Vinita Singh-Gupta

Down-regulation of Apurinic/Apyrimidinic Endonuclease 1/Redox Factor-1 Expression by Soy Isoflavones Enhances Prostate Cancer Radiotherapy In vitro and In vivo

We previously showed that genistein, the major bioactive component of soy isoflavones, acts as a radiosensitizer and potentiates prostate tumor cell killing by radiation in vitro and in animal tumor models in vivo. However, when given alone in vivo, pure genistein promoted increased lymph node metastasis, which was not observed with a soy isoflavone mixture consisting of genistein, daidzein, and glycitein. In this study, we show that soy inhibit tumor cell growth and potentiates radiation-induced cell killing in vitro like pure genistein. In an orthotopic model, combining soy isoflavones with tumor irradiation inhibited prostate tumor growth. To determine the molecular mechanisms by which soy isoflavones potentiate radiotherapy, we investigated apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) and nuclear factor kappaB (NF-kappaB), two signaling molecules involved in survival pathways. Soy isoflavones decreased APE1/Ref-1 expression in vitro, whereas radiation up-regulated it. Pretreatment with soy isoflavones followed by radiation inhibited APE1/Ref-1 expression. APE1/Ref-1 decrease correlated with decreased DNA-binding activity of NF-kappaB mediated by soy isoflavones and radiation, thus promoting cell killing. In vivo treatment of prostate tumors with soy isoflavones and radiation down-regulated APE1/Ref-1 protein expression and NF-kappaB activity, confirming the molecular alterations observed in vitro. The down-regulation of APE1/Ref-1 and NF-kappaB by isoflavones, in vitro and in vivo, supports our hypothesis that these markers represent biological targets of isoflavones. Indeed, a 2-fold increase in APE1/Ref-1 expression, obtained by cDNA transfection, resulted in a 2-fold increase in NF-kappaB DNA-binding activity, and both of which were down-regulated by soy isoflavones, confirming the cross-talk between these molecules and, in turn, causing radiosensitization.

Radiation‐induced HIF‐1α cell survival pathway is inhibited by soy isoflavones in prostate cancer cells

We previously showed that treatment of prostate cancer cells with soy isoflavones and radiation resulted in greater cell killing in vitro, and caused downregulation of NF‐κB and APE1/Ref‐1. APE1/Ref‐1 functions as a redox activator of transcription factors, including NF‐κB and HIF‐1α. These molecules are upregulated by radiation and implicated in radioresistance of cancer cells. We extended our studies to investigate the role of HIF‐1α survival pathway and its upstream Src and STAT3 molecules in isoflavones and radiation interaction. Radiation induced phosphorylation of Src and STAT3 leading to induction of HIF‐1α. Genistein, daidzein or a mixture of soy isoflavones did not activate this pathway. These data were observed both in PC‐3 (AR‐) and C4‐2B (AR+) androgen‐independent cell lines. Pretreatment with isoflavones inhibited Src/STAT3/HIF‐1α activation by radiation and nuclear translocation of HIF‐1α. These findings correlated with decreased expression of APE1/Ref‐1 and DNA binding activity of HIF‐1α and NF‐κB. In APE1/Ref‐1 cDNA transfected cells, radiation caused a greater increase in HIF‐1α and NF‐κB activities but this effect was inhibited by pretreatment with soy prior to radiation. Transfection experiments indicate that APE1/Ref‐1 inhibition by isoflavones impairs the radiation‐induced transcription activity of NF‐κB and HIF‐1α. This mechanism could result in the inhibition of genes essential for tumor growth and angiogenesis, as demonstrated by inhibition of VEGF production and HUVECs tube formation. Our novel findings suggest that the increased responsiveness to radiation mediated by soy isoflavones could be due to pleiotropic effects of isoflavones blocking cell survival pathways induced by radiation including Src/STAT3/HIF‐1α, APE1/Ref‐1 and NF‐κB.

Soy isoflavones sensitize cancer cells to radiotherapy

Soy isoflavones are dietary compounds isolated from soybeans, which are safe for human use and have mild anti-cancer properties. Soy isoflavones inhibit the activity of transcription factors and genes essential for tumor cell proliferation, invasion, and neovascularization, and it appears that soy isoflavones may enhance the effectiveness of conventional therapies against cancer. Soy isoflavones could be an effective complementary treatment given that they inhibit the survival signaling pathways of various cancer cells through altered activation of APE1/Ref-1, NF-κB, and HIF-1α, which are genes essential for tumor cell survival, tumor growth, and angiogenesis, thus making such cells more sensitive to radiotherapy. Studies in which soy isoflavones were given in conjunction with radiotherapy to prostate cancer patients suggest that soy isoflavones might also mitigate the adverse effects of radiation on normal tissues, probably by acting as antioxidants. These observations open new avenues for exploiting soy isoflavones as supplements to conventional therapies.

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.

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.

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.

Abstract LB-358: Soy improves radiotherapy for lung tumors and mitigates radiation-induced injury to lung tissue.

Radiotherapy of locally-advanced non-small cell lung cancer (NSCLC) is limited by pneumonitis and fibrosis caused by radiation-induced injury to normal lung tissue. We have investigated the effect of a high thoracic dose of radiation combined with soy isoflavones in a pre-clinical model of orthotopic A549 human lung carcinoma xenografts in nude mice. Mice bearing established lung tumors (>300µm) were pretreated with soy for 3 days starting day 15 after A549 cell injection, and then received 10 Gy irradiation administered to the whole lung. Soy treatment, given orally at 1mg/day (50mg/kg) was continued for 5 days per week for 4 more weeks, then lungs were perfused with formalin and processed for histology. The effect of the therapy on the tumor cells, the surrounding tumor microenvironment and lung tissue structures was further analyzed using novel immunostaining and quantitative techniques. Compared to large invasive lung tumor nodules (up to 1-2 x 10 6 µm 2 ) in untreated mice, the combined therapy caused a significant inhibition of tumor progression in lungs resulting in few residual small tumor nodules (0.02-0.07 x 10 6 µm 2 , p