A Novel Mechanism of High Dose Radiation Sensitization by Metformin.

Introduction: Metformin, the most widely used treatment for diabetes, is lethal to cancer cells and increases in toxicity when used in combination with radiation. In addition to various molecular and metabolic mechanisms that have been previously proposed, the studies presented provide evidence of an additional, novel mechanism of sensitization following high dose radiotherapy; the magnitude of sensitization depends on the microenvironmental levels of glucose and oxygen which are in turn affected by high dose radiation. Methods: Cancer cells (A549 and MCF7) were studied in vitro under various controlled conditions. Endpoints included clonogenic cell survival and ROS expression measured by DHE and DCFDA. CD1 nu/nu athymic mice implanted with A549 cells received metformin alone (200 mg/kg, i.p.), radiation alone (15 Gy) or a combination of metformin and radiation; the effect of treatment sequence on efficacy was assessed by tumor growth delay and histology. In a separate set of experiments, tumor blood flow was measured using a tracer clearance technique using SPECT after the administration of metformin alone, radiation alone and the combined treatment. Results: In vivo, metformin provided equally effective tumor growth delay when given 24 h after radiation as when given 1 h or 4 h before radiation, an observation not previously reported and, in fact, unexpected based on published scientific literature. When drug followed radiation, the tumors were histologically characterized by massive cellular necrosis. In vitro, cancer cells when glucose depleted and/or hypoxic were preferentially killed by metformin, in a drug dose dependent manner. A549 cells exposed to 5.0 mM of metformin was reduced seven fold in survival when in a glucose deprived as compared to a low-glucose medium (0 vs. 1.0 g/L). Finally, using a SPECT detector to follow the washout of a radioactive tracer, it was shown that a high single dose of radiosurgery (15 Gy) could dramatically inhibit blood flow and presumably diminish glucose and oxygen. Discussion: Insight into the best timing of drug and radiation administration is gained through an understanding of the mechanisms of interaction. A new mechanism of metformin sensitization by high dose radiation is proposed based on the blood flow, glucose and oxygen.

Time-dependent inhibition of pan-inflammatory cytokines mitigates radiation-induced skin injury in mice.

Radiation injury to skin poses substantial morbidity risks in the curative treatment of cancers and is also of concern in the context of radiological attack or nuclear accident scenarios. Late effects can be severe and are frequently characterized by subcutaneous fibrosis and morbidity. These experiments presented here assess the potential of MW01-2-151SRM (MW-151), a novel small-molecule inhibitor of microglial activation and associated proinflammatory cytokine/chemokine production, as a mitigator of radiation-induced skin injury. Groups of C57BL/6 mice received focal irradiation of the right hind leg at a dose of 30 Gy. Therapy was initiated either on day 3, day 7 or day 14 postirradiation and maintained subsequently for 21 days by intraperitoneal injections administered three times per week. The primary end point was skin injury, which was assessed three times a week for at least 60 days postirradiation and scored using a semi-quantitative scale. Secondary end points measured at selected times included histology (primarily H&E) and immunofluorescence labeling of various macrophage (F4-80) and inflammatory (TGF-ß, TNF-α, MMP9) markers. Relative to untreated controls, mitigation of radiation-induced skin injury in mice receiving MW-151 was highly dependent on the timing of therapy initiation. Initiation on day 3 postirradiation had no discernable effect, whereas mitigating effects were maximal following initiation on day 7 and present to a lesser degree following initiation on day 14. The response to MW-151 therapy in individual animals was essentially all-or-none and the relative benefits associated with the timing of therapy initiation primarily reflected differences in the number of responders. These data support the hypothesis that proinflammatory cytokines/chemokines play complex roles in orchestrating the response to radiation-induced skin injury and suggest that there is a critical period during which they initiate the pathogenesis resulting in late effects.

Selective inhibition of microglia-mediated neuroinflammation mitigates radiation-induced cognitive impairment.

Cognitive impairment precipitated by irradiation of normal brain tissue is commonly associated with radiation therapy for treatment of brain cancer, and typically manifests more than 6 months after radiation exposure. The risks of cognitive impairment are of particular concern for an increasing number of long-term cancer survivors. There is presently no effective means of preventing or mitigating this debilitating condition. Neuroinflammation mediated by activated microglial cytokines has been implicated in the pathogenesis of radiation-induced cognitive impairment in animal models, including the disruption of neurogenesis and activity-induced gene expression in the hippocampus. These pathologies evolve rapidly and are associated with relatively subtle cognitive impairment at 2 months postirradiation. However, recent reports suggest that more profound cognitive impairment develops at later post-irradiation time points, perhaps reflecting a gradual loss of responsiveness within the hippocampus by the disruption of neurogenesis. We hypothesized that inhibiting neuroinflammation using MW01-2-151SRM (MW-151), a selective inhibitor of proinflammatory cytokine production, might mitigate these deleterious radiation effects by preserving/restoring hippocampal neurogenesis. MW-151 therapy was initiated 24 h after 10 Gy whole-brain irradiation (WBI) administered as a single fraction and maintained for 28 days thereafter. Proinflammatory activated microglia in the dentate gyrus were assayed at 2 and 9 months post-WBI. Cell proliferation and neurogenesis in the dentate gyrus were assayed at 2 months post-WBI, whereas novel object recognition and long-term potentiation were assayed at 6 and 9 months post-WBI, respectively. MW-151 mitigated radiation-induced neuroinflammation at both early and late time points post-WBI, selectively mitigated the deleterious effects of irradiation on hippocampal neurogenesis, and potently mitigated radiation-induced deficits of novel object recognition consolidation and of long-term potentiation induction and maintenance. Our results suggest that transient administration of MW-151 is sufficient to partially preserve/restore neurogenesis within the subgranular zone and to maintain the functional integrity of the dentate gyrus long after MW-151 therapy withdrawal.

Mechanisms of radiation-induced skin injury and implications for future clinical trials.

PURPOSE: To summarize current knowledge regarding mechanisms of radiation-induced skin injury and medical countermeasures available to reduce its severity. Advances in radiation delivery using megavoltage and intensity modulated radiation therapy have permitted delivery of higher doses of radiation to well-defined tumor target tissues. Although skin is not a radiation dose-limiting tissue, injury to skin poses substantial morbidity risks in the curative treatment of cancers, especially when radiation is administered in combination with chemotherapy. In the continuum of radiation-induced skin injury, late effects are most severe being characterized by sub-cutaneous fibrosis and morbidity. The principal pathogenesis is initiated by depletion of acutely responding epithelial tissues and damage to vascular endothelial microvessels. Emerging concepts of radiation- induced skin injury suggest that the recovery of stromal stem cells and tissue repair remain chronically impaired by long-lived free radicals, reactive oxygen species, and pro-inflammatory cytokines/chemokines resulting in progressive damage after radiation exposure. CONCLUSIONS: As pathways underlying the cellular and molecular mechanisms of radiation-induced skin injury are becoming better understood, novel approaches are being developed for mitigating or treating the associated pathogenesis.

Plerixafor, a CXCR4 antagonist, mitigates skin radiation-induced injury in mice.

Even with modern 3D conformal treatments skin radiation injury can be an inadvertent complication associated with clinical radiotherapy particularly at tissue folds. It is also of concern in the context of a radiological terrorism incident or accident, since skin irradiation lowers the lethal dose of whole body radiation. We hypothesize that radiation-induced skin injury originates from a loss of stem and progenitor cells, accompanied by excessive ROS production and proinflammatory cytokines. Plerixafor, a CXCR-4 antagonist, is one of the most efficient bone marrow stem cell mobilizers and these studies were designed to experimentally assess the potential of Plerixafor to reduce skin radiation injury. The right hind legs of groups of C57BL/6 mice were exposed to radiation alone or in combination with Plerixafor. Plerixafor was administered intraperitoneally at a dose of 5 mg/kg given in two doses separated by two days and started either on day 0, 4, 7, 15 or 24 after irradiation. The primary end point was skin injury, which was assessed three times a week for at least 2 months using a semi-quantitative scale. Secondary end points measured at selected time points included histology (primarily H&E) and cytokine levels (TGF-ß and TNF-α). The acute and late skin injury in mice receiving Plerixafor was highly dependent on the timing of administration of the drug. The maximum benefit was observed when the drug was started 1 week after radiation exposure, and earlier or later administration of the drug decreased its efficacy. Secondary damage end points (cytokine levels and histologically assessed tissue thickness) provided confirmatory observations. In an attempt to gain insight into the effect of timing of administration of the agent on the mitigation effect, the ligand to CXCR4, stromal derived factor, SDF-1, was measured as a function of time after radiation exposure. Expression of SDF-1 monitored in skin as a function of time after a 30 Gy radiation exposure suggested a strong correlation between timing of administration of Plerixafor and expression of SDF-1 in irradiated skin: optimum drug administration timing coincided with maximal SDF-1 expression in the skin of irradiated mice. This report presents the first observation that CXCR4 antagonist improves both acute and late skin response to radiation exposure.

Combined atorvastatin and ramipril mitigate radiation-induced impairment of dentate gyrus neurogenesis.

Whole brain irradiation (WBI) is commonly administered therapeutically and is routinely associated with late delayed radiation injuries, manifesting as severe and irreversible cognitive impairment. Neural progenitors within the subgranular zone (SGZ) of the dentate gyrus are among the most radiosensitive cell types in the adult brain and are known to participate in hippocampal plasticity and normal cognitive function. These progenitors and the specialized SGZ microenvironment required for neuronal differentiation are the source of neurogenic potential in the adult dentate gyrus, and provide a continuous supply of immature neurons which may then migrate into the adjacent granule cell layer to become mature granule cell neurons. The extreme radiosensitivity of these progenitors and the SGZ microenvironment implicate them as potentially significant contributors to radiation-induced cognitive impairment. Previous reports suggest that statin drugs may be neuroprotective and may promote neurogenesis within the SGZ following both traumatic and ischemic brain injury. Here we investigate whether atorvastatin might similarly protect progenitors and/or preserve neurogenic potential within the SGZ when administered following radiation injury. We also investigate whether such mitigating effects might be enhanced by administering atorvastatin in combination with the angiotensin converting enzyme (ACE) inhibitor, ramipril, which has previously been shown to produce subtle mitigating effects in this context. Atorvastatin was administered to adult male Fisher 344 rats beginning 24 h post-WBI at doses of 10 and 15 Gy, and maintained daily until sacrifice at 12 weeks post-WBI. Combined atorvastatin and ramipril (atorvastatin + ramipril) were administered according to the same protocol following WBI doses of 10 Gy. Progenitor proliferation, neuronal differentiation, and microglial activation were assayed immunohistochemically. Our results indicate that chronic administration of atorvastatin is relatively ineffective as a mitigator of radiation injury in this context, whereas atorvastatin + ramipril appear to interact synergistically to potently and selectively mitigate radiation-induced disruption of neurogenic signaling within SGZ microenvironment.

Antioxidant diet supplementation starting 24 hours after exposure reduces radiation lethality.

Antioxidants mitigate radiation-induced lethality when started soon after radiation exposure, a delivery time that may not be practical due to difficulties in distribution and because the oral administration of such agents may require a delay beyond the prodromal stage of the radiation syndrome. We report the unexpected finding that antioxidant supplementation starting 24 h after total-body irradiation resulted in better survival than antioxidant supplementation started soon after the irradiation. The antioxidant dietary supplement was l-selenomethionine, sodium ascorbate, N-acetyl cysteine, alpha-lipoic acid, alpha-tocopherol succinate, and co-enzyme Q10. Total-body irradiation with 8 Gy in the absence of antioxidant supplementation was lethal by day 16. When antioxidant supplementation was started soon after irradiation, four of 14 mice survived. In contrast, 14 of 18 mice receiving antioxidant supplementation starting 24 h after irradiation were alive and well 30 days later. The numbers of spleen colonies and blood cells were higher in mice receiving antioxidant supplementation starting 24 h after irradiation than in mice receiving radiation alone. A diet supplemented with antioxidants administered starting 24 h after total-body irradiation improved bone marrow cell survival and mitigated lethality, with a radiation protection factor of approximately 1.18.

Ramipril mitigates radiation-induced impairment of neurogenesis in the rat dentate gyrus.

BACKGROUND: Sublethal doses of whole brain irradiation (WBI) are commonly administered therapeutically and frequently result in late delayed radiation injuries, manifesting as severe and irreversible cognitive impairment. Neural progenitors within the subgranular zone (SGZ) of the dentate gyrus are among the most radiosensitive cell types in the adult brain and are known to participate in hippocampal plasticity and normal cognitive function. These progenitors and the specialized SZG microenvironment required for neuronal differentiation are the source of neurogenic potential in the adult dentate gyrus, and provide a continuous supply of immature neurons which may then migrate into the adjacent granule cell layer to become mature granule cell neurons. The extreme radiosensitivity of these progenitors and the SGZ microenvironment suggests the hippocampus as a prime target for radiation-induced cognitive impairment. The brain renin-angiotensin system (RAS) has previously been implicated as a potent modulator of neurogenesis within the SGZ and selective RAS inhibitors have been implicated as mitigators of radiation brain injury. Here we investigate the angiotensin converting enzyme (ACE) inhibitor, ramipril, as a mitigator of radiation injury in this context. METHODS: Adult male Fisher 344 rats received WBI at doses of 10 Gy and 15 Gy. Ramipril was administered beginning 24 hours post-WBI and maintained continuously for 12 weeks. RESULTS: Ramipril produced small but significant reductions in the deleterious effects of radiation on progenitor proliferation and neuronal differentiation in the rat dentate gyrus following 10 Gy-WBI, but was not effective following 15 Gy-WBI. Ramipril also reduced the basal rate of neurogenesis within the SGZ in unirradiated control rats. CONCLUSIONS: Our results indicate that chronic ACE inhibition with ramipril, initiated 24 hours post-irradiation, may reduce apoptosis among SGZ progenitors and/or inflammatory disruption of neurogenic signaling within SGZ microenvironment, and suggest that angiotensin II may participate in maintaining the basal rate of granule cell neurogenesis.

Does hyperthermia increase adenoviral transgene expression or dissemination in tumors?

PURPOSE: Viral vectors used for cancer gene therapy are usually delivered by direct intratumoral administration. We studied the role of hyperthermia (HT) in vitro and in vivo in an attempt to achieve higher transfection rates (especially, larger volume of spread). MATERIALS AND METHODS: Replication-deficient adenoviruses containing either the human sodium-iodide symporter (Ad5-CMV-hNIS) or green fluorescent protein (Ad5-CMV-eGFP) as reporter genes were used. For in vitro studies, human lung cancer A549 cells were transfected with the virus and assayed for hNIS expression by radioactive pertechnetate uptake or green fluorescence activity using a gamma-counter or fluoroscopy respectively in the presence and absence of HT. For in vivo studies, A549 tumors were established intramuscularly in CD1 athymic mice. The adenoviral constructs (10(10) viral particles/tumor) were injected intratumorally when the tumors reached 10-11 mm in diameter. Different timing sequences of HT were examined and viral spread was assessed using technetium-autoradiography or GFP-fluorescence microscopy. RESULTS: In the in vitro studies, A549 cells infected with the adenoviral construct did not show any difference in gene expression level in the presence or absence of HT. In vivo, the effect of HT on the volume of gene expression in A549 tumors was highly variable with some groups of mice showing better spread in the presence of HT and others showing reduced spread with HT. CONCLUSION: Improvements in intratumoral adenoviral spread in response to hyperthermia were not consistently observed in a mouse tumor model using two quantitative endpoints of gene expression.

Radiation-induced skin injury in the animal model of scleroderma: implications for post-radiotherapy fibrosis.

BACKGROUND: Radiation therapy is generally contraindicated for cancer patients with collagen vascular diseases (CVD) such as scleroderma due to an increased risk of fibrosis. The tight skin (TSK) mouse has skin which, in some respects, mimics that of patients with scleroderma. The skin radiation response of TSK mice has not been previously reported. If TSK mice are shown to have radiation sensitive skin, they may prove to be a useful model to examine the mechanisms underlying skin radiation injury, protection, mitigation and treatment. METHODS: The hind limbs of TSK and parental control C57BL/6 mice received a radiation exposure sufficient to cause approximately the same level of acute injury. Endpoints included skin damage scored using a non-linear, semi-quantitative scale and tissue fibrosis assessed by measuring passive leg extension. In addition, TGF-beta1 cytokine levels were measured monthly in skin tissue. RESULTS: Contrary to our expectations, TSK mice were more resistant (i.e. 20%) to radiation than parental control mice. Although acute skin reactions were similar in both mouse strains, radiation injury in TSK mice continued to decrease with time such that several months after radiation there was significantly less skin damage and leg contraction compared to C57BL/6 mice (p < 0.05). Consistent with the expected association of transforming growth factor beta-1 (TGF-beta1) with late tissue injury, levels of the cytokine were significantly higher in the skin of the C57BL/6 mouse compared to TSK mouse at all time points (p < 0.05). CONCLUSION: TSK mice are not recommended as a model of scleroderma involving radiation injury. The genetic and molecular basis for reduced radiation injury observed in TSK mice warrants further investigation particularly to identify mechanisms capable of reducing tissue fibrosis after radiation injury.

Mitigation of radiation-induced skin injury by AAV2-mediated MnSOD gene therapy.

BACKGROUND: Radiation-induced, long-lived free radicals, reactive oxygen species and pro-inflammatory cytokines have been implicated in the resultant tissue injury after exposure to ionizing radiation. METHODS: An approach designed to reduce the damaging effects of reactive oxidants employs metalloenzymes of superoxide dismutase (SOD), such as MnSOD. Recombinant adeno-associated virus 2 (AAV2) provides safe and long-term expression in humans. We tested the effectiveness of AAV2-MnSOD-hrGFP, a vector expressing MnSOD and green fluorescent protein (GFP) in preclinical models. RESULTS: Infection of cultured cells with AAV2-MnSOD-hrGFP showed enhanced expression of MnSOD and GFP. Sustained expression of GFP was achieved for at least 1 month in vivo following administration of AAV2-MnSOD-hrGFP to subcutaneous tissue of C57BL/6J mice. A single subcutaneous injection of AAV2-MnSOD-hrGFP significantly mitigated acute skin injury following single dose of irradiation of either 30 or 35 Gy. CONCLUSIONS: The proof-of-concept demonstrated in the present study together with the known safety profile in humans indicate that AAV-mediated MnSOD expression has potential countermeasure utility against normal tissue injury following radiation therapy or radiological accident.

Histone deacetylase inhibitors protect against and mitigate the lethality of total-body irradiation in mice.

It was hypothesized that histone deacetylase (HDAC) inhibitors may increase survival after total-body irradiation (TBI) based on previous reports demonstrating that HDAC inhibitors stimulate the proliferation of bone marrow stem cells. Using the time for mice to lose 20% or more of their weight as the end point, two HDAC inhibitors, valproic acid and trichostatin-A, were found to reduce lethality in a dose-dependent manner. HDAC inhibitors were effective at reducing lethality when given either 24 h before or 1 h after TBI. The results indicate that HDAC inhibitors have potential for protecting against and mitigating radiation-induced lethality.

Reporter gene imaging using radiographic contrast from nonradioactive iodide sequestered by the sodium-iodide symporter.

The hypothesis that the human sodium-iodide symporter, NIS, can be used to detect NIS expression using standard radiological techniques was tested using adenoviral transduced NIS expression in human tumor xenografts grown in mice and in a naive dog prostate. Nonradioactive iodide was administered systemically to animals that 1-3 days previously had received a local injection of a replication-competent adenovirus expressing NIS under the control of the CMV promoter. The distribution of radiopacity was assessed in mouse tumors using micro-CT and a clinical X-ray machine and in the prostate of an anesthetized dog using a clinical spiral CT. Iodide sequestration and NIS expression were measured using X-ray spectrochemical analysis and fluorescence microscopy, respectively. Radiographic contrast due to NIS gene expression that was observed indicates the technique has potential for use in preclinical rodent tumor studies but probably lacks sensitivity for human use.

Differential radiation effect in tumor and normal tissue after treatment with ramipril, an angiotensin-converting enzyme inhibitor.

The angiotensin-converting enzyme inhibitor, ramipril, has been shown to mitigate radiation injury in normal tissues. Using A549 cell xenografts grown in athymic mice, we measured the effect of ramipril on radiation damage to tumors. Ramipril did not alter tumor response to radiation despite different times of drug administration with respect to radiation delivery (drug started 2 weeks before or immediately after irradiation). In contrast, using the same dose, ramipril reduced normal tissue radiation injury (30 Gy x 2 or 6 Gy x 10) as assessed by a semi-quantitative scale of skin damage and relative leg contraction. The results indicate that ramipril could offer therapeutic gain due to its different effect on normal tissues and tumors.

Mitigation of radiation-induced optic neuropathy in rats by ACE inhibitor ramipril: importance of ramipril dose and treatment time.

PURPOSE: Radiation-induced optic nerve damage was reduced by ramipril, a prodrug angiotensin-converting enzyme inhibitor (ACEI). This study was to determine the optimum dose and administration time of ramipril for mitigating radiation-induced optic neuropathy. MATERIALS AND METHOD: Adult Fischer 344 male rats were treated with a single dose radiation 30 Gy by using radiosurgical technique. After irradiation, the animals were randomly assigned into groups of different ramipril doses and administration time; control (no treatment), radiation alone, radiation+ramipril in different doses and starting times of drug. Ramipril was given 0.5-1.5 mg/kg/day and AT1R blocker Losartan 20 mg/kg/day in drinking water for 180 days. Functional endpoint with visual evoked potential (VEP) and anatomical endpoint with gross and histological analysis with immunohistochemical (IHC) stain were used. RESULTS: Normal VEP measurements in un-irradiated rats were 46.2+/-7.9 ms. There was no change of VEP value until 4 months, but was lengthened to 188.1+/-58.7 ms at 6 months after radiation. By ramipril treatment with the dose of 1.5 mg starting at 2 weeks after radiation, VEP was significantly shortened to 105.7+/-88.5 ms at 6 months. Gross and microscopic structure of the irradiated optic nerve was well preserved in the ramipril-treated group. CONCLUSION: Ramipril can mitigate the radiation-induced optic nerve damage and preserve the functional integrity of the nerve. The results support early treatment with a high dose of ramipril after radiation.

A quantitative method for measuring gene expression magnitude and volume delivered by gene therapy vectors.

This study describes a quantitative method to measure the magnitude and distribution of gene expression following local delivery of an adenoviral vector containing the human sodium iodide symporter (hNIS) reporter gene into the canine prostate. Following systemic administration of Na(99m)TcO(4), autoradiographs of prostate sections depicting hNIS-dependent (99m)TcO(4)(-) uptake were digitized and stacked to produce a three-dimensional reconstruction of gene expression. Frequency histograms reflecting hNIS gene expression magnitude and volume were used to quantify hNIS function. The method demonstrated submillimeter resolution allowing for precise measurements of gene expression magnitude and volume in vivo. The method developed here could be applied to other reporter gene systems in which the readout can be digitized from thin tissue sections.

Modification of radiation injury by ramipril, inhibitor of angiotensin-converting enzyme, on optic neuropathy in the rat.

Inhibitors of angiotensin-converting enzyme (ACE) have been used to reduce radiation-induced normal tissue injury. The present study was carried out to determine whether ramipril, one of the inhibitors of ACE, would ameliorate radiation-induced brain damage, using a well-characterized optic neuropathy model in the rat, one of the most critical and radiosensitive structures in the brain. The brains of adult Fischer rats were irradiated stereotactically with 30 Gy using a single collimated beam. Six months after irradiation and 1.5 mg/kg day(-1) ramipril (started 2 weeks after irradiation), rats were assessed for optic nerve damage functionally, using visual evoked potential, and histologically. Results show that ramipril conferred significant modification of radiation injury, since rats receiving radiation alone showed a threefold lengthening in the mean peak latency in the visual evoked potential, whereas 75% of rats receiving radiation followed by ramipril had evoked potentials that resembled those of normal untreated control rats. The histology of irradiated and ramipril-treated optic nerves appeared nearly normal, while there was significant demyelination in both optic nerves of irradiated rats. The study represents the first demonstration of prophylaxis of radiation injury by a carboxyl-containing ACE inhibitor, providing a pharmacological strategy designed to reduce radiation-induced normal tissue damage.

Arsenic trioxide enhances radiation response of 9L glioma in the rat brain.

Arsenic trioxide (ATO) at low doses induces leukemia cells to undergo apoptosis and at higher doses causes blood flow to solid tumors to shut down. To determine whether a potential synergistic interaction exists between ATO at the non-toxic dose level in the rat and radiation, the present study was carried out with orthotopic 9L malignant gliomas growing in the brains of rats. Animals died within 50 days of treatment when 12-day-old 9L gliomas growing in the brain of Fischer rats were treated with either the drug alone (8 mg/kg) or radiation alone (25 Gy). In contrast, the overall tumor cure rate exceeded 50% at a follow-up time of 120 days after the combined treatment with radiation and ATO. Long-term surviving animals showed no clinical or disproportionately enhanced histopathological changes in the brain parenchyma. Early changes in tumor physiology showed that the vascular leakage of FITC-dextran conjugates was apparent within 8 h of drug administration. Last, the use of diffusion magnetic resonance imaging as an early surrogate marker of therapeutic efficacy corroborated the effects of drug with and without radiation on brain histology and animal survival.

Synergistic interaction with arsenic trioxide and fractionated radiation in locally advanced murine tumor.

We have shown previously that arsenic trioxide (ATO) preferentially shutsdown tumor blood flow, leading to pronounced cell death in the central part of the solid tumor with a minimal effect on the surrounding normal tissues. On the basis of the histopathological and tumor perfusion studies, we hypothesized that the tumor control rate of locally advanced solid tumors would increase after the combined treatment of ATO and radiation relative to either radiation or ATO alone. The antitumor action and quantitative tumor perfusion studies were carried out with locally advanced methylcholanthrene-induced fibrosarcoma grown in BALB/c mice. The s.c. methylcholanthrene-induced fibrosarcoma leg tumors were treated with ATO alone (10 mg/kg), radiation alone (30 Gy), or drug plus radiation together. Radiation alone and drug alone delayed the growth of the tumor by a few days compared with untreated tumors. In contrast, when radiation and drug were given together, the tumor growth delay was longer than 1 month, resulting local tumor cure of 55%. The fractionated radiation combined with ATO showed a similar pronounced tumor growth delay relative to the drug alone or radiation alone. Sustained reduction in tumor blood flow after the combined treatment measured using a rubidium uptake method paralleled enhanced tumor response. There was an immediate 10-fold increase in the production of tumor necrosis factor-alpha in the tumor tissue after the drug treatment, concomitant with the onset of prompt reduction of the tumor blood flow. The present results indicate that tumor response was better with combined treatment than with either treatment alone, suggesting that ATO has potential as an adjuvant to radiotherapy.