Endothelial nitric oxide synthase rs1799983 gene polymorphism is associated with the risk of developing intracranial aneurysm.

PURPOSE: The intracranial aneurysm (IA) rupture is associated with a subarachnoid hemorrhage. One third of patients die, and one third remain depend for daily activities. Genetic factors are crucial in the formation and clinical evolution of IAs. Multiple loci have been associated with AIs, much of them implicating multiple pathways related to vascular endothelial maintenance and extracellular matrix integrity. Thus, the aim of our study was to characterize whether polymorphisms in genes implicated in the vascular endothelial maintenance could modify the risk of developing IAs. SUBJECTS AND METHODS: We have studied 176 patients with IA recruited in the Service of Neurosurgery at the University Hospital of Valladolid (Spain) and a control group if 150 sex-matched healthy subjects. Clinical variables were collected from each patient. We have analyzed VEGFA rs833061, VEGFR2 rs2071559, endothelin rs5370, endoglin rs3739817, and eNOS rs1799983 polymorphisms. RESULTS: Our results showed that allele T of the eNOS rs1799983 polymorphism is correlated with decreased risk of developing the disease; thus, allele G of the eNOS rs1799983 polymorphism increased the risk of developing IA. CONCLUSION: The association of eNOS rs1799983 polymorphism with the risk to suffer IA reinforces the hypothesis that genetic variants in eNOS gene could be crucial in the pathogenesis of IA.

Telomere Length as a New Risk Marker of Early-Onset Colorectal Cancer.

Early-onset colorectal cancer (EOCRC; age younger than 50 years) incidence has been steadily increasing in recent decades worldwide. The need for new biomarkers for EOCRC prevention strategies is undeniable. In this study, we aimed to explore whether an aging factor, such as telomere length (TL), could be a useful tool in EOCRC screening. The absolute leukocyte TL from 87 microsatellite stable EOCRC patients and 109 healthy controls (HC) with the same range of age, was quantified by Real Time Quantitative PCR (RT-qPCR). Then, leukocyte whole-exome sequencing (WES) was performed to study the status of the genes involved in TL maintenance (hTERT, TERC, DKC1, TERF1, TERF2, TERF2IP, TINF2, ACD, and POT1) in 70 sporadic EOCRC cases from the original cohort. We observed that TL was significantly shorter in EOCRC patients than in healthy individuals (EOCRC mean: 122 kb vs. HC mean: 296 kb; p < 0.001), suggesting that telomeric shortening could be associated with EOCRC susceptibility. In addition, we found a significant association between several SNPs of hTERT (rs79662648), POT1 (rs76436625, rs10263573, rs3815221, rs7794637, rs7784168, rs4383910, and rs7782354), TERF2 (rs251796 and rs344152214), and TERF2IP (rs7205764) genes and the risk of developing EOCRC. We consider that the measurement of germline TL and the status analysis of telomere maintenance related genes polymorphisms at early ages could be non-invasive methods that could facilitate the early identification of individuals at risk of developing EOCRC.

Familial component of early-onset colorectal cancer: opportunity for prevention.

BACKGROUND: Individuals with a non-syndromic family history of colorectal cancer are known to have an increased risk. There is an opportunity to prevent early-onset colorectal cancer (age less than 50 years) (EOCRC) in this population. The aim was to explore the proportion of EOCRC that is preventable due to family history of colorectal cancer. METHODS: This was a retrospective multicentre European study of patients with non-hereditary EOCRC. The impact of the European Society of Gastrointestinal Endoscopy (ESGE), U.S. Multi-Society Task Force (USMSTF), and National Comprehensive Cancer Network (NCCN) guidelines on prevention and early diagnosis was compared. Colorectal cancer was defined as potentially preventable if surveillance colonoscopy would have been performed at least 5 years before the age of diagnosis of colorectal cancer, and diagnosed early if colonoscopy was undertaken between 1 and 4 years before the diagnosis. RESULTS: Some 903 patients with EOCRC were included. Criteria for familial colorectal cancer risk in ESGE, USMSTF, and NCCN guidelines were met in 6.3, 9.4, and 30.4 per cent of patients respectively. Based on ESGE, USMSTF, and NCCN guidelines, colorectal cancer could potentially have been prevented in 41, 55, and 30.3 per cent of patients, and diagnosed earlier in 11, 14, and 21.1 per cent respectively. In ESGE guidelines, if surveillance had started 10 years before the youngest relative, there would be a significant increase in prevention (41 versus 55 per cent; P = 0.010). CONCLUSION: ESGE, USMSTF, and NCCN criteria for familial colorectal cancer were met in 6.3, 9.4, and 30.4 per cent of patients with EOCRC respectively. In these patients, early detection and/or prevention could be achieved in 52, 70, and 51.4 per cent respectively. Early and accurate identification of familial colorectal cancer risk and increase in the uptake of early colonoscopy are key to decreasing familial EOCRC.

Panobinostat Synergistically Enhances the Cytotoxicity of Microtubule Destabilizing Drugs in Ovarian Cancer Cells.

Ovarian cancer (OC) is one of the most common gynecologic neoplasia and has the highest mortality rate, which is mainly due to late-stage diagnosis and chemotherapy resistance. There is an urgent need to explore new and better therapeutic strategies. We have previously described a family of Microtubule Destabilizing Sulfonamides (MDS) that does not trigger multidrug-mediated resistance in OC cell lines. MDS bind to the colchicine site of tubulin, disrupting the microtubule network and causing antiproliferative and cytotoxic effects. In this work, a novel microtubule-destabilizing agent (PILA9) was synthetized and characterized. This compound also inhibited OC cell proliferation and induced G2/M cell cycle arrest and apoptosis. Interestingly, PILA9 was significantly more cytotoxic than MDS. Here, we also analyzed the effect of these microtubule-destabilizing agents (MDA) in combination with Panobinostat, a pan-histone deacetylase inhibitor. We found that Panobinostat synergistically enhanced MDA-cytotoxicity. Mechanistically, we observed that Panobinostat and MDA induced α-tubulin acetylation and that the combination of both agents enhanced this effect, which could be related to the observed synergy. Altogether, our results suggest that MDA/Panobinostat combinations could represent new therapeutic strategies against OC.

Chloroquine-Induced DNA Damage Synergizes with Nonhomologous End Joining Inhibition to Cause Ovarian Cancer Cell Cytotoxicity.

Ovarian cancer (OC) is the most lethal gynecological malignancy; therefore, more effective treatments are urgently needed. We recently reported that chloroquine (CQ) increased reactive oxygen species (ROS) in OC cell lines (OCCLs), causing DNA double-strand breaks (DSBs). Here, we analyzed whether these lesions are repaired by nonhomologous end joining (NHEJ), one of the main pathways involved in DSB repair, and if the combination of CQ with NHEJ inhibitors (NHEJi) could be effective against OC. We found that NHEJ inhibition increased the persistence of γH2AX foci after CQ-induced DNA damage, revealing an essential role of this pathway in the repair of the lesions. NHEJi decreased the proliferation of OCCLs and a strong in vitro synergistic effect on apoptosis induction was observed when combined with CQ. This effect was largely abolished by the antioxidant N-Acetyl-L-cysteine, revealing the critical role of ROS and DSB generation in CQ/NHEJi-induced lethality. We also found that the NHEJ efficiency in OCCLs was not affected by treatment with Panobinostat, a pan-histone deacetylase inhibitor that also synergizes with CQ in OCCLs by impairing homologous recombination. Accordingly, the triple combination of CQ-NHEJi-Panobinostat exerted a stronger in vitro synergistic effect. Altogether, our data suggest that the combination of these drugs could represent new therapeutic strategies against OC.

Identification of a truncated ß1-chimaerin variant that inactivates nuclear Rac1.

ß1-chimaerin belongs to the chimaerin family of GTPase-activating proteins (GAPs) and is encoded by the CHN2 gene, which also encodes the ß2- and ß3-chimaerin isoforms. All chimaerin isoforms have a C1 domain that binds diacylglycerol as well as tumor-promoting phorbol esters and a catalytic GAP domain that inactivates the small GTPase Rac. Nuclear Rac has emerged as a key regulator of various cell functions, including cell division, and has a pathological role by promoting tumorigenesis and metastasis. However, how nuclear Rac is regulated has not been fully addressed. Here, using several approaches, including siRNA-mediated gene silencing, confocal microscopy, and subcellular fractionation, we identified a nuclear variant of ß1-chimaerin, ß1-Δ7p-chimaerin, that participates in the regulation of nuclear Rac1. We show that ß1-Δ7p-chimaerin is a truncated variant generated by alternative splicing at a cryptic splice site in exon 7. We found that, unlike other chimaerin isoforms, ß1-Δ7p-chimaerin lacks a functional C1 domain and is not regulated by diacylglycerol. We found that ß1-Δ7p-chimaerin localizes to the nucleus via a nuclear localization signal in its N terminus. We also identified a key nuclear export signal in ß1-chimaerin that is absent in ß1-Δ7p-chimaerin, causing nuclear retention of this truncated variant. Functionally analyses revealed that ß1-Δ7p-chimaerin inactivates nuclear Rac and negatively regulates the cell cycle. Our results provide important insights into the diversity of chimaerin Rac-GAP regulation and function and highlight a potential mechanism of nuclear Rac inactivation that may play significant roles in pathologies such as cancer.

Methoxy and bromo scans on N-(5-methoxyphenyl) methoxybenzenesulphonamides reveal potent cytotoxic compounds, especially against the human breast adenocarcinoma MCF7 cell line.

Thirty seven N-(5-methoxyphenyl)-4-methoxybenzenesulphonamide with methoxy or/and bromo substitutions (series 1-4) and with different substituents on the sulphonamide nitrogen have been synthesised. 21 showed sub-micromolar cytotoxicity against HeLa and HT-29 human tumour cell lines, and were particularly effective against MCF7. The most potent series has 2,5-dimethoxyanilines, especially the 4-brominated compounds 23-25. The active compounds inhibit microtubular protein polymerisation at micromolar concentrations, thus pointing at tubulin as the target. Co-treatment with the MDR inhibitor verapamil suggests that they are not MDR substrates. Compound 25 showed nanomolar antiproliferative potency. It severely disrupts the microtubule network in cells and arrests cells at the G2/M cell-cycle phase, thus confirming tubulin targeting. 25 triggered apoptotic cell death, and induced autophagy. Docking studies suggest binding in a distinct way to the colchicine site. These compounds are promising new antitumor agents acting on tubulin.

Microtubule Destabilizing Sulfonamides as an Alternative to Taxane-Based Chemotherapy.

Pan-Gyn cancers entail 1 in 5 cancer cases worldwide, breast cancer being the most commonly diagnosed and responsible for most cancer deaths in women. The high incidence and mortality of these malignancies, together with the handicaps of taxanes-first-line treatments-turn the development of alternative therapeutics into an urgency. Taxanes exhibit low water solubility that require formulations that involve side effects. These drugs are often associated with dose-limiting toxicities and with the appearance of multi-drug resistance (MDR). Here, we propose targeting tubulin with compounds directed to the colchicine site, as their smaller size offer pharmacokinetic advantages and make them less prone to MDR efflux. We have prepared 52 new Microtubule Destabilizing Sulfonamides (MDS) that mostly avoid MDR-mediated resistance and with improved aqueous solubility. The most potent compounds, N-methyl-N-(3,4,5-trimethoxyphenyl-4-methylaminobenzenesulfonamide 38, N-methyl-N-(3,4,5-trimethoxyphenyl-4-methoxy-3-aminobenzenesulfonamide 42, and N-benzyl-N-(3,4,5-trimethoxyphenyl-4-methoxy-3-aminobenzenesulfonamide 45 show nanomolar antiproliferative potencies against ovarian, breast, and cervix carcinoma cells, similar or even better than paclitaxel. Compounds behave as tubulin-binding agents, causing an evident disruption of the microtubule network, in vitro Tubulin Polymerization Inhibition (TPI), and mitotic catastrophe followed by apoptosis. Our results suggest that these novel MDS may be promising alternatives to taxane-based chemotherapy in chemoresistant Pan-Gyn cancers.

Establishment of a conditional Nomo1 mouse model by CRISPR/Cas9 technology.

The Nomo1 gene mediates a wide range of biological processes of importance in embryonic development. Accordingly, constitutive perturbation of Nomo1 function may result in myriad developmental defects that trigger embryonic lethality. To extend our understanding of Nomo1 function in postnatal stages and in a tissue-specific manner, we generated a conditional knockout mouse model of Nomo1. To achieve this, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology in C57Bl/6J mouse zygotes to generate a new mouse model in which exon 3 of the Nomo1 gene is specifically flanked (or floxed) by LoxP sites (Nomo1f/f). Nomo1f/f mouse embryonic fibroblasts were transduced with a Cre adenovirus and efficiently recombined between LoxP sites. Genomic and expression studies in Nomo1-transduced MEFs demonstrated that the Nomo1 exon 3 is ablated. Western blot assay showed that no protein or early truncated protein is produced. In vivo assay crossing Nomo1f/f mouse with a Msi1-CRE transgenic mouse corroborated the previous findings and it showed Nomo1 exon 3 deletion at msi1+ cell compartment. This short technical report demonstrates that CRISPR/Cas9 technology is a simple and easy method for creating conditional mouse models. The Nomo1f/f mouse will be useful to researchers who wish to explore the role of Nomo1 in any developmental stage or in a tissue-specific manner.

The impact of vascular burden on behavioural and psychological symptoms in older adults with dementia: the BEVASDE study.

OBJECTIVES: Behavioural and psychological symptoms (BPS) worsen quality of life and increase institutionalization in dementia, but the relationship between BPS and vascular burden on neuroimaging is unclear. Our aim is to explore whether the profile of BPS differs between patients with large-vessel or cortical vascular dementia (cVaD), small-vessel or subcortical vascular dementia (sVaD) and Alzheimer's disease (AD). METHODS: The BEVASDE study comprised 806 demented patients (cVaD-136, sVaD-184, AD-486) recruited from outpatient consultations in Salamanca and Avila, Spain. The Clinical Dementia Rating Scale (CDR) and the 12-item Neuropsychiatric Inventory (NPI) were used to evaluate dementia severity and BPS. RESULTS: BPS were reported in 98.5%, 97.3% and 96.9% of the cVaD, sVaD and AD cases, respectively. The median NPI score was 36 in both cVaD and sVaD and 34 in AD, with a median number of four symptoms per patient. The most frequent disorders were depression (64.4%), apathy (61.8%) and sleep disturbance (60.5%). Multivariate regression analyses after controlling for possible confounders showed a higher risk of euphoria (p = 0.011), apathy (p = 0.007), irritability (p = 0.002) and sleep disturbance (p = 0.020) in cVaD than in AD and more apathy (p = 0.0001) and irritability (p = 0.0001) in sVaD than in AD. In contrast, AD subjects had a higher risk of delusions (p = 0.007) and hallucinations (p = 0.023) than patients with cVaD as well as more aberrant motor behaviour than both cVaD (p = 0.0001) and sVaD (p = 0.003). CONCLUSION: BPS are common in dementia and may help in differential diagnosis of the various subtypes. We should inquire about them and treat as necessary.