Christopher K. Yunker

Soy isoflavones augment radiation effect by inhibiting APE1/ref-1 DNA repair activity in non-small cell lung cancer

Introduction: Soy isoflavones sensitize cancer cells to radiation both in vitro and in vivo. To improve the effect of radiotherapy for non-small cell lung cancer, we assessed the potential of using a complementary approach with soy isoflavones. Methods: Human A549 non-small cell lung cancer cells were treated with soy isoflavones, radiation, or both and tested for cell growth. DNA double-strand breaks (DSBs) were detected by immunostaining for &ggr;-H2AX foci. Expressions of &ggr;-H2AX, HIF-1&agr;, and APE1/Ref-1 were assessed by Western blots. DNA-binding activities of HIF-1&agr; and NF-&kgr;B transcription factors were analyzed by electrophoretic mobility shift assay. Results: Soy isoflavones increased A549 cell killing induced by radiation. Multiple &ggr;-H2AX foci were detectable at 1 hour after radiation but decreased at 24 hours after radiation. Soy isoflavones also caused DNA DSBs, but &ggr;-H2AX foci increased over time. Soy isoflavones and radiation caused an increase in &ggr;-H2AX foci, which persisted at 24 hours, indicating both increased DNA damage and inhibition of repair. Soy isoflavones inhibited the radiation-induced activity of the DNA repair/redox enzyme APE1/Ref-1 and the transcription factors NF-&kgr;B and HIF-1&agr;. E3330, which inhibits the redox activity of APE1/Ref-1, did not alter the repair of radiation-induced DSBs. Methoxyamine, which inhibits APE1/Ref-1 DNA repair activity, partly blocked the decrease in radiation-induced DSBs at 24 hours, suggesting partial mitigation of radiation-induced DNA repair akin to the effect of soy combined with radiation, in agreement with cytotoxic assays. Conclusions: Inhibition of APE1/Ref-1 DNA repair activity by soy could be involved in the mechanism by which soy alters DNA repair and leads to cell killing.

Soy isoflavones radiosensitize lung cancer while mitigating normal tissue injury

BACKGROUND We have demonstrated that soy isoflavones radiosensitize cancer cells. Prostate cancer patients receiving radiotherapy (RT) and soy tablets had reduced radiation toxicity to surrounding organs. We have now investigated the combination of soy with RT in lung cancer (NSCLC), for which RT is limited by radiation-induced pneumonitis. METHODS Human A549 NSCLC cells were injected i.v. in nude mice to generate lung tumor nodules. Lung tumor-bearing mice were treated with left lung RT at 12 Gy and with oral soy treatments at 1mg/day for 30 days. Lung tissues were processed for histology. RESULTS Compared to lung tumor nodules treated with soy isoflavones or radiation, lung tissues from mice treated with both modalities showed that soy isoflavones augmented radiation-induced destruction of A549 lung tumor nodules leading to small residual tumor nodules containing degenerating tumor cells with large vacuoles. Soy isoflavones decreased the hemorrhages, inflammation and fibrosis caused by radiation in lung tissue, suggesting protection of normal lung tissue. CONCLUSIONS Soy isoflavones augment destruction of A549 lung tumor nodules by radiation, and also mitigate vascular damage, inflammation and fibrosis caused by radiation injury to normal lung tissue. Soy could be used as a non-toxic complementary approach to improve RT in NSCLC.

Differential effect of soy isoflavones in enhancing high intensity radiotherapy and protecting lung tissue in a pre-clinical model of lung carcinoma

BACKGROUND Radiotherapy of locally-advanced non-small cell lung cancer is limited by radiation-induced pneumonitis and fibrosis. We have further investigated the role of soy isoflavones to improve the effect of a high intensity radiation and reduce lung damage in a pre-clinical lung tumor model. METHODS Human A549 NSCLC cells were injected i.v. in nude mice to generate a large tumor burden in the lungs. Mice were treated with lung irradiation at 10 Gy and with oral soy. The therapy effect on the tumor cells and surrounding lung tissue was analyzed on lung sections stained with H&E, Ki-67 and Massons Trichrome. Pneumonitis and vascular damage were evaluated by measurements of alveolar septa and immunofluorescent staining of vessel walls. RESULTS Combined soy and radiation caused a significantly stronger inhibition of tumor progression compared to each modality alone in contrast to large invasive tumor nodules seen in control mice. At the same time, soy reduced radiation injury in lung tissue by decreasing pneumonitis, fibrosis and protecting alveolar septa, bronchioles and vessels. CONCLUSIONS These studies demonstrate a differential effect of soy isoflavones on augmenting tumor destruction induced by radiation while radioprotecting the normal lung tissue and support using soy to alleviate radiotoxicity in lung cancer.

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.

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 F4/80+CD11c− 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.

Axitinib Improves Radiotherapy in Murine Xenograft Lung Tumors

A third of patients with non-small cell lung cancer (NSCLC) present with un-resectable stage III locally advanced disease and are currently treated by chemo-radiotherapy but the median survival is only about 21 months. Using an orthotopic xenograft model of lung carcinoma, we have investigated the combination of radiotherapy with the anti-angiogenic drug axitinib (AG-013736, Pfizer), which is a small molecule receptor tyrosine kinase inhibitor that selectively targets the signal transduction induced by VEGF binding to VEGFR receptors. We have tested the combination of axitinib with radiotherapy in nude mice bearing human NSCLC A549 lung tumors. The therapy effect was quantitatively evaluated in lung tumor nodules. The modulation of radiation-induced pneumonitis, vascular damage and fibrosis by axitinib was assessed in lung tissue. Lung irradiation combined with long-term axitinib treatment was safe resulting in minimal weight loss and no vascular injury in heart, liver and kidney tissues. A significant decrease in the size of lung tumor nodules was observed with either axitinib or radiation, associated with a decrease in Ki-67 staining and a heavy infiltration of inflammatory cells in tumor nodules. The lungs of mice treated with radiation and axitinib showed a complete response with no detectable residual tumor nodules. A decrease in pneumonitis, vascular damage and fibrosis were observed in lung tissues from mice treated with radiation and axitinib. Our studies suggest that axitinib is a potent and safe drug to use in conjunction with radiotherapy for lung cancer that could also act as a radioprotector for lung tissue by reducing pneumonitis and fibrosis.

Abstract B185: Induction of specific immunity to MUC1 antigen by tumor irradiation and cancer vaccines in murine tumor models

We have previously demonstrated that tumor irradiation potentiates cancer vaccines using genetic modification of tumor cells in murine tumor models. We showed that prior tumor irradiation enhanced the response of mice to intratumoral gene therapy with an IL-2 adenovector for non-immunogenic tumors. To investigate whether tumor irradiation augments the immune response to a specific tumor antigen, we have now tested the efficacy of tumor irradiation to enhance the therapeutic effect of MVA-MUC1-IL2 cancer vaccine (Transgene TG4010) for the treatment of murine renal adenocarcinoma Renca cells transfected with MUC1. Established subcutaneous (s.c.) Renca-MUC1 tumors (18mm3) were irradiated with 8 Gy on day 11 and peritumoral s.c. injections of MVA-MUC1-IL2 vector were administered at 106- 107 PFU on day 12 and 20. Tumor growth delays were monitored by tumor measurements and histological responses were evaluated following treatment with radiation alone, vector alone, radiation + MVA-MUC1-IL2 vector or radiation + MVA empty vector. Histological evaluation of tumors at an early time point, by 2-3 weeks after radiation and cancer vaccine, revealed that tumors treated with radiation and vaccine showed extensive areas of necrosis due to complete tumor destruction. Intense hemorrhages were observed due to disruption of tumor vasculature. Small areas of remaining tumor showed apoptotic tumor cells and degenerating giant tumor cells typical of radiation-induced changes. At the periphery of the tumor and infiltrating the tumor, we observed large bands of inflammatory cells including lymphocytes and macrophages as well as fibroblasts spindled shaped cells. Immuno-histochemical staining with CD45 leukocyte marker and F4/80 macrophage marker confirmed extensive infiltration of leukocytes and macrophages at the periphery and inside of areas of tumor destruction. These alterations were not as pronounced with radiation alone suggesting a drastic effect of the combined radiation + vaccine therapy on the tumor microenvironment. Radiation induced tumor growth delays for about 15 days but longer tumor growth delays of 30-35 days were observed with radiation + MVA-MUC1-IL2. By day 55, over 30% of the mice treated with radiation + MVA-MUC1-IL2 had a complete response with no sign of tumor whereas no responders were observed with either radiation alone or cancer vaccine alone. Complete responders were immune to rechallenge with Renca-MUC1 cells. These findings suggest that tumor irradiation given prior to cancer vaccine augments a specific immune response targeted at a specific tumor antigen that results in specific tumor immunity. The mechanisms of interaction between tumor irradiation and gene-mediated immunotherapy could include radiation-induced alterations in the tumor microenvironment, as those observed histologically in our studies, which could facilitate a more effective anti-tumor immune response. These include radiation-induced apoptosis and necrosis of tumor cells causing tumor-debulking and release of tumor antigens for APC presentation. Inflammatory cells mobilized in the tumor by radiation-induced tissue damage could subsequently be activated by the immune response triggered by the cancer vaccine. These studies support investing further pre-clinical and clinical efforts to combine radiotherapy with cancer vaccines for the treatment of cancer. Citation Format: Gilda Gali Hillman, Matthew D. Fountain, Shoshana E. Rothstein, Lyndsey Reich, Lisa Abernathy, Christopher K. Yunker, Fulvio Lonardo, Philippe Slos. Induction of specific immunity to MUC1 antigen by tumor irradiation and cancer vaccines in murine tumor models. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B185.

Abstract 277: Radioprotection of lung tissue involves modulation of radiation-induced macrophage activation by soy isoflavones

Radiation therapy for lung cancer is limited by radiation toxicity to lung tissue that results from a chronic inflammatory response, leading to pneumonitis and fibrosis. We have reported in preclinical mouse models that treatment with soy isoflavones mitigates inflammatory cytokines and fibrosis, but the cellular mediators of radioprotection remain unclear. Macrophages possess the plasticity to respond to environmental stressors in tissues that functionally may range from proinflammatory to immunosuppressive phenotypes. We hypothesize that soy isoflavones mediate radioprotection of normal lung tissue by modifying macrophage phenotype and function in irradiated lungs. In this study, we investigate the role of lung macrophage subsets in radioprotection of lung tissue by soy. BALB/c mice received a single 10 Gy dose of thoracic irradiation with soy isoflavones given orally at 1 mg per day, prior-to and continuously after radiation for up to 18 weeks. Bronchoalveolar lavage (BAL) fluid and lungs were harvested at early and late time points post-radiation. Differential cell counts on BAL fluid cytospins were performed and ratios of enlarged, foamy macrophages and smaller macrophages were calculated. At 4 weeks, lungs were dissociated into single cell suspensions and cells were stained with anti-CD45, anti-F4/80, and anti-CD11c fluorescent antibodies to analyze interstitial (F4/80+CD11c-) and alveolar (F4/80+CD11c+) lung tissue macrophages by flow cytometry. In situ M1 macrophages were detected by immunohistochemical staining in lung tissue sections for the pan-macrophage marker F4/80 and the M1 macrophage activation marker NOS2. At 18 weeks after radiation, there was a significant increase in the percentage of enlarged, foamy macrophages in BAL fluid to 79.0±6.7% compared with 14.6±5.3% in control (p = 0.0003). Soy significantly inhibited this radiation-induced increase to 33.1±8.3% compared with radiation alone (p = 0.0091). In situ staining of F4/80 and NOS2 in lung tissue sections revealed an increase of activated M1 macrophages caused by radiation in contrast to relatively low NOS2 levels in lungs of mice treated with radiation and soy or control. Soy significantly reduced the percentage of F4/80+CD11c- interstitial macrophage (p = 0.0313) in lung tissue post-radiation. F4/80+CD11c+ alveolar macrophages in lungs are significantly decreased (p = 0.0074) after radiation, however soy did not have a significant effect (p = 0.7160). These data indicate that radiation-induced proinflammatory M1 macrophage activation is inhibited by soy. Further studies are ongoing to clarify the role of interstitial and alveolar macrophages in radiation-induced lung inflammation and its regulation by soy. These findings suggest that soy modulation of the macrophage subset functions in response to radiation may play a critical role in soy-mediated radioprotective effects in lungs. Citation Format: Lisa M. Abernathy, Matthew D. Fountain, John M. David, Christopher K. Yunker, Michael C. Joiner, Gilda G. Hillman. Radioprotection of lung tissue involves modulation of radiation-induced macrophage activation by soy isoflavones. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 277. doi:10.1158/1538-7445.AM2015-277

New DCE-MRI parameters to quantify the vascular changes induced by sunitinib treatment in renal carcinoma tumors

To develop new dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) parameters to quantify the vascular effects of different doses of the antiangiogenic drug sunitinib on renal cell carcinoma (RCC) kidney tumors in mice. Mice bearing established RCC xenograft tumors were treated with sunitinib doses of 10, 20 or 40 mg/kg/day (SU10, SU20 or SU40 respectively) or treated with vehicle only (control). New DCE parameters, contrast agent uptake to the peak (AUCtp), time to peak concentration (TTP), washout slope (Nslope) and full width half maximum (FWHM), were obtained from T1-weighted images. These parameters were quantified for tumor-bearing kidneys and normal kidneys. Treatments with SU20 and SU40 caused increased perfusion in the tumor core compared to control and SU10. Kidney tumors treated with SU20 had an almost identical pattern of contrast agent uptake rate, peak and clearance as those observed in normal kidneys. The effect of SU20 on normal kidneys was milder than that observed with SU40. Treatment with SU40 caused increased contrast agent uptake by the cortex of the normal kidneys compared to the normal kidneys in control and SU10. FWHM also provided new information about the effect of different treatment doses and showed that kidney tumors treated with SU20 have almost the same values of FWHM as the normal kidneys in control mice. The new DCE parameters, including AUCtp, Nslope and FWHM, have the potential to give a precise description of the treatment effect not only in the whole mouse kidney but also in different regions inside the kidney.