Innate Immune Pathways Associated with Lung Radioprotection by Soy Isoflavones.

INTRODUCTION: Radiation therapy for lung cancer causes pneumonitis and fibrosis. Soy isoflavones protect against radiation-induced lung injury, but the mediators of radioprotection remain unclear. We investigated the effect of radiation on myeloid-derived suppressor cells (MDSCs) in the lung and their modulation by soy isoflavones for a potential role in protection from radiation-induced lung injury. METHODS: BALB/c mice (5-6 weeks old) received a single 10 Gy dose of thoracic irradiation and soy isoflavones were orally administrated daily before and after radiation at 1 mg/day. Arginase-1 (Arg-1) and nuclear factor κB (NF-κB) p65 were detected in lung tissue by western blot analysis and immunohistochemistry. Lung MDSC subsets and their Arg-1 expression were analyzed by flow cytometry. Cytokine levels in the lungs were measured by ELISA. RESULTS: At 1 week after radiation, CD11b+ cells expressing Arg-1 were decreased by radiation in lung tissue yet maintained in the lungs treated with radiation and soy isoflavones. Arg-1 was predominantly expressed by CD11b+Ly6ClowLy6G+ granulocytic MDSCs (gr-MDSCs). Arg-1 expression in gr-MDSCs was reduced by radiation and preserved by supplementation with soy isoflavones. A persistent increase in Arg-1+ cells was observed in lung tissue treated with combined radiation and soy isoflavones at early and late time points, compared to radiation alone. The increase in Arg-1 expression mediated by soy isoflavones could be associated with the inhibition of radiation-induced activation of NF-κB and the control of pro-inflammatory cytokine production demonstrated in this study. CONCLUSION: A radioprotective mechanism of soy isoflavones may involve the promotion of Arg-1-expressing gr-MDSCs that could play a role in downregulation of inflammation and lung radioprotection.

Radiation injury to cardiac arteries and myocardium is reduced by soy isoflavones.

OBJECTIVE: The negative effects of incidental radiation on the heart and its vessels, particularly in the treatment of locally advanced non-small cell lung cancer, esophageal cancer, left-sided breast cancer, and lymphoma, are known. Late cardiac events induced by radiotherapy including coronary artery disease, ischemia, congestive heart failure, and myocardial infarction can manifest months to years after radiotherapy. We have previously demonstrated that soy isoflavones mitigate inflammatory responses induced in lungs by thoracic irradiation resulting in decreased vascular damage, inflammation, and fibrosis. In the current study, we investigate the use of soy isoflavones to protect cardiac vessels and myocardium from radiation injury. METHODS: Mice received a single dose of 10-Gy thoracic irradiation and daily oral treatment with soy isoflavones. At different time points, hearts were processed for histopathology studies to evaluate the effect of soy isoflavones on radiation-induced damage to cardiac vessels and myocardium. RESULTS: Radiation damage to arteries and myocardium was detected by 16 weeks after radiation. Soy isoflavones given in conjunction with thoracic irradiation were found to reduce damage to the artery walls and radiation-induced fibrosis in the myocardium. CONCLUSION: Our histopathological findings suggest a radioprotective role of soy isoflavones to prevent cardiac injury. This approach could translate to the use of soy isoflavones as a safe complement to thoracic radiotherapy with the goal of improving the overall survival in patients whose cancer has been successfully controlled by the radiotherapy but who otherwise succumb to heart toxicity.

Radiotherapy and MVA-MUC1-IL-2 vaccine act synergistically for inducing specific immunity to MUC-1 tumor antigen.

BACKGROUND: We previously demonstrated that tumor irradiation potentiates cancer vaccines using genetic modification of tumor cells in murine tumor models. To investigate whether tumor irradiation augments the immune response to MUC1 tumor antigen, we have tested the efficacy of tumor irradiation combined with an MVA-MUC1-IL2 cancer vaccine (Transgene TG4010) for murine renal adenocarcinoma (Renca) cells transfected with MUC1. METHODS: Established subcutaneous Renca-MUC1 tumors were treated with 8 Gy radiation on day 11 and peritumoral injections of MVA-MUC1-IL2 vector on day 12 and 17, or using a reverse sequence of vaccine followed by radiation. Growth delays were monitored by tumor measurements and histological responses were evaluated by immunohistochemistry. Specific immunity was assessed by challenge with Renca-MUC1 cells. Generation of tumor-specific T cells was detected by IFN-γ production from splenocytes stimulated in vitro with tumor lysates using ELISPOT assays. RESULTS: Tumor growth delays observed by tumor irradiation combined with MVA-MUC1-IL-2 vaccine were significantly more prolonged than those observed by vaccine, radiation, or radiation with MVA empty vector. The sequence of cancer vaccine followed by radiation two days later resulted in 55-58% complete responders and 60% mouse long-term survival. This sequence was more effective than that of radiation followed by vaccine leading to 24-30% complete responders and 30% mouse survival. Responding mice were immune to challenge with Renca-MUC1 cells, indicating the induction of specific tumor immunity. Histology studies of regressing tumors at 1 week after therapy, revealed extensive tumor destruction and a heavy infiltration of CD45+ leukocytes including F4/80+ macrophages, CD8+ cytotoxic T cells and CD4+ helper T cells. The generation of tumor-specific T cells by combined therapy was confirmed by IFN-γ secretion in tumor-stimulated splenocytes. An abscopal effect was measured by rejection of an untreated tumor on the contralateral flank to the tumor treated with radiation and vaccine. CONCLUSIONS: These findings suggest that cancer vaccine given prior to local tumor irradiation augments an immune response targeted at tumor antigens that results in specific anti-tumor immunity. These findings support further exploration of the combination of radiotherapy with cancer vaccines for the treatment of cancer.

Soy Isoflavones Promote Radioprotection of Normal Lung Tissue by Inhibition of Radiation-Induced Activation of Macrophages and Neutrophils.

INTRODUCTION: Radiation therapy for lung cancer is limited by toxicity to normal lung tissue that results from an inflammatory process, leading to pneumonitis and fibrosis. Soy isoflavones mitigate inflammatory infiltrates and radiation-induced lung injury, but the cellular immune mediators involved in the radioprotective effect are unknown. METHODS: Mice received a single dose of 10 Gy radiation delivered to the lungs and daily oral treatment of soy isoflavones. At different time points, mice were either processed to harvest bronchoalveolar lavage fluid for differential cell counting and lungs for flow cytometry or immunohistochemistry studies. RESULTS: Combined soy and radiation led to a reduction in infiltration and activation of alveolar macrophages and neutrophils in both the bronchoalveolar and lung parenchyma compartments. Soy treatment protected F4/80CD11c interstitial macrophages, which are known to play an immunoregulatory role and are decreased by radiation. Furthermore, soy isoflavones reduced the levels of nitric oxide synthase 2 expression while increasing arginase-1 expression after radiation, suggesting a switch from proinflammatory M1 macrophage to an anti-inflammatory M2 macrophage phenotype. Soy also prevented the influx of activated neutrophils in lung caused by radiation. CONCLUSIONS: Soy isoflavones inhibit the infiltration and activation of macrophages and neutrophils induced by radiation in lungs. Soy isoflavones-mediated modulation of macrophage and neutrophil responses to radiation may contribute to a mechanism of resolution of radiation-induced chronic inflammation leading to radioprotection of lung tissue.

Radiation-Induced Esophagitis is Mitigated by Soy Isoflavones.

INTRODUCTION: Lung cancer patients receiving radiotherapy present with acute esophagitis and chronic fibrosis, as a result of radiation injury to esophageal tissues. We have shown that soy isoflavones alleviate pneumonitis and fibrosis caused by radiation toxicity to normal lung. The effect of soy isoflavones on esophagitis histopathological changes induced by radiation was investigated. METHODS: C57BL/6 mice were treated with 10 Gy or 25 Gy single thoracic irradiation and soy isoflavones for up to 16 weeks. Damage to esophageal tissues was assessed by hematoxylin-eosin, Masson's Trichrome and Ki-67 staining at 1, 4, 10, and 16 weeks after radiation. The effects on smooth muscle cells and leukocyte infiltration were determined by immunohistochemistry using anti-αSMA and anti-CD45, respectively. RESULTS: Radiation caused thickening of esophageal tissue layers that was significantly reduced by soy isoflavones. Major radiation alterations included hypertrophy of basal cells in mucosal epithelium and damage to smooth muscle cells in muscularis mucosae as well as disruption of collagen fibers in lamina propria connective tissue with leukocyte infiltration. These effects were observed as early as 1 week after radiation and were more pronounced with a higher dose of 25 Gy. Soy isoflavones limited the extent of tissue damage induced by radiation both at 10 and 25 Gy. CONCLUSION: Soy isoflavones have a radioprotective effect on the esophagus, mitigating the early and late effects of radiation injury in several esophagus tissue layers. Soy could be administered with radiotherapy to decrease the incidence and severity of esophagitis in lung cancer patients receiving thoracic radiation therapy.

Radioprotection of lung tissue by soy isoflavones.

INTRODUCTION: Radiation-induced pneumonitis and fibrosis have restricted radiotherapy for lung cancer. In a preclinical lung tumor model, soy isoflavones showed the potential to enhance radiation damage in tumor nodules and simultaneously protect normal lung from radiation injury. We have further dissected the role of soy isoflavones in the radioprotection of lung tissue. METHODS: Naive Balb/c mice were treated with oral soy isoflavones for 3 days before and up to 4 months after radiation. Radiation was administered to the left lung at 12 Gy. Mice were monitored for toxicity and breathing rates at 2, 3, and 4 months after radiation. Lung tissues were processed for histology for in situ evaluation of response. RESULTS: Radiation caused damage to normal hair follicles, leading to hair loss in the irradiated left thoracic area. Supplementation with soy isoflavones protected mice against radiation-induced skin injury and hair loss. Lung irradiation also caused an increase in mouse breathing rate that was more pronounced by 4 months after radiation, probably because of the late effects of radiation-induced injury to normal lung tissue. However, this effect was mitigated by soy isoflavones. Histological examination of irradiated lungs revealed a chronic inflammatory infiltration involving alveoli and bronchioles and a progressive increase in fibrosis. These adverse effects of radiation were alleviated by soy isoflavones. CONCLUSION: Soy isoflavones given pre- and postradiation protected the lungs against adverse effects of radiation including skin injury, hair loss, increased breathing rates, inflammation, pneumonitis and fibrosis, providing evidence for a radioprotective effect of soy.

Impaired dendritic cell function in aging leads to defective antitumor immunity.

We recently reported that bone marrow-derived dendritic cells (DC) from aged miced are less effective than their young counterparts in inducing the regression of B16-ovalbumin (OVA) melanomas. To examine the underlying mechanisms, we investigated the effect of aging on DC tumor antigen presentation and migration. Although aging does not affect the ability of DCs to present OVA peptide((257-264)), DCs from aged mice are less efficient than those from young mice in stimulating OVA-specific T cells in vitro. Phenotypic analysis revealed a selective decrease in DC-specific/intracellular adhesion molecule type-3-grabbing nonintegrin (DC-SIGN) level in aged DCs. Adoptive transfer experiments showed defective in vivo DC trafficking in aging. This correlates with impaired in vitro migration and defective CCR7 signaling in response to CCL21 in aged DCs. Interestingly, vaccination of young mice using old OVA peptide((257-264))-pulsed DCs (OVA PP-DC) resulted in impaired activation of OVA-specific CD8(+) T cells in vivo. Effector functions of these T cells, as determined by IFN-gamma production and cytotoxic activity, were similar to those obtained from mice vaccinated with young OVA PP-DCs. A decreased influx of intratumor CD8(+) T cells was also observed. Importantly, although defective in vivo migration could be restored by increasing the number of old DCs injected, the aging defect in DC tumor surveillance and OVA-specific CD8(+) T-cell induction remained. Taken together, our findings suggest that defective T-cell stimulation contributes to the observed impaired DC tumor immunotherapeutic response in aging.