Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress

Introduction: The etiology of radiation-induced erectile dysfunction (ED) is complex and multifactorial, and it appears to be mainly atherogenic. Aim: To focus on vascular aspects of radiation-induced ED and to elucidate whether the protective effects of sildenafil are mediated by attenuation of oxidative stress and apoptosis in the endothelial cells. Methods: Bovine aortic endothelial cells (BAECs), with or without pretreatment of sildenafil (5 μM at 5 minutes before radiation), were used to test endothelial dysfunction in response to external beam radiation at 10–15 Gy. Generation of reactive oxygen species (ROS) was studied. Extracellular hydrogen peroxide (H2O2) was measured using the Amplex Red assay and intracellular H2O2 using a fluorescent sensor. In addition, ROS superoxide (O2•-) was measured using a O2•- chemiluminescence enhancer. Both H2O2 and O2•- are known to reduce the bioavailability of nitric oxide, which is the most significant chemical mediator of penile erection. Generation of cellular peroxynitrite (ONOO−) was measured using a chemiluminescence assay with the PNCL probe. Subsequently, we measured the activation of acid sphingomyelinase (ASMase) enzyme by radioenzymatic assay using [14C-methylcholine] sphingomyelin as substrate, and the generation of the proapoptotic C16-ceramide was assessed using the diacylglycerol kinase assay. Endothelial cells apoptosis was measured as a readout of these cells’ dysfunction. Main Outcome Measures: Single high-dose radiation therapy induced NADPH oxidases (NOXs) activation and ROS generation via the proapoptotic ASMase/ceramide pathway. The radio-protective effect of sildenafil on BAECs was due to inhibition of this pathway. Results: Here, we demonstrate for the first time that radiation activated NOXs and induced generation of ROS in BAECs. In addition, we showed that sildenafil significantly reduced radiation-induced O2•- and as a result there was reduction in the generation of peroxynitrite in these cells. Subsequently, sildenafil protected the endothelial cells from radiation therapy-induced apoptosis. Strengths and Limitations: This is the first study demonstrating that single high-dose radiation therapy induced NOXs activation, resulting in the generation of O2•- and peroxynitrite in endothelial cells. Sildenafil reduced ROS generation by inhibiting the ASMase/ceramide pathway. These studies should be followed in an animal model of ED. Conclusions: This study demonstrated that sildenafil protects BAECs from radiation-induced oxidative stress by reducing NOX-induced ROS generation, thus resulting in decreased endothelial dysfunction. Therefore, it provides a potential mechanism to better understand the atherogenic etiology of postradiation ED. Wortel RC, Mizrachi A, Li H, et al. Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress. J Sex Med 2019;16:1721–1733. © 201

Endothelial Membrane Remodeling Is Obligate for Anti-Angiogenic Radiosensitization during Tumor Radiosurgery

While there is significant interest in combining anti-angiogenesis therapy with conventional anti-cancer treatment, clinical trials have as of yet yielded limited therapeutic gain, mainly because mechanisms of anti-angiogenic therapy remain to a large extent unknown. Currently, anti-angiogenic tumor therapy is conceptualized to either "normalize" dysfunctional tumor vasculature, or to prevent recruitment of circulating endothelial precursors into the tumor. An alternative biology, restricted to delivery of anti-angiogenics immediately prior to single dose radiotherapy (radiosurgery), is provided in the present study.Genetic data indicate an acute wave of ceramide-mediated endothelial apoptosis, initiated by acid sphingomyelinase (ASMase), regulates tumor stem cell response to single dose radiotherapy, obligatory for tumor cure. Here we show VEGF prevented radiation-induced ASMase activation in cultured endothelium, occurring within minutes after radiation exposure, consequently repressing apoptosis, an event reversible with exogenous C(16)-ceramide. Anti-VEGFR2 acts conversely, enhancing ceramide generation and apoptosis. In vivo, MCA/129 fibrosarcoma tumors were implanted in asmase(+/+) mice or asmase(-/-) littermates and irradiated in the presence or absence of anti-VEGFR2 DC101 or anti-VEGF G6-31 antibodies. These anti-angiogenic agents, only if delivered immediately prior to single dose radiotherapy, de-repressed radiation-induced ASMase activation, synergistically increasing the endothelial apoptotic component of tumor response and tumor cure. Anti-angiogenic radiosensitization was abrogated in tumors implanted in asmase(-/-) mice that provide apoptosis-resistant vasculature, or in wild-type littermates pre-treated with anti-ceramide antibody, indicating that ceramide is necessary for this effect.These studies show that angiogenic factors fail to suppress apoptosis if ceramide remains elevated while anti-angiogenic therapies fail without ceramide elevation, defining a ceramide rheostat that determines outcome of single dose radiotherapy. Understanding the temporal sequencing of anti-angiogenic drugs and radiation enables optimized radiosensitization and design of innovative radiosurgery clinical trials

Cellular shear adhesion force measurement and simultaneous imaging by atomic force microscope

This paper presents a sensitive and fast cellular shear adhesion force measurement method using an atomic force microscope (AFM). In the work, the AFM was used both as a tool for the imaging of cells on the nano-scale and as a force sensor for the measurement of the shear adhesion force between the cell and the substrate. After the cell imaging, the measurement of cellular shear adhesion forces was made based on the different positions of the cell on the nano-scale. Moreover, different pushing speeds of probe and various locations of cells were used in experiments to study their influences. In this study, the measurement of the cell adhesion in the upper portion of the cell is different from that in the lower portion. It may reveal that the cancer cells have the metastasis tendency after cultured for 16 to 20 hours, which is significant for preventing metastasis in the patients diagnosed with early cancer lesions. Furthermore, the cellular shear adhesion forces of two types of living cancer cells were obtained based on the measurements of AFM cantilever deflections in the torsional and vertical directions. The results demonstrate that the shear adhesion force of cancer cells is twice as much as the same type of cancer cells with TRAIL. The method can also provide a way for the measurement of the cellular shear adhesion force between the cell and the substrate, and for the simultaneous exploration of cells using the AFM imaging and manipulatio

Expression of the proapoptotic protein Bid is an adverse prognostic factor for radiotherapy outcome in carcinoma of the cervix

The Bcl-2 family of apoptotic regulators is thought to play an essential role in cancer development and influence the sensitivity of tumour cells to radiotherapy. Bid is an abundantly expressed Bcl-2 family protein playing a central role in various pathways of apoptosis by integrating and converging signals at the mitochondria. The relevance of apoptotic modulation by Bcl-2 and related proteins in tumour development and radiation response for human tumours remains undefined. Therefore, a study was made regarding the expression of Bid in patients with locally advanced cervix carcinoma who received radiotherapy. Bid expression was assessed using immunohistochemistry in pretreatment archival biopsies from 98 patients. The data were correlated with clinicopathologic characteristics and treatment outcome. Pretreatment tumour radiosensitivity data were available for 60 patients. Strong Bid expression was associated with a patient age less than the median of 52 years (P=0.034) and poor metastasis-free survival. In multivariate analysis, after allowing for stage, Bid expression was a significant prognostic factor for both disease-specific and metastasis-free survival (P=0.026). It is concluded that strong tumour Bid expression is associated with poor outcome following radiotherapy regardless of intrinsic tumour cell radiosensitivity, and is adverse prognostic for disease-specific and metastasis-free survival in younger patients

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

BACKGROUND: Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. METHODOLOGY/PRINCIPAL FINDINGS: We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. CONCLUSIONS/SIGNIFICANCE: These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity

Identification of Novel Functional Inhibitors of Acid Sphingomyelinase

We describe a hitherto unknown feature for 27 small drug-like molecules, namely functional inhibition of acid sphingomyelinase (ASM). These entities named FIASMAs (Functional Inhibitors of Acid SphingoMyelinAse), therefore, can be potentially used to treat diseases associated with enhanced activity of ASM, such as Alzheimer's disease, major depression, radiation- and chemotherapy-induced apoptosis and endotoxic shock syndrome. Residual activity of ASM measured in the presence of 10 µM drug concentration shows a bimodal distribution; thus the tested drugs can be classified into two groups with lower and higher inhibitory activity. All FIASMAs share distinct physicochemical properties in showing lipophilic and weakly basic properties. Hierarchical clustering of Tanimoto coefficients revealed that FIASMAs occur among drugs of various chemical scaffolds. Moreover, FIASMAs more frequently violate Lipinski's Rule-of-Five than compounds without effect on ASM. Inhibition of ASM appears to be associated with good permeability across the blood-brain barrier. In the present investigation, we developed a novel structure-property-activity relationship by using a random forest-based binary classification learner. Virtual screening revealed that only six out of 768 (0.78%) compounds of natural products functionally inhibit ASM, whereas this inhibitory activity occurs in 135 out of 2028 (6.66%) drugs licensed for medical use in humans

Gene therapy for carcinoma of the breast: Pro-apoptotic gene therapy

The dysregulation of apoptosis contributes in a variety of ways to the malignant phenotype. It is increasingly recognized that the alteration of pro-apoptotic and anti-apoptotic molecules determines not only escape from mechanisms that control cell cycle and DNA damage, but also endows the cancer cells with the capacity to survive in the presence of a metabolically adverse milieu, to resist the attack of the immune system, to locally invade and survive despite a lack of tissue anchorage, and to evade the otherwise lethal insults induced by drugs and radiotherapy. A multitude of apoptosis mediators has been identified in the past decade, and the roles of several of them in breast cancer have been delineated by studying the clinical correlates of pathologically documented abnormalities. Using this information, attempts are being made to correct the fundamental anomalies at the genetic level. Fundamental to this end are the design of more efficient and selective gene transfer systems, and the employment of complex interventions that are tailored to breast cancer and that are aimed concomitantly towards different components of the redundant regulatory pathways. The combination of such genetic modifications is most likely to be effective when combined with conventional treatments, thus robustly activating several pro-apoptotic pathways

Genetic variants associated with mosaic Y chromosome loss highlight cell cycle genes and overlap with cancer susceptibility.

The Y chromosome is frequently lost in hematopoietic cells, which represents the most common somatic alteration in men. However, the mechanisms that regulate mosaic loss of chromosome Y (mLOY), and its clinical relevance, are unknown. We used genotype-array-intensity data and sequence reads from 85,542 men to identify 19 genomic regions (P < 5 × 10-8) that are associated with mLOY. Cumulatively, these loci also predicted X chromosome loss in women (n = 96,123; P = 4 × 10-6). Additional epigenome-wide methylation analyses using whole blood highlighted 36 differentially methylated sites associated with mLOY. The genes identified converge on aspects of cell proliferation and cell cycle regulation, including DNA synthesis (NPAT), DNA damage response (ATM), mitosis (PMF1, CENPN and MAD1L1) and apoptosis (TP53). We highlight the shared genetic architecture between mLOY and cancer susceptibility, in addition to inferring a causal effect of smoking on mLOY. Collectively, our results demonstrate that genotype-array-intensity data enables a measure of cell cycle efficiency at population scale and identifies genes implicated in aneuploidy, genome instability and cancer susceptibility.This research has been conducted using the UK Biobank Resource under Application Number 9905. This work was supported by the UK Medical Research Council (Unit Programme numbers MC_UU_12015/1 and MC_UU_12015/2). Research in the S. Jackson laboratory is funded by Cancer Research UK (CRUK; programme grant C6/A18796), with Institute core funding provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S. Jackson receives salary from the University of Cambridge, supplemented by CRUK

Reevaluation of the pH, ferning and nile blue sulphate staining methods in pregnant women with premature rupture of the fetal membranes

Peer Reviewe

Self-control and SAT outcomes: Evidence from two national field studies.

Self-control is often thought to be synonymous with willpower, defined as the direct modulation of impulses in order to do what is best in the long-run. However, research has also identified more strategic approaches to self-control that require less effort than willpower. To date, field research is lacking that compares the efficacy of willpower to strategic self-control for consequential and objectively measured real-world outcomes. In collaboration with the College Board, we surveyed two national samples of high school students about how they motivated themselves to study for the SAT college admission exam. In Study 1 (N = 5,563), compared to willpower, strategic self-control predicted more hours of SAT practice and higher SAT scores, even when controlling for prior PSAT scores. Additionally, the more self-control strategies students deployed, the higher their SAT scores. Consistent with dose-response curves in other domains, there were positive albeit diminishing marginal returns to additional strategies. Mediation analyses suggest that the benefits of self-control strategies to SAT scores was fully explained by increased practice time. These results were confirmed in Study 2, a preregistered replication with N = 14,259 high school students. Compared to willpower, strategic self-control may be especially beneficial in facilitating the pursuit of goals in high-stakes, real-world situations