The radioprotectant nano-genistein enhances radiotherapy efficacy of lung tumors in mice.

Background: Radiotherapy for non-small cell lung cancer (NSCLC) can be dose-limiting due to treatment-related toxicities. Genistein has been shown to be a robust radioprotective agent in preclinical models. A novel genistein oral nanosuspension formulation (nano-genistein) has demonstrated efficacy in mitigating radiation-induced lung damage in preclinical animal models. However, while those studies have confirmed that nano-genistein can protect normal lung tissue from radiation-induced toxicities, no studies have assessed the effect of nano-genistein on lung tumors. Here, we evaluated the impact of nano-genistein on the efficacy of radiation treatment of lung tumors in a mouse xenograft model. Methods: Two separate studies were conducted utilizing human A549 cells implanted either dorsally within the upper torso or in the flank. Daily oral administration of nano-genistein (200 or 400 mg/kg/day) occurred prior to and after exposure to a single dose of thoracic or abdominal 12.5 Gy radiation. Tumor growth was monitored twice weekly, nano-genistein treatment continued for up to 20 weeks and histopathology of tissues was completed post euthanasia. Results: Continuous nano-genistein dosing was safe across all study groups in both studies. Animals receiving nano-genistein better maintained body weight following irradiation compared to corresponding vehicle treated animals. Animals that received nano-genistein also had reduced tumor growth and improved normal lung histopathology compared to those receiving vehicle suggesting that nano-genistein does not protect tumors from radiotherapy but is radioprotective of the lungs. There were no treatment-related histopathological findings noted in the skin adjacent to the tumor, esophagus, or uterus. Conclusions: These results, including the safety following extended dosing, support the continued evaluation of nano-genistein as an adjunctive treatment for patients with NSCLC undergoing radiotherapy and serve as the basis of a phase 1b/2a multicenter clinical trial.

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.

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.

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.

The cardiovascular responses to mu opioid agonist and antagonist in conscious normal and obese rats.

Beta-endorphin decreases blood pressure in normal rats but increases blood pressure in obese rats. Since beta-endorphins can bind both mu opioid and kappa-opioid receptors we investigated the effect of a mu specific receptor agonist, D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and a mu specific antagonist, D-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) on cardiovascular responses in conscious control and obese rats. Rats were also implanted with telemetry transmitters and intracerebroventricular (ICV) cannulas for recording and peptide administration. The mu agonist, DAMGO, increased blood pressure (BP) in control rats. DAMGO also increased BP in obese rats but only at high concentrations. The heart rate responses paralleled the MAP responses. CTAP, the mu antagonist, paradoxically increased the MAP in both control and obese rats. The responsiveness to the mu agonist and antagonist was greater in controls. In other animals the brains were excised and the ventral medial hypothalamic area removed and mu receptor expression determined using PCR. The expression of mu opioid receptors was increased in obese rats. We conclude that the mu opioids can stimulate cardiovascular responses, but the excitatory responsiveness was not increased in conscious obese rats.

Hypothalamic agouti-related protein immunoreactivity in food-restricted, obese, and insulin-treated animals: evidence for glia cell localization.

Agouti-related protein (AGRP) has been implicated in the regulation of metabolic balance. Overexpression of this peptide leads to obesity. Its activity is mediated via the melanocortin-4 (MC-4) receptor where it acts as an MC-4 receptor antagonist. In this study, we characterized the AGRP brain distribution and cellular localization in control, food-restricted, obese, and insulin-treated rats using immunohistochemistry. AGRP immunostaining was found selectively in regions of the arcuate and ventromedial hypothalamic nuclei. These regions were stained less intensely in food-restricted rats than in controls. AGRP-positive cells in the hypothalamus of obese animals were three times more numerous than in control rats. Also, insulin treatment acted to decrease AGRP immunostaining. Analysis of AGRP cellular localization demonstrated its presence in the cytoplasm of numerous small (7-12 microm) cell bodies of putative protoplasmic astrocytes as well as in nerve fibers. Glia fibrillary acidic protein (GFAP) immunostaining of sections adjacent to those stained for AGRP revealed astrocytes with morphology similar to AGRP-positive cells. A few AGRP-positive nerve cell bodies were also found in the arcuate nucleus of obese rats. We conclude that AGRP hypothalamic content is decreased by fasting and intracerebroventricular (i.c.v.) insulin treatment and increased in obesity. In addition to its presence in nerve fibers, AGRP localization in astroglia-like cells suggests a possible role for these elements in its synthesis or its sequestration from the neuronal compartment.

High fat feeding is associated with increased blood pressure, sympathetic nerve activity and hypothalamic mu opioid receptors.

Obesity and high fat diets are associated with an increased prevalence of diabetes, cardiovascular disease, and hypertension. However, the mechanism(s) linking obesity and high fat diet to these metabolic and cardiovascular disorders are not fully elucidated. Leptin stimulates the formation of pro-opiomelanocortin and its products. The stimulation of the central nervous system (CNS) opioids and their receptors is associated with an increase in cardiovascular dynamics. In this study we hypothesized that obesity changed the CNS opioids and their receptors that could play a role in altered cardiovascular and autonomic nervous regulation in obesity. Male Wistar rats were fed either a high fat (HF) or regular chow (control) diet. After 12 weeks, rats were anesthetized and instrumented to record mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). A blood sample was collected and plasma glucose, insulin, leptin, beta-endorphins were measured. The brains were subsequently processed for immunohistochemistry and in situ hybridization. The HF rats were larger and had a greater percentage of body fat. Leptin and insulin levels were also higher in the HF animals. Basal MAP and RSNA were significantly higher in HF rats. Additionally, immunohistochemistry and in situ hybridization demonstrated that HF rats had increased hypothalamus mu opioid receptors compared to controls. These studies suggest that HF feeding is associated with increased body fat, plasma leptin, insulin, and hypothalamic mu opioid receptors. The increased mu opioid receptors may contribute to the higher MAP and RSNA observed in HF animals.

Insulin mediated hemodynamic responses in spontaneous hypertensive rats (SHRs): effect of chromosome 4 gene transfer.

The spontaneous hypertensive rat (SHR) is a widely studied model of essential hypertension and has been reported to exhibit alterations in carbohydrate and lipid metabolism. Genetic linkage studies implicated that SHR carries deletion variant of Cd36 gene of chromosome 4, the gene that encodes fatty acid transporter. Thus it could be possible that primary genetic defect in SHR is compromised tissue utilization of fatty acid that would form the basis for the pathogenesis of hyperinsulinemia, insulin resistance and insulin-mediated responses. We measured both the hemodynamic and metabolic responses to insulin in SHR in comparison with the chromosome congenic spontaneous hypertensive rats (cSHRs) (rats in which piece of chromosome 4 containing wild type Cd36 was integrated into the SHR genome). A bolus infusion of insulin increased iliac conductance and decreased blood pressure in Wistar Kyoto (WKY) rats. However, in SHR insulin did not reduce blood pressure as in WKY but after about 15 min it significantly enhanced blood pressure and reduced iliac conductance. Whereas in cSHR insulin did not reduce blood pressure as in WKY rats. However, pressor responses to insulin were eliminated by chromosome 4 gene transfer. Glucose clearance was significantly slower in both SHR and cSHR. Glucose tolerance test revealed that SHR are hyperinsulinemic and insulin resistant. These findings indicate that transfer of segment of chromosome 4 from Brown Norway rats onto spontaneous hypertensive background eliminates hyperinsulinemia and pressor effects of insulin.

The effects of beta-endorphin (beta-END) on cardiovascular and behavioral dynamics in conscious rats.

Beta-endorphin (beta-END) a product of the proopiomelanocortin (POMC) has been demonstrated to play a role in the regulation of metabolic and autonomic responses. Recent studies have suggested the involvement of the endogenous opioid system in cardiovascular control. Previous studies conducted in our laboratory using anesthetized animals investigated the actions of beta-END and other POMC derived peptides on sympathetic and cardiovascular dynamics. In this study, we determined both the acute and chronic effects of beta-END on cardiovascular and behavioral dynamics in conscious unrestrained rats using radio-telemetry. Animals were instrumented with a radio-telemetry transmitter in the abdominal cavity and the attached catheter inserted into the femoral artery for recording of cardiovascular dynamics and activity. They were subsequently implanted with intracerebroventricular (ICV) cannulas. The acute ICV administration of beta-END significantly increased the mean arterial pressure (MAP) and heart rate (HR) compared to controls. The cardiovascular responses returned toward control levels after 2 h. In contrast, the chronic infusion of beta-END significantly decreased the MAP and HR during both the active and inactive phase. Chronic beta-END administration also decreased physical activity. Food intake was increased initially and later declined and water consumption followed a similar pattern. We conclude that in the conscious unrestrained animal the acute administration of beta-END increases MAP and HR while the chronic infusion of beta-END decreases MAP, HR, physical activity, and stimulate a short-term increase in food and water intake.

Chronic nitric oxide deficiency is associated with altered leutinizing hormone and follicle-stimulating hormone release in ovariectomized rats.

Nitric oxide (NO) synthase (NOS) has been found in the gonadotrophs and folliculo-stellate cells of the anterior pituitary. Previous observations from our laboratory suggest that NO may play a role in regulating gonadotropin secretion. Because estrogen secretion by the ovary can influence gonadotropin secretion, we investigated the hypothesis that chronic in vivo NO deficiency has a direct estrogen-independent effect on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. Chronic NO deficiency was induced by adding an NOS inhibitor, N-nitro-L-arginine (L-NNA, 0.6 g/l) to the drinking water of ovariectomized (OVX) rats. The control OVX rats were untreated. After 6-8 weeks, the animals were sacrificed, and the pituitaries were removed and perfused continuously for 4 hr in the presence of pulsatile gonadotropin-releasing hormone (GnRH, 500 ng/pulse) every 30 min. S-Nitroso-L-acetyl penicillamine (SNAP, an NO donor, 0.1 mM) or L-nitro-arginine methyl ester (L-NAME, an NOS inhibitor, 0.1 mM) was added to the media and perfusate samples were collected at 10-min intervals. GnRH-stimulated LH and FSH levels were significantly lower in pituitaries from OVX/NO-deficient pituitaries compared with pituitaries from the OVX control group. The addition of SNAP significantly decreased LH and FSH secretion by pituitaries from OVX control animals, but significantly increased their secretion by pituitaries from the OVX/NO-deficient animals. L-NAME also suppressed LH and FSH secretion by pituitaries from the OVX control animals and stimulated their release by pituitaries from the NO-deficient/OVX animals. Immunohistochemistry of frontal sections through the hypothalamus demonstrated that OVX/NO deficiency is associated with increased GnRH in the median eminence. We conclude that NO has a chronic stimulatory effect on LH and FSH release and the subsequent altered secretory responsiveness to NO agonist or antagonist is the result of chronic NO suppression.