N-hydroxy-amide analogues of MHC-class I peptide ligands with nanomolar binding affinities

A novel class of major histocompatibility complex class I (MHC-I) ligands containing an N-hydroxy-amide bond was designed on the basis of the natural epitope SIINFEKL, and synthesized on solid phase. The capacity of these compounds to bind to the MHC-I molecule H-2K^b and to induce T cell responses was analysed in comparison with the corresponding glycine containing variant of SIINFEKL. Binding to the MHC molecule was diminished by the N-hydroxy group at positions 2 and 3 of the oligomer and improved in the case of positions 4, 5, 6 and 7. No change was seen for position 1. The efficacy of T cell stimulation was strongly reduced by the modification of all positions except for position 1. A complete loss of activity was found for the N-hydroxy variant in positions 4 and 6. N-Hydroxy amide-containing peptides displayed an enhanced stability to enzymatic degradation. This new class of MHC ligand can become instrumental as immunomodulatory reagent in various disease situations. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Functional expression of the transient receptor potential channel TRPA1, a sensor for toxic lung inhalants,in pulmonary epithelial cells

The cation channel TRPA1 functions as a chemosensory protein and is directly activated by a number of noxious inhalants. A pulmonary expression of TRPA1 has been described in sensory nerve endings and its stimulation leads to the acceleration of inflammatory responses in the lung. Whereas the function of TRPA1 in neuronal cells is well defined, only few reports exist suggesting a role in epithelial cells. The aim of the present study was therefore (1) to evaluate the expression of TRPA1 in pulmonary epithelial cell lines, (2) to characterize TRPA1-promoted signaling in these cells, and (3) to study the extra-neuronal expression of this channel in lung tissue sections. Our results revealed that the widely used alveolar type II cell line A549 expresses TRPA1 at the mRNA and protein level. Furthermore, stimulating A549 cells with known TRPA1 activators (i.e., allyl isothiocyanate) led to an increase in intracellular calcium levels, which was sensitive to the TRPA1 blocker ruthenium red. Investigating TRPA1 coupled downstream signaling cascades it was found that TRPA1 activation elicited a stimulation of ERK1/2 whereas other MAP kinases were not affected. Finally, using epithelial as well as neuronal markers in immunohistochemical approaches, a non-neuronal TRPA1 protein expression was detected in distal parts of the porcine lung epithelium, which was also found examining human lung sections. TRPA1-positive staining co-localized with both epithelial and neuronal markers underlining the observed epithelial expression pattern. Our findings of a functional expression of TRPA1 in pulmonary epithelial cells provide causal evidence for a non-neuronal TRPA1-mediated control of inflammatory responses elicited upon TRPA1-mediated registration of toxic inhalants in vivo.     

Enhancing the fungicidal activity of amphotericin B via vacuole disruption by benzyl isothiocyanate,a cruciferous plant constituent

Amphotericin B (AmB), a typical polyene macrolide antifungal agent, is widely used to treat systemic mycoses. In the present study, we show that the fungicidal activity of AmB was enhanced by benzyl isothiocyanate (BITC), a cruciferous plant-derived compound, in the budding yeast, Saccharomyces cerevisiae. In addition to forming a molecular complex with ergosterol present in fungal cell membranes to form K⁺-permeable ion channels, AmB has been recognized to mediate vacuolar membrane disruption resulting in lethal effects. BITC showed no effect on AmB-induced plasma membrane permeability; however, it amplified AmB-induced vacuolar membrane disruption in S. cerevisiae. Furthermore, the BITC-enhanced fungicidal effects of AmB significantly decreased cell viability due to the disruption of vacuoles in the pathogenic fungus Candida albicans. The application of the combinatorial antifungal effect of AmB and BITC may aid in dose reduction of AmB in clinical antifungal therapy and consequently decrease side effects in patients. These results also have significant implications for the development of vacuole-targeting chemotherapy against fungal infections.     

Garlic allyl derivatives interact with membrane lipids to modify the membrane fluidity

As a novel approach to the mode of medicinal action of garlic, its constituents were comparatively studied with respect to their interactions with membrane lipids to modify the membrane fluidity. Allyl derivatives rigidified tumor cell and platelet model membranes consisting of unsaturated phospholipids and cholesterol at 20–500 μM with the potency being diallyl trisulfide (DATS) > diallyl disulfide (DADS) by preferentially acting on the hydrocarbon cores of lipid bilayers. They were also effective in rigidifying candida cell model membranes prepared with ergosterol and phospholipids at 100–500 μM with the potency being DADS > DATS > diallyl sulfide (DAS), but not bacteria cell model membranes without ergosterol. Alliin, a precursor of these DASs, was not active on any membranes at 500 μM. Both relative intensity and selectivity in membrane effects correlated with those in antiproliferative, antiplatelet and antimicrobial effects. In cell culture experiments, membrane-active DASs inhibited the growth of tumor cells cultured for 24 and 48 h at 20–500 μM to show the potency being DATS > DADS, together with rigidifying cell membranes by acting on their deeper regions more intensively. However, membrane-inactive allyl derivatives were not growth-inhibitory on tumor cells. The membrane lipid interactions of DASs appear to be one of possible mechanisms underlying different effects of garlic.     

Pyrogallol inhibits the growth of lung cancer Calu-6 cells via caspase-dependent apoptosis

Pyrogallol (PG) is a polyphenol compound and a known O₂⁻ generator. We evaluated the effects of PG on the growth and apoptosis of human pulmonary adenocarcinoma Calu-6 cells. PG decreased the viability of Calu-6 cells in a dose- and time-dependent manner. The induction of apoptosis by PG was accompanied by the loss of mitochondrial membrane potential (ΔΨ_m), cytochrome c release from mitochondria and activation of caspase-3 and caspase-8. All tested caspase inhibitors, especially the pan-caspase inhibitor (Z-VAD), markedly rescued Calu-6 cells from PG-induced cell death. Rescue was accompanied by inhibition of caspase-3 activation and PARP cleavage. Treatment with Z-VAD also prevented the loss of mitochondrial membrane potential (ΔΨ_m). In conclusion, PG inhibits the growth of Calu-6 cells via caspase-dependent apoptosis.     

SAHA treatment overcomes the anti-apoptotic effects of Bcl-2 and is associated with the formation of mature PML nuclear bodies in human leukemic U937 cells

Bcl-2 protects tumor cells from the apoptotic effects of various antineoplastic agents. Increased expression of Bcl-2 has been associated with poor response to chemotherapy in various malignancies, including leukemia. Therefore, bypassing the resistance conferred by anti-apoptotic factors such as Bcl-2 represents an attractive therapeutic strategy against cancer cells, including leukemic cells. We undertook this study to examine whether SAHA (suberoylanilide hydroxamic acid) overcomes the resistance by Bcl-2 in human leukemic cells, with a specific focus on the involvement of PML-NBs. Experiments were conducted with Bcl-2-overexpressing human leukemic U937 cells. Since we previously demonstrated that overexpression of Bcl-2 attenuates resveratrol-induced apoptosis in human leukemic U937 cells, resveratrol-treated U937 cells were used as a negative control. The present study indicates that SAHA at 1-7 μM, the dose range known to induce apoptosis in various cancer cells, overcomes the anti-apoptotic effects of Bcl-2 in Bcl-2-overexpressing human leukemic U937 cells. Notably, we observed that SAHA-induced formation of mature promyelocytic leukemia (PML) nuclear bodies (NBs) correlates with overcoming the anti-apoptotic effects of Bcl-2 in human leukemic U937 cells. Thus, PML protein and the formation of mature PML-NBs could be considered as therapeutic targets that could help bypass the resistance to apoptosis conferred by Bcl-2. Elucidating exactly how PML regulates Bcl-2 will require further work.     

Niemann-Pick C1 protein transports copper to the secretory compartment from late endosomes where ATP7B resides

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.     

Epithelial-mesenchymal transition of ovarian tumor cells induces an angiogenic monocyte cell population

Vasculogenesis, or recruitment of progenitors able to differentiate into endothelial-like cells, may provide an important contribution to neovessel formation in tumors. However, the factors involved in the vasculogenic process and in particular the role of the epithelial-mesenchymal transition of tumor cells have not yet been investigated. We found a CD14⁺/KDR⁺ angiogenic monocyte population in undifferentiated ovarian tumors, significantly increased in the corresponding tumor metastasis. In vitro, monocyte differentiation into CD14⁺/KDR⁺ cells was induced by conditioned media from the primary ovarian tumor cells expressing a mesenchymal phenotype. In contrast, the ovarian tumor cell line SKOV3 expressing an epithelial phenotype was unable to stimulate the differentiation of monocytes into CD14⁺/KDR⁺ cells. When an epithelial-mesenchymal transition was induced in SKOV3, they acquired this differentiative ability. Moreover, after mesenchymal transition pleiotrophin expression by SKOV3 was increased and conversely its blockade significantly reduced monocyte differentiation. The obtained CD14⁺/KDR⁺ cell population showed the expression of endothelial markers, increased the formation of capillary-like structures by endothelial cells and promoted the migration of ovarian tumor cells in vitro. In conclusion, we showed that the epithelial-mesenchymal transition of ovarian tumor cells induced differentiation of monocytes into the pro-angiogenic CD14⁺/KDR⁺ population and thus it may provide a tumor microenvironment that favours vasculogenesis and metastatization of the ovarian cancer.     

fps/fes knockout mice display a lactation defect and the fps/fes tyrosine kinase is a component of E-cadherin-based adherens junctions in breast epithelial cells during lactation

The fps/fes proto-oncogene encodes a cytoplasmic protein-tyrosine kinase implicated in vesicular trafficking and cytokine and growth factor signaling in hematopoietic, neuronal, vascular endothelial and epithelial lineages. Genetic evidence has suggested a tumor suppressor role for Fps/Fes in breast and colon. Here we used fps/fes knockout mice to investigate potential roles for this kinase in development and function of the mammary gland. Fps/Fes expression was induced during pregnancy and lactation, and its kinase activity was dramatically enhanced. Milk protein and fat composition from nursing fps/fes-null mothers was normal; however, pups reared by them gained weight more slowly than pups reared by wild-type mothers. Fps/Fes displayed a predominantly dispersed punctate intracellular distribution which was consistent with vesicles within the luminal epithelial cells of lactating breast, while a small fraction co-localized with β-catenin and E-cadherin on their basolateral surfaces. Fps/Fes was found to be a component of the E-cadherin adherens junction (AJ) complex; however, the phosphotyrosine status of β-catenin and core AJ components in fps/fes-null breast tissue was unaltered, and epithelial cell AJs and gland morphology were intact. We conclude that Fps/Fes is not essential for the maintenance of epithelial cell AJs in the lactating breast but may instead play important roles in vesicular trafficking and milk secretion.