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Exposure to high levels of radiation can cause acute, long-term health effects, such as acute radiation syndrome, cancer, and cardiovascular disease. This is an important occupational hazard in different fields, such as the aerospace and healthcare industry, as well as a crucial burden to overcome to boost space applications and exploration. Protective bulky equipment made of heavy metals is not suitable for many advanced purporses, such as mobile devices, wearable shields, and manned spacecrafts. In the latter case, the in-space manufacturing of protective shields is highly desirable and remains an unmet need. Composites made of polymers and high atomic number fillers are potential means for radiation protection due to their low weight, good flexibility, and good processability. In the present work, we developed electrospun composites based on polycaprolactone (polymer matrix) and tungsten powder for application as shielding materials. Electrospinning is a versatile technology that is easily scalable at an industrial level and allows obtaining very lightweight, flexible sheet materials for wearables. By controlling tungsten powder size, we engineered homogeneous, stable and processable suspensions to fabricate radiation composite shielding sheets. The shielding capability was assessed by an in vivo model on prototype composite sheets containing 80 w% of W filler in a polycaprolactone (PCL) fibrous matrix by means of irradiation tests (X-rays) on mice. The obtained results are promising; as expected, the shielding effectivity of the developed composite material increases with the thickness/number of stacked layers. It is worth noting that a thin barrier consisting of 24 layers of the innovative shielding material reduces the extent of apoptosis by 1.5 times compared to the non-shielded mice.

Apolipoprotein E (ApoE) is a lipid carrier in both the peripheral and the central nervous systems (CNSs). Lipid-loaded ApoE lipoprotein particles bind to several cell surface receptors to support membrane homeostasis and brain injury repair. In the brain, ApoE is produced predominantly by astrocytes, but it is also abundantly expressed in most neurons of the CNS. In this study, we addressed the role of ApoE in the hippocampus in mice, focusing on its role in response to radiation injury. To this aim, 8-week-old, wild-type, and ApoE-deficient (ApoE -/- ) female mice were acutely whole-body irradiated with 3 Gy of X-rays (0.89 Gy/min), then sacrificed 150 days post-irradiation. In addition, age-matching ApoE -/- females were chronically whole-body irradiated (20 mGy/d, cumulative dose of 3 Gy) for 150 days at the low dose-rate facility at the Institute of Environmental Sciences (IES), Rokkasho, Japan. To seek for ApoE-dependent modification during lineage progression from neural stem cells to neurons, we have evaluated the cellular composition of the dentate gyrus in unexposed and irradiated mice using stage-specific markers of adult neurogenesis. Our findings indicate that ApoE genetic inactivation markedly perturbs adult hippocampal neurogenesis in unexposed and irradiated mice. The effect of ApoE inactivation on the expression of a panel of miRNAs with an established role in hippocampal neurogenesis, as well as its transcriptional consequences in their target genes regulating neurogenic program, have also been analyzed. Our data show that the absence of ApoE -/- also influences synaptic functionality and integration by interfering with the regulation of mir-34a, mir-29b, and mir-128b, leading to the downregulation of synaptic markers PSD95 and synaptophysin mRNA. Finally, compared to acute irradiation, chronic exposure of ApoE null mice yields fewer consequences except for the increased microglia-mediated neuroinflammation. Exploring the function of ApoE in the hippocampus could have implications for developing therapeutic approaches to alleviate radiation-induced brain injury.

Cystic lesions of the anterior head and neck region are a challenging and frequent finding on cytological smears. The scant amount of cellular material in cystic slides poses the greatest difficulty to interpretation, so that frequently they are diagnosed as inadequate or with minimal cellular component. Despite the majority of cystic lesions being benign, a minor portion consist of malignant cystic entities. In these latter cases, the evidence of very scant malignant cells can be misdiagnosed and/or underestimated, leading to a false negative diagnosis. Many papers have already described and detailed the range of possible benign and malignant cystic lesions in head and neck. In the current review we have focused on the less common entities that often lead to serious misinterpretation., (© 2023 John Wiley & Sons Ltd.)

Cystic lesions of the salivary glands are very uncommon entities. However, on occasion, some neoplasms of the salivary glands show a cystic component, which may be predominant or only partially cystic. Basal cell adenoma, canalicular adenoma, oncocytoma, sebaceous adenoma, intraductal papilloma, epithelial-myoepithelial carcinoma, intraductal carcinoma, and secretory carcinoma are such cystic entities. Cystic degeneration and necrosis, which can develop within solid tumours, represent another possibility. The ability to recognise this type of lesion is a challenge in diagnostic cytology because hypocellular fluid is frequently recovered. Furthermore, evaluating all of the differential diagnoses for cystic lesions of the salivary glands is helpful in obtaining the correct diagnosis. Herein, we evaluate the various types of cystic lesions within the salivary glands., (© 2023 John Wiley & Sons Ltd.)

Pelvic radiation disease (PRD), a frequent side effect in patients with abdominal/pelvic cancers treated with radiotherapy, remains an unmet medical need. Currently available preclinical models have limited applications for the investigation of PRD pathogenesis and possible therapeutic strategies. In order to select the most effective irradiation protocol for PRD induction in mice, we evaluated the efficacy of three different locally and fractionated X-ray exposures. Using the selected protocol (10 Gy/day × 4 days), we assessed PRD through tissue (number and length of colon crypts) and molecular (expression of genes involved in oxidative stress, cell damage, inflammation, and stem cell markers) analyses at short (3 h or 3 days after X-ray) and long (38 days after X-rays) post-irradiation times. The results show that a primary damage response in term of apoptosis, inflammation, and surrogate markers of oxidative stress was found, thus determining a consequent impairment of cell crypts differentiation and proliferation as well as a local inflammation and a bacterial translocation to mesenteric lymph nodes after several weeks post-irradiation. Changes were also found in microbiota composition, particularly in the relative abundance of dominant phyla, related families, and in alpha diversity indices, as an indication of dysbiotic conditions induced by irradiation. Fecal markers of intestinal inflammation, measured during the experimental timeline, identified lactoferrin, along with elastase, as useful non-invasive tools to monitor disease progression. Thus, our preclinical model may be useful to develop new therapeutic strategies for PRD treatment.

Cell communication via exosomes is capable of influencing cell fate in stress situations such as exposure to ionizing radiation. In vitro and in vivo studies have shown that exosomes might play a role in out-of-target radiation effects by carrying molecular signaling mediators of radiation damage, as well as opposite protective functions resulting in resistance to radiotherapy. However, a global understanding of exosomes and their radiation-induced regulation, especially within the context of an intact mammalian organism, has been lacking. In this in vivo study, we demonstrate that, compared to sham-irradiated (SI) mice, a distinct pattern of proteins and miRNAs is found packaged into circulating plasma exosomes after whole-body and partial-body irradiation (WBI and PBI) with 2 Gy X-rays. A high number of deregulated proteins (59% of WBI and 67% of PBI) was found in the exosomes of irradiated mice. In total, 57 and 13 miRNAs were deregulated in WBI and PBI groups, respectively, suggesting that the miRNA cargo is influenced by the tissue volume exposed to radiation. In addition, five miRNAs (miR-99b-3p, miR-200a-3p, miR-200a, miR-182-5p, miR-182) were commonly overexpressed in the exosomes from the WBI and PBI groups. In this study, particular emphasis was also given to the determination of the in vivo effect of exosome transfer by intracranial injection in the highly radiosensitive neonatal cerebellum at postnatal day 3. In accordance with a major overall anti-apoptotic function of the commonly deregulated miRNAs, here, we report that exosomes from the plasma of irradiated mice, especially in the case of WBI, prevent radiation-induced apoptosis, thus holding promise for exosome-based future therapeutic applications against radiation injury.

Recent epidemiological findings and reanalysis of historical data suggest lens opacities resulting from ionizing radiation exposures are likely induced at lower doses than previously thought. These observations have led to ICRP recommendations for a reduction in the occupational dose limits for the eye lens, as well as subsequent implementation in EU member states. The EU CONCERT LDLensRad project was initiated to further understand the effects of ionizing radiation on the lens and identify the mechanism(s) involved in radiation-induced cataract, as well as the impact of dose and dose-rate. Here, we present the results of a long-term study of changes to lens opacity in male and female adult mice from a variety of different genetic (radiosensitive or radioresistant) backgrounds, including mutant strains Ercc2 and Ptch1, which were assumed to be susceptible to radiation-induced lens opacities. Mice received 0.5, 1 and 2 Gy 60Co gamma-ray irradiation at dose rates of 0.063 and 0.3 Gy min-1. Scheimpflug imaging was used to quantify lens opacification as an early indicator of cataract, with monthly observations taken postirradiation for an 18-month period in all strains apart from 129S2, which were observed for 12 months. Opacification of the lens was found to increase with time postirradiation (with age) for most mouse models, with ionizing radiation exposure increasing opacities further. Sex, dose, dose rate and genetic background were all found to be significant contributors to opacification; however, significant interactions were identified, which meant that the impact of these factors was strain dependent. Mean lens density increased with higher dose and dose rate in the presence of Ercc2 and Ptch1 mutations. This project was the first to focus on low (<1 Gy) dose, multiple dose rate, sex and strain effects in lens opacification, and clearly demonstrates the importance of these experimental factors in radiobiological investigations on the lens. The results provide insight into the effects of ionizing radiation on the lens as well as the need for further work in this area to underpin appropriate radiation protection legislation and guidance., (©2022 by Radiation Research Society. All rights of reproduction in any form reserved.)

Epidemiological studies suggest an increased incidence and risk of cataract after low-dose (<2 Gy) ionizing radiation exposures. However, the biological mechanism(s) of this process are not fully understood. DNA damage and repair are thought to have a contributing role in radiation-induced cataractogenesis. Recently we have reported an inverse dose-rate effect, as well as the low-dose response, of DNA damage and repair in lens epithelial cells (LECs). Here, we present further initial findings from two mutated strains (Ercc2+/- and Ptch1+/-) of mice, both reportedly susceptible to radiation-induced cataract, and their DNA damage and repair response to low-dose and low-dose-rate gamma rays. Our results support the hypothesis that the lens epithelium responds differently to radiation than other tissues, with reported radiation susceptibility to DNA damage not necessarily translating to the LECs. Genetic predisposition and strain(s) of mice have a significant role in radiation-induced cataract susceptibility., (©2022 by Radiation Research Society. All rights of reproduction in any form reserved.)

Experimental mouse studies are important to gain a comprehensive, quantitative and mechanistic understanding of the biological factors that modify individual risk of radiation-induced health effects, including age at exposure, dose, dose rate, organ/tissue specificity and genetic factors. In this study, neonatal Ptch1+/- mice bred on CD1 and C57Bl/6 background received whole-body irradiation at postnatal day 2. This time point represents a critical phase in the development of the eye lens, cerebellum and dentate gyrus (DG), when they are also particularly susceptible to radiation effects. Irradiation was performed with γ rays (60Co) at doses of 0.5, 1 and 2 Gy, delivered at 0.3 Gy/min or 0.063 Gy/min. Wild-type and mutant mice were monitored for survival, lens opacity, medulloblastoma (MB) and neurogenesis defects. We identified an inverse genetic background-driven relationship between the radiosensitivity to induction of lens opacity and MB and that to neurogenesis deficit in Ptch1+/- mutants. In fact, high incidence of radiation-induced cataract and MB were observed in Ptch1+/-/CD1 mutants that instead showed no consequence of radiation exposure on neurogenesis. On the contrary, no induction of radiogenic cataract and MB was reported in Ptch1+/-/C57Bl/6 mice that were instead susceptible to induction of neurogenesis defects. Compared to Ptch1+/-/CD1, the cerebellum of Ptch1+/-/C57Bl/6 mice showed increased radiosensitivity to apoptosis, suggesting that differences in processing radiation-induced DNA damage may underlie the opposite strain-related radiosensitivity to cancer and non-cancer pathologies. Altogether, our results showed lack of dose-rate-related effects and marked influence of genetic background on the radiosensitivity of Ptch1+/-mice, supporting a major contribution of individual sensitivity to radiation risk in the population., (©2022 by Radiation Research Society. All rights of reproduction in any form reserved.)

One harmful long-term effect of ionizing radiation is cataract development. Recent studies have been focused on elucidating the mechanistic pathways involved in this pathogenesis. Since accumulating evidence has established a role of microRNAs in ocular diseases, including cataract, the goal of this work was to determine the microRNA signature of the mouse lens, at short time periods postirradiation, to understand the mechanisms related to radio-induced cataractogenesis. To evaluate the differences in the microRNA profiles, 10-week-old Patched1 heterozygous (Ptch1+/-) mice, bred onto two different genetic backgrounds (CD1 and C57Bl/6J), received whole-body 2 Gy γ-ray irradiation, and 24 h later lenses were collected. Next-generation sequencing and bioinformatics analysis revealed that genetic background markedly influenced the list of the deregulated microRNAs and the mainly predicted perturbed biological functions of 2 Gy irradiated Ptch1+/- mouse lenses. We identified a subset of microRNAs with a contra-regulated expression between strains, with a key role in regulating Toll-like receptor (TLR)-signaling pathways. Furthermore, a detailed analysis of miRNome data showed a completely different DNA damage response in mouse lenses 24 h postirradiation, mainly mediated by a marked upregulation of p53 signaling in Ptch1+/-/C57Bl/6J lenses that was not detected on a CD1 background. We propose a strict interplay between p53 and TLR signaling in Ptch1+/-/C57Bl/6J lenses shortly after irradiation that could explain both the resistance of this strain to developing lens opacities and the susceptibility of CD1 background to radiation-induced cataractogenesis through activation of epithelial-mesenchymal transition., (©2022 by Radiation Research Society. All rights of reproduction in any form reserved.)

The advent of new 'omics' techniques determined a massive boost in the measurement of the whole spectra of molecules within cells, favoring promising new radiobiological studies at low doses. The main aim of this work was to assess the radiation-induced perturbations of miRNA profiles and their temporal dynamics. Human Umbilical Vein Endothelial Cells were irradiated with low doses of γ-rays. At different time points post-irradiation, cells were harvested and miRNAs isolated. A full mapping of the miRNA sequences via Next-Generation-Sequencing analysis was performed followed by bioinformatic analyses. Pathway enrichment analyses on the differentially expressed miRNAs focused both on the averaged effects of different doses over the 24-h experiment and on the altered temporal dynamics of the miRNA profiles. These complementary analyses provided a picture of the dose- and time-dependent miRNAs responses, allowing to better explore the candidate biomarkers linked to radiation exposures and their corresponding pathways and functions., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

DSBs are harmful lesions produced through endogenous metabolism or by exogenous agents such as ionizing radiation, that can trigger genomic rearrangements. We have recently shown that exposure to 2 Gy of X-rays has opposite effects on the induction of Shh-dependent MB in NHEJ- and HR-deficient Ptch1 +/- mice. In the current study we provide a comprehensive link on the role of HR/NHEJ at low doses (0.042 and 0.25 Gy) from the early molecular changes through DNA damage processing, up to the late consequences of their inactivation on tumorigenesis. Our data indicate a prominent role for HR in genome stability, by preventing spontaneous and radiation-induced oncogenic damage in neural precursors of the cerebellum, the cell of origin of MB. Instead, loss of DNA-PKcs function increased DSBs and apoptosis in neural precursors of the developing cerebellum, leading to killing of tumor initiating cells, and suppression of MB tumorigenesis in DNA-PKcs -/- /Ptch1 +/- mice. Pathway analysis demonstrates that DNA-PKcs genetic inactivation confers a remarkable radiation hypersensitivity, as even extremely low radiation doses may deregulate many DDR genes, also triggering p53 pathway activation and cell cycle arrest. Finally, by showing that DNA-PKcs inhibition by NU7441 radiosensitizes human MB cells, our in vitro findings suggest the inclusion of MB in the list of tumors beneficiating from the combination of radiotherapy and DNA-PKcs targeting, holding promise for clinical translation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Medulloblastoma (MB) is the most common pediatric brain tumor, comprising four distinct molecular variants, one of which characterized by activation of the Sonic Hedgehog (SHH) pathway, driving 25-30% of sporadic MB. SHH-dependent MBs arise from granule cell precursors (GCPs), are fatal in 40-70% of cases and radioresistance strongly contributes to poor prognosis and tumor recurrence. Patched1 heterozygous (Ptch1 +/- ) mice, carrying a germ-line heterozygous inactivating mutation in the Ptch1 gene, the Shh receptor and negative regulator of the pathway, are uniquely susceptible to MB development after radiation damage in neonatal cerebellum. Here, we irradiated ex-vivo GCPs isolated from cerebella of neonatal WT and Ptch1 +/- mice. Our results highlight a less differentiated status of Ptch1-mutated cells after irradiation, influencing DNA damage response. Increased expression levels of pluripotency genes Nanog, Oct4 and Sal4, together with greater clonogenic potential, clearly suggest that radiation induces expansion of the stem-like cell compartment through cell-reprogramming and self-renewal maintenance, and that this mechanism is strongly dependent on Nanog. These results contribute to clarify the molecular mechanisms that control radiation-induced Shh-mediated tumorigenesis and may suggest Nanog as a potential target to inhibit for adjuvant radiotherapy in treatment of SHH-dependent MB.

Along with their role in the maintenance of nuclear architecture, nuclear lamins also control genomic stability, DNA damage repair, transcription, cell proliferation, differentiation and senescence. Recent reports reveal that prelamin A-processing defects play a role in cancer development by impacting on transcription of key players in the maintenance of the genome stability, including RAD51. Here, we performed a 'proof of concept' study evaluating the role of prelamin A and RAD51 expression in clinical outcome of cervical cancer patients. We analyzed biomarker expression by immunohistochemistry in tumor material from locally advanced cervical cancer (LACC) patients (n=66) and correlated data with clinicopathological parameters and with response to neoadjuvant chemoradiation (CT/RT). In LACC patients who underwent neoadjuvant CT/RT the percentage of cases showing high prelamin A levels was greater in patients who completely responded to treatment (25 of 40, 62.5%) than in patients with macroscopic residual tumor (6 of 26, 23.1%, p=0.0024). Conversely, patients showing high RAD51 expression were less likely to respond to treatment (14 of 26, 53.8%) than were those with low protein levels (12 of 40, 30%, p=0.072). Only prelamin A retained an independent role in predicting response to treatment (p=0.003), while RAD51 approached statistical significance (p=0.07). Notably, high RAD51 expression highly significantly predicted poor outcome, emerging as an independent prognostic factor for disease free survival (p=0.038), while approaching statistical significance for overall survival (p=0.09). Our findings provide a framework for future prospective studies investigating molecular predictors of response to neoadjuvant chemoradiotherapy in LACC patients., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no competing interest.

It has historically been accepted that incorrectly repaired DNA double strand breaks (DSBs) are the principal lesions of importance regarding mutagenesis, and long-term biological effects associated with ionizing radiation. However, radiation may also cause dysregulation of epigenetic processes that can lead to altered gene function and malignant transformation, and epigenetic alterations are important causes of miRNAs dysregulation in cancer.Patched1 heterozygous (Ptch1+/-) mice, characterized by aberrant activation of the Sonic hedgehog (Shh) signaling pathway, are a well-known murine model of spontaneous and radiation-induced medulloblastoma (MB), a common pediatric brain tumor originating from neural granule cell progenitors (GCPs). The high sensitivity of neonatal Ptch1+/- mice to radiogenic MB is dependent on deregulation of the Ptch1 gene function. Ptch1 activates a growth and differentiation programme that is a strong candidate for regulation through the non-coding genome. Therefore we carried out miRNA next generation sequencing in ex vivo irradiated and control GCPs, isolated and purified from cerebella of neonatal WT and Ptch1+/- mice. We identified a subset of miRNAs, namely let-7 family and miR-17~92 cluster members, whose expression is altered in GCPs by radiation alone, or by synergistic interaction of radiation with Shh-deregulation. The same miRNAs were further validated in spontaneous and radiation-induced MBs from Ptch1+/- mice, confirming persistent deregulation of these miRNAs in the pathogenesis of MB.Our results support the hypothesis that miRNAs dysregulation is associated with radiosensitivity of GCPs and their neoplastic transformation in vivo.

While most of the evidence for radiation-induced late health effects relates to cancer, there has been increasing interest recently in the development of non-cancer diseases, including lens opacity, observed in populations exposed to low-dose radiation. In a recent study, we reported that mice heterozygous for the Patched1 (Ptch1) gene represented a novel and powerful animal model for this disorder, and a useful tool for investigating the mechanisms of radiogenic cataract development. Given the ongoing and considerable uncertainty in allowable lens dose levels and the existence of a threshold for the development of cataracts, we tested the effects of a decreasing range of radiation doses (2 Gy, 1 Gy and 0.5 Gy X rays) by irradiating groups of Ptch1(+/-) mice at 2 days of age. Our findings showed that at this dose range, acute exposure of this highly susceptible mouse model did not induce macroscopically detectable cataracts, and only the 2 Gy irradiated mice showed microscopic alterations of the lens. Molecular analyses performed to evaluate the induction of epithelial-mesenchymal transition (EMT) and subsequent fibrotic alterations in mouse lens cells also indicated the existence of a dose threshold for such effects in the mouse model used. The mechanisms of cataractogenesis remain unclear, and further experimental studies are essential to elucidate those mechanisms specific for cataract initiation and development after irradiation, as well as the underlying genetic factors controlling cataract susceptibility.

Mutations in DNA repair pathways are frequent in human cancers. Hence, gaining insights into the interaction of DNA repair genes is key to development of novel tumor-specific treatment strategies. In this study, we tested the functional relationship in development and oncogenesis between the homologous recombination (HR) factor Rad54 and Parp-1 , a nuclear enzyme that plays a multifunctional role in DNA damage signaling and repair. We introduced single or combined Rad54 and Parp-1 inactivating germline mutations in Ptc1 heterozygous mice, a well-characterized model of medulloblastoma, the most common malignant pediatric brain tumor. Our study reveals that combined inactivation of Rad54 and Parp-1 causes a marked growth delay culminating in perinatallethality, providing for the first time evidence of synthetic lethal interactions between Rad54 and Parp-1 in vivo . Although the double mutation hampered investigation of Rad54 and Parp-1 interactions in cerebellum tumorigenesis, insights were gained by showing accumulation of endogenous DNA damage and increased apoptotic rate in granule cell precursors (GCPs). A network-based approach to detect differential expression of DNA repair genes in the cerebellum revealed perturbation of p53 signaling in Rad54 -/- / Parp-1 -/- / Ptc1 +/- , and MEFs from combined Rad54/Parp-1 mutants showed p53/p21-dependent typical senescent features. These findings help elucidate the genetic interplay between Rad54 and Parp-1 by suggesting that p53/p21-mediated apoptosis and/or senescence may be involved in synthetic lethal interactions occurring during development and inhibition of tumor growth., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflict of interest.

Age-related cataract is the most common cause of visual impairment. Moreover, traumatic cataracts form after injury to the eye, including radiation damage. We report herein that sonic hedgehog (Shh) signaling plays a key role in cataract development and in normal lens response to radiation injury. Mice heterozygous for Patched 1 (Ptch1), the Shh receptor and negative regulator of the pathway, develop spontaneous cataract and are highly susceptible to cataract induction by exposure to ionizing radiation in early postnatal age, when lens epithelial cells undergo rapid expansion in the lens epithelium. Neonatally irradiated and control Ptch1(+/-) mice were compared for markers of progenitors, Shh pathway activation, and epithelial-to-mesenchymal transition (EMT). Molecular analyses showed increased expression of the EMT-related transforming growth factor β/Smad signaling pathway in the neonatally irradiated lens, and up-regulation of mesenchymal markers Zeb1 and Vim. We further show a link between proliferation and the stemness property of lens epithelial cells, controlled by Shh. Our results suggest that Shh and transforming growth factor β signaling cooperate to promote Ptch1-associated cataract development by activating EMT, and that the Nanog marker of pluripotent cells may act as the primary transcription factor on which both signaling pathways converge after damage. These findings highlight a novel function of Shh signaling unrelated to cancer and provide a new animal model to investigate the molecular pathogenesis of cataract formation., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: The male-to-female sex ratio for medulloblastoma (MB) is approximately 1.5∶1, female gender being also a favorable prognostic factor. This study aimed at evaluating the impact of gender on MB tumorigenesis., Methods: In vitro activity of 17β-estradiol (E2), DPN [2,3-bis(4-hydroxyphenyl)-propionitrile, a selective estrogen receptor β (ERβ)-agonist], PPT [4,4',4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, a selective ERα-agonist] or DHT (5 alpha-dihydrotestosterone) was evaluated in three human MB cell lines. D283 Med cells were transplanted into athymic mice., Results: A significant expression of ERβ, with little or no ERα, and low AR (androgen receptor) was found in MB cell lines. The compounds tested did not affect cell proliferation. In vivo, we observed a significantly lower growth of D283 Med in nude female mice compared to males. At microscopic examination, tumors from females showed a shift towards differentiation, as evaluated by lower nestin, and higher NSE (neuron-specific enolase) and GFAP (glial fibrillary acidic protein) expression compared to males. Tumors from females also showed lower Ki67 and p53 expression. The wild-type ERβ protein (ERβ1) was lost in male tumors, while it was a permanent feature in females, and a strong negative correlation was found between Ki67 and ERβ1 expression. Conversely, tumor levels of ERβ2 and ERβ5 did not significantly differ between genders. Increased levels of cyclin-dependent kinase inhibitor p21 were observed in females, suggesting that estrogen may decrease tumor growth through blocking cell cycle progression. An inhibition of the insulin-like growth factor I (IGF-I) signaling was also evident in females., Conclusion: We provides mechanistic evidence supporting the idea that ERβ1 signaling may have pro-differentiation and tumor suppressive function in medulloblastomas.

Neural stem cells are highly susceptible to radiogenic DNA damage, however, little is known about their mechanisms of DNA damage response (DDR) and the long-term consequences of genotoxic exposure. Patched1 heterozygous mice (Ptc1(+/-)) provide a powerful model of medulloblastoma (MB), a frequent pediatric tumor of the cerebellum. Irradiation of newborn Ptc1(+/-) mice dramatically increases the frequency and shortens the latency of MB. In this model, we investigated the mechanisms through which multipotent neural progenitors (NSCs) and fate-restricted progenitor cells (PCs) of the cerebellum respond to DNA damage induced by radiation, and the long-term developmental and oncogenic consequences. These responses were assessed in mice exposed to low (0.25 Gy) or high (3 Gy) radiation doses at embryonic day 13.5 (E13.5), when NSCs giving rise to the cerebellum are specified but the external granule layer (EGL) has not yet formed, or at E16.5, during the expansion of granule PCs to form the EGL. We found crucial differences in DDR and apoptosis between NSCs and fate-restricted PCs, including lack of p21 expression in NSCs. NSCs also appear to be resistant to oncogenesis from low-dose radiation exposure but more vulnerable at higher doses. In addition, the pathway to DNA repair and the pattern of oncogenic alterations were strongly dependent on age at exposure, highlighting a differentiation-stage specificity of DNA repair pathways in NSCs and PCs. These findings shed light on the mechanisms used by NSCs and PCs to maintain genome integrity during neurogenesis and may have important implications for radiation risk assessment and for development of targeted therapies against brain tumors., (Copyright © 2013 AlphaMed Press.)

Purpose: To investigate the tissue dependence in transmission of abscopal radiation signals and their oncogenic consequences in a radiosensitive mouse model and to explore the involvement of gap junction intercellular communication (GJIC) in mediating radiation tumorigenesis in off-target mouse skin., Methods and Materials: Patched1 heterozygous (Ptch1(+/-)) mice were irradiated at postnatal day 2 (P2) with 10 Gy of x-rays. Individual lead cylinders were used to protect the anterior two-thirds of the body, whereas the hindmost part was directly exposed to radiation. To test the role of GJICs and their major constituent connexin43 (Cx43), crosses between Ptch1(+/-) and Cx43(+/-) mice were similarly irradiated. These mouse groups were monitored for their lifetime, and skin basal cell carcinomas (BCCs) were counted and recorded. Early responses to DNA damage - Double Strand Breaks (DSBs) and apoptosis - were also evaluated in shielded and directly irradiated skin areas., Results: We report abscopal tumor induction in the shielded skin of Ptch1(+/-) mice after partial-body irradiation. Endpoints were induction of early nodular BCC-like tumors and macroscopic infiltrative BCCs. Abscopal tumorigenesis was significantly modulated by Cx43 status, namely, Cx43 reduction was associated with decreased levels of DNA damage and oncogenesis in out-of-field skin, suggesting a key role of GJIC in transmission of oncogenic radiation signals to unhit skin., Conclusions: Our results further characterize the nature of abscopal responses and the implications they have on pathologic processes in different tissues, including their possible underlying mechanistic bases., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The prognostic relevance of estrogen (ER) and progesterone receptor (PR) expression in endometrioid endometrial cancer is still controversially discussed. The present study has focused on the evaluation of the prognostic value of ERα, ERβ1, ERβ2, and PR in this histotype. Specifically, we were interested in evaluating whether the relative level of ER subtype-specific expression (in terms of a ratio ERα/ERβ1 and ERα/ERβ2) would predict clinical outcome better than their absolute levels in patients with endometrioid endometrial cancer. To this end, protein content was assessed by immunohistochemistry in a group of 121 cases and staining was analyzed in relation to clinicopathologic variables, disease-free survival and overall survival. Results obtained have demonstrated that none of the biological markers analyzed possess an independent prognostic role with regard to disease-free survival. Multivariate analysis of overall survival has shown that ERα alone is not an independent prognostic indicator in patients with endometrioid endometrial cancer (hazard ratio [HR]; 0.5; 95% confidence interval [CI], 0.09-3.0; P = .5). On the other hand, an ERα/ERβ1 ratio of 1 or less or an ERα/ERβ2 ratio of 1 or less has proved to be independently associated with a higher risk of death (HR, 6.4 [95% CI, 1.0-40.6; P = .04] and 9.7 [95% CI, 1.1-85.3; P = .04], respectively) along with age, tumor stage, and Ki-67. In conclusion, we report here that the ERα/ERβ1 and ERα/ERβ2 expression ratios are independent prognostic markers of survival in endometrioid endometrial cancer; these findings suggest that phenotyping these interacting markers conjointly may better predict patient survival than each individual marker alone., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: To investigate the dose and spatial dependence of abscopal radiation effects occurring in vivo in the mouse, along with their tumorigenic potential in the central nervous system (CNS) of a radiosensitive mouse model., Methods and Materials: Patched1 (Ptch1)(+/-) mice, carrying a germ-line heterozygous inactivating mutation in the Ptch1 gene and uniquely susceptible to radiation damage in neonatal cerebellum, were exposed directly to ionizing radiation (1, 2, or 3 Gy of x-rays) or treated in a variety of partial-body irradiation protocols, in which the animals' head was fully protected by suitable lead cylinders while the rest of the body was exposed to x-rays in full or in part. Apoptotic cell death was measured in directly irradiated and shielded cerebellum shortly after irradiation, and tumor development was monitored in lifetime groups. The same endpoints were measured using different shielding geometries in mice irradiated with 3 or 10 Gy of x-rays., Results: Although dose-dependent cell death was observed in off-target cerebellum for all doses and shielding conditions tested, a conspicuous lack of abscopal response for CNS tumorigenesis was evident at the lowest dose of 1 Gy. By changing the amount of exposed body volume, the shielding geometry could also significantly modulate tumorigenesis depending on dose., Conclusions: We conclude that interplay between radiation dose and exposed tissue volume plays a critical role in nontargeted effects occurring in mouse CNS under conditions relevant to humans. These findings may help understanding the mechanisms of long-range radiation signaling in harmful effects, including carcinogenesis, occurring in off-target tissues., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Recent evidence links aberrant activation of Hedgehog (Hh) signaling with the pathogenesis of several cancers including medulloblastoma, glioblastoma, melanoma as well as pancreas, colorectal, and prostate carcinomas. Here we investigated the role of the transcription factor Gli1 in ovarian cancer. To this end, the expression profile of Gli1 was examined in normal ovaries, ovarian tumors, and ovarian cancer cell lines, and the in vitro effects of a specific Hh-pathway blocker, KAAD-cyclopamine, or a specific Gli1 inhibitor (GANT58) on cell proliferation and on Hh target gene expression were also assessed. Results obtained showed that epithelial cells in ovarian cancer tissue express significantly higher levels of nuclear Gli1 than in normal ovarian tissue, where the protein was almost undetectable. In addition, multivariate analysis showed that nuclear Gli1 was independently associated to poor survival in advanced serous ovarian cancer patients (HR = 2.2, 95%CI 1.0-5.1, p = 0.04). In vitro experiments demonstrated Gli1 expression in the three ovarian carcinoma cell lines tested, A2780, SKOV-3 and OVCAR-3. Remarkably, although KAAD-cyclopamine led to decreased cell proliferation, this treatment did not inhibit hedgehog target gene expression in any of the three ovarian cancer cell lines, suggesting that the inhibition of cell proliferation was a nonspecific or toxic effect. In line with these data, no differences on cell proliferation were observed when cell lines were treated with GANT58. Overall, our clinical data support the role of Gli1 as a prognostic marker in advanced serous ovarian cancer and as a possible therapeutic target in this disease. However, our in vitro findings draw attention to the need for selection of appropriate experimental models that accurately represent human tumor for testing future therapies involving Hh pathway inhibitors.

Two types of ovarian carcinomas are distinguished with respect to morphology, biology, and clinical course, and are designated as Type I and Type II tumors. However, placement of clear cell carcinomas into one of these 2 groups has been problematic as they exhibit morphologic, molecular, and clinical features that do not entirely resemble either Type I or Type II tumors. The present study aimed at better elucidating the clinicopathologic and immunohistochemical features of clear cell carcinomas, in comparison with the 2 main broad categories. To this end, a panel of classic clinicopathologic and immunohistochemical parameters, including estrogen receptor α (ERα), ERβ, progesterone receptor, Ki67, p53, and HER2/neu was evaluated in 71 Type I, 157 Type II, and 21 clear cell carcinomas. Overall, findings from the present study support the idea that ovarian clear cell carcinomas are neither Type I nor Type II carcinomas of the ovary; indeed, results obtained showed that similarities between clear cell carcinomas and Type I were limited to the patient's age, tumor dimension, incidence of lymph node and extranodal metastases, and p53 labeling index, whereas the patient's age and incidence of extranodal metastases were the only parameters comparable with the Type II group. The hormonal receptor profile of clear cell carcinomas was characterized by low expression of nuclear ERα and progesterone receptor, and by almost exclusively nuclear ERβ immunopositivity, features significantly different from both Type I and II tumors. Finally, the percentage of HER2/neu-positive samples in clear cell carcinomas was 10- and 2.5-fold higher than Type I and Type II ovarian tumors, respectively. In conclusion, our study provides insights into clear cell carcinoma that could help in explaining its unique prognostic features, and, eventually, in orienting toward new therapeutic options.

In this study, we investigated the prognostic value of metastasis tumor antigen 1 expression in 81 untreated patients with ovarian cancer. The expression of metastasis tumor antigen 1 was evaluated by immunohistochemistry, and staining was analyzed in relation to clinicopathologic variables, disease-free survival, and overall survival. High expression of metastasis tumor antigen 1 was found to be associated with advanced stage (I/II versus III/IV, P = .02) and with worse response to first-line treatment (P = .03). Cases with high metastasis tumor antigen 1 expression showed a lower disease-free survival compared with cases with low expression (P = .02). In multivariate analysis of disease-free survival, metastasis tumor antigen 1 overexpression retained an independent negative prognostic role (P = .04), when considered together with histotype, stage of disease, residual tumor at surgery, and chemosensitivity. The evaluation of the prognostic relevance of metastasis tumor antigen 1 in late-stage disease showed that overexpression was a prognostic factor for poor disease-free survival and overall survival in this subset of patients, in both univariate and multivariate models. These findings indicate that metastasis tumor antigen 1 overexpression can be used as a predictor of clinical outcome in patients with ovarian cancer and therefore may represent a new prognostic marker., (Copyright © 2012 Elsevier Inc. All rights reserved.)

While it is well documented that the mitogenic actions of estrogens are critical in the development and progression of human breast and some gynecologic cancers, only latest data demonstrate a crucial involvement of estrogen-signaling in the carcinogenesis of non-classical estrogen target tissues, as colon, prostate, lung, skin, and brain. Only recently it has also been found out that the biological effects of estrogens are mediated by two distinct estrogen receptors (ERs), ERα and ERβ, and that their relative levels in a given cell are important determinants of response to estradiol and selective estrogen receptor modulators. Indeed, although ERα and ERβ have similar structure, they produce different effects, and there is currently increasing evidence that, for some tumors, an imbalanced ERβ expression might play a pivotal role in tumor development and progression. However, the prognostic value, the potential significance in predicting response to endocrine therapy, and, eventually, the utility of ERβ as a therapeutic target need to be assessed in large-scale and prospective clinical studies. This review examines the experimental and clinical evidences for a role of ERβ in carcinogenesis of classical and nonclassical estrogen target tissues. If anomalies of ERβ expression could be demonstrated to represent a critical step in the development and progression of some types of cancers, its re-expression by genetic engineering, as well as the use of targeted ERβ therapies would constitute new important therapeutic approaches.

Aims: To investigate the prognostic value of cytoplasmic oestrogen receptor beta (ERβ) expression in a series of untreated patients with non-human papillomavirus (HPV)-related vulvar cancer., Methods and Results: Immunohistochemistry was carried out using a polyclonal rabbit anti-human ERβ antibody. The nuclear and cytoplasmic expression of ERβ was evaluated in 33 patients. Cytoplasmic immunoreactivity was correlated with histopathological and molecular parameters (Ki67, p21), disease-free survival (DFS) and overall survival (OS). The expression of cytoplasmic ERβ was found to be associated with grade (P=0.006), while no association was found with any of the remaining variables examined. Cases with high cytoplasmic ERβ expression showed lower DFS and OS compared to cases with low cytoplasmic ERβ (P=0.007, P=0.01, respectively). There was also a progressive decline in both the DFS and OS with increasing tumour size (P=0.05, P=0.07, respectively) and with increasing depth of infiltration (P=0.14, P=0.07, respectively). On multivariate analysis, only tumour size and cytoplasmic ERβ staining retained an independent negative prognostic role for DFS and OS., Conclusions: The assessment of cytoplasmic ERβ expression could be helpful to identify poor prognosis in elderly patients with non-HPV-related vulvar squamous cell carcinoma (SCC)., (© 2011 Blackwell Publishing Limited.)

Objective: In this study we investigated the prognostic value of estrogen receptor α (ERα), ERβ and progesterone receptor (PR) expression in 58 untreated advanced serous ovarian cancer patients. The study also included 12 macroscopically and histopathologically normal ovaries., Materials and Methods: Protein expression was evaluated by immunohistochemistry, and antibody staining detected in both the nuclear and cytoplasmic compartments was taken into account. Immunopositivity was analyzed in relation to tumor clinicopathological variables, disease-free survival (DFS), and overall survival (OS)., Results: Epithelial cells in ovarian cancer tissue showed significantly lower levels of nuclear ERβ and PR, but not ERα, than in normal ovarian tissue. In the case of ERβ, however, while normal ovarian epithelium exhibited almost exclusively strong nuclear staining, ovarian cancer tissue mostly showed cytoplasmic immunopositivity. Nuclear ERα and ERβ expression were not associated with clinical outcome. Conversely, any cytoplasmic ERβ expression was an independent unfavorable prognostic factor for DFS, a finding approaching statistical significance also for OS. These data suggest that, in advanced serous ovarian cancer, cytoplasmic ERβ signaling may be more important for patient survival than its nuclear signaling. In the case of PR, positivity was an independent favorable prognostic factor for DFS., Conclusions: These novel findings, that need to be confirmed in a large prospective trial, suggest that additional prognostic, and possibly therapeutic opportunities may be available in advanced serous ovarian cancer., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: Hyaluronan (HA)-receptors (mainly CD44 and RHAMM) are overexpressed in a wide variety of cancers including ovarian tumors, and HA-bioconjugates have been developed to enhance selective entry of cytotoxic drugs into HA receptor-expressing cancerous cells. Here, we evaluated the potential application of a new HA-paclitaxel bioconjugate, ONCOFID-P, for intraperitoneal (IP) treatment of ovarian cancer., Methods: In vitro cytotoxic effect of ONCOFID-P was first assessed on CD44(+) OVCAR-3 and SKOV-3 human ovarian cancer cell lines. Studies were performed in female Balb/c athymic mice IP implanted with OVCAR-3 or SKOV-3 and treated with IP ONCOFID-P, and IP and intravenous (IV) free paclitaxel, at their maximum tolerated dose (MTD 168, 80 and 80 mg/kg, total dose, respectively). The potential detrimental effect of the IP ONCOFID-P and IP free paclitaxel on hematopoiesis was also assessed on peripheral blood, bone marrow and spleen., Results: Results show that ONCOFID-P cytotoxicity against both OVCAR-3 and SKOV-3 cell lines was somewhat less effective than free paclitaxel. Conversely, in in vivo experiments, IP treatment with ONCOFID-P was overall more effective than IV and IP free paclitaxel in inhibiting intra-abdominal tumor dissemination, abrogating ascites, prolonging survival and curing mice. ONCOFID-P and IP free paclitaxel were equivalent in terms of myelotoxicity, although the former was administered at a two-fold higher dose., Conclusions: Present data strongly support the development of ONCOFID-P for locoregional treatment of ovarian cancer.

Aims: The most common vulvar squamous cell carcinoma (conventional SCC) occurs in elderly women and develops following a human papillomavirus (HPV)-negative pathway. Because the highest incidence of conventional SCC is observed in patients with low oestrogen levels (postmenopausal women), the aim was to investigate whether hormonal factors could play a role in the development of cancer., Methods and Results: The expression profile of oestrogen receptor α (ERα), ERβ and progesterone receptor (PR) in a section containing both normal and tumour tissue, as well as the SCC-associated vulvar lesion, was evaluated in 34 elderly patients. Also, as recent studies have identified E-cadherin as a novel transcriptional target of oestrogen signalling, the modulation of this epithelial-mesenchymal transition (EMT) marker was studied. Finally, the expression of the proliferation marker Ki67 and of the apoptotic marker p53 was assessed. Results showed that changes in both ERα and ERβ expression characterize the transition from normal epithelium to cancer in patients with vulvar SCC: ERα was lost in cancer while ERβ decreased, mainly showing cytoplasmic localization. A reduction in the expression of E-cadherin was also observed in tumours, compared to normal epithelium., Conclusions: The data put the ER signalling pathway into the spotlight as a potentially important factor in vulvar carcinogenesis., (© 2011 Blackwell Publishing Limited.)

Medulloblastoma (MB) is the most common pediatric tumor of the CNS, representing ∼20% of all childhood CNS tumors. Although in recent years many molecular mechanisms that control MB development have been clarified, the effects of biological factors such as sex on this tumor remain to be explained. Epidemiological data, in fact, indicate a significant difference in the incidence of MB between the 2 sexes, with considerably higher susceptibility of males than females. Besides this different susceptibility, female sex is also a significant favorable prognostic factor in MB, with girls having a much better outcome. Despite these literature data, there has been little investigation into estrogen influence on MB development. In our study, we evaluated how hormone deficiency resulting from ovariectomy and hormone replacement influences the development of early and advanced MB stages in Patched1 heterozygous mice, a well-characterized mouse model of radiation-induced MB. Susceptibility to MB development was significantly increased in ovariectomized Ptch1(+/-) females and restored to levels observed in control mice after estrogen replacement. We next investigated the molecular mechanisms by which estrogen might influence tumor progression and show that ERβ, but not ERα, is involved in modulation of MB development by estrogens. Finally, our study shows that a functional interaction between estrogen- and IGF-I-mediated pathways may be responsible for the effects observed., (Copyright © 2010 UICC.)

Background: Chronic anal fissure (CAF) is a painful condition that is unlikely to resolve with conventional conservative management. Previous studies have reported that topical treatment of CAF with glyceryl trinitrate (GTN) reduces pain and promotes healing, but optimal treatment duration is unknown., Methods: To assess the effect of different treatment durations on CAF, we designed a prospective randomized trial comparing 40 versus 80 days with twice daily topical 0.4% GTN treatment (Rectogesic, Prostrakan Group). Chronicity was defined by the presence of both morphological (fibrosis, skin tag, exposed sphincter, hypertrophied anal papilla) and time criteria (symptoms present for more than 2 months or pain of less duration but similar episodes in the past). A gravity score (1 = no visible sphincter; 2 = visible sphincter; 3 = visible sphincter and fibrosis) was used at baseline. Fissure healing, the primary endpoint of the study, maximum pain at defecation measured with VAS and maximum anal resting pressure were assessed at baseline and at 14, 28, 40 and 80 days. Data was gathered at the end of the assigned treatment., Results: Of 188 patients with chronic fissure, 96 were randomized to the 40-day group and 92 to the 80-day group. Patients were well matched for sex, age, VAS and fissure score. There were 34 (19%) patients who did not complete treatment, 18 (10%) because of side effects. Of 154 patients who completed treatment, 90 (58%) had their fissures healed and 105 (68%) were pain free. There was no difference in healing or symptoms between the 40- and the 80-day group. There was no predictor of fissure healing. A low fissure gravity score correlated with increased resolution of pain (P < 0.05) and improvement of VAS score (P < 0.05) on both univariate and multivariate analysis. A lower baseline resting pressure was associated with better pain resolution on univariate analysis (P < 0.01). VAS at defecation and fissure healing significantly improved until 40 days (P < 0.001), while the difference between 40 and 80 days was not significant., Conclusion: We found no benefits in treating CAF with topical GTN for 80 days compared to 40 days. Fissure healing and VAS improvement continue until 6 weeks of treatment but are unlikely thereafter.

This study was aimed at evaluating the potential application of benzophenanthridine alkaloids, sanguinarine and cheleritrine, in the therapy of melanoma cancer. In vitro antiproliferative activity of sanguinarine was higher than that of cheleritrine against the B16 melanoma 4A5 cells. Both agents were able to produce DNA breaks, and the DNA unwinding assay showed that they act as DNA intercalating agents. Sanguinarine was selected for determination of its in vivo preclinical efficacy. Oral treatment with sanguinarine reduced the tumor burden in a transplantable murine tumor grown in a syngeneic host (B16 melanoma 4A5 in C57BL/6 mice), and in a human tumor xenograft grown in immunodeficient mice (A375 human melanoma in athymic nude mice). In A375 tumors a significant decrease in the proliferation marker Ki67, and a reduction in the activated mitogen-activated protein kinases (p-p44/42 MAPK), and in protein kinase B (pAKT) were also observed. Three out of eleven A375-bearing treated mice were tumor-free at the end of treatment, and did not develop any tumor after a further, treatment-free, observation period of 60 days. Sanguinarine also showed a striking antiangiogenic activity in mice. Data from the present study support the concept that sanguinarine can be effective in melanoma skin cancer.

Patched1 heterozygous mice (Ptch1(+/-)) are useful for basal cell carcinoma (BCC) studies, being remarkably susceptible to BCC induction by ultraviolet or ionizing radiation. Analogously, skin carcinogenesis-susceptible (Car-S) mice are elective for studies of papilloma and squamous cell carcinoma (SCC) induction. We previously reported a striking effect of gender on BCC induction in Ptch1(+/-) mice, with total resistance of females; likewise, Car-S females show increased skin tumor resistance relative to males. Here, we investigated the protective role of endogenous estrogen in skin keratinocyte tumorigenesis. Control (CN) and ovariectomized Ptch1(+/-) or Car-S females were irradiated for BCC induction or topically treated with chemical carcinogens for SCC induction. Susceptibility to BCC or SCC was dramatically increased in ovariectomized Ptch1(+/-) and Car-S females and restored to levels observed in males. Remarkably, progression of initially benign papillomas to malignant SCC occurred only in ovariectomized Car-S females. We explored the mechanisms underlying tumor progression and report overexpression of estrogen receptor (ER)-alpha, downregulation of ERbeta and upregulation of cyclin D1 in papillomas from ovariectomized Car-S relative to papillomas from CN females. Thus, an imbalanced ERalpha/ERbeta expression may be associated with estrogen-mediated modulation of non-melanoma skin carcinogenesis, with a key role played by cyclin D1. Our findings underscore a highly protective role of endogenous estrogen against skin tumorigenesis by diverse agents in two independent mouse models of skin cancer.

Objective: This study was designed to compare the effects of 17beta-estradiol (17beta-E2) and a phytoestrogen-containing soy extract on the immune system in an ovariectomized rat model of menopause. Specifically, T- and B-lymphocyte subsets, the balance of type 1 and 2 immune responses in the mesenteric lymph nodes, and serum levels of different classes of immunoglobulin were examined as study endpoints., Design: Ovariectomized rats were treated with either the phytoestrogen-containing soy extract (50 or 100 mg/kg/day PO), 17beta-E2 (0.5 mg/kg/day PO), or vehicle; a sham control was included in the study. After the rats were killed, mesenteric lymph nodes and blood samples were collected. B- and T (CD4 and CD8)-lymphocyte subsets in mesenteric lymph nodes were evaluated by flow cytometry analysis. Cytokine-producing T lymphocytes were identified within each T-lymphocyte subset as TH1 (interferon-gamma CD4), TH2 (interleukin-4 CD4), TC1 (interferon-gamma CD8), and TC2 (interferon-4 CD8) lymphocytes. Serum levels of immunoglobulin classes were determined by enzyme-linked immunosorbent assay., Results: There were no differences in the proportions of B lymphocytes and CD4 and CD8 T lymphocytes among groups. Treatment with 17beta-E2 and phytoestrogen-containing soy extract induced a reduction in TH1 and TC1 lymphocytes paralleled by a slight, nonsignificant, increase in the frequency of TH2. Data expressed as TH1/TH2 and TC1/TC2 ratios depicted a significant polarization of local immunity toward a humoral response. Evaluation of immunoglobulin serum levels did not show any significant difference among groups., Conclusions: Here we show that estrogens and soy phytochemicals similarly polarize the immune system toward a type 2 immune response in a preclinical model of menopause; our data draw attention to the crucial need to evaluate in clinical studies the potential side effects on the immune system of the complex soy products that are actually consumed in the postmenopausal setting.

This study investigated the effects of a phytoestrogen-containing standardized soy extract (SSE) on the growth of nonsmall cell lung cancer (NSCLC; A549) xenografts in female athymic mice. Tumor-bearing mice received either sterile water or SSE [50 or 100 mg/(kg x d), per os], 5 d/wk, until the mean tumor weight in each group was at least 900 mg. Treatment with SSE reduced tumor growth in the high-dose group compared with control (P < 0.01); tumors in both treated groups had reduced proliferation and greater apoptosis compared with controls (P < 0.05). SSE treatment also induced diffuse central necrosis, reducing the viable tissue mass within the tumor. Whereas tumor levels of epidermal growth factor receptor were comparable in control and treated mice, the expression of phosphorylated protein kinase B (p-Akt) was lower in tumors of mice treated with 100 mg SSE/(kg x d) than in controls (P < 0.01). The protein level of phosphorylated mitogen-activated protein kinase also tended to be lower (P = 0.07) in this group than in controls. Estrogen receptor (ER)alpha and ERbeta were present in tumors, but their expression levels did not differ among groups. Serum insulin-like growth factor-1 concentrations also were not affected by the treatments. In conclusion, we found that soy phytochemicals slow the in vivo growth of NSCLC xenografts; the modulation of the Akt-signaling pathway observed in tumors of SSE-treated mice may have a role in the activity observed. Our research provides further support for the concept that consumption of phytoestrogens may be effective in delaying lung cancer progression.

The present study aimed at improving our understanding of the effects of 17beta-estradiol and phytoestrogens on the uterine tissue, by evaluating tissue-specific modulation of molecules related to cell-cycle control and angiogenesis. Specifically, the uterine expression of Ki67, peroxisome proliferator-activated receptor gamma (PPARgamma), and vascular endothelial growth factor receptor-2 (VEGFR-2), was examined by immunohistochemical analysis. Ovariectomized (OVX) rats were treated with either the vehicle, a phytoestrogen- containing soy extract (SSE) (100 mg/kg/day pos), or 17beta-estradiol (0.5 mg/kg/day pos); a sham control group (SHAM) was also included in the study. At necropsy, uteri were weighed, collected, and subsequently processed for histopathology or immunohistochemistry. SSE-treated rats did not show any significant change either in the weight or in histological features of the uterus when compared to OVX controls; the epithelial expression of proliferation marker Ki67 was seen to be significantly reduced, in comparison to both SHAM and OVX rats. Conversely, 17beta-estradiol significantly increased uterine weight, induced hyperplasia in the majority of rats, and enhanced Ki67 epithelial expression. The regulation of PPARgamma expression, reduced after ovariectomy, was similar in SSE- and 17beta-estradiol-treated rats, showing a further significant decrease in stromal immunostaining, in comparison to OVX controls. VEGFR-2 epithelial immunostaining, slightly reduced following ovariectomy, was highly increased on 17beta-estradiol treatment, while following SSE, the pattern of staining observed was similar to that of OVX controls. Data from this study show that PPARgamma and VEGFR-2 represent additional targets by which sex steroid estrogen and plant-derived phytoestrogens may, at certain doses, differentially regulate endometrial functions.

The study reported here was designed to determine whether a phytoestrogen-containing soy extract (SSE) could negate/overwhelm the inhibitory effects of ICI 182 780 on the growth of estrogen-sustained human breast cancer xenografts (MCF-7), in ovariectomized athymic mice. As expected, estradiol-supplemented tumors did not grow over the study period in ICI 182 780-treated females; concomitant administration of 50 mg/kg per day SSE slightly potentiated the inhibitory activity of the drug, while at 100 mg/kg per day, SSE partially negated ICI 182 780 activity. In keeping with these in vivo outcomes, we observed that the level of cyclin D1 (and progesterone receptor) in MCF-7 xenografts was considerably reduced by ICI 182 780, an effect enhanced by concomitant treatment with 50 SSE, but reduced by the higher dosage (i.e. 100 mg/kg per day). Thrombospondin-1 (TSP-1) and kallikrein 6 (KLK6) levels were also reduced following ICI 182 780, although to a lesser degree; again, combined anti-estrogen and SSE produced a dose-dependent regulation in TSP-1 and KLK6 tumor level, with a further reduction in the mRNA gene expression at 50 SSE (compared with ICI 182 780) and a partial reversion of the drug-induced down-regulation at 100 mg/kg per day. No modulation was detected in the serum concentration of IGF-1 (a potent mitogen for estrogen receptor-positive breast cancer cell lines) either upon treatment with ICI 182 780 or concomitant administration of the anti-estrogen with SSE. In conclusion, results from this study raise concerns about the consumption of isoflavone supplements in conjunction with ICI 182 780 therapy, in postmenopausal women with estrogen-dependent breast cancer.

Leptin is a hormone which controls fat metabolism. Leptin plasma levels and adipose tissue mRNA expression were measured in cancer patients. Plasma levels were correlated with TNM staging, cachexia parameters, tumour markers and hormones. Breast and colorectal cancer patients showed blood plasma levels of insulin, TNF-alpha and tumour markers higher than controls. Breast cancer patients, but not colorectal cancer patients, had plasma levels and adipose tissue expression of leptin significantly higher than controls associated with elevated values of estrogen- and progesterone-receptors. These data suggest the possible use of leptin as a clinical marker.