Hyperglycemia exacerbates colon cancer malignancy through hexosamine biosynthetic pathway.

Hyperglycemia is a common feature of diabetes mellitus, considered as a risk factor for cancer. However, its direct effects in cancer cell behavior are relatively unexplored. Herein we show that high glucose concentration induces aberrant glycosylation, increased cell proliferation, invasion and tumor progression of colon cancer. By modulating the activity of the rate-limiting enzyme, glutamine-fructose-6-phosphate amidotransferase (GFAT), we demonstrate that hexosamine biosynthetic pathway (HBP) is involved in those processes. Biopsies from patients with colon carcinoma show increased levels of GFAT and consequently aberrant glycans' expression suggesting an increase of HBP flow in human colon cancer. All together, our results open the possibility that HBP links hyperglycemia, aberrant glycosylation and tumor malignancy, and suggest this pathway as a potential therapeutic target for colorectal cancer.

Antimicrobial activity of synthetic bornyl benzoates against Trypanosoma cruzi.

We report here for the first time the in vitro effects of (1S,2R,4S)-1,7,7-trimethyl-bicyclo[2·2·1]heptan-2-yl-3',4',5'-trimethoxy benzoate (1) and (1S,2R,4S)-1,7,7-trimethyl-bicyclo[2·2·1]heptan-2-yl benzoate (2) on the growth and ultrastructure of Trypanosoma cruzi. These two synthetic compounds exerted an antiproliferative effect on the epimastigote forms of the parasite. The ICs(50/72h) of two synthetic L-bornyl benzoates, 1 and 2, was 10·1 and 12·8 µg/ml, respectively. Both compounds were more selective against epimastigotes than HEp-2 cells. Ultrastructural analysis revealed intense cytoplasmic vacuolization and the appearance of cytoplasmic materials surrounded by membranes. The treatment of peritoneal macrophages with compounds 1 and 2 caused a significant decrease in the number of T. cruzi-infected cells. L-Bornyl benzoate derivatives may serve as a potential source for the development of more effective and safer chemotherapeutic agents against T. cruzi infections.

Effects of (-) mammea A/BB isolated from Calophyllum brasiliense leaves and derivatives on mitochondrial membrane of Leishmania amazonensis.

We have previously demonstrated antileishmanial activity on Leishmania amazonensis of the natural (1-2), synthetic (7) and derivatives of coumarin (-) mammea A/BB (3-6) isolated from the dichloromethane extract of Calophyllum brasiliense leaves. The aim of the present study was to evaluate morphological and ultrastructural alterations in Leishmania amazonensis induced by these compounds. In promastigote forms, all seven compounds produced significant morphological and ultrastructural alterations, as revealed by scanning and transmission electron microscopy. The compound 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one (3), the most active antileishmanial with LD50 of 0.9 µM), induced cell shrinkage and a rounded appearance of the cells. Parasites incubated in the presence of compound (3) showed ultrastructural changes, such as the appearance of mitochondrial swelling with a reduction in the density of the mitochondrial matrix and the presence of vesicles inside the mitochondrion, indicating damage and significant change in this organelle; abnormal chromatin condensation, alterations in the nuclear envelope, intense atypical cytoplasmic vacuolization, and the appearance of autophagic vacuoles were also observed. In addition, the compound (3) may be acting to depolarize the mitochondrial membrane potential of the cells, leading to death of the parasite.

Giardia disrupts the arrangement of tight, adherens and desmosomal junction proteins of intestinal cells.

Giardia duodenalis is a parasitic protozoan that causes diarrhea and other symptoms which together constitute a disease known as giardiasis. Although the disease has been well defined, the mechanisms involving the establishment of the infection have not yet been fully elucidated. In this study, we show that after 24h of interaction between parasites and intestinal Caco-2 cells, there was an alteration of the paracellular permeability, as observed by an approximate 42% of reduction in the transepithelial electrical resistance and permeation to ruthenium red, which was concomitant with ultrastructural changes. Nevertheless, epithelium viability was not affected. We also demonstrate that there was no change in expression of junctional proteins (tight and adherens) but that the distribution of these proteins in Caco-2 cells after parasite adhesion was significantly altered, as observed via laser scanning confocal microscopy 3D reconstruction. The present work shows that adhesion of Giardia duodenalis trophozoites to intestinal cells in vitro induces disturbances of the tight, adherens and desmosomal junctions.

Effects of serine protease inhibitors on viability and morphology of Leishmania (Leishmania) amazonensis promastigotes.

To investigate the importance of serine proteases in Leishmania amazonensis promastigotes, we analyzed the effects of classical serine protease inhibitors and a Kunitz-type inhibitor, obtained from sea anemone Stichodactyla helianthus (ShPI-I), on the viability and morphology of parasites in culture. Classical inhibitors were selected on the basis of their ability to inhibit L. amazonensis serine proteases, previously described. The N-tosyl-L: -phenylalanine chloromethyl ketone (TPCK) and benzamidine (Bza) inhibitors, which are potential Leishmania proteases inhibitors, in all experimental conditions reduced the parasite viability, with regard to time dependence. On the other hand, N-tosyl-lysine chloromethyl ketone (TLCK) did not significantly affect the parasite viability, as it was poor Leishmania enzymes inhibitor. Ultrastructural analysis demonstrated that both Bza and TPCK induced changes in the flagellar pocket region with membrane alteration, including bleb formation. However, TPCK effects were more pronounced than those of Bza in Leishmania flagellar pocket in plasma membrane, and intracellular vesicular bodies was visualized. ShPI-I proved to be a powerful inhibitor of L. amazonensis serine proteases and the parasite viability. The ultrastructural alterations caused by ShPI-I were more dramatic than those induced by the classical inhibitors. Vesiculation of the flagellar pocket membrane, the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole, and alterations of promastigotes shape resulted.

Golgi complex disassembly caused by light-activated calphostin C involves MAPK and PKA.

We examined the participation of MAPK and PKA in the Golgi complex disassembly caused by light-activated Calphostin C in HT-29 cells. When these cells were incubated with Calphostin C, fragmentation and dispersal of the Golgi complex was observed as assessed by immunofluorescence microscopy. Electron microscopy analysis showed that clusters of vesicles and large tubule-vesicular membrane structures, resembling the Golgi remnants present in mitotic cells, substituted the Golgi stacks. In addition, Calphostin C treatment caused inhibition of the endocytic route. We confirmed that the Golgi disassembly was not due to PKC inhibition, and suggested, based on the use of specific inhibitors, that other kinases are involved. It was shown that pretreatment with PD98059 and H-89, both inhibitors of MAPK and PKA, respectively, prior to incubation with Calphostin C, caused blockade of the Golgi disassembly, as well as the inhibition of the endocytic pathway caused by this drug. This finding supports the existence of a novel mechanism by which MAPK and PKA may regulate the Golgi breakdown caused by Calphostin C in HT-29 cells.

Claudins: multifunctional players in epithelial tight junctions and their role in cancer.

The molecular architecture of tight junctions has been a subject of extensive studies that have shown tight junctions to be composed of many peripheral and integral membrane proteins. Claudins have been considered the main tight junction-forming proteins; however, the role they play in a series of pathophysiological events, including human carcinoma development, is only now beginning to be understood. Increasing evidence from in vitro and in vivo studies have identified the influence of claudins on tight junction structure and function, although claudins also participate in cellular contexts other than tight junctions. The aim of this review is to summarize and discuss the conceptual framework concerning claudins, focusing on the involvement of these proteins in epithelial cell polarity establishment, paracellular transport control, signal transduction and tumorigenesis.

Morphological and molecular alterations at the junctional complex in irradiated human colon adenocarcinoma cells, Caco-2.

PURPOSE: Ionizing radiation is one of the main modalities used in the treatment of colorectal cancer. Despite a number of epigenetic or non-targeted effects of radiation exposure that have been described, the effect of radiation on cell-cell adhesion in the epithelium has been less studied. We report morphological and molecular alterations induced by ionizing radiation at the junctional complex level of human colon cancer Caco-2 cells. MATERIALS AND METHODS: Cells were irradiated with doses of 2, 5 or 10 Gy and the effects on the junctional complex were monitored for different times after irradiation. Alterations of tight and adherens junction components were observed by measuring the transepithelial electrical resistance, by immunofluorescence and immunoblotting and electron microscopy analyses. RESULTS: Ionizing radiation caused alterations in the junctional complex, as evidenced by: (a) a decrease in the transepithelial electrical resistance, (b) alterations in the pattern of the distribution of junctional proteins as observed for E-cadherin, occludin, and zonula occludens 1 (ZO-1), but with minor changes in claudin-1 localization, and (c) wide spaces between opposed cells. These effects were dose and time-dependent since minor doses of irradiation caused a reversible effect on E-cadherin distribution and transepithelial electrical resistance. CONCLUSIONS: The results obtained show that ionizing radiation caused redistribution of the main junctional proteins E-cadherin, occludin and ZO-1 with minor changes for claudin-1, leading to disassembly of the junctional complex and loss of its functionality in Caco-2 cells. The molecular mechanisms responsible for these events need further elucidation.

Subcellular localization of an extracellular serine protease in Leishmania (Leishmania) amazonensis.

Extracellular proteolytic activity was detected in a Leishmania ( L.) amazonensis culture supernatant and a 56-kDa protein was purified using (NH4)2SO4 precipitation followed by affinity chromatography on aprotinin-agarose. A rabbit serum obtained against the 56-kDa extracellular serine protease was used in order to analyze its location in L. ( L.) amazonensis parasites. Immunocytochemistry studies revealed that the enzyme is mainly found in the flagellar pocket and cytoplasmic vesicles of promastigote forms, whereas in amastigotes, it is located in electron-dense structures resembling megasomes. These results indicate that the 56-kDa serine protease is released into the extracellular environment through the flagellar pocket; and its intracellular location suggests either a correlated enzymatic activity or intracellular trafficking.

Increase of melanogenesis by retinoic acid: an ultrastructural and morphometric study.

Melanin is a dark pigment protecting the skin against UV radiation in some organisms. Studies on invasion and metastasis using retinoic acid as inhibitor agent are well known, but its role in melanin production (melanogenesis), especially at ultrastructural level and using morphometry were not well studied. In the present study, we analyzed the effects of retinoic acid on the melanosomes in B16F10 melanoma cells. These organelles were identified and quantified using routine electron microscopy and the specific HMB45 antibody. Other approaches such as immunofluorescence, and flow cytometry were also used. Our results indicated that retinoic acid increased the melanogenesis process in B16F10 melanoma cells. Furthermore, this work also provided evidence that this substance interferes at the subcellular level altering the numerical density of melanosomes, as well as the relative volume of the nucleus and nucleolus. In addition, the cells displayed altered morphology and an increase in the percentage of the relative volume of melanosomes, mainly the stages II-III and IV, leading to melanin formation. Furthermore, a decrease in the cells number after retinoic acid treatment was also observed.

Assembly and functional analysis of tight junction in a colon adenocarcinoma cell line: effect of glucose depletion.

We describe morphologic and biochemical changes in the colonic epithelial HCT-116 cell line following depletion of glucose from the culture medium. Cultured cells under permissive differentiation conditions (inosine-supplemented glucose-free medium) exhibited, after confluence, an enterocytic differentiation, in contrast to cells grown under standard culture conditions, where they remain in an undifferentiated state. The differentiated phenotype was characterized by the presence of a monolayer of polarized cells displaying an apical tight junction, and by the presence of alkaline phosphatase, a well known brush border marker. We demonstrated that the formed tight junctions were functional using the following criteria: a) labeling of the junctions with antibodies recognizing the tight juntion proteins occludin and ZO-1, as observed by immunofluorescence and immunoblotting analysis; b) characteristic organization of the tight junction strands, as observed in freeze-fracture replicas; c) increase ofthe transepithelial resistance across the monolayer; d) not permeation of the ruthenium red stain across the tight junction, and e) presence of the hyperphosphorylated form of occludin.

Isolation and characterization of the Golgi complex of the protozoan Trypanosoma cruzi.

In this study the Golgi complex of the epimastigote forms of Trypanosoma cruzi were isolated and characterized. Using well-controlled sonication to rupture the cells and centrifugation on a discontinuous sucrose density gradient, a highly enriched Golgi fraction was obtained. The Golgi fraction contained most of the beta-galactosyltransferase (beta-Gal transferase) and UDP-N-acetyl-glucosamine: polypeptide-alpha-N-acetyl-glucosaminyltransferase (O-alpha-GlcNAc transferase) activities with minimal contamination of other organelles, as observed by enzymatic assays and electron microscopy analysis. To characterize the Golgi from T. cruzi cells further, it was incubated with a monoclonal antibody against a 58 kDa protein involved in the association of the Golgi complex with microtubules in mammalian cells. Immunofluorescence microscopy showed that the 58 kDa protein is localized in the T. cruzi Golgi region, a result confirmed by high resolution scanning electron microscopy immunocytochemistry. Thus, our results show, for the first time, that the beta-Gal transferase, the O-alpha-GlcNAc transferase and the 58 kDa protein are present in the Golgi complex of T. cruzi and are novel biochemical markers which can be used in the characterization of this organelle in T. cruzi.

Evidence that increased tyrosine phosphorylation causes disassembly of adherens junctions but does not perturb paracellular permeability in Caco-2 cells.

In this study, we report on the apparent effect of increased tyrosine phosphorylation events on the assembly and integrity of adherens junctions (AJs) and on paracellular permeability in Caco-2 cells. Cell monolayers were incubated with the phosphotyrosine phosphatase inhibitor vanadate/H2O2. Addition of this compound to monolayer resulted in disruption of the AJs, as revealed by electron microscopy and by a loss of membrane association of the AJ-associated protein uvomorulin/E-cadherin (U/E-c). However, tight junctions (TJs) were unaltered, as determined by measuring the transepithelial resistance (Rt), by ruthenium red labeling, as seen by transmission electron microscopy, and the distribution of TJ strands as seen in freeze-fracture replicas and by hyperphosphorylation of triton-insoluble occludin. Also examination of vanadate/H2O2 treated cells indicated a specific increase in AJ-associated phosphotyrosine residues as evaluated by immunofluorescence microscopy, but no modification of F-actin distribution, as revealed by confocal laser scanning microscopy analysis. To verify that modulation of AJs was indeed related to tyrosine phosphorylation, we tested a range of distinct protein kinase inhibitors. Of the three inhibitors tested (tyrphostin 25, genistein and staurosporine), tyrphostin 25 completely blocked the effects of vanadate/ H2O2 on assembly and integrity of AJs, redistribution of U/E-c and phosphotyrosine labeling. Our results indicate that, after addition of vanadate/H2O2 to Caco-2 monolayers, specific tyrosine phosphorylation of proteins cause disruption of AJs, but no modifications of the TJs' structure and functionality. These observations suggest that, in contrast to what happens with epithelial cells, TJs and AJs of Caco-2 cells are regulated by independent mechanisms.