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PURPOSE Chordoma is a locally aggressive tumor that most commonly affects the base of the skull/clivus, cervical, and sacral spine. Conventional radiotherapy (RT), cannot be safely increased further to improve disease control due to the risk of toxicity to the surrounding critical structures. Tumor-targeted hyperthermia (HT) combined with Proton Beam Radiation Therapy (PBRT) is known to act as a potent radiosensitizer in cancer control. In this study, we investigated whether PBRT efficacy for chordoma can be enhanced in combination with HT as a radiosensitizer. MATERIAL AND METHODS Human chordoma cell lines, U-CH2 and Mug-chor1 were treated in vitro with HT followed by PBRT with variable doses. The colony-forming assay was performed, and dose-response was characterized by linear-quadratic model fits. HSP-70 and Brachyury (TBXT) biomarkers for chordoma aggression levels were quantified by western blot analysis. Gene microarray analysis was performed by U133 Arrays. Pathway Analysis was also performed using IPA bioinformatic software. RESULTS Our findings in both U-CH2 and Mug-Chor1 cell lines demonstrate that hyperthermia followed by PBRT has an enhanced cell killing effect when compared with PBRT-alone (p

Prostate cancer (PCa) treatment with radiation therapy (RT) has an excellent cure rate. However, Radiation-induced Erectile Dysfunction (RiED) is a common and irreversible toxicity impacting quality of life, and there is no FDA approved specific drug for RiED. We previously showed that prostate RT increased RhoA/ROCK signaling in the cavernous nerve (CN) and penile tissues, which may lead to RiED in rats. In this study, we investigated whether RhoA/ROCK pathway inhibition by a specific inhibitor called Hydroxyfasudil (HF) can improve RiED in our well-established rat model.Male Sprague-Dawley rats were randomized to the following groups: sham-RT, HF-only, RT-only, and RT + HF. Rats were either exposed to a single dose of 25 Gy prostate-confined RT or a sham procedure. 10 mg/kg HF or normal saline was injected intraperitoneally. Erectile function was evaluated by intracavernosal pressure (ICP) and mean arterial pressure (MAP) measurements at week 14 post-RT. Cavernous nerve (CN) injury was evaluated by transmission electron microscopy (TEM), and penile tissue fibrosis by Masson trichrome staining (MT).We have found that the HF treatment prior to RT showed significant (p 0.001) improvement in ICP/MAP ratio, area under the curve, and maximum ICP value, compared to RT-alone rats. Furthermore, RT + HF treated rats exhibited increased CN myelination and decreased axonal atrophy, comparted to RT-only. HF treatment showed significantly decreased penile tissue fibrosis (p 0.05) compared to RT-alone treated rats.Our results provide the first preclinical evidence that targeting RhoA/ROCK pathway by HF may provide a novel therapeutic option for the treatment of RiED.

To assess whether BIO 300, a synthetic genistein nanosuspension, improves the therapeutic index in prostate cancer treatment by preventing radiation-induced erectile dysfunction (ED) without reducing tumor radiosensitivity.Male Sprague-Dawley rats were exposed to 25 Gy of 220-kV prostate-confined x-rays. Animals were randomized to receive sham radiation therapy (RT), RT alone, RT with daily BIO 300 at 2 experimental dosing regimens, or RT with daily genistein. Erectile response was evaluated over time. Penile shaft tissue was harvested for histologic analyses. Murine xenograft studies using prostate cancer cell lines determined the effects of BIO 300 dosing on RT efficacy.Prostate-confined RT significantly decreased apomorphine-induced erectile response (P.05 vs sham RT). Erection frequency in animals receiving prophylactic treatment with BIO 300 starting 3 days before RT was similar to sham controls after RT. Treatment with synthetic genistein did not mitigate loss in erectile frequency. At week 14, post-RT treatment with BIO 300 resulted in significantly higher quality of erectile function compared with both the RT arm and the RT arm receiving genistein starting 3 days before irradiation (P.05). In hormone-sensitive and insensitive prostate tumor-bearing mice, BIO 300 administration did not negatively affect radiation-induced tumor growth delay.BIO 300 prevents radiation-induced ED, measured by erection frequency, erectile function, and erection quality, when administered 3 days before RT and continued daily for up to 14 weeks. Data also suggest that BIO 300 administered starting 2 hours after RT mitigates radiation-induced ED. Data provide strong nonclinical evidence to support clinical translation of BIO 300 for mitigation of ED while maintaining treatment response to RT.

Caveolin-1 is a membrane protein highly expressed in many tumors and plays an important role in tumor progression and metastasis. This review describes the structure of the Caveolin-1 protein and its pre-clinical and clinical significance, demonstrating that Caveolin-1 is a novel biomarker for radioresistance which has the promising potential to improve the clinical outcome of cancer patients undergoing radiation treatment.Targeted radiation therapy has shown immense benefits for cancer treatment. However, one of the major challenges for effective clinical outcome of radiation therapy for cancer patients is the development of radioresistance during radiation treatment. As a consequence, radiation therapy becomes a less effective modality for successful clinical outcome. Furthermore, a radioresistant tumor has the ability to repair its genome, and therefore becomes more aggressive and metastasizes. The plausible mechanisms for tumor radioresistance include the rapid DNA repair, somatic mutations in tumor oncogenes, aberrant activation of kinase pathways, and changes in the tumor microenvironment including tumor hypoxia, tumor vasculature, and cancer stem cells. Caveolin-1 is significantly upregulated in certain cancer cells and aberrantly mediates downstream signaling mechanisms. Notably, numerous recent research reports have shown the role of Caveolin-1 in tumor radioresistance and poor treatment outcome. Thus, Caveolin-1 could be a novel prognostic biomarker to monitor tumor radioresistance in cancer patients undergoing radiation therapy.Caveolin-1 has the promising potential to become a novel prognostic biomarker to monitor tumor radioresistance and radiation response specifically in the prostate, pancreas, and lung cancer.

Purpose To investigate the outcome of suppression of the renin angiotensin system using captopril combined with an antioxidant (Eukarion [EUK]-207) for mitigation of radiation-induced lung damage in rats. Methods and Materials The thoracic cavity of female Sprague-Dawley rats was irradiated with a single dose of 11 Gy. Treatment with captopril at a dose of 40 mg/kg/d in drinking water and EUK-207 given by subcutaneous injection (8 mg/kg daily) was started 1 week after irradiation (PI) and continuing until 14 weeks PI. Breathing rate was monitored until the rats were killed at 32 weeks PI, when lung fibrosis was assessed by lung hydroxyproline content. Lung levels of the cytokine transforming growth factor-β1 and macrophage activation were analyzed by immunohistochemistry. Oxidative DNA damage was assessed by 8-hydroxy-2-deoxyguanosine levels, and lipid peroxidation was measured by a T-BARS assay. Results The increase in breathing rate in the irradiated rats was significantly reduced by the drug treatments. The drug treatment also significantly decreased the hydroxyproline content, 8-hydroxy-2-deoxyguanosine and malondialdehyde levels, and levels of activated macrophages and the cytokine transforming growth factor-β1 at 32 weeks. Almost complete mitigation of these radiation effects was observed by combining captopril and EUK-207. Conclusion Captopril and EUK-207 can provide mitigation of radiation-induced lung damage out to at least 32 weeks PI after treatment given 1-14 weeks PI. Overall the combination of captopril and EUK-207 was more effective than the individual drugs used alone.

OBJECTIVE: Non-ablative or mild hyperthermia (HT) has been shown in preclinical (and clinical) studies as a localized radiosensitizer that enhances the tumoricidal effects of radiation. Most preclinical in vivo HT studies use subcutaneous tumor models which do not adequately represent clinical conditions (e.g. proximity of normal/critical organs) or replicate the tumor microenvironment—both of which are important factors for eventual clinical translation. The purpose of this work is to demonstrate proof-of-concept of locoregional radiosensitization with superficially applied, radiofrequency (RF)-induced HT in an orthotopic mouse model of prostate cancer. METHODS: In a 4-arm study, 40 athymic male nude mice were inoculated in the prostate with luciferase-transfected human prostate cancer cells (PC3). Tumor volumes were allowed to reach 150–250 mm(3) (as measured by ultrasound) following which, mice were randomized into (i) control (no intervention); (ii) HT alone; (iii) RT alone; and (iv) HT + RT. RF-induced HT was administered (Groups ii and iv) using the Oncotherm LAB EHY-100 device to achieve a target temperature of 41 °C in the prostate. RT was administered ~30 min following HT, using an image-guided small animal radiotherapy research platform. In each case, a dual arc plan was used to deliver 12 Gy to the target in a single fraction. One animal from each cohort was euthanized on Day 10 or 11 after treatment for caspase-9 and caspase-3 Western blot analysis. RESULTS: The inoculation success rate was 89%. Mean tumor size at randomization (~16 days post-inoculation) was ~189 mm(3) . Following the administration of RT and HT, mean tumor doubling times in days were: control = 4.2; HT = 4.5; RT = 30.4; and HT + RT = 33.4. A significant difference (p = 0.036) was noted between normalized nadir volumes for the RT alone (0.76) and the HT + RT (0.40) groups. Increased caspase-3 expression was seen in the combination treatment group compared to the other treatment groups. CONCLUSION: These early results demonstrate the successful use of external mild HT as a localized radiosensitizer for deep-seated tumors. ADVANCES IN KNOWLEDGE: We successfully demonstrated the feasibility of administering external mild HT in an orthotopic tumor model and demonstrated preclinical proof-of-concept of HT-based localized radiosensitization in prostate cancer radiotherapy.

Radioprotection and mitigation effects of the antioxidants, Eukarion (EUK)-207, curcumin, and the curcumin analogs D12 and D68, on radiation-induced DNA damage or lipid peroxidation in murine skin were investigated. These antioxidants were studied because they have been previously reported to protect or mitigate against radiation-induced skin reactions.DNA damage was assessed using two different assays. A cytokinesis-blocked micronucleus (MN) assay was performed on primary skin fibroblasts harvested from the skin of C3H/HeJ male mice 1 day, 1 week and 4 weeks after 5 Gy or 10 Gy irradiation. Local skin or whole body irradiation (100 kVp X-rays or caesium (Cs)-137 γ-rays respectively) was performed. DNA damage was further quantified in keratinocytes by immunofluorescence staining of γ-histone 2AX (γ-H2AX) foci in formalin-fixed skin harvested 1 hour or 1 day post-whole body irradiation. Radiation-induced lipid peroxidation in the skin was investigated at the same time points as the MN assay by measuring malondialdehyde (MDA) with a Thiobarbituric acid reactive substances (TBARS) assay.None of the studied antioxidants showed significant mitigation of skin DNA damage induced by local irradiation. However, when EUK-207 or curcumin were delivered before irradiation they provided some protection against DNA damage. In contrast, all the studied antioxidants demonstrated significant mitigating and protecting effects on radiation-induced lipid peroxidation at one or more of the three time points after local skin irradiation.Our results show no evidence for mitigation of DNA damage by the antioxidants studied in contrast to mitigation of lipid peroxidation. Since these agents have been reported to mitigate skin reactions following irradiation, the data suggest that changes in lipid peroxidation levels in skin may reflect developing skin reactions better than residual post-irradiation DNA damage in skin cells. Further direct comparison studies are required to confirm this inference from the data.

Prostate cancer is one of the most prevalent cancers and the second leading cause of cancer-related deaths in men in the United States. A large number of patients undergo radiation therapy (RT) as a standard care of treatment; however, RT causes erectile dysfunction (radiation-induced erectile dysfunction; RiED) because of late side effects after RT that significantly affects quality of life of prostate cancer patients. Within 5 years of RT, approximately 50% of patients could develop RiED. Based on the past and current research findings and number of publications from our group, the precise mechanism of RiED is under exploration in detail. Recent investigations have shown prostate RT induces significant morphologic arterial damage with aberrant alterations in internal pudendal arterial tone. Prostatic RT also reduces motor function in the cavernous nerve which may attribute to axonal degeneration may contributing to RiED. Furthermore, the advances in radiogenomics such as radiation induced somatic mutation identification, copy number variation and genome-wide association studies has significantly facilitated identification of biomarkers that could be used to monitoring radiation-induced late toxicity and damage to the nerves; thus, genomic- and proteomic-based biomarkers could greatly improve treatment and minimize arterial tissue and nerve damage. Further, advanced technologies such as proton beam therapy that precisely target tumor and significantly reduce off-target damage to vital organs and healthy tissues. In this review, we summarize recent advances in RiED research and novel treatment modalities for RiED. We also discuss the possible molecular mechanism involved in the development of RiED in prostate cancer patients. Further, we discuss various readily available methods as well as novel strategies such as stem cell therapies, shockwave therapy, nerve grafting with tissue engineering, and nutritional supplementations might be used to mitigate or cure sexual dysfunction following radiation treatment.

Radiation-induced inflammation and production of reactive oxygen species (ROS) play a critical role in normal tissue response. In this study we have examined some aspects of these effects in lung and skin.The superoxide dismutase (SOD) catalase mimetic, EUK-207, and genistein, an isoflavone with anti-inflammatory properties, were given post-irradiation and micronuclei (MN) formation was determined in cells derived from irradiated lung and skin. Changes in breathing rate were measured using a plethysmograph following irradiation of C57Bl6 mice knocked out for tumor necrosis factor (TNF)-alpha or its receptors, TNFR1/2, or treated with endotoxin (lipopolysaccharide - LPS).Both EUK-207 and genistein given after irradiation caused a large reduction in MN levels observed in lung cells during 14 weeks post-irradiation but ceasing treatment resulted in a rebound in MN levels at 28 weeks post-irradiation. In contrast, treatment with EUK-207 was largely ineffective in reducing MN observed in skin cells post-irradiation. Knock-out of TNF-alpha resulted in a reduced increase in breathing rate (peak at 12 weeks post-irradiation) relative to wild-type and TNFR1/2 knock-out. Treatment with LPS 1 h post-irradiation also reduced the increase in breathing rate.The increase in MN in lung cells after treatment with EUK-207 or genistein was stopped suggests that continuing ROS production contributes to DNA damage in lung cells over prolonged periods. That this effect was not seen in skin suggests this mechanism is less prominent in this tissue. The reduced level of radiation pneumonitis (as monitored by breathing rate changes) in animals knocked out for TNF-alpha suggests that this cytokine plays a significant role in inducing inflammation in lung following irradiation. The similar effect observed following LPS given post-irradiation suggests the possibility that such treatment modifies the long-term cyclic inflammatory response following irradiation in lungs.

We examined the effects of genistein and/or Eukarion (EUK)-207 on radiation-induced lung damage and investigated whether treatment for 0-14 weeks (wks) post-irradiation (PI) would mitigate late lung injury.The lungs of female Sprague-Dawley (SD) rats were irradiated with 10 Gy. EUK-207 was delivered by infusion and genistein was delivered as a dietary supplement starting immediately after irradiation (post irradiation [PI]) and continuing until 14 wks PI. Rats were sacrificed at 0, 4, 8, 14 and 28 wks PI. Breathing rate was monitored and lung fibrosis assessed by lung hydroxyproline content at 28 wks. DNA damage was assessed by micronucleus (MN) assay and 8-hydroxy-2-deoxyguanosine (8-OHdG) levels. The expression of the cytokines Interleukin (IL)-1α, IL-1β, IL-6, Tumor necrosis factor (TNF)-α and Transforming growth factor (TGF)-β1, and macrophage activation were analyzed by immunohistochemistry.Increases in breathing rate observed in the irradiated rats were significantly reduced by both drug treatments during the pneumonitis phase and the later fibrosis phase. The drug treatments decreased micronuclei (MN) formation from 4-14 wks but by 28 wks the MN levels had increased again. The 8-OHdG levels were lower in the drug treated animals at all time points. Hydroxyproline content and levels of activated macrophages were decreased at 28 wks in all drug treated rats. The treatments had limited effects on the expression of the cytokines.Genistein and EUK-207 can provide partial mitigation of radiation-induced lung damage out to at least 28 wks PI even after cessation of treatment at 14 wks PI.

Exposure of civilian populations to radiation due to accident, war or terrorist act is an increasing concern. The lung is one of the more radiosensitive organs that may be affected in people receiving partial-body irradiation and radiation injury in lung is thought to be associated with the development of a prolonged inflammatory response. Here we examined how effectively damage to the lung can be mitigated by administration of drugs initiated at different times after radiation exposure and examined response in adolescent animals for comparison with the young adult animals that we had studied previously. We studied the mitigation efficacy of the isoflavone genistein (50 mg/kg) and the salen-Mn superoxide dismutase-catalase mimetic EUK-207 (8 mg/kg), both of which have been reported to scavenge reactive oxygen species and reduce activity of the NFkB pathway. The drugs were given by subcutaneous injection to 6- to 7-week-old Fisher rats daily starting either immediately or 2 weeks after irradiation with 12 Gy to the whole thorax. The treatment was stopped at 28 weeks post irradiation and the animals were assessed for levels of inflammatory cytokines, activated macrophages, oxidative damage and fibrosis at 48 weeks post irradiation. We demonstrated that both genistein and EUK-207 delayed and suppressed the increased breathing rate associated with pneumonitis. These agents also reduced levels of oxidative damage (50–100%), levels of TGF-β1 expression (75–100%), activated macrophages (20–60%) and fibrosis (60–80%). The adolescent rats developed pneumonitis earlier following irradiation of the lung than did the adult rats leading to greater severe morbidity requiring euthanasia (~37% in adolescents vs. ~10% in young adults) but the extent of the mitigation of the damage was similar or slightly greater.

Background and purpose Inflammatory and fibrogenic processes play a crucial role in the radiation-induced injury in the lung. The aim of the present study was to examine whether additive LPS exposure in the lung (to simulate respiratory infection) would affect pneumonitis or fibrosis associated with lung irradiation. Material and methods Wildtype C57Bl/6J (WT-C57) and TNFα, TNFR1 and TNFR2 knockout ( −/− ) mice, in C57Bl/6J background, were given whole thorax irradiation (10Gy) with or without post-irradiation intratracheal administration of LPS (50μg/mice). Functional deficit was examined by measuring breathing rate at various times after treatment. Real-time Reverse Transcription–Polymerase Chain Reaction (RT–PCR) and immunohistochemistry were used to analyze the protein expression and m-RNA of Interleukin-1 alpha (IL-1α), Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), Tumour Necrosis Factor alpha (TNFα) and Transforming Growth Factor beta (TGFβ) in the lung at various times after treatment. Inflammatory cells were detected by Mac-3 (macrophages) and Toluidine Blue (mast cells) staining. Collagen content was estimated by hydroxyproline (total collagen) and Sircol assay (soluble collagen). Levels of oxidative damage were assessed by 8 - hydroxy-2-deoxyguanosine (8-OHdG) staining. Results LPS exposure significantly attenuated the breathing rate increases following irradiation of WT-C57, TNFR1 −/− and TNFR2 −/− mice and to a lesser extent in TNFα −/− mice. Collagen content was significantly reduced after LPS treatment in WT-C57, TNFR1 −/− and TNFα −/− mice and there was a trend in TNFR2 −/− mice. Similarly there were lower levels of inflammatory cells and cytokines in the LPS treated mice. Conclusions This study reveals a mitigating effect of early exposure to LPS on injury caused by irradiation on lungs of C57Bl mice. The results suggest that immediate infection post irradiation may not impact lung response negatively in radiation-accident victims, however, further studies are required in different animal models, and with specific infectious agents, to confirm and extend our findings.

There is a serious need to develop effective mitigators against accidental radiation exposures. In radiation accidents, many people may receive nonuniform whole-body or partial-body irradiation. The lung is one of the more radiosensitive organs, demonstrating pneumonitis and fibrosis that are believed to develop at least partially because of radiation-induced chronic inflammation. Here we addressed the crucial questions of how damage to the lung can be mitigated and whether the response is affected by irradiation to the rest of the body. We examined the widely used dietary supplement genistein given at two dietary levels (750 or 3750 mg/kg) to Fischer rats irradiated with 12 Gy to the lung or 8 Gy to the lung + 4 Gy to the whole body excluding the head and tail (whole torso). We found that genistein had promising mitigating effects on oxidative damage, pneumonitis and fibrosis even at late times (36 weeks) when drug treatment was initiated 1 week after irradiation and stopped at 28 weeks postirradiation. The higher dose of genistein showed no greater beneficial effect. Combined lung and whole-torso irradiation caused more lung-related severe morbidity resulting in euthanasia of the animals than lung irradiation alone.

We investigated whether genistein could protect the lung from radiation-induced injury. We hypothesized that genistein would reduce the levels of inflammatory cytokines and ROS after irradiation and therefore lead to reduced DNA damage and functional deficits. Whole lungs of Sprague-Dawley rats were irradiated with 18 Gy at approximately 0.5 Gy/min. At 28 weeks a micronucleus assay was used to examine DNA damage and, using immunohistochemical analysis, expression of IL-1alpha, IL-1beta, IL-6, TNF-alpha and TGF-beta, macrophage activation, oxidative stress (8-OHdG) and collagen levels were measured. A TBARS assay was used to measure the level of malondialdehyde. Functional damage was assessed by measuring the breathing rate of the rats over the course of the experiment. The increase in breathing rate after irradiation was damped in rats receiving genistein during the phase of pneumonitis (6-10 weeks), and there was a 50-80-day delay in lethality in this group. Genistein treatment also decreased the levels of the inflammatory cytokines TNF-alpha, IL-1beta and TGF-beta and led to a reduction in collagen content, a reduction in 8-OHdG levels, and complete protection against DNA damage measured in surviving rats at 28 weeks after irradiation. These results demonstrates that genistein treatment can provide partial protection against the early (pneumonitis) effects of lung irradiation and reduce the extent of fibrosis, although not sufficiently to prevent lethality at the radiation dose used in this study.