Potent growth suppressive activity of curcumin in human breast cancer cells: Modulation of Wnt/β-catenin signaling

Abnormal activation of the Wnt/β-catenin signaling pathway and subsequent upregulation of β-catenin driven downstream targets—c-Myc and cyclin D1 is associated with development of breast cancer. The objective of our study was to determine if curcumin could modulate the key elements of Wnt pathway in breast cancer cells; an effect that might underscore its usefulness for chemoprevention/treatment of this malignancy. Curcumin showed a cytotoxic effect on MCF-7 cells with 50% inhibitory concentration (IC₅₀) of 35 μM; while IC₅₀ for MDA-MB-231 cells was 30 μM. Treatment with low cytostatic dose of 20 μM curcumin showed G₂/M arrest in both breast cancer cells. The effect of curcumin (20 μM) treatment on expression of Wnt/β-catenin pathway components in breast cancer cells (MCF-7 and MDA-MB-231) was analyzed by immunofluorescence and Western blotting. Curcumin was found to effectively inhibit the expression of several Wnt/β-catenin pathway components—disheveled, β-catenin, cyclin D1 and slug in both MCF-7 and MDA-MB-231. Immunofluorescence analysis showed that curcumin markedly reduced the nuclear expression of disheveled and β-catenin proteins. Further, the protein levels of the positively regulated β-catenin targets—cyclin D1 and slug, were downregulated by curcumin treatment. The expression levels of two integral proteins of Wnt signaling, GSK3β and E-cadherin were also altered by curcumin treatment. In conclusion, our data demonstrated that the efficacy of curcumin in inhibition of cell proliferation and induction of apoptosis might occur through modulation of β-catenin pathway in human breast cancer cells.     

Molecular mechanics studies of dermorphin

Molecular mechanical simulations have been carried out on dermorphin. Presence of D-Ala2 at the N-terminus and L-Pro6 residue at the C-terminus indicated the probability of beta-turns. From the stereochemical considerations, three types- II', III' and V' - for the beta-turn at the N-terminus of the peptide and two types-I and III- for the C-terminus side of the peptide are possible. In our molecular mechanics calculations, we considered six folded and one extended conformations for dermorphin to asses the relative stabilities. Three of the six folded conformations are lower in energy and have the following general feature-similar in energy, three hydrogen bonds, semirigid beta-sheet segment and favorable Tyr1-Tyr5 interaction. The presence of beta-sheet structure might play a role in mu-receptor selective interaction of dermorphin.     

Involvement of residues 147VYYEIGK153 in binding of lethal factor to protective antigen of Bacillus anthracis

Anthrax toxin is a complex of protective antigen (PA, 735 aa), lethal factor (LF, 776 aa), and edema factor (EF, 767 aa). PA binds to cell surface receptors and is cleaved by cell surface proteases into PA63, while LF and EF compete for binding to PA63. The PA63-LF/EF complex is internalized into the cytosol and causes different pathogenic responses in animals and cultured cells. 1-300 amino acid residues of LF have been viewed as the region responsible for the high affinity binding of LF to PA. Amino acid analysis of LF and EF revealed a common stretch of 7 amino acids (147VYYEIGK153). In the present study, each amino acid of this stretch was replaced by alanine at a time. Y148A, Y149A, I151A, and K153A mutants were found to be deficient in their ability to lyse J774A.1 cells and their binding ability to PA63 was drastically reduced. We propose that these four amino acids play a crucial role in the process of binding of LF to PA63.     

Cribriform plate of ethmoid, olfactory bulb and olfactory acuity in forty species of bats

Relationships between the cribriform plate of the ethmoid, the olfactory bulb, and olfactory acuity were explored using material from 13 of the 17 bat families. All megachiropteran cribriform plates were entirely perforated. In contrast, microchiropteran plates showed distinct perforated portions dorsally and nonperforated portions ventrally. The plates of frugivorous species had more foramina than those of insectivorous ones. Bats with mixed dietary habits were intermediate. Our data suggest that the Chilonycterinae were originally frugivorous, and have only secondarily reverted to an insectivorous diet. Trend analyses show that wherever dietary preference appears to favor a more acute sense of smell, bulb diameter tends to be larger. In general, frugivorous bats tend to have bulbs exceeding 2 mm in diameter; insectivorous bats tend to have bulb diameters of 2 mm or less. The number of foramina in the plates and total cribriform plate area tends to increase as a function of bulb area, but the plate area the foramina occupied increases as a function of bulb volume. The ratio of the size of the bulb to the size of the cerebral hemisphere does not predict olfactory acuity in bats.     

Fungal functional ecology: bringing a trait‐based approach to plant‐associated fungi

Fungi play many essential roles in ecosystems. They facilitate plant access to nutrients and water, serve as decay agents that cycle carbon and nutrients through the soil, water and atmosphere, and are major regulators of macro‐organismal populations. Although technological advances are improving the detection and identification of fungi, there still exist key gaps in our ecological knowledge of this kingdom, especially related to function . Trait‐based approaches have been instrumental in strengthening our understanding of plant functional ecology and, as such, provide excellent models for deepening our understanding of fungal functional ecology in ways that complement insights gained from traditional and ‐omics‐based techniques. In this review, we synthesize current knowledge of fungal functional ecology, taxonomy and systematics and introduce a novel database of fungal functional traits (Fun^Fun). Fun^Fun is built to interface with other databases to explore and predict how fungal functional diversity varies by taxonomy, guild, and other evolutionary or ecological grouping variables. To highlight how a quantitative trait‐based approach can provide new insights, we describe multiple targeted examples and end by suggesting next steps in the rapidly growing field of fungal functional ecology.     

Depigmenting effect of Xanthohumol from hop extract in MNT-1 human melanoma cells and normal human melanocytes

Xanthohumol (XH) is the most abundant prenylated flavonoid found in the hop plant (Humulus lupulus L.) and has previously been shown to have depigmenting effects in B16F10 mouse melanoma cells; however, studies of its depigmenting efficacy in human melanocytes are still lacking. In this work, we explored the effects of XH on melanogenesis in MNT-1 human melanoma cells and normal human melanocytes from darkly-pigmented skin (HEM-DP). XH was screened for cytotoxicity over 48 h, and subsequently tested on melanogenesis in MNT-1 cells. XH was further tested in HEM-DP cells for melanin synthesis and melanosome export; dendricity was quantitated to assess effects on melanosome export. Melanosome degradation was studied in human keratinocytes (HaCaT). Our results showed that XH inhibited melanin synthesis in MNT-1 cells at 30 μM but increased intracellular tyrosinase activity without affecting ROS levels. In HEM-DP cells, XH robustly suppressed cellular tyrosinase activity at nontoxic concentrations (2.5–5 μM) without any effect on melanin synthesis. However, XH inhibited melanosome export by reducing dendrite number and total dendrite length. Further testing in HaCaT cells demonstrated that XH induced melanosome degradation at low micromolar concentrations without any cytotoxicity. In summary, our results demonstrate that XH at low micromolar concentrations might hold promise as a potent inhibitor of human pigmentation by primarily targeting melanin export and melanin degradation. Further studies to elucidate the signaling mechanisms of action of melanosome export inhibition by XH and in vivo efficacy are warranted.     

Corticotropin-releasing Factor Receptor 2 Mediates Sex-Specific Cellular Stress Responses

Although females suffer twice as much as males from stress-related disorders, sex-specific participating and pathogenic cellular stress mechanisms remain uncharacterized. Using corticotropin-releasing factor receptor 2–deficient (Crhr2^−/− ) and wild-type (WT) mice, we show that CRF receptor type 2 (CRF₂) and its high-affinity ligand, urocortin 1 (Ucn1), are key mediators of the endoplasmic reticulum (ER) stress response in a murine model of acute pancreatic inflammation. Ucn1 was expressed de novo in acinar cells of male, but not female WT mice during acute inflammation. Upon insult, acinar Ucn1 induction was markedly attenuated in male but not female Crhr2^−/− mice. Crhr₂^−/− mice of both sexes show exacerbated acinar cell inflammation and necrosis. Electron microscopy showed mild ER damage in WT male mice and markedly distorted ER structure in Crhr2^−/− male mice during pancreatitis. WT and Crhr2^−/− female mice showed similarly distorted ER ultrastructure that was less severe than distortion seen in Crhr2^−/− male mice. Damage in ER structure was accompanied by increased ubiquitination, peIF2, and mistargeted localization of vimentin in WT mice that was further exacerbated in Crhr2^−/− mice of both sexes during pancreatitis. Exogenous Ucn1 rescued many aspects of histological damage and cellular stress response, including restoration of ER structure in male WT and Crhr2^−/−mice, but not in females. Instead, females often showed increased damage. Thus, specific cellular pathways involved in coping and resolution seem to be distinct to each sex. Our results demonstrate the importance of identifying sex-specific pathogenic mechanisms and their value in designing effective therapeutics.     

Targeting RET to induce medullary thyroid cancer cell apoptosis: an antagonistic interplay between PI3K/Akt and p38MAPK/caspase-8 pathways

Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy.     

185 Vascular endothelial growth factor receptor-2 (VEGFR2): structure and functional relationship

Vascular endothelial growth factor (VEGF), is expressed in the vicinity of sprouting vessels and its receptor (VEGF-R2/Flk-1/kdr) on the angioblasts and new vessels, and both are required for vasculogenesis and angiogenesis. VEGFR2, also called as KDR or Flk-1, is identified as an early marker for endothelial cell progenitors, whose expression is restricted to endothelial cells in vivo. VEGFR2 consists of extracellular (7-Ig-like sub-domains), transmembrane and cytoplasmic domains. In order to understand the structure–functional relationship and signal transduction process of VEGFR2, we have examined their amino acid sequences from a wide range of species including mammals, birds, Zebrafish and also computed the phylogenetic tree, secondary and domain structures. Phylogeny constructed using Maximum Parsimony tree software MEGA-5 version suggested an interesting sequence similarity between Zebrafish and Gallus, closeness between human, rat, horse and pig. Strong homology in amino acids sequences was observed between the species, such as human, Macaca mulatta, gorilla, etc, and small variations in Zebrafish and zebrafinch. The Arg and Asp residues which are involved in forming salt bridges are evolutionarily conserved from Zebrafish to human in D7 domain of VEGFR2, indicating their functional importance in VEGFR activity. Amino acids, tyrosine in the extracellular loops and cysteines involved in disulphide bridges of VEGFR2, are highly conserved suggesting their importance during ligand binding, the details of which will be discussed.