Sulfated glycosphingolipid as mediator of phagocytosis: SM4s enhances apoptotic cell clearance and modulates macrophage activity

Sulfoglycolipids are present on the surface of a variety of cells. The sulfatide SM4s is increased in lung, renal, and colon cancer and is associated with an adverse prognosis, possibly due to a low immunoreactivity of the tumor. As macrophages significantly contribute to the inflammatory infiltrate in malignancies, we postulated that SM4s may modulate macrophage function. We have investigated the effect of SM4s on the uptake of apoptotic tumor cells, macrophage cytokine profile, and receptor expression. Using flow cytometry and microscopic analyses, we found that coating apoptotic murine carcinoma cells from the colon and kidney with SM4s promoted their phagocytosis by murine macrophages up to 3-fold ex vivo and in vivo. This increased capacity was specifically inhibited by preincubation of macrophages with oxidized or acetylated low density lipoprotein and maleylated albumin, indicating involvement of scavenger receptors in this interaction. The uptake of SM4s-coated apoptotic cells significantly enhanced macrophage production of TGF-beta1, expression of P-selectin, and secretion of IL-6. These data suggest that SM4s within tumors may promote apoptotic cell removal and alter the phenotype of tumor-associated macrophages.     

Dictyopteris undulata extract induces apoptosis in human colon cancer cells

The present study investigated the cytotoxic and apoptotic effects of an ethanol extract derived from the marine brown alga Dictyopteris undulata against human colon adenocarcinoma cells. The Dictyopteris undulata extract (DUE) showed cytotoxic activity against SW480 cells in a dose-dependent manner, with 50% inhibition of cell viability at a concentration of 40 μg/mL. DUE also induced programmed cell death in SW480 cells, as evidenced by apoptotic body formation, DNA fragmentation, an increase in the population of apoptotic sub-G1 phase cells, and mitochondrial membrane depolarization. Moreover, DUE significantly modulated the expression of apoptosisassociated proteins, resulting in a decrease in B cell lymphoma-2 expression and an increase in Bcl-2-associated X protein expression, as well as the activation of caspase-9 and caspase-3. Furthermore, DUE showed apoptotic cell death in two other colon cancer cell lines, SNU407 and HT29. These observations suggest that DUE may prove useful as a therapeutic agent for the attenuation of colon cancer.     

Toluidine Blue Staining of Coccidia in Cultured Animal Cells

Cultured animal cells infected with various species of Eimeria (coccidia) from chickens are washed in Hanks balanced salt solution (HBSS) and fixed in 5% formalin in HBSS. The fixed cultures are washed briefly in distilled water to remove HBSS salts and then dehydrated in a series of mixtures of 40 to 50% ethanol with increasing concentrations of tertiary butanol (TB) and decreasing concentrations of distilled water. Cultures are placed for 1 min in a mixture of 2 parts ethanol :TB (25:75) and 1 part 0.05% toluidine blue O in McIlvaine buffer (pH 6.0), followed by 1 min in 0.05% toluidine blue O in McIlvaine buffer (pH 6.0). The stained cultures are dipped for 1-2 sec in TB, allowed to dry and mounted permanently on slides. Cover-slip cultures fixed and stained by this procedure 8 years ago have not faded or discolored. The alcohol mixtures, formalin in HBSS, stain and buffer can be prepared in large volumes and stored indefinitely. The staining procedure has proven to be rapid and dependable with a variety of cell types in monolayer cultures in research and teaching applications.     

Prodigiosin-induced apoptosis in human colon cancer cells

Prodigiosin is a red pigment produced by various bacteria including Serratia marcescens. Colorectal cancer is one of the most frequent malignancies and one of the most frequent causes of cancer death in the Western world. Its treatment is far from satisfactory and the challenge to oncologists is to find novel chemical entities with less toxicity and greater effectiveness than those used in current chemotherapy. Here we characterize the apoptotic action of prodigiosin in colon cancer cells. DLD-1 and SW-620 human colon adenocarcinoma cells, NRK and Swiss-3T3 nonmalignant cells were assayed by the MTT assay, fragmentation pattern of DNA, Hoechst 33342 staining and study of PARP cleavage by Western blot, in order to characterize the prodigiosin-induced apoptosis. Prodigiosin was purified and its structure was confirmed. Metastatic SW-620 cells were more sensitive to prodigiosin (IC50: 275 nM) than DLD-1. We did not observe a significant decrease in the viability of NRK cells. We confirmed that prodigiosin induces apoptosis in both cancer cell lines by the characteristic DNA laddering pattern and condensed nuclei or apoptotic bodies identified by fluorescence microscopy. These results indicate that prodigiosin induces apoptosis in colon cancer cells.     

Inhibitory effects of norcantharidin against human lung cancer cell growth and migration

The effects of norcantharidin (NCTD), an anticancer drug in China, on the growth and migration in human lung cancer cells were investigated by in vitro and ex vivo assays. NCTD significantly inhibited the in vitro and ex vivo growth of human non-small cell lung cancer A549 cells in dose- and time-dependent manners. Western blot analysis indicated that NCTD dose-dependently down-regulated the expression of anti-apoptotic protein Bcl-2 and up-regulated the level of pro-apoptotic protein Bax, eventually leading the reduction of ratio of Bcl-2/Bax proteins in A549 cells. Moreover, NCTD significantly suppressed the A549 cell migration in the case of without reducing the cell viability. More importantly, NCTD significantly enhanced the anticancer activity of anticancer agents such as trichostatin A (the histone deacetylase inhibitor), celecoxib (the inhibitor of cyclooxygenase-2) and lovastatin (the inhibitor of HMG-CoA reductase) by strongly reducing the viability and/or the ratio of Bcl-2/Bax protein in A549 cells. Our findings suggest that NCTD may have the wide therapeutic and/or adjuvant therapeutic application in the treatment of human lung cancer.     

Effects of matrine against the growth of human lung cancer and hepatoma cells as well as lung cancer cell migration

The purpose of this study is to investigate in vitro and ex vivo effects of matrine on the growth of human lung cancer and hepatoma cells and the cancer cell migration as well as the expressions of related proteins in the cancer cells. Matrine significantly inhibited the in vitro and ex vivo growth of human non-small cell lung cancer A549 and hepatoma SMMC-7721 cells. Matrine induced the apoptosis in A549 and SMMC-7721 cells. Western blot analysis indicated that matrine dose-dependently down-regulated the expression of anti-apoptotic protein Bcl-2 and up-regulated the level of pro-apoptotic protein bax, eventually leading the reduction of ratios of Bcl-2/Bax proteins in A549 and SMMC-7721 cells. Furthermore, matrine significantly suppressed the A549 cell migration without reducing the cell viability. In addition, matrine dramatically reduced the secretion of vascular endothelial growth factor A in A549 cells. More importantly, matrine markedly enhanced the anticancer activity of anticancer agent trichostatin A (the histone deacetylase inhibitor) by strongly reducing the viability and/or the ratio of Bcl-2/Bax protein in A549 cells. Our findings suggest that matrine may have the broad therapeutic and/or adjuvant therapeutic application in the treatment of human non-small cell lung cancer and hepatoma.     

Vascular endothelial growth factor enhances macrophage clearance of apoptotic cells

Efficient clearance of apoptotic cells from the lung by alveolar macrophages is important for the maintenance of tissue structure and function. Lung tissue from humans with emphysema contains increased numbers of apoptotic cells and decreased levels of vascular endothelial growth factor (VEGF). Mice treated with VEGF receptor inhibitors have increased numbers of apoptotic cells and develop emphysema. We hypothesized that VEGF regulates apoptotic cell clearance by alveolar macrophages (AM) via its interaction with VEGF receptor 1 (VEGF R1). Our data show that the uptake of apoptotic cells by murine AMs and human monocyte-derived macrophages is inhibited by depletion of VEGF and that VEGF activates Rac1. Antibody blockade or pharmacological inhibition of VEGF R1 activity also decreased apoptotic cell uptake ex vivo. Conversely, overexpression of VEGF significantly enhanced apoptotic cell uptake by AMs in vivo. These results indicate that VEGF serves a positive regulatory role via its interaction with VEGF R1 to activate Rac1 and enhance AM apoptotic cell clearance.     

Human 8-oxoguanine DNA glycosylase suppresses the oxidative stress induced apoptosis through a p53-mediated signaling pathway in human fibroblasts

Human 8-oxoguanine DNA glycosylase (hOGG1) is the main defense enzyme against mutagenic effects of cellular 7,8-dihydro-8-oxoguanine. In this study, we investigated the biological role of hOGG1 in DNA damage-related apoptosis induced by hydrogen peroxide (H(2)O(2))-derived oxidative stress. The down-regulated expression of hOGG1 by its small interfering RNA prominently triggers the H(2)O(2)-induced apoptosis in human fibroblasts GM00637 and human lung carcinoma H1299 cells via the p53-mediated apoptotic pathway. However, the apoptotic responses were specifically inhibited by hOGG1 overexpression. The p53-small interfering RNA transfection into the hOGG1-deficient GM00637 markedly inhibited the H(2)O(2)-induced activation of p53-downstream target proteins such as p21, Noxa, and caspase-3/7, which eventually resulted in the increased cell viability. Although the cell viability of hOGG1-knockdown H1299 p53 null cells was similar to that of the hOGG1 wild-type H1299, after the overexpression of p53 the hOGG1-knockdown H1299 showed the significantly decreased cell viability compared with that of the hOGG1 wild-type H1299 at the same experimental condition. Moreover, the array comparative genome hybridization analyses revealed that the hOGG1-deficient GM00637 showed more significant changes in the copy number of large regions of their chromosomes in response to H(2)O(2) treatment. Therefore, we suggest that although p53 is a major modulator of apoptosis, hOGG1 also plays a pivotal role in protecting cells against the H(2)O(2)-induced apoptosis at the upstream of the p53-dependent pathway to confer a survival advantage to human fibroblasts and human lung carcinomas through maintaining their genomic stability.     

Peri-epithelial origin of prostanoids in the human colon

The biology of prostanoids in the normal human colon is only beginning to be understood. We used in situ and in vitro techniques to define the lineage, number, per cell enzyme content, and epithelial functional effect of prostaglandin-generating cells, identified by the presence of cyclooxygenase 1 (COX 1). Immunohistochemical results were quantitated densitometrically, and cell surface staining in situ was verified by flow cytometry of isolated cells and by Western blotting. Three populations of COX 1(+) mucosal cells were identified, based on their morphology and local distribution in human mucosa; these were in the intra-epithelial, crypt apical, and lamina propria regions, with each containing a similar amount of COX 1 protein on a per cell basis. The most numerous were COX 1(+) mononuclear cells in the lamina propria, identified as CD3(+) T lymphocytes, both in situ and ex vivo. In toto, 21% of lamina propria mononuclear cells were COX 1(+), and over 50% of these cells were CD3(+) T cells. Findings were similar in the colon with mild-moderate inflammation due to ulcerative colitis. Using established surface markers, intra-epithelial and crypt apical COX 1(+) cells were non-lymphoid CD45(+) leukocytes; neither IgA (B-lymphocytes) nor alpha-smooth muscle actin (myelofibroblasts) was co-expressed on these COX 1(+) cells. Examining the effect of a major product of COX 1 in an in vitro system of human colonic epithelial monolayers, prostaglandin E(2) (PGE(2)) in low concentration (10(-6) M) enhanced epithelial barrier function and partially protected epithelia from the barrier-disruptive consequences of a pro-inflammatory cytokine, IFN-gamma. We conclude that the human colon contains three tiers of cell types for local synthesis of prostanoids, distinguishable by their location, morphology, and cell lineage. Further, maintenance of the barrier function of colonic epithelium may be added to other cell functions in mucosa regulated, in part, by prostanoids.     

Differential influence of butyrate concentration on proximal and distal colonic mucosa in rats born germ-free and associated with a strain of Clostridium paraputrificum

In vivo influence of butyrate in colonic mucosa was studied using a model of gnotobiotic rats monoassociated with a Clostridium paraputrificum. Rats were fed a diet containing increasing amounts of non-digestible carbohydrates, the fermentation of which led to modulated amounts of butyrate in the large intestine. In the proximal colon, the increase in the butyrate concentration alters crypt depth and the number of mucus-containing cells; the increase in butyrate was highly correlated with the number of neutral-mucin-containing cells. Conversely, in the distal colon, no relation was found between the increase in butyrate concentration and crypt depth or number of mucin-containing cells. In both the proximal and distal colon, the mitotic index remained unchanged. In conclusion, in vivo production of physiological quantities of butyrate had a trophic effect on proximal colonic mucosa, but did not influence the distal epithelium.     

Effects of freeze-drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm

Freeze-drying sperm is an alternative to cryopreservation. Although sperm from various species has been freeze-dried, there are few reports for bovine sperm. The primary objective of this study was to evaluate the protective effect of various freeze-drying media on the structural and functional components of bovine sperm. The media tested were composed of TCM 199 with Hanks salts supplemented with 10% fetal calf serum (FCS) and TCM 199 with Hanks salts supplemented with 10% FCS and 0.2 M trehalose and EGTA solution. The efficiency of each medium on the preservation of freeze-dried sperm structures was evaluated with conventional and electron microscopy, DNA integrity was analyzed by a TUNEL assay, and fertilizing ability of lyophilized sperm was determined with ICSI. Although the plasma membrane was damaged in all media tested, mitochondria were similarly preserved in all freeze-drying treatments. The acrosome was best preserved in the media that contained trehalose (other treatments also conserved this structure). In contrast, media containing EGTA or trehalose most effectively preserved the nuclei in freeze-dried sperm, with only 2 and 5%, respectively, of cells with fragmented DNA. Furthermore, sperm conserved with these media also had higher (P<0.05) rates of sperm head decondensation (32.5 and 27.5%), pronucleus formation (37.5 and 45.0%) and blastocyst formation (19.4 and 18.3%) than medium supplemented with FCS (15.0, 20.0 and 10.2%, respectively). In conclusion, media with EGTA and trehalose adequately protected bovine sperm during freeze-drying by preserving the viability of their nuclei.     

Apoptosis in extracorporeal preserved inguinal fat flaps of the rat

Fat cells are fragile cells with a short life span outside the body. Ways to reduce cell death in a biochemical way are almost unknown due to scarce information on the type of cellular death that is induced in fat tissue. This study was designed to investigate the apoptotic pathways of fat tissue in a permanent perfusion bioreactor system with the Hannover preservation solution and the Eurocollins solution in fat flaps of rats. In Lewis rats, the inguinal adipofascial flaps were elevated bilaterally and placed in a bioreactor at 37°C. To detect caspases 3, 8, 9 and 12, immunofluorescence stains of fat tissue specimen were analysed at several time points after preservation of flaps were placed in Hannover solution and Eurocollins solution for 10 days. An additional visual assessment of viability by a calcein based life/dead test was performed. It revealed a superior viability of the adipose tissue preserved in Hannover solution. Immunofluorescence staining demonstrated that apoptotic pathways via mitochondria, endoplasmatic reticulum and death receptors were activated, as Caspases 8, 9 and 12 were detected. Caspase 3 as an effector in the common apoptotic pathway was detected as well. Adipose tissue preserved at 37°C ex vivo in a bioreactor system undergoes apoptosis. Immunofluorescence examination of the fat tissue preserved ex vivo revealed that apoptotic pathways via mitochondria, endoplasmatic reticulum and death receptors are being activated. Significantly less activation of Caspase 3, 8, 9 and 12 in flaps preserved in Hannover solution in comparison to Eurocollins was found, supporting the anti apoptotic characteristics of Hannover solution. Based on these findings, further research to modify the apoptotic pathways to ameliorate viability of fat tissue can be performed.     

An ex vivo method for studying mucus formation, properties, and thickness in human colonic biopsies and mouse small and large intestinal explants

The colon mucus layers minimize the contact between the luminal flora and the epithelial cells, and defects in this barrier may lead to colonic inflammation. We now describe an ex vivo method for analysis of mucus properties in human colon and mouse small and large intestine. Intestinal explants were mounted in horizontal perfusion chambers. The mucus surface was visualized by adding charcoal particles on the apical side, and mucus thickness was measured using a micropipette. Mucus thickness, adhesion, and growth rate were recorded for 1 h. In mouse and human colon, the ability of the mucus to act as a barrier to beads the size of bacteria was also evaluated. Tissue viability was monitored by transepithelial potential difference. In mouse ileum, the mucus could be removed by gentle aspiration, whereas in colon ～40 μm of the mucus remained attached to the epithelial surface. Both mouse and human colon had an inner mucus layer that was not penetrated by the fluorescent beads. Spontaneous mucus growth was observed in human (240 μm/h) and mouse (100 μm/h) colon but not in mouse ileum. In contrast, stimulation with carbachol induced a higher mucus secretion in ileum than colon (mouse ileum: Δ200 μm, mouse colon: Δ130 μm, human colon: Δ140 μm). In conclusion, while retaining key properties from the mucus system in vivo, this setup also allows for studies of the highly dynamic mucus system under well-controlled conditions.     

Survival of Saccharomyces cerevisiae Y5 during starvation in the presence of osmotic supports

The viability of starved or dying Saccharomyces cerevisiae Y5 cells was extended for up to 1,500 h by adding osmotic supports (0.6 M sorbitol or mannitol) to the starvation media. Replacement of these polyols with 0.3 M KCl, 0.5 M MgSO4, or 0.2 M MgCl2 postponed the death of cells incubated in N-free medium, but accelerated it in distilled water.     

Survival of Saccharomyces cerevisiae Y5 during starvation in the presence of osmotic supports.

The viability of starved or dying Saccharomyces cerevisiae Y5 cells was extended for up to 1,500 h by adding osmotic supports (0.6 M sorbitol or mannitol) to the starvation media. Replacement of these polyols with 0.3 M KCl, 0.5 M MgSO4, or 0.2 M MgCl2 postponed the death of cells incubated in N-free medium, but accelerated it in distilled water.     

Role ofBifidobacterium longum in the induction of apoptotic deletion in the human enterocyte-like Caco-2 cell line

Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced byB. longum. This has been valuedin vitro, performing the incubation of threeB. longum strains with enterocyte-like Caco-2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells withB. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherentB. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues.     

Regulation of cell death and survival in intestinal intraepithelial lymphocytes.

Intraepithelial lymphocytes (IEL) of the small murine bowel represent a unique population of mostly CD8(+) T lymphocytes that reside within the epithelial cell layer of the intestinal mucosa. The close interaction with epithelial cells appears to be crucial for IEL survival since isolation and ex vivo culture induces massive apoptosis in this lymphocyte population. Here, we provide evidence that this form of IEL cell death may be mediated at least in part by endogenously produced glucocorticoids since adrenalectomy or treatment of mice with a glucocorticoid receptor antagonist significantly enhanced ex vivo survival of IEL. We further demonstrate that ex vivo activation of IEL induces upregulation of anti-apoptotic gene products, compensates for the lack of survival cytokines and rescues from apoptotic cell death. Thus, similar to thymocytes and T cell hybridomas, IEL survival may be regulated by the antagonistic action of TCR activation and glucocorticoids.     

GHRH antagonist causes DNA damage leading to p21 mediated cell cycle arrest and apoptosis in human colon cancer cells

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo. High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCT-116 colon cancer cells with 10μM GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OTM) and loss of inner mitochondrial membrane potential (?Ψm). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADP-ribose)polymerase (PARP) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21Waf1/Cip1 was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and PI staining assays. In vivo, JMR-132 decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic mice up to 75% (p     

Solid tissues can be manipulated ex vivo and used as vehicles for gene therapy

BACKGROUND: Organ fragments can be cultured for weeks in vitro if they are prepared of microscopic thickness and if the basic organ structure is preserved. Such organ fragments, which we termed micro-organs (MOs), express in culture endogenous tissue-specific gene products. We have exploited this methodology to engineer MOs ex vivo by gene transfer. METHODS: MOs prepared from spleen, lung, colon and skin were infected using: herpes simplex type-1, adeno virus, vaccinia virus and murine leukemia virus (MuLV), carrying the reporter gene beta-galactosidase. RESULTS: All four viral vectors infected MOs in culture, with adeno infection giving significantly higher values. After optimization, high levels of expression (> 15% positive cells), comparable to those obtained with the adeno construct, were also obtained using the MuLV construct both in vitro and after implantation into syngeneic hosts. After implantation, the engineered tissue was found to remain localized, become vascularized, and to express the transduced gene for several months. CONCLUSIONS: The system can be used to study interactions between viruses and tissues both ex vivo and in vivo. Furthermore, the approach proposes a novel platform for ex vivo gene therapy. Such engineered structures could be used as autologous biological pumps for continuous secretion in vivo of gene products of clinical importance.