Oxidative and non-oxidative DNA damage and cardiovascular disease

Evidence for the association of DNA damage with cardiovascular disease has been obtained from in vitro cell culture models, experimental cardiovascular disease and analysis of samples obtained from humans with disease. There is general acceptance that several factors associated with the risk of developing cardiovascular disease cause oxidative damage to DNA in cell culture models with both nuclear and mitochondrial DNA as targets. Moreover, evidence obtained over the past 10 years points to a possible mechanistic role for DNA damage in experimental atherosclerosis culminating in recent studies challenging the assumption that DNA damage is merely a biomarker of the disease process. This kind of mechanistic insight provides a renewed impetus for further studies in this area.     

Immunochemical detection of oxidatively damaged DNA

Abstract

Oxidatively damaged DNA is implicated in various diseases, including neurodegenerative disorders, cancer, diabetes, cardiovascular and inflammatory diseases as well as aging. Several methods have been developed to detect oxidatively damaged DNA. They include chromatographic techniques, the Comet assay, ³²P-postlabelling and immunochemical methods that use antibodies to detect oxidized lesions. In this review, we discuss the detection of 8-oxo-7,8-dihydro-29-deoxyguanosine (8-oxodG), the most abundant oxidized nucleoside. This lesion is frequently used as a marker of exposure to oxidants, including environmental pollutants, as well as a potential marker of disease progression. We concentrate on studies published between the years 2000 and 2011 that used enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry to detect 8-oxodG in humans, laboratory animals and in cell lines. Oxidative damage observed in these organisms resulted from disease, exposure to environmental pollutants or from in vitro treatment with various chemical and physical factors.     

Studies on the Host-Specific Pathotoxins Produced by Helminthosporium maydis,race T

Two host-specific pathotoxins, Band 1- and Band 2-toxins, were isolated from the toxin complex obtained from the culture broth and mycelial mat of Helminthosporium maydis, race T, the fungus causing Southern corn blight disease. Chemical and spectrometric studies showed them to be polyketo-polyhydroxy compounds with C₄₁ carbon chains. Band 2-toxin is identical to Band 1-toxin except that Band 2 toxin has an additional hydroxyl group in place of one of the keto-group of Band 1-toxin.     

Combined HASPIN and mTOR inhibition is synergistic against KRAS-driven carcinomas

BackgroundOncogenic mutations in the KRAS gene are very common in human cancers, resulting in cells with well-characterized selective advantages. For more than three decades, the development of effective therapeutics to inhibit KRAS-driven tumorigenesis has proved a formidable challenge and KRAS was considered ‘undruggable’. Therefore, multi-targeted therapy may provide a reasonable strategy for the effective treatment of KRAS-driven cancers. Here, we assess the efficacy and mechanistic rationale for combining HASPIN and mTOR inhibition as a potential therapy for cancers carrying KRAS mutations.MethodsWe investigated the synergistic effect of a combination of mTOR and HASPIN inhibitors on cell viability, cell cycle, cell apoptosis, DNA damage, and mitotic catastrophe using a panel of human KRAS-mutant and wild-type tumor cell lines. Subsequently, the human transplant models were used to test the therapeutic efficacy and pharmacodynamic effects of the dual therapy.ResultsWe demonstrated that the combination of mTOR and HASPIN inhibitors induced potent synergistic cytotoxic effects in KRAS-mutant cell lines and delayed the growth of human tumor xenograft. Mechanistically, we showed that inhibiting of mTOR potentiates HASPIN inhibition by preventing the phosphorylation of H3 histones, exacerbating mitotic catastrophe and DNA damage in tumor cell lines with KRAS mutations, and this effect is due in part to a reduction in VRK1.ConclusionsThese findings indicate that increased DNA damage and mitotic catastrophe are the basis for the effective synergistic effect observed with mTOR and HASPIN inhibition, and support the clinical evaluation of this dual therapy in patients with KRAS-mutant tumors.     

In vitro DNA binding activity and molecular docking reveals pierisin-5 as an anti-proliferative agent against gastric cancer

Abstract

Pierisin-5 is a DNA dependent ADP ribosyltransferase (ADRT) protein from the larvae of Indian cabbage white butterfly, Pieris canidia. Interestingly, Pierisin-5 ADP-ribosylates the DNA as a substrate, but not the protein and subsequently persuades apoptotic cell death in human cancer cells. This has led to the investigation on the DNA binding activity of Pierisin-5 using in vitro and in silico approaches in the present study. However, both the structure and the mechanism of ADP-ribosylation of pierisin-5 are unknown. In silico modeled structure of the N-terminal ADRT catalytic domain interacted with the minor groove of B-DNA for ribosylation with the help of β-NAD⁺ which lead to a structural modification in DNA (DNA adduct). The possible interaction between calf thymus DNA (CT-DNA) and purified pierisin-5 protein was studied through spectral–spatial studies and the blue shift and hyperchromism in the UV–Visible spectra was observed. The DNA adduct property of pierisin-5 protein was validated by in vitro cytotoxic assay on human gastric (AGS) cancer cell lines. Our study is the first report of the mechanism of DNA binding property of pierisin-5 protein which leads to the induction of cytotoxicity and apoptotic cell death against cancer cell lines.

Communicated by Ramaswamy H. Sarma     

Carbohydrate-based heteronuclear complexes as topoisomerase Iα inhibitor: approach toward anticancer chemotherapeutics

Due to the critical role of cellular enzymes necessary for cell proliferation by deciphering topological hurdles in the process of DNA replication, topoisomerases have been one of the major targets in the anticancer drug development area. A need, therefore, arises for new metallodrugs that specifically recognizes DNA and inhibits the activity of topoisomerase enzymes, herein, we report the synthesis and characterization of new metal-based glycoconjugate entities containing heterobimetallic core Cu^II–Sn^IV (1) and Ni^II–Sn^IV (2) derived from N-glycoside ligand (L). The optimized structure of complex 1 and other significant vibrational modes have been explained using dispersion corrected B3LYP/DFT calculations. In vitro DNA binding profile of the L and both the complexes 1 and 2 were done by various biophysical studies. Complex 1 breaks pBR322 DNA via a hydrolytic means which was validated by T4 DNA enzymatic assay. To get a mechanistic insight of mode of action topoisomerase I (Topo I) inhibition assay was carried out. Also, we have taken the help of molecular modeling studies in accordance with experimental findings. In vitro cytotoxicity of the complex 1 was evaluated against a panel of cancer cells which exhibited remarkably good anticancer activity (GI₅₀ values <10 μg/ml). Moreover, intracellular localization of the complex 1 was visualized by confocal microscopy against HeLa cells.     

Loss of p21 expression in senescent cells after DNA damage accompanied with increase of miR-93 expression and reduced p53 interaction with p21 gene promoter

To answer what is a critical event for higher incidence of tumor development in old than young individuals, primary culture of human diploid fibroblasts were employed and DNA damage was induced by doxorubicin or X-ray irradiation. Response to the damage was different between young and old cells; loss of p21^sdi1 expression in spite of p53^S15 activation in old cells along with [³H]thymidine and BrdU incorporation, but not in young cells. The phenomenon was confirmed by other tissue fibroblasts obtained from different donor ages. Induction of miR-93 expression and reduced p53 binding to p21 gene promoter account for loss of p21^sdi1 expression in senescent cells after DNA damage, suggesting a mechanism of in vivo carcinogenesis in aged tissue without repair arrest.     

Molecular Structure,Conformation and Interactions of Antitumor Antibiotic Cyanonaphthridinomycin,a Covalent Binder of DNA

Abstract

X-ray, NMR and molecular modeling studies on cyanonaphthridinomycin (C₂₂ H₂₆N₄O₅), a DNA binding antibiotic, have been carried out to study the structure, conformation and interactions with DNA. The crystals belong to the space group P₂₁ with the cell dimensions of a = 5.934(1), b = 20.684(4), c = 16.866(3)A γ = 90.9° and Z = 4(two molecules/asymmetric unit). The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.087 for 4061 reflections. The conformation of the molecule is compared with that of naphthridinomycin. There are differences in the orientation of the methoxyl group and the saturated oxazole ring. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. Molecular mechanics studies were carried out to obtain the energy minimized structure and its comparison with X-ray and NMR results. Molecular modelling studies were performed to propose models for drug-DNA interactions. Both partial intercalation and groove-binding models have been proposed.     

In-vitro cytotoxic and genotoxic effects of arsenic trioxide on human leukemia (HL-60) cells using the MTT and alkaline single cell gel electrophoresis (Comet) assays

Although arsenic trioxide (ATO) has been the subject of toxicological research, in vitro cytotoxicity and genotoxicity studies using relevant cell models and uniform methodology are not well elucidated. Hence, the aim of the present study was to evaluate the cytotoxicity and genotoxicity induced by ATO in a human leukemia (HL-60) cell line using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and alkaline single cell gel electrophoresis (Comet) assays, respectively. HL-60 cells were treated with different doses of ATO for 24 h prior to cytogenetic assessment. Data obtained from the MTT assay indicated that ATO significantly (P < 0.05) reduced the viability of HL-60 cells in a dose-dependent manner, showing a LD₅₀ value of 6.4 ± 0.6 μg/mL. Data generated from the comet assay also indicated a significant dose-dependent increase in DNA damage in HL-60 cells associated with ATO exposure. We observed a significant increase (P < 0.05) in comet tail-length, tail arm and tail moment, as well as in percentages of DNA cleavage at all doses tested, showing an evidence of ATO-induced genotoxic damage in HL-60 cells. This study confirms that the comet assay is a sensitive and effective method to detect DNA damage caused by heavy metals like arsenic. Taken together, our findings suggest that ATO exposure significantly (P < 0.05) reduces cellular viability and induces DNA damage in HL-60 cells as assessed by MTT and alkaline single cell gel electrophoresis assays, respectively.     

基于响应面分析的高抗原活性猪水肿病大肠杆菌疫苗培养基的研制及效果评价

【背景】猪水肿病大肠杆菌引发的疾病造成了很大的危害,但现有培养基存在培养密度低的问题。【目的】研制出高抗原活性猪水肿病大肠杆菌疫苗培养基。【方法】以常用的市售猪水肿培养基为对照,通过单因素试验、爬坡试验(Plackett-Burman, PB)、响应面(Box-Behnken, BB)试验对猪水肿培养基进行响应面优化,得到猪水肿培养基最优配方。以响应面试验得到的培养基培养猪水肿病大肠杆菌,评价不同培养时间点菌株的抗原活性,制作灭活疫苗,进行动物免疫保护试验。【结果】对研制的培养基进行扩大培养验证,发现扩大培养得到的菌株活菌数可达5×10⁹ CFU/mL以上,约为对照组的2倍。制备的灭活疫苗效价可达1:140 000,并在9 h时抗原蛋白效价达到最高。【结论】本研究研制出的疫苗培养基显著提高了猪大肠杆菌菌体密度,并可提高菌体抗原活性,为猪水肿病灭活疫苗的制备提供了技术指引。     

Experimental approaches to free radicals and ageing

Abstract

Probucol, a clinically used cholesterol lowering and antioxidant drug, was investigated for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) plus hydrogen peroxide (H₂O₂). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H₂O₂-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Fe-NTA is associated with a high incidence of renal adenocarcinoma in rodents. Lipid peroxidation and DNA damage are the principal manifestation of Fe-NTA induced toxicity, which could be mitigated by probucol. Incubation of renal microsomal membrane and/or calf thymus DNA with H₂O₂ (40 mM) in the presence of Fe-NTA (0.1 mM) induces renal microsomal lipid peroxidation and DNA damage to about 2.4-fold and 5.9-fold, respectively, as compared to control (P < 0.05). Induction of renal microsomal lipid peroxidation and DNA damage was inhibited by probucol in a concentration-dependent manner. In lipid peroxidation protection studies, probucol treatment showed a concentration-dependent inhibition (10–34% inhibition; P <0.05) of Fe-NTA plus H₂O₂-induced lipid peroxidation as measured by thiobarbituric acid reacting species' (TBARS) formation in renal microsomes. Similarly, in DNA damage protection studies, probucol treatment also showed a concentration-dependent strong inhibition (36–71% inhibition; P < 0.05) of DNA damage. From these studies, it was concluded that probucol inhibits peroxidation of microsomal membrane lipids and DNA damage induced by Fe-NTA plus H₂O₂. However, because the lipid peroxidation and DNA damage studied here are regarded as early markers of carcinogenesis, we suggest that probucol may be developed as a cancer chemopreventive agent against renal carcinogenesis and other adverse effects of Fe-NTA exposure in experimental animals, in addition to being a cholesterol-lowering drug, useful for the control of hypercholestrolemia.     

Utilizing proteomics to understand and define hypertension: where are we and where do we go?

Introduction: Hypertension is a complex and multifactorial cardiovascular disorder. With different mechanisms contributing to a different extent to an individual’s blood pressure, the discovery of novel pathogenetic principles of hypertension is challenging. However, there is an urgent and unmet clinical need to improve prevention, detection, and therapy of hypertension in order to reduce the global burden associated with hypertension-related cardiovascular diseases.

Areas covered: Proteomic techniques have been applied in reductionist experimental models including angiotensin II infusion models in rodents and the spontaneously hypertensive rat in order to unravel mechanisms involved in blood pressure control and end organ damage. In humans proteomic studies mainly focus on prediction and detection of organ damage, particularly of heart failure and renal disease. While there are only few proteomic studies specifically addressing human primary hypertension, there are more data available in hypertensive disorders in pregnancy, such as preeclampsia. We will review these studies and discuss implications of proteomics on precision medicine approaches.

Expert commentary: Despite the potential of proteomic studies in hypertension there has been moderate progress in this area of research. Standardized large-scale studies are required in order to make best use of the potential that proteomics offers in hypertension and other cardiovascular diseases.     

依赖于端粒延长替代机制的胶质瘤临床前模型及应用现状

胶质瘤是中枢神经系统最常见的恶性肿瘤,主要起源于神经胶质细胞。由于胶质瘤具有高度侵袭性的特点,其死亡率在各种癌症中名列前茅。因此,新的治疗方法和新的药物开发对于胶质瘤的治疗意义十分重大。在大约30%的胶质瘤中,端粒的维持并不是通过端粒酶的激活延长的,而是通过端粒延长替代机制（ALT）来维持和延长端粒。然而,由于目前对于ALT胶质瘤细胞系以及临床前的ALT胶质瘤模型的研究还比较匮乏,从而制约了ALT胶质瘤的机制研究。因此,本篇综述在此探讨了目前发现的ALT胶质瘤细胞系及ALT胶质瘤动物模型,以及这些模型在临床前研究中发挥的作用和最新的进展。     

Evaluation of antiproliferative activities and apoptosis induction caused by copper(II)–benzothiazolesulfonamide complexes in Jurkat T lymphocytes and Caco-2 cells

Two copper(II) complexes with a benzothiazolesulfonamide ligand, [Cu(L)₂(py)₂] (1) and [Cu(en)₂(L)₂] (2) [HL is N-2-(4-methylbenzothiazole)toluenesulfonamide, py is pyridine, en is ethylenediamine], were prepared and then characterized with the aid of X-ray crystallography and spectroscopy. Whereas the copper(II) ion in 1 presents a square-planar geometry, in 2 it has a distorted octahedral environment. In addition, although the ligand is monodentate in both complexes, it exhibits different coordination behavior in each, interacting through the benzothiazole nitrogen atom in 1 and through the sulfonamide nitrogen atom in 2. The propensity for binding of 1 and 2 to calf thymus DNA was studied by thermal denaturation, viscosimetry, and cyclic voltammetry. The ability of the complexes to cleave DNA was studied in vitro through ascorbate activation and was tested by monitoring the expression of the yEGFP gene containing the RAD54 reporter. Moreover, their antiproliferative activity was verified in two cellular models: yeast and human tumor cells in culture. While 1 was found to be the more active cleaving agent in vitro, 2 showed a higher propensity for inflicting DNA damage at the cellular level. The biological studies carried out with human tumor cells, namely, colon adenocarcinoma Caco-2 cells (HTB-37) and leukemia Jurkat T lymphocytes (TIB-152), confirmed that both compounds inhibit the growth of these cell lines, although 2 is more effective. This difference is associated with the latter compound’s greater ability to induce cell death by apoptosis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fine particulate matter air pollution and atherosclerosis: Mechanistic insights

BackgroundAtherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous plaque in the arteries. Its etiology is very complicated and its risk factors primarily include genetic defects, smoking, hyperlipidemia, hypertension, lack of exercise, and infection. Recent studies suggest that fine particulate matter (PM_2.5) air pollution may also contribute to the development of atherosclerosis.Scope of reviewThe present review integrates current experimental evidence with mechanistic pathways whereby PM_2.5 exposure can promote the development of atherosclerosis.Major conclusionsPM_2.5-mediated enhancement of atherosclerosis is likely due to its pro-oxidant and pro-inflammatory effects, involving multiple organs, different cell types, and various molecular mediators.General significanceStudies about the effects of PM_2.5inhalation on atherosclerosis may yield a better understanding of the link between air pollution and major cardiovascular diseases, and provide useful information for policy makers to determine acceptable levels of PM_2.5 air quality. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.     

The influence of cardiovascular risk factors on bone marrow mesenchymal stromal cell fitness

Background aimsIn the past, cell transplantation strategies for the treatment of heart failure have shown promising results in experimental and clinical studies. Bone marrow (BM)-derived stem cells represent the most frequently used cell population. Within this heterogeneous cell population, mesenchymal stromal cells (MSC) have been identified to induce therapeutic effects, mainly through paracrine mechanisms. Because of their low frequency in native tissues, in vitro cell culture expansion is mandatory prior to transplantation. We sought to identify patient-specific cardiovascular risk factors influencing the proliferative potential of MSC.MethodsBM aspirates from 51 patients undergoing elective cardiac surgery were analyzed for MSC frequency and cell culture expansion potential. Fibroblastic colony-forming units (CFU-F) were quantified for culture conditions applying autologous (AS) or fetal bovine serum (FBS) and different basic media. Univariate and multivariate analyzes were performed in order to determine the impact of patient-specific factors on CFU-F numbers.ResultsExpanded MSC showed a specific immune phenotype and displayed adipogenic, chondrogeneic and osteogeneic differentiation potential. CFU-F numbers did not differ under AS or FBS supplementation. Elevated numbers of mononuclear cells, diabetes mellitus, steroid treatment, chronic obstructive pulmonary disease, renal failure, high euroSCORE and impaired left ventricular function were significant determinants for higher CFU-F numbers.ConclusionsThe impact of specific cardiovascular risk factors on MSC fitness could be determined. These results may help to establish patient profiling in order to identify patients suitable for autologous MSC transplantation, and might lead to the identification of disease-related mechanisms of stem cell activation.     

Chemically-induced unscheduled DNA synthesis in cultures of adult human epidermal keratinocytes

Skin is a major target organ for many experimental carcinogens that exist in our environment and the majority of previous carcinogenicity studies have utilised animal derived models. In view of the fact, that many of these environmental chemicals exhibit species- and tissue-specific metabolism, a human skin tissue derived model would be a distinct advantage. Squamous epithelial carcinoma is a predominant form of skin cancer in man and, in theory, human epidermal keratinocytes present an appropriate target cell to employ as an in vitro system to study epidermal carcinogenesis. This report demonstrates the valuable potential of human keratinocyte cultures as a suitable model for mechanistic studies on factors which may influence DNA damage and, hence, the subsequent development of cancer in human epidermis.Keratinocytes were serially cultivated from adult human skin samples and maintained in culture for at least 3 passages. Tertiary cultures, isolated from 3 separate individuals, were exposed to the direct-acting experimental carcinogen, methyl methanesulfonate (CAS No. 66-27-3), and benzo[a]pyrene (CAS No. 50-32-8), which requires metabolic activation. DNA repair was assessed by a quantitative autoradiographic technique.Methyl methanesulfonate and benzo[a]pyrene both elicited a dose-related increase in unscheduled DNA synthesis in cultures prepared from each individual. Inter-individual variation in the response was observed for each chemical, but this was greater in the case of benzo[a]pyrene, which indicates inter-individual variation in both xenobiotic metabolism activity and DNA repair capacity.     

Epigenetics in hyperhomocysteinemic states. A special focus on uremia

Aim of this article is to review the topic of epigenetic control of gene expression, especially regarding DNA methylation, in chronic kidney disease and uremia. Hyperhomocysteinemia is considered an independent cardiovascular risk factor, although the most recent intervention studies utilizing folic acid are negative. The accumulation of homocysteine in blood leads to an intracellular increase of S-adenosylhomocysteine (AdoHcy), a powerful competitive methyltransferase inhibitor, which is itself considered a predictor of cardiovascular events. The extent of methylation inhibition of each individual methyltransferase depends on the methyl donor S-adenosylmethionine (AdoMet) availability, on the [AdoMet]/[AdoHcy] ratio, and on the individual Km value for AdoMet and Ki for AdoHcy. DNA methyltransferases are among the principal targets of hyperhomocysteinemia, as studies in several cell culture and animal models, as well as in humans, almost unequivocally show. In vivo, DNA methylation may be also influenced by various factors in different tissues, for example by rate of cell growth, folate status, etc. and importantly inflammation.     

Fitting experimental data to models that use morphological data from public databases

Ideally detailed neuron models should make use of morphological and electrophysiological data from the same cell. However, this rarely happens. Typically a modeler will choose a cell morphology from a public database, assign standard values for R _a, C _m, and other parameters and then do the modeling study. The assumption is that the model will produce results representative of what might be obtained experimentally. To test this assumption we developed models of CA1 hippocampal pyramidal neurons using 4 different morphologies obtained from 3 public databases. The multiple run fitter in NEURON was used to fit parameter values in each of the 4 morphological models to match experimental data recorded from 19 CA1 pyramidal cells. Fits with fixed standard parameter values produced results that were generally not representative of our experimental data. However, when parameter values were allowed to vary, excellent fits were obtained in almost all cases, but the fitted parameter values were very different among the 4 reconstructions and did not match standard values. The differences in fitted values can be explained by very different diameters, total lengths, membrane areas and volumes among the reconstructed cells, reflecting either cell heterogeneity or issues with the reconstruction data. The fitted values compensated for these differences to make the database cells and experimental cells more similar electrotonically. We conclude that models using fully reconstructed morphologies need to be calibrated with experimental data (even when morphological and electrophysiological data come from the same cell), model results should be generated with multiple reconstructions, morphological and experimental cells should come from the same strain of animal at the same age, and blind use of standard parameter values in models that use reconstruction data may not produce representative experimental results. Action Editor: Steve Redman