Quantitative Ultrasound Characterization of Tumor Cell Death: Ultrasound-Stimulated Microbubbles for Radiation Enhancement

The aim of this study was to assess the efficacy of quantitative ultrasound imaging in characterizing cancer cell death caused by enhanced radiation treatments. This investigation focused on developing this ultrasound modality as an imaging-based non-invasive method that can be used to monitor therapeutic ultrasound and radiation effects. High-frequency (25 MHz) ultrasound was used to image tumor responses caused by ultrasound-stimulated microbubbles in combination with radiation. Human prostate xenografts grown in severe combined immunodeficiency (SCID) mice were treated using 8, 80, or 1000 µL/kg of microbubbles stimulated with ultrasound at 250, 570, or 750 kPa, and exposed to 0, 2, or 8 Gy of radiation. Tumors were imaged prior to treatment and 24 hours after treatment. Spectral analysis of images acquired from treated tumors revealed overall increases in ultrasound backscatter intensity and the spectral intercept parameter. The increase in backscatter intensity compared to the control ranged from 1.9±1.6 dB for the clinical imaging dose of microbubbles (8 µL/kg, 250 kPa, 2 Gy) to 7.0±4.1 dB for the most extreme treatment condition (1000 µL/kg, 750 kPa, 8 Gy). In parallel, in situ end-labelling (ISEL) staining, ceramide, and cyclophilin A staining demonstrated increases in cell death due to DNA fragmentation, ceramide-mediated apoptosis, and release of cyclophilin A as a result of cell membrane permeabilization, respectively. Quantitative ultrasound results indicated changes that paralleled increases in cell death observed from histology analyses supporting its use for non-invasive monitoring of cancer treatment outcomes.     

Dll4-Notch Signalling Blockade Synergizes Combined Ultrasound-Stimulated Microbubble and Radiation Therapy in Human Colon Cancer Xenografts

Tumour vasculature acts as an essential lifeline for tumour progression and facilitates metastatic spread. Novel vascular targeting strategies aiming to sustain vascular shutdown could potentially induce substantial damage, resulting in a significant tumour growth delay. We investigated the combination of two novel complementary vascular targeting agents with radiation therapy in a strategy aiming to sustain vascular disruption. Experiments were carried out with delta-like ligand 4 (Dll4) blockade (angiogenesis deregulator) treatment administered in combination with a radiation-based vascular destruction treatment in a highly aggressive well-perfused colon cancer tumour line implanted in female athymic nude mice. Tumours were treated with permutations of radiation, ultrasound-stimulated microbubbles (USMB) and Dll4 monoclonal antibody (mAb). Tumour vascular response was assessed with three-dimensional power Doppler ultrasound to measure active flow and immunohistochemistry. Tumour response was assessed with histochemical assays and longitudinal measurements of tumour volume. Our results suggest a significant tumour response in animals treated with USMB combined with radiation, and Dll4 mAb, leading to a synergistic tumour growth delay of up to 24 days. This is likely linked to rapid cell death within the tumour and a sustained tumour vascular shutdown. We conclude that the triple combination treatments cause a vascular shutdown followed by a sustained inhibition of angiogenesis and tumour cell death, leading to a rapid tumour vascular-based ‘collapse’ and a significant tumour growth delay.     

Targeting integrins and PI3K/Akt-mediated signal transduction pathways enhances radiation-induced anti-angiogenesis

The integrins and PI3K/Akt are important mediators of the signal transduction pathways involved in tumor angiogenesis and cell survival after exposure to ionizing radiation. Selective targeting of either integrins or PI3K/Akt can radiosensitize tumors. In this study, we tested the hypothesis that the combined inhibition of integrin alphanubeta3 by cRGD and PI3K/Akt by LY294002 would significantly enhance radiation-induced inhibition of angiogenesis by vascular endothelial cells. Treatment with cRGD inhibited the adhesion and tube formation of human umbilical vein endothelial cells (HUVECs). The inhibitory effect was further increased when cRGD and LY294002 were applied simultaneously. Both radiation and cRGD induced Akt phosphorylation, up-regulated COX2 expression, and increased PGE2 production in HUVECs. Treatment with LY294002 effectively inhibited radiation- and cRGD-induced Akt phosphorylation and up-regulation of COX2 and increased apoptosis of HUVECs. The combined use of cRGD and LY294002 enhanced radiation-induced cell killing. The clonogenic survival of HUVECs was decreased from 34% with 2 Gy radiation to 4% with these agents combined. These results demonstrate that combined use of ionizing radiation, cRGD and LY294002 inhibited multiple signaling transduction pathways involved in tumor angiogenesis and enhanced radiation-induced effects on vascular endothelial cells.     

Microbeam radiation therapy alters vascular architecture and tumor oxygenation and is enhanced by a galectin-1 targeted anti-angiogenic peptide

In this study, we sought to determine the therapeutic potential of variably sized (50 μm or 500 μm wide, 14 mm tall) parallel microbeam radiation therapy (MRT) alone and in combination with a novel anti-angiogenic peptide, anginex, in mouse mammary carcinomas (4T1)--a moderately hypoxic and radioresistant tumor with propensity to metastasize. The fraction of total tumor volume that was directly irradiated was approximately 25% in each case, but the distance between segments irradiated by the planar microbeams (width of valley dose region) varied by an order of magnitude from 150-1500 μm corresponding to 200 μm and 2000 μm center-to-center inter-microbeam distances, respectively. We found that MRT administered in 50 μm beams at 150 Gy was most effective in delaying tumor growth. Furthermore, tumor growth delay induced by 50 μm beams at 150 Gy was virtually indistinguishable from the 500 μm beams at 150 Gy. Fifty-micrometer beams at the lower peak dose of 75 Gy induced growth delay intermediate between 150 Gy and untreated tumors, while 500 μm beams at 75 Gy were unable to alter tumor growth compared to untreated tumors. However, the addition of anginex treatment increased the relative tumor growth delay after 500 μm beams at 75 Gy most substantially out of the conditions tested. Anginex treatment of animals whose tumors received the 50 μm beams at 150 Gy also led to an improvement in growth delay from that induced by the comparable MRT alone. Immunohistochemical staining for CD31 (endothelial cells) and αSMA (smooth muscle pericyte-associated blood vessels as a measure of vessel normalization) indicated that vessel density was significantly decreased in all irradiated groups and pericyte staining was significantly increased in the irradiated groups on day 14 after irradiation. The addition of anginex treatment further decreased the mean vascular density in all combination treatment groups and further increased the amount of pericyte staining in these tumors. Finally, evidence of tumor hypoxia was found to decrease in tumors analyzed at 1-14 days after MRT in the groups receiving 150 Gy peak dose, but not 75 Gy peak dose. Our results suggest that tumor vascular damage induced by MRT at these potentially clinically acceptable peak entrance doses may provoke vascular normalization and may be exploited to improve tumor control using agents targeting angiogenesis.     

Low Dose Fractionated Radiation Potentiates the Effects of Taxotere in Nude Mice Xenografts of Squamous Cell Carcinoma of Head and Neck

This study evaluated the combined effect of Low Dose Fractionated Radiation (LDFRT) and Taxotere (TXT) therapy on the growth of SCCHN (squamous cell carcinoma of head and neck; SQ-20B, a p53 mutant SCCHN cell line) tumors in a nude mouse model to exploit the increased hyper radiation sensitivity (HRS) phenomenon present in G2M cell cycle phase when induced by low doses of radiation that was demonstrated in in-vitro settings. Seventy-eight animals were randomized into one control group and 5 treatment groups (treatments were administered weekly for six weeks). Tumor regression was observed in all the groups, however, tumor regression was not significant in 2 Gy or TXT or 2 Gy plus TXT treated groups when compared to control group. The tumor regression was significant in both the LDFRT group (p     

Antitumor Effects of Combining Docetaxel (Taxotere) with the Antivascular Action of Ultrasound Stimulated Microbubbles

Ultrasound stimulated microbubbles (USMB) are being investigated for their potential to promote the uptake of anticancer agents into tumor tissue by exploiting their ability to enhance microvascular permeability. At sufficiently high ultrasound transmit amplitudes it has also recently been shown that USMB treatments can, on their own, induce vascular damage, shutdown blood flow, and inhibit tumor growth. The objective of this study is to examine the antitumor effects of ‘antivascular’ USMB treatments in conjunction with chemotherapy, which differs from previous work which has sought to enhance drug uptake with USMBs by increasing vascular permeability. Conceptually this is a strategy similar to combining vascular disrupting agents with a chemotherapy, and we have selected the taxane docetaxel (Taxotere) for evaluating this approach as it has previously been shown to have potent antitumor effects when combined with small molecule vascular disrupting agents. Experiments were conducted on PC3 tumors implanted in athymic mice. USMB treatments were performed at a frequency of 1 MHz employing sequences of 50 ms bursts (0.00024 duty cycle) at 1.65 MPa. USMB treatments were administered on a weekly basis for 4 weeks with docetaxel (DTX) being given intravenously at a dose level of 5 mg/kg. The USMB treatments, either alone or in combination with DTX, induced an acute reduction in tumor perfusion which was accompanied at the 24 hour point by significantly enhanced necrosis and apoptosis. Longitudinal experiments showed a modest prolongation in survival but no significant growth inhibition occurred in DTX–only and USMB-only treatment groups relative to control tumors. The combined USMB-DTX treatment group produced tumor shrinkage in weeks 4–6, and significant growth inhibition and survival prolongation relative to the control (p<0.001), USMB-only (p<0.01) and DTX-only treatment groups (p<0.01). These results suggest the potential of enhancing the antitumor activity of docetaxel by combining it with antivascular USMB effects.     

Anti-tumor activities of the angiogenesis inhibitors interferon-inducible protein-10 and the calreticulin fragment vasostatin

Tumor growth depends upon an adequate supply of oxygen and nutrients achieved through angiogenesis and maintenance of an intact tumor vasculature. Therapy with individual agents that target new vessel formation or existing vessels has suppressed experimental tumor growth, but rarely resulted in the eradication of tumors. We therefore tested the combined anti-tumor activity of vasostatin and interferon-inducible protein-10 (IP-10), agents that differently target the tumor vasculature. Vasostatin, a selective and direct inhibitor of endothelial cell proliferation, significantly reduced Burkitt tumor growth and tumor vessel density. IP-10, an "angiotoxic" chemokine, caused vascular damage and focal necrosis in Burkitt tumors. When combined, vasostatin plus IP-10 reduced tumor growth more effectively than each agent alone, but complete tumor regression was not observed. Microscopically, these tumors displayed focal necrosis and reduction in vessel density. Combination therapy with the inhibitors of angiogenesis vasostatin and IP-10 is effective in reducing the rate of tumor growth but fails to induce tumor regression, suggesting that curative treatment may require supplemental drugs targeting directly the tumor cells.     

The effect of hyperthermia on radiation-induced carcinogenesis

Ten groups of mice were exposed to either a single (30 Gy) or multiple (six fractions of 6 Gy) X-ray doses to the leg. Eight of these groups had the irradiated leg made hyperthermic for 45 min immediately following the X irradiation to temperatures of 37 to 43 degrees C. Eight control groups had their legs made hyperthermic with a single exposure or six exposures to heat as the only treatment. In mice exposed to radiation only, the postexposure subcutaneous temperature was 36.0 +/- 1.1 degrees C. Hyperthermia alone was not carcinogenic. At none of the hyperthermic temperatures was the incidence of tumors in the treated leg different from that induced by X rays alone. The incidence of tumors developing in anatomic sites other than the treated leg was decreased in mice where the leg was exposed to hyperthermia compared to mice where the leg was irradiated. A systemic effect of local hyperthermia is suggested to account for this observation. In mice given single X-ray doses and hyperthermia, temperatures of 37, 39, or 41 degrees C did not influence radiation damage as measured by the acute skin reactions. A hyperthermic temperature of 43 degrees C potentiated the acute radiation reaction (thermal enhancement factor 1.1). In the group subjected to hyperthermic temperatures of 37 or 39 degrees C and X rays given in six fractions, the skin reaction was no different from that of the group receiving X rays alone. Hyperthermic temperatures of 41 and 43 degrees C resulted in a thermal enhancement of 1.16 and 1.36 for the acute skin reactions. From Day 50 to Day 600 after treatment, the skin reactions showed regular fluctuations with a 150-day periodicity. Following a fractionated schedule of combined hyperthermia and X rays, late damage to the leg was less than that following X irradiation alone. Mice subjected to X rays and hyperthermic temperatures of 41 and 43 degrees C had a lower median survival time than the mice treated with hyperthermia alone. This effect was not associated with tumor incidence.     

Advantage of combining NLCQ-1 (NSC 709257) with radiation in treatment of human head and neck xenografts

NLCQ-1 (NSC 709257), a hypoxia-selective cytotoxin that targets DNA through weak intercalation, was investigated for efficacy in combination with single or fractionated radiotherapy of human head and neck xenografts. A staged tumor experiment was performed in tumor-bearing female athymic nude mice that were locally irradiated with or without NLCQ-1. Tumor hypoxia was assessed by immunohistochemistry for pimonidazole adducts in tumors of varying weight. Fractionated radiation, depending on the dose, was administered either once daily for 4 days or once daily for 4 days followed by a 7-day rest and repeat. NLCQ-1 was administered i.p. at 15 mg/kg alone or 45 min before each radiation dose. Hypoxia (1-52%) was detected in all tumors and was positively correlated with tumor size. NLCQ-1 alone resulted in about 10 days of tumor growth delay, measured at sixfold the tumor's original size, without causing toxicity. All combination treatments with NLCQ-1 were more effective than treatments with radiation alone. Radiation at 1 Gy given once daily for 4 days on days 20 and 30 caused 3.5 days of tumor growth delay, whereas in combination with NLCQ-1 it caused 14.5 days of growth delay. Radiation at 5 Gy given in two doses 10 days apart resulted in 3.5 days of tumor growth delay, whereas more than 20 additional days of delay were observed in combination with NLCQ-1. Radiation given as a single dose of 10 Gy resulted in about 7 days of tumor growth delay, whereas in combination with NLCQ-1 about 30 additional days of delay were seen. These results suggest a significant advantage in combining radiation with NLCQ-1 in treatment of human head and neck tumors, which are known to have hypoxic areas.     

Radiation-induced vascular damage in tumors: implications of vascular damage in ablative hypofractionated radiotherapy (SBRT and SRS)

We have reviewed the studies on radiation-induced vascular changes in human and experimental tumors reported in the last several decades. Although the reported results are inconsistent, they can be generalized as follows. In the human tumors treated with conventional fractionated radiotherapy, the morphological and functional status of the vasculature is preserved, if not improved, during the early part of a treatment course and then decreases toward the end of treatment. Irradiation of human tumor xenografts or rodent tumors with 5-10 Gy in a single dose causes relatively mild vascular damages, but increasing the radiation dose to higher than 10 Gy/fraction induces severe vascular damage resulting in reduced blood perfusion. Little is known about the vascular changes in human tumors treated with high-dose hypofractionated radiation such as stereotactic body radiotherapy (SBRT) or stereotactic radiosurgery (SRS). However, the results for experimental tumors strongly indicate that SBRT or SRS of human tumors with doses higher than about 10 Gy/fraction is likely to induce considerable vascular damages and thereby damages the intratumor microenvironment, leading to indirect tumor cell death. Vascular damage may play an important role in the response of human tumors to high-dose hypofractionated SBRT or SRS.     

Modification of the effects of hyperthermia and neutron radiation on the activity of acid phosphatase in CaNT tumors.

Acid phosphatase activity was measured in implanted murine CaNT tumors of varying volumes. There is a clear monotonically increasing relation between acid phosphatase activity and tumor volume. Also the tumors were subjected to either induced artificial hypoxia or hyperthermia (41.0 degrees C) alone, or combined with neutron irradiation (3.8 Gy). Changes in the activity of this enzyme following radiation damage could reflect tissue damage associated with metabolic disturbances. The effect on enzyme activity after sequential hyperthermia and neutron irradiation is not synergistic, as is shown in the quantitative experimental data. This implies that the mechanisms of heat damage differ from that of neutron beam damage, as reflected by acid phosphatase activity. The CaNT tumor was also shown to be thermosensitive after administration of mitoxantrone. Finally, the role of exogenous ATP was shown to provide heat protection by modification of those thermal effects resulting in the activity of acid phosphatase. The augmentation of this hydrolytic enzyme probably represents initial metabolic damage in the tumor after different modalities of radiation alone, or combined with mitoxantrone and exogenous ATP.     

Low-level X-radiation effects on functional vascular changes in Syrian hamster cheek pouch epithelium during hydrocarbon carcinogenesis

Effects of repeated low-level X radiation on functional microvascular changes in hamster cheek pouch epithelium during and following carcinogenesis by 7,12-dimethylbenz[a]anthracene (DMBA) were studied. Prior studies showed enhancement of such carcinogenesis by repeated 20 rad head and neck X-radiation exposures, and it was proposed that one possible mechanism was radiogenic alteration of the functional microvasculature in a manner which favored subsequent tumor development. Hamsters were treated with either radiation, DMBA, radiation + DMBA, or no treatment. Animals were sacrificed at 3-week intervals from 0 to 39 weeks after treatments began. Pouch vascular volume and permeability changes were studied by fractional distributions of radiotracers and were analyzed by a variety of statistical methods which explored the vascular parameters, treatment types, elapsed time, presence of the carcinogen, and histopathologic changes. All treatments resulted in significant changes in vascular volume with time, while only DMBA treatments alone resulted in significant changes in vascular permeability with time. Prior to the appearances of frank neoplasms, volumetric changes in DMBA only and radiation only groups were similar, while volume changes in DMBA + radiation groups increased slowly to a peak later than in other groups and then declined steadily to levels similar to the radiation only group. As in prior studies, there were significant vascular volume differences between DMBA and DMBA + radiation groups of tumor-bearing cheek pouches. DMBA maxima were significantly higher than those of DMBA + radiation. Radiation significantly affected DMBA-associated vascular volume and permeability changes during carcinogenesis. Several possible explanations for the relationship of these changes to the enhancement of DMBA carcinogenesis include: radiation blocking normal capillary proliferative and/or dilatory responses to inflammation secondary to neoplastic changes; radiation-induced focal increases in the pericapillary connective tissue histohematic barrier, stimulating angiogenesis but reducing nutrient diffusion; radiation exposures sensitizing vascular endothelium to subsequent angiogenic stimulation from premalignant tissues; DMBA vascular and epithelial effects partially or completely blocking radiation effects on epithelial and/or endothelial cells; and radiation damage to vessel walls partially or fully inhibiting normal physiologic mechanisms of repairing DMBA damage to the vessels.     

HZE ⁵⁶Fe-ion irradiation induces endothelial dysfunction in rat aorta: role of xanthine oxidase

Ionizing radiation has been implicated in the development of significant cardiovascular complications. Since radiation exposure is associated with space exploration, astronauts are potentially at increased risk of accelerated cardiovascular disease. This study investigated the effect of high atomic number, high-energy (HZE) iron-ion radiation on vascular and endothelial function as a model of space radiation. Rats were exposed to a single whole-body dose of iron-ion radiation at doses of 0, 0.5 or 1 Gy. In vivo aortic stiffness and ex vivo aortic tension responses were measured 6 and 8 months after exposure as indicators of chronic vascular injury. Rats exposed to 1 Gy iron ions demonstrated significantly increased aortic stiffness, as measured by pulse wave velocity. Aortic rings from irradiated rats exhibited impaired endothelial-dependent relaxation consistent with endothelial dysfunction. Acute xanthine oxidase (XO) inhibition or reactive oxygen species (ROS) scavenging restored endothelial-dependent responses to normal. In addition, XO activity was significantly elevated in rat aorta 4 months after whole-body irradiation. Furthermore, XO inhibition, initiated immediately after radiation exposure and continued until euthanasia, completely inhibited radiation-dependent XO activation. ROS production was elevated after 1 Gy irradiation while production of nitric oxide (NO) was significantly impaired. XO inhibition restored NO and ROS production. Finally, dietary XO inhibition preserved normal endothelial function and vascular stiffness after radiation exposure. These results demonstrate that radiation induced XO-dependent ROS production and nitroso-redox imbalance, leading to chronic vascular dysfunction. As a result, XO is a potential target for radioprotection. Enhancing the understanding of vascular radiation injury could lead to the development of effective methods to ameliorate radiation-induced vascular damage.     

Inhibitory effect of 5-FU loaded ultrasound microbubbles on tumor growth and angiogenesis

The anti-neovascularization treatment is one of the effective strategies for tumor molecular target therapy. At present, the target and effect of the anti-neovascularization treatment is limited, and it is urgent to establish a new vascular targeting strategy to effectively treat tumors. In this work, we used high intensity focused ultrasound (HIFU) combined with targeted microbubbles to establish a molecular targeted ultrasound response microbubble for neovascular cells. Furthermore, the effects of drug loaded microbubbles on neovascularization and tumor cells were studied. The tumor vascular targeted and ultrasound-responsive microbubbles of 5-FU@DLL4-MBs were prepared by the thin-film dispersion method. The size and zeta potential of 5-FU@DLL4-MBs was about 1248 nm and −9.1 mV. 5-FU@DLL4-MBs released 5-FU showed an ultrasound-responsive manner, and had better vascular-targeting ability. Furthermore, the 5-FU@DLL4-MBs showed the strongest cytotoxic effect on HUVECs or HepG-2 cells and can be effectively internalized into the HUVECs cells. Thus, 5-FU@DLL4-MBs combined with HIFU can be considered as a potential method for antitumor angiogenesis in the future.     

Modification of radiation damage in the canine kidney by hyperthermia: a histologic and functional study

The purpose of this study was to evaluate the effect of hyperthermia on the histologic and functional response of the canine kidney, a late-responding normal tissue, to irradiation. Both kidneys were irradiated. Radiation was delivered in single doses of 0, 10, or 15 Gy. Whole-body hyperthermia was used to produce renal kidney temperatures approximating 42.0 degrees C for 60 min. Thirty-six beagles were placed randomly in the following six treatment groups: control, whole-body hyperthermia alone, 10 Gy alone, 10 Gy + whole-body hyperthermia, 15 Gy alone, and 15 Gy + whole-body hyperthermia. Renal histologic and functional changes were assessed at 1 to 9 months after therapy. No changes were seen in glomerular filtration rate or renal tissue volumes in control or hyperthermia alone groups. Renal vascular and glomerular volumes were not affected significantly by any combination of hyperthermia and/or radiation. In all groups receiving radiation, glomerular filtration rate decreased, percentage renal tubular volume decreased, and interstitial volume increased significantly after therapy. The magnitude of these changes in the functional and histologic response of the kidney and the latent period before expression of this damage were dependent on radiation dose. However, hyperthermia did not modify expression of radiation damage in the kidney based on glomerular filtration rate and histologic quantification of renal tissue components.     

The antiangiogenic agents SU5416 and SU6668 increase the antitumor effects of fractionated irradiation.

Angiogenesis is critical for tumor development, growth and metastasis. The vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) and their tyrosine kinase receptors are major regulators of angiogenesis. Radiation induces the production of VEGF, FGF and PDGF in many tumor cells. We hypothesized that inhibition of the function of these growth factors could inhibit tumor angiogenesis and thereby enhance the efficacy of radiation therapy. To test this hypothesis, we used the small molecule inhibitors SU5416 (an inhibitor for Vegf receptor) and SU6668 (an inhibitor for Vegf, Fgf and Pdgf receptors) alone and in combination with fractionated irradiation to treat C3H mice bearing SCC VII carcinomas. The SCC VII tumors express Vegf, Fgf2 (also known as bFGF), Pdgf and their associated receptors. Animals were given either SU5416 or SU6668 daily before or after irradiation (2 Gy per fraction per day for 5 days). The results from these experiments demonstrate that administration of either SU5416 or SU6668 without radiation delayed tumor growth. Administration of SU5416 at a dose of 25 mg/kg per day (the maximum tolerated effective dose) inhibited tumor growth by 17.9% on day 7 (P < 0.05 compared to untreated control mice) and produced an average tumor growth delay time of 0.5-2.0 days. When combined with fractionated irradiation, administration of SU5416 increased the inhibition of tumor growth to 50-53% on day 7 and the tumor growth delay time to 5.7-6.5 days (P < 0.001 compared with SU5416 alone; P < or = 0.05 compared with radiation alone). SU6668 alone inhibited tumor growth in a dose-dependent manner. Administration of SU6668 at a dose of 75 mg/kg per day (a suboptimal dose) inhibited tumor growth by 36% on day 7 and produced an average tumor growth delay time of 3.3 +/- 1.4 days. The combination of SU6668 with fractionated radiation increased inhibition of tumor growth to 66-70% and the tumor growth delay time from 3.3 days to 11.9 days (P < or = 0.001 compared with either radiation alone or SU6668 alone). Administration of these agents before or after irradiation produced similar results (P = 0.40 for SU5416; P = 0.98 for SU6668). SU5416 or SU6668 alone or in combination with radiation was very well tolerated with little or no toxicity. These results suggest that inhibition of Vegf, Fgf and Pdgf receptor function by SU5416 and SU6668 can enhance the efficacy of irradiation. The targeting of multiple tyrosine kinase receptors by SU6668 is more effective than inhibition of the Vegf receptor alone by SU5416 for the enhancement of tumor cell killing by fractionated irradiation.     

Single exposure to radiation produces early anti-angiogenic effects in mouse aorta

Radiation exposure can increase the risk for many non-malignant physiological complications, including cardiovascular disease. We have previously demonstrated that ionizing radiation can induce endothelial dysfunction, which contributes to increased vascular stiffness. In this study, we demonstrate that gamma radiation exposure reduced endothelial cell viability or proliferative capacity using an in vitro aortic angiogenesis assay. Segments of mouse aorta were embedded in a Matrigel-media matrix 1 day after mice received whole-body gamma irradiation between 0 and 20 Gy. Using three-dimensional phase contrast microscopy, we quantified cellular outgrowth from the aorta. Through fluorescent imaging of embedded aortas from Tie2GFP transgenic mice, we determined that the cellular outgrowth is primarily of endothelial cell origin. Significantly less endothelial cell outgrowth was observed in aortas of mice receiving radiation of 5, 10, and 20 Gy radiation, suggesting radiation-induced endothelial injury. Following 0.5 and 1 Gy doses of whole-body irradiation, reduced outgrowth was still detected. Furthermore, outgrowth was not affected by the location of the aortic segments excised along the descending aorta. In conclusion, a single exposure to gamma radiation significantly reduces endothelial cell outgrowth in a dose-dependent manner. Consequently, radiation exposure may inhibit re-endothelialization or angiogenesis after a vascular injury, which would impede vascular recovery.     

Inhibition of Intestinal Epithelial Apoptosis Improves Survival in a Murine Model of Radiation Combined Injury

World conditions place large populations at risk from ionizing radiation (IR) from detonation of dirty bombs or nuclear devices. In a subgroup of patients, ionizing radiation exposure would be followed by a secondary infection. The effects of radiation combined injury are potentially more lethal than either insult in isolation. The purpose of this study was to determine mechanisms of mortality and possible therapeutic targets in radiation combined injury. Mice were exposed to IR with 2.5 Gray (Gy) followed four days later by intratracheal methicillin-resistant Staphylococcus aureus (MRSA). While either IR or MRSA alone yielded 100% survival, animals with radiation combined injury had 53% survival (p = 0.01). Compared to IR or MRSA alone, mice with radiation combined injury had increased gut apoptosis, local and systemic bacterial burden, decreased splenic CD4 T cells, CD8 T cells, B cells, NK cells, and dendritic cells, and increased BAL and systemic IL-6 and G-CSF. In contrast, radiation combined injury did not alter lymphocyte apoptosis, pulmonary injury, or intestinal proliferation compared to IR or MRSA alone. In light of the synergistic increase in gut apoptosis following radiation combined injury, transgenic mice that overexpress Bcl-2 in their intestine and wild type mice were subjected to IR followed by MRSA. Bcl-2 mice had decreased gut apoptosis and improved survival compared to WT mice (92% vs. 42%; p<0.01). These data demonstrate that radiation combined injury results in significantly higher mortality than could be predicted based upon either IR or MRSA infection alone, and that preventing gut apoptosis may be a potential therapeutic target.     

Combined action of X-rays and nonylphenol on mouse sperm

The aim of this study was to assess the effects of 2-weeks’ X-ray and/or nonylphenol (NP) exposure on male mice’s sperm count and quality. Pzh:SFIS mice were exposed to X-rays (0.05 Gy, 0.10 Gy, 0.20 Gy) or to nonylphenol (25 mg/kg bw, 50 mg/kg bw, 100 mg/kg bw) or to both agents (0.05 Gy + 25 mg/kg bw NP, 0.10 Gy + 50 mg/kg bw NP). At 24 h and 5 weeks after the end of exposure the sperm count, morphology and frequency of DNA damage in the male germ cells were estimated. Each agent alone diminished sperm count and morphology. The dose of 0.05 Gy of X-rays decreased the frequency of DNA damage. Combined exposure to lower doses of both agents significantly improved sperm morphology and decreased the level of DNA damage compared to one agent alone. Combined exposure to higher doses reduced the frequency of DNA damage compared to the effect of the appropriate dose of NP. Results of combined exposure to low doses of both agents suggest that 0.05 Gy of X-rays stimulate the DNA damagecontrol system and in consequence repair of DNA caused by X-rays and NP. It may be correlated with increased antioxidant capacity.     

Low dose fractionated radiation potentiates the effects of taxotere in nude mice xenografts of squamous cell carcinoma of head and neck

This study evaluated the combined effect of Low Dose Fractionated Radiation (LDFRT) and Taxotere (TXT) therapy on the growth of SCCHN (squamous cell carcinoma of head and neck; SQ-20B, a p53 mutant SCCHN cell line) tumors in a nude mouse model to exploit the increased hyper radiation sensitivity (HRS) phenomenon present in G(2)/M cell cycle phase when induced by low doses of radiation that was demonstrated in in vitro settings. Seventy-eight animals were randomized into one control group and 5 treatment groups (treatments were administered weekly for six weeks). Tumor regression was observed in all the groups, however, tumor regression was not significant in 2 Gy or TXT or 2 Gy plus TXT treated groups when compared to control group. The tumor regression was significant in both the LDFRT group (p < 0.0043) and LDFRT + TXT group (p < 0.0006) when compared to other groups. A significantly prolonged tumor growth delay was observed in LDFRT group (p < 0.0081). Importantly, in combination of TXT and LDFRT, no tumor regrowth was observed in 12 out of 13 mice since LDFRT + TXT treatment caused a sustained regression of tumors for 9 weeks. Molecular analysis of resected tumor specimens demonstrated that Bax levels were elevated with concomitant increase in cytochrome c release to the cytosol of the treatment Group VI. These findings strongly suggest that LDFRT can be used in combination with TXT to potentiate the effects of drug on tumor regression through an apoptotic mode of death. Furthermore, the G(2)/M cell cycle arrest by TXT appears to be an important component of the enhanced apoptotic effect of TXT + LDFRT combined treatment.