Ukrain, a plant derived semi-synthetic compound, exerts antitumor effects against murine and human breast cancer and induce protective antitumor immunity in mice

Despite the recent advances in anti-cancer therapies, breast cancer accounts for the highest percentage of estimated new cases among female cancer patients. The anti-cancer drug Ukrain, a plant-derived semi-synthetic compound, has been shown to be effective in a variety of tumor models including colon, brain, ovarian, melanoma and lymphoma. However, the direct cytotoxic effects of Ukrain have yet to be investigated in breast cancer models. Aim: Herein, we investigated the in vitro and in vivo cytotoxicity of Ukrain using murine (4T07 and TUBO) and human (SKBR-3) breast cancer cell lines. Methods: Cells were treated with varying concentrations of Ukrain for up to 72 h and analyzed for viability by trypan blue exclusion, apoptosis by intracellular caspase 3 and Annexin V staining, and proliferative potential by a clonogenic assay. Female BALB/c mice were challenged subcutaneously (s.c.) with 4T07-RG cells and administered 5 mg/kg or 12.5 mg/kg body weight Ukrain intravenously (i.v.) on the same day and 3 days later. Protective immune responses were determined following re-challenge of tumor-free mice 35 days post primary challenge. Results: Ukrain exposure induced apoptosis in a dose and time-dependent manner with 50 µg/mL Ukrain leading to >50% cell death after 48 h exposure for all three breast cancer cell lines. Ukrain administration (12.5 mg/kg) led to significant inhibition of 4T07 tumor growth in vivo and sustained protective anti-tumor immunity following secondary challenge. Conclusion: Our findings demonstrate the in vitro and in vivo cytotoxic effects of Ukrain on breast cancer cells and may provide insight into designing Ukrain-based therapies for breast cancer patients

Immune Complex-Induced, Nitric Oxide-Mediated Vascular Endothelial Cell Death by Phagocytes Is Prevented with Decoy FcyReceptors

Autoimmune vasculitis is an endothelial inflammatory disease that results from the deposition of immune-complexes (ICs) in blood vessels. The interaction between Fcgamma receptors (FcyRs) expressed on inflammatory cells with ICs is known to cause blood vessel damage. Hence, blocking the interaction of ICs and inflammatory cells is essential to prevent the IC-mediated blood vessel damage. Thus we tested if uncoupling the interaction of FcyRs and ICs prevents endothelium damage. Herein, we demonstrate that dimeric FcyR-Igs prevented nitric oxide (NO) mediated apoptosis of human umbilical vein endothelial cells (HUVECs) in an in vitro vasculitis model. Dimeric FcyR-Igs significantly inhibited the IC-induced upregulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release by murine monocytic cell line. However, FcyR-Igs did not affect the exogenously added NO-induced upregulation of pro-apoptotic genes such as Bax (15 fold), Bak (35 fold), cytochrome-C (11 fold) and caspase-3 (30 fold) in HUVECs. In conclusion, these data suggest that IC-induced NO could be one of the major inflammatory mediator promoting blood vessel inflammation and endothelial cell death during IC-mediated vasculitis which can be effectively blocked by dimeric decoy FcyRs

Comparison of proteomic profiles of the venoms of two of the \u27Big Four\u27 snakes of India, the Indian Cobra (Naja naja) andthe common krait (Bungarus caeruleus), and analyses of their toxins

Snake venoms are mixtures of biologically-active proteins and peptides, and several studies have described the characteristics of some of these toxins. However, complete proteomic profiling of the venoms of many snake species has not yet been done. The Indian cobra (Naja naja) and common krait (Bungarus caeruleus) are elapid snake species that are among the ‘Big Four’ responsible for the majority of human snake envenomation cases in India. As understanding the composition and complexity of venoms is necessary for successful treatment of envenomation in humans, we utilized three different proteomic profiling approaches to characterize these venoms: i) one-dimensional SDS-PAGE coupled with in-gel tryptic digestion and electrospray tandem mass spectrometry (ESI-LC-MS/MS) of individual protein bands; ii) in-solution tryptic digestion of crude venoms coupled with ESI-LC-MS/MS; and iii) separation by gel-filtration chromatography coupled with tryptic digestion and ESI-LC-MS/MS of separated fractions. From the generated data, 81 and 46 different proteins were identified from N. naja and B. caeruleus venoms, respectively, belonging to fifteen different protein families. Venoms from both species were found to contain a variety of phospholipases A2 and three-finger toxins, whereas relatively higher numbers of snake venom metalloproteinases were found in N. naja compared to B. caeruleus venom. The analyses also identified less represented venom proteins including L-amino acid oxidases, cysteine-rich secretory proteins, 5’-nucleotidases and venom nerve growth factors. Further, Kunitz-type serine protease inhibitors, cobra venom factors, phosphodiesterases, vespryns and aminopeptidases were identified in the N. naja venom, while acetylcholinesterases and hyaluronidases were found in the B. caeruleus venom. We further analyzed protein coverage (Lys/Arg rich and poor regions as well as potential glycosylation sites) using in-house software. These studies expand our understanding of the proteomes of the venoms of these two medically-important species

Reappraisal of Vipera aspis Venom Neurotoxicity

BACKGROUND: The variation of venom composition with geography is an important aspect of intraspecific variability in the Vipera genus, although causes of this variability remain unclear. The diversity of snake venom is important both for our understanding of venomous snake evolution and for the preparation of relevant antivenoms to treat envenomations. A geographic intraspecific variation in snake venom composition was recently reported for Vipera aspis aspis venom in France. Since 1992, cases of human envenomation after Vipera aspis aspis bites in south-east France involving unexpected neurological signs were regularly reported. The presence of genes encoding PLA(2) neurotoxins in the Vaa snake genome led us to investigate any neurological symptom associated with snake bites in other regions of France and in neighboring countries. In parallel, we used several approaches to characterize the venom PLA(2) composition of the snakes captured in the same areas. [br/] METHODOLOGY/PRINCIPAL FINDINGS: We conducted an epidemiological survey of snake bites in various regions of France. In parallel, we carried out the analysis of the genes and the transcripts encoding venom PLA(2)s. We used SELDI technology to study the diversity of PLA(2) in various venom samples. Neurological signs (mainly cranial nerve disturbances) were reported after snake bites in three regions of France: Languedoc-Roussillon, Midi-Pyrénées and Provence-Alpes-Côte d'Azur. Genomes of Vipera aspis snakes from south-east France were shown to contain ammodytoxin isoforms never described in the genome of Vipera aspis from other French regions. Surprisingly, transcripts encoding venom neurotoxic PLA(2)s were found in snakes of Massif Central region. Accordingly, SELDI analysis of PLA(2) venom composition confirmed the existence of population of neurotoxic Vipera aspis snakes in the west part of the Massif Central mountains. [br/] CONCLUSIONS/SIGNIFICANCE: The association of epidemiological studies to genetic, biochemical and immunochemical analyses of snake venoms allowed a good evaluation of the potential neurotoxicity of snake bites. A correlation was found between the expression of neurological symptoms in humans and the intensity of the cross-reaction of venoms with anti-ammodytoxin antibodies, which is correlated with the level of neurotoxin (vaspin and/or ammodytoxin) expression in the venom. The origin of the two recently identified neurotoxic snake populations is discussed according to venom PLA(2) genome and transcriptome data

An interaction map of circulating metabolites, immune gene networks, and their genetic regulation

Background: Immunometabolism plays a central role in many cardiometabolic diseases. However, a robust map of immune-related gene networks in circulating human cells, their interactions with metabolites, and their genetic control is still lacking. Here, we integrate blood transcriptomic, metabolomic, and genomic profiles from two population-based cohorts (total N = 2168), including a subset of individuals with matched multi-omic data at 7-year follow-up. Results: We identify topologically replicable gene networks enriched for diverse immune functions including cytotoxicity, viral response, B cell, platelet, neutrophil, and mast cell/basophil activity. These immune gene modules show complex patterns of association with 158 circulating metabolites, including lipoprotein subclasses, lipids, fatty acids, amino acids, small molecules, and CRP. Genome-wide scans for module expression quantitative trait loci (mQTLs) reveal five modules with mQTLs that have both cis and trans effects. The strongest mQTL is in ARHGEF3 (rs1354034) and affects a module enriched for platelet function, independent of platelet counts. Modules of mast cell/basophil and neutrophil function show temporally stable metabolite associations over 7-year follow-up, providing evidence that these modules and their constituent gene products may play central roles in metabolic inflammation. Furthermore, the strongest mQTL in ARHGEF3 also displays clear temporal stability, supporting widespread trans effects at this locus. Conclusions: This study provides a detailed map of natural variation at the blood immunometabolic interface and its genetic basis, and may facilitate subsequent studies to explain inter-individual variation in cardiometabolic disease.Peer reviewe

A neurotoxic phospholipase A2 variant: Isolation and characterization from eastern regional Indian cobra (Naja naja) venom

CM-Sephadex C-25 column chromatography profile of Indian cobra (Naja naja) venom from eastern region showed a distinct and a dominant phospholipase peak, peak-10, while it was not seen in either southern or western venom samples. Peak-10 was subjected to CM-Sephadex C-25 and Sephadex G-50 column chromatography to isolate NN-X-PLA2. NN-X-PLA2 is a single chain protein with the relative molecular weight of 10kDa by SDS-PAGE. It was toxic to mice with an LD50 value 0.098mg/kg body weight (i.p.) and the mice exhibited acute neurotoxic symptoms. Upon indirect stimulation, it inhibited the twitching of frog's gastrocnemius muscle in a dose dependent manner. NN-X-PLA2 was weakly anticoagulant and devoid of cytotoxicity, myotoxicity, hemorrhage, edema inducing, and directlytic activities and effects on platelet aggregation process. Upon chemical modification independently with p-bromophenacyl bromide and acetic anhydride, NN-X-PLA2 lost both enzymatic and toxic properties

Variation in biochemical and pharmacological properties of Indian cobra (Naja naja naja) venom due to geographical distribution

Indian cobra (Naja naja naja) venom obtained from three different geographical regions was studied in terms of electrophoretic pattern, biochemical and pharmacological activities. SDS-PAGE banding pattern revealed significant variation in the protein constituents of the three regional venoms. The eastern venom showed highest indirect hemolysis and hyaluronidase activity. In contrast, western and southern venoms were rich in proteolytic activity. All the three regional venoms were devoid of p-tosyl-L-arginine methyl ester hydrolysing activity. The eastern venom was found to be most lethal among the three regional venoms. The lethal potency varied as eastern > western > southern regional venoms. In addition, all the three regional venoms showed marked variations in their ability to induce symptoms/signs of neurotoxicity, myotoxicity, edema and effect on plasma coagulation process. Polyclonal antiserum prepared against the venom of eastern region cross-reacted with both southern and western regional venoms, but varied in the extent of cross-reactivity by ouchterlony immunodiffusion and ELISA

Resveratrol increases catecholamine synthesis in macrophages: implications on obesity.

Sympathetic activation of white adipocytes results in the transdifferentiation of white adipocytes into brown like thermogenic adipocytes called beige adipocytes. White adipose tissue (WAT) is composed of several cell types including macrophages; activation of anti-inflammatory M2 macrophages was recently shown to induce beiging of WAT in rodent models. M2 macrophages secrete catecholamines which play a predominant role in the induction of beiging in WAT. In the current study, we demonstrate the novel anti-obesity effects of resveratrol (RES), a phytoalexin, mediated through catecholamine synthesis in RAW 264.7 macrophage cell line. RES significantly increased the mRNA expression of tyrosine hydroxylase, phenylalanine N-methyl transferase, dopamine beta hydroxylase and phenylalanine hydroxylase expression in these cells. In addition, RES increased arginase, a marker for M2 phenotype and decreased LPS-induced iNOS (inducible nitric oxide synthase), a marker for M1 phenotype. Furthermore RES decreased nitric oxde production from LPS-treated RAW264.7 cells. To demonstrate the effect of RES on catecholamine secretion, we examined the cell culture supernatant from RES treated RAW264.7 cells for catecholamine levels. The results indicate that RES significantly increased catecholamine production in RAW264.7 cells. Finally, upregulation of thermogenic markers was seen in mature 3T3-L1 adipocytes incubated with conditioned media from RAW264.7 cells treated with RES. These studies suggest a novel anti-obesity mechanism for RES by increasing catecholamine production in macrophages