold), but the picture turned upside down when NF-jB was pre-activated by bacterial lipopolysaccharide (LPS) to imitate its increased basal activity in tumors. Instead of radiosensitization, PAO and lactacystin drastically impaired (by >2 times) the radioresponse of hypoxic EMT-6 cells through disruption of NF-jB signaling towards the iNOS gene [37]. Taken together, our findings suggest that the radiosensitizing effects of NF-jB inhibitors may be seriously compromised through iNOS, one of its downstream targets. Secondly, this counteraction may be unmasked only in hypoxia, which is a major cause of tumor radioresistance. Finally, the benefit of radiosensitization obtained through activated NF-jB signaling towards the iNOS gene (2- to 2.5-fold) is much more promising than that caused by NF-jB inhibition (1.4-fold). This comparison encouraged us to look for a good pre-clinical candidate to activate the NF-jB/ iNOS/NO pathway.

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Lipid A analogs and hypoxic tumor cell radiosensitization During the last decade, the mechanism and profile of the NF-jB signaling pathway has been clarified in many types of mammalian cells, activated by diverse stimuli such as TNF-a, IL-1b and LPS. Perhaps one of the best studied and widely used stimuli was and still remains to be LPS, the major component of the outer membrane of gram-negative bacteria. This endotoxin and immunostimulator is known to activate monocytes/macrophages through the Toll-like receptor (TLR)4/MyD88–receptor complex, which finally results in NF-jB controlled secretion of a variety of pro-inflammatory mediators including NO, TNF-a and IL-6 [38]. An attempt to use LPS in cancer immunotherapy was not successful, since strong septic-like toxicity could not be prevented at a dose as low as 4 ng/kg [39]. Because of that, the bioactive component of LPS, lipid A, was isolated and its simplified synthetic analogs MPL (monophosphoryl lipid A), ONO-4007 and OM-174 showed promising antitumor effects in experimental models [40–44]. These derivatives appeared to be much better tolerated in preclinical and clinical studies, while preserving the immunomodulating activities of LPS [45]. The questions we raised were: (1) Is lipid A as active as LPS in iNOS induction and radiosensitization? (2) Does lipid A cause radiosensitization at plasma relevant concentrations? (3) Does lipid A activate iNOS in EMT-6 tumor cells through NF-jB signaling? As we deduced from the LPS chemistry, lipid A did closely mimic the activity of LPS to induce iNOS in hypoxia but not in normoxia. What was similar and predictable, the range of radiosensitizing effects up to 2.5-fold was observed in hypoxic but not aerobic EMT-6 tumor cells [46]. What was different and worthy to mention, lipid A displayed a 30–100 times less potency and hence was routinely used at 3–30 lg/ml. Such concentrations were, however, plasma achievable in cancer patients, as phase I trials with ONO-4007 and OM-174 have shown [47,48]. What was unclear and worthy to explore is the role the of the NF-jB signaling pathway towards the iNOS gene, considering the considerable shift in active concentrations. Two conclusions emerged from our transfection experiments using an NF-jB tandem and an array of plasmids containing either deletions or mutations at critical points in the two regions of the iNOS promoter that bind transcription factors. First, the role of NF-jB is crucial in the lipid A-induced transcriptional activation of iNOS, since the activity of the NF-jB tandem and the wild type iNOS promoter was significantly increased. Second, lipid A-driven signaling predominantly targets the proximal but not distal NF-jB binding site of the iNOS promoter, which fits to the background of LPS [29,49]. Therefore, the difference between LPS and lipid A observed in our experiments presumably reflected a modulated affinity of lipid A for the TLR4 receptor, as a result of the carbohydrate core removal from LPS. The similar if not identical profile of lipid A/LPS activities was very helpful for us to profit from the extensive background on the very toxic, and sometimes fatal, substance LPS in the domain of immunology. Why we turned to immunology being convinced radiobiologists is explained in the next paragraph.

although, the inflammatory mediators TNF-a and NO have been repeatedly used as biochemicals to radiosensitize tumor cells [21,23,24,52,53]. We decided to explore whether the proinflammatory tumor infiltrate may be exploited for tumor cell radiosensitization. As a first step, the macrophage-like RAW 264.7 cell line was used to model the interaction between pro-inflammatory and tumor cells [54]. RAW 264.7 macrophages were activated with plasma-relevant concentrations of lipid A, and the conditioned medium was screened for cytokine secretion by ELISA and for the oxidative NO metabolite nitrite. The use of RAW 264.7 cells appeared to be a valid experimental model, since the spectrum of released mediators was generally matching that of native macrophages [55]. Indeed, TNF-a and IL-6 were produced at the highest rates. Both cytokines are known to be co-expressed by monocytes/macrophages during the acute phase of inflammation and immunity. Second, IL-12 and IL-18 were produced as well, which illuminates paracrine signaling from macrophages to T/NK cells, leading to the secondary production of IFN-c [56]. This cascade was omitted in our model, which was based on a pure macrophage like cell line. Two possible mechanisms of radiosensitization were identified: (a) indirect, through induction of the iNOS/NO pathway in EMT-6 cells by macrophage-released mediators, and (b) direct, through induction of the iNOS/NO pathway in RAW 264.7 macrophages and diffusion of NO to bystander tumor cells (Fig. 4). Macrophages and hypoxia appear to be independent partners that co-localize in the same tumor regions [50,51]. Indeed, hypoxia may up-regulate the iNOS expression in tumor cells only if a basal level of iNOS or a high transcriptional inducibility of iNOS is displayed, because of the genetic pattern of the tumor cells. Let us name such a pattern as iNOS-positive, which seems to be true for at least some types of tumors [57–65]. What about iNOS-negative tumors or tumor cell subpopulations, wherein the iNOS/NO pathway is genetically silenced? In such a case, neither hypoxia nor macrophage-released cytokines look promising anymore. This is the situation wherein macrophages are supposed to make the difference, since they are an alternative source of NO. To our knowledge, an iNOS-negative pattern of macrophages has little if any chance to exist, regarding that macrophages are naturally programmed to deliver a high output of NO upon proper activation. It appears from our experimental data, that this activation may require IFN-c next to lipid A, to benefit from their synergism based on the cooperative signaling of NF-jB and JAK/STAT towards the iNOS promoter [29]. To study the possible cooperation between immune and tumor cells through interferon-c, we modeled the immune cells in the proinflammatory infiltrate by splenocytes, which contain both macrophages and IFN-c producing T/NK-cells. Stimu-

lipid A analogues

Macrophages T/NK T/NK--cells

Role of the proinflammatory tumor infiltrate in radioresponse Solid tumors contain a complex network of inflammatory cells (e.g., macrophages, T/NK-cells), which are re-programmed to stimulate (rather than to inhibit) tumorigenesis, through the secretion of several growth and pro-angiogenic factors [50,51]. Such a mechanism was described for breast, cervix and bladder carcinomas, wherein an increased density of tumor-associated macrophages was correlated with poor prognosis. In the past, an idea to exploit inflammatory cells in tumor immunotherapy was regularly revisited but never realized. Surprisingly, an idea to exploit inflammatory cells in tumor radiotherapy was even never explored,

NO

tumor cells

cytokines

iNOS mediated NO production in tumor cells

Fig. 4. Activated macrophages may radiosensitize hypoxic tumor cells by two iNOS-dependent mechanisms: (a) directly through the release of NO, or (b) indirectly through the secretion of pro-inflammatory mediators which in turn activate NO production within tumor cells.

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lation of splenocytes by the lipid A analog OM-174 resulted in the production of IL-12, IL-18 and IFN-c. This caused up to 2.1-fold radiosensitization of EMT-6 tumor cells, which was associated with the iNOS-mediated production of NO. Both iNOS activation and radiosensitization were counteracted by neutralizing antibodies against IFN-c or against IL-12/18 [66]. Lipid A analogs appear therefore be a potential new class of tumor cell radiosensitizers through activation of the IFN-c/iNOS pathway. In conclusion, NO is a potent hypoxic cell radiosensitizer, but at high levels. Hence, the required doses of NO donors cannot be used in the clinic because of vasoactive complications. Our laboratory demonstrated that iNOS, activated by pro-inflammatory cytokines, is capable of radiosensitizing tumor cells through endogenous production of NO, at non-toxic extracellular concentrations. We observed that this radiosensitizing effect is transcriptionally controlled by hypoxia and by NF-jB. Tumor-associated immune cells may contribute to the iNOS-mediated radiosenisitization by the generation of pro-inflammatory cytokines and NO, which may diffuse towards bystander tumor cells. Our findings indicate a rationale for combining immunostimulatory and radiosensitizing strategies in the future. References [1] P. Howard-Flanders, D. Moore, The time interval after pulsed irradiation within which injury to bacteria can be modified by dissolved oxygen. I. A search for an effect of oxygen 0.02 second after pulsed irradiation, Radiat. Res. 9 (1958) 422– 437. [2] E.J. Hall, Radiobiology for the Radiologist, Lippincott Williams & Wilkins, 2000. [3] R.H. Thomlinson, L.H. Gray, The histological structure of some human lung cancers and the possible implications for radiotherapy, Br. J. Cancer 9 (1955) 539–549. [4] J.M. Brown, Evidence for acutely hypoxic cells in mouse tumours, and a possible mechanism of reoxygenation, Br. J. Radiol. 52 (1979) 650–656. [5] D.J. Chaplin, P.L. Olive, R.E. Durand, Intermittent blood flow in a murine tumor: radiobiological effects, Cancer Res. 47 (1987) 597–601. [6] P. Vaupel, K. Schlenger, C. Knoop, M. Hockel, Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized O2 tension measurements, Cancer Res. 51 (1991) 3316–3322. [7] A.W. Fyles, M. Milosevic, R. Wong, M.C. Kavanagh, M. Pintilie, A. Sun, W. Chapman, W. Levin, L. Manchul, T.J. Keane, R.P. Hill, Oxygenation predicts radiation response and survival in patients with cervix cancer, Radiother. Oncol. 48 (1998) 149–156. [8] P. Stadler, A. Becker, H.J. Feldmann, G. Hansgen, J. Dunst, F. Wurschmidt, M. Molls, Influence of the hypoxic subvolume on the survival of patients with head and neck cancer, Int. J. Radiat. Oncol. Biol. Phys. 44 (1999) 749–754. [9] J.M. Brown, W.R. Wilson, Exploiting tumour hypoxia in cancer treatment, Nat. Rev. Cancer 4 (2004) 437–447. [10] Radiotherapy and hyperbaric oxygen. Report of a Medical Research Council Working Party. Lancet 2 (1978). 881-884. [11] D.A. Fein, W.R. Lee, A.L. Hanlon, J.A. Ridge, C.J. Langer, W.J. Curran Jr., L.R. Coia, Pretreatment hemoglobin level influences local control and survival of T1-T2 squamous cell carcinomas of the glottic larynx, J. Clin. Oncol. 13 (1995) 2077– 2083. [12] J. Overgaard, H.S. Hansen, L. Specht, M. Overgaard, C. Grau, E. Andersen, J. Bentzen, L. Bastholt, O. Hansen, J. Johansen, L. Andersen, J.F. Evensen, Five compared with six fractions per week of conventional radiotherapy of squamous-cell carcinoma of head and neck: DAHANCA 6 and 7 randomised controlled trial, Lancet 362 (2003) 933–940. [13] M. Henke, R. Laszig, C. Rube, U. Schafer, K.D. Haase, B. Schilcher, S. Mose, K.T. Beer, U. Burger, C. Dougherty, H. Frommhold, Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: randomised, double-blind, placebo-controlled trial, Lancet 362 (2003) 1255– 1260. [14] J.H. Kaanders, L.A. Pop, H.A. Marres, I. Bruaset, F.J. van den Hoogen, M.A. Merkx, A.J. van der Kogel, ARCON: experience in 215 patients with advanced headand-neck cancer, Int. J. Radiat. Oncol. Biol. Phys. 52 (2002) 769–778. [15] G.E. Adams, M.S. Cooke, Electron-affinic sensitization. I. A structural basis for chemical radiosensitizers in bacteria, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 15 (1969) 457–471. [16] S. Dische, Chemical sensitizers for hypoxic cells: a decade of experience in clinical radiotherapy, Radiother. Oncol. 3 (1985) 97–115. [17] W.A. Denny, W.R. Wilson, Tirapazamine: a bioreductive anticancer drug that exploits tumour hypoxia, Expert. Opin. Investig. Drugs 9 (2000) 2889–2901. [18] D.A. Wink, J.B. Mitchell, Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide, Free Radic. Biol. Med. 25 (1998) 434–456. [19] P. Howard-Flanders, Effect of nitric oxide on the radiosensitivity of bacteria, Nature 180 (1957) 1191–1192.

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