ATRA promotes alpha tocopherol succinate-induced apoptosis in freshly isolated leukemic cells from chronic myeloid leukemic patients

We investigated the in vitro efficacy of all-trans retinoic acid (ATRA) and alpha-tocopherol succinate (α-TS) alone and in combination on the induction of cell death in freshly isolated leukemic cells obtained from chronic myeloid leukemia (CML) patients. In vitro cytotoxicity and induction of lipid peroxidation by ATRA (10 μM) and α-TS (25 or 50 μM) were evaluated in primary leukemic cells by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and malondialdehyde formation respectively. Treatment of leukemic cells with α-TS alone or in combination with ATRA significantly (P < 0.05) decreased the cell viability in a concentration and time dependent manner as compared to peripheral blood mononuclear cells obtained from normal healthy controls. Lipid peroxidation was enhanced by 98% (P < 0.05) on combined treatment of cells with ATRA (10 μM) and α-TS (50 μM). ATRA alone did not enhance the externalization of phosphatidyl serine as studied by annexin-V binding using fluorescence activated cell sorter analysis, whereas in combination with α-TS it increased to 400% at 12 h. The treatment of leukemic cells to combination of ATRA with α-TS significantly decreased (P < 0.05) mitochondrial membrane potential and enhanced lysosomal destabilization. The combination of these drugs also increased mitochondrial and cytosolic reactive oxygen species (ROS) production, nitric oxide levels, and caspase-3 activity significantly and caused DNA fragmentation at 24 h in a concentration dependent manner in the leukemic cells. Our data suggest that ATRA in combination with α-TS efficiently induces apoptosis in leukemic cells, which may be a useful therapeutic modality in CML patients.     

Tumor Necrosis Factor-α +489G/A gene polymorphism is associated with chronic obstructive pulmonary disease

Background  

Chronic obstructive pulmonary disease (COPD) is characterized by a chronic inflammatory process, in which the pro-inflammatory cytokine Tumor Necrosis Factor (TNF)-α is considered to play a role. In the present study the putative involvement of TNF-α gene polymorphisms in pathogenesis of COPD was studied by analysis of four TNF-α gene polymorphisms in a Caucasian COPD population.     

In vitro induction of tumor necrosis factor-α by ochratoxin A (OTA) from rat liver: role of Kupffer cells

Tumor necrosis factor-α (TNF-α) is released from blood-free perfused rat liver by the fungal metabolite ochratoxin A. Here we have identified Kupffer cells as the sole source of OTA-mediated cytokine release. If single cell preparation of Kupffer cells, hepatocytes, or sinusoidal endothelial cells were prepared from rat livers, only Kupffer cells released TNF-α upon incubation with 2.5 μmol/l OTA. OTA failed to induce TNF-α release in the blood-free perfused isolated rat liver when Kupffer cells were blockedin vitro by 15 μmol/l gadolinium chloride. When rats were pretreatedin vivo with the Kupffer cell depleting clodronate liposomes, OTA-mediated TNF-α release was abrogated in the isolated perfused liver model.     

Activation of cloned BK<Subscript>Ca</Subscript> channels in nitric oxide-induced apoptosis of HEK293 cells

The large conductance Ca²⁺-activated K⁺ (BK_Ca) channels are highly expressed in vascular smooth muscle cells (VSMCs) and play an essential role in the regulation of various physiological functions. Besides its electrophysiological function in vascular relaxation, BK_Ca has also been reported to be implicated in nitric oxide (NO)-induced apoptosis of VSMCs. However, the molecular mechanism is not clear and has not been determined on cloned channels. The present study was designed to clarify whether activation of cloned BK_Ca channel was involved in NO-induced apoptosis in human embryonic kidney 293 (HEK293) cell. The cDNA encoding the α-subunit of BK_Ca channel, hSloα, was transiently transfected into HEK293 cells. The apoptotic death in HEK-hSloα cells was detected using immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, MTT test, and flow cytometry assays. Whole-cell and single-channel characteristics of HEK-hSloα cells exhibited functional features similar to native BK_Ca channel in VSMCs. Exposuring of HEK- hSloα cells to S-nitroso-N-acetyl-penicillamine increased the hSloα channel activities of whole-cell and single-channel, and then increased percentage of cells undergoing apoptosis. However, blocking hSloα channels with 1 mM tetraethylammonia or 100 nM iberiotoxin significantly decreased the NO-induced apoptosis, whereas 30 μM NS1619, the specific agonist of BK_Ca, independently increased hSloα currents and induced apoptosis. These results indicated that activation of cloned BK_Ca channel was involved in NO-induced apoptosis of HEK293 cells.     

Taurine chloramine modulates cytokine production by human peripheral blood mononuclear cells

Summary.  The effect of taurine (Tau) and taurine chloramine (Tau-Cl) on the production of TNF-α, IL-1β, and IL-6 by peripheral blood mononuclear cells of healthy volunteers was examined. Cells were stimulated with bacterial lipopolysaccharide (LPS) in the presence of either Tau or Tau-Cl. After 24 h culture the cytokine concentrations were measured in both culture supernatants (secreted) and cell lysates (cell-associated) using ELISA. In LPS-stimulated cells Tau-Cl inhibited both the secreted and cell-associated IL-1β and IL-6, while exerted dual effect on TNF-α production: raising it slightly at low and reducing at higher concentration. By contrast, Tau had no significant effect on the cytokine production. These results indicate that Tau-Cl modulates synthesis of pro-inflammatory cytokines, and therefore it may play a role in the initiation and propagation of immune response. Received November 29, 2001 Accepted January 18, 2002 Published online August 30, 2002 Acknowledgments This research was supported by grants from the State Committee for Scientific Research of Poland (No 4 P05B 01018) and the Institute of Rheumatology (No I/14). The Institute of Rheumatology is supported by a core grant from the State Committee for Scientific Research of Poland. Authors' address: Ewa Kontny, Ph.D., Department of Pathophysiology and Immunology, Institute of Rheumatology, Spartanska 1, 02-637 Warsaw, Poland, E-mail: zpatiir@warman.com.pl Abbreviations: Tau, taurine; Tau-Cl, taurine chloramine; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-1β, interleukin 1β; IL-6, interleukin 6; PBMC, peripheral blood mononuclear cells     

CCN2 (Connective Tissue Growth Factor) is essential for extracellular matrix production and integrin signaling in chondrocytes

The matricellular protein CCN2 (Connective Tissue Growth Factor; CTGF) is an essential mediator of ECM composition, as revealed through analysis of Ccn2 deficient mice. These die at birth due to complications arising from impaired endochondral ossification. However, the mechanism(s) by which CCN2 mediates its effects in cartilage are unclear. We investigated these mechanisms using Ccn2 ^−/− chondrocytes. Expression of type II collagen and aggrecan were decreased in Ccn2 ^−/− chondrocytes, confirming a defect in ECM production. Ccn2 ^−/− chondrocytes also exhibited impaired DNA synthesis and reduced adhesion to fibronectin. This latter defect is associated with decreased expression of α5 integrin. Moreover, CCN2 can bind to integrin α5β1 in chondrocytes and can stimulate increased expression of integrin α5. Consistent with an essential role for CCN2 as a ligand for integrins, immunofluorescence and Western blot analysis revealed that levels of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK)1/2 phosphorylation were reduced in Ccn2 ^−/− chondrocytes. These findings argue that CCN2 exerts major effects in chondrocytes through its ability to (1) regulate ECM production and integrin α5 expression, (2) engage integrins and (3) activate integrin-mediated signaling pathways.     

In vitro analysis of the melanoma/endothelium interaction increasing the release of soluble intercellular adhesion molecule 1 by endothelial cells

Melanoma cells constitutively release intercellular adhesion molecule 1 (ICAM-1) as soluble ICAM-1 (sICAM-1), and its levels are elevated in melanoma patients and correlate with disease progression. However, this correlation is not absolute, suggesting that specific characteristics of neoplastic cells and/or ICAM-1-positive non-neoplastic cells may influence the amounts of circulating sICAM-1. In this study, we found a weak correlation (r = 0.55; r ² = 0.3) between sICAM-1 release by 40 metastatic melanomas (36 primary cultures and 4 cell lines), and ICAM-1 expression on neoplastic cells. In addition, melanoma-secreted interleukin-1α (IL-1α) (1/40) but not vascular endothelial growth factor (VEGF) (29/40), significantly (P < 0.05) up-regulated the shedding of sICAM-1 by human umbilical vein endothelial cells (HUVEC). This was completely abolished by IL-1α/β neutralizing antibodies both at the protein and mRNA level. Altogether, our results suggest that (i) the extent of sICAM-1 release is distinctive for individual melanomas and can be independent of ICAM-1 expression; (ii) tumor endothelia may sustain levels of sICAM-1 in selected melanomas; (iii) melanoma-released VEGF does not affect ICAM-1 expression and sICAM-1 release by HUVEC. Melanoma-derived sICAM-1 inhibits cell-mediated cytotoxicity of melanoma cells; therefore, constitutive levels of sICAM-1 release and IL-1α secretion by individual melanomas can differentially influence tumor progression and the clinical effectiveness of cytotoxic-cell-based vaccines. Received: 15 October 1998 / Accepted: 17 February 1999     

Cytokine expression due to Helicobacter pylori in a tissue culture model

Helicobacter pylori, in recent years, has been recognized as the major causative agent in chronic gastritis and peptic ulcer disease in humans. H. pylori is a ubiquitous organism, with at least half of the world’s population infected. Of those individuals with peptic ulcer disease, it is estimated that 90% of cases are caused by H. pylori. Currently, the efficacy of therapies is starting to decline due to increasing resistance rates, especially towards clarithromycin. Due to this, new therapies are needed to combat this bacterium. It is hypothesized that cytokine release (especially interleukin-1β, -6, -8, and TNF-α) due to H. pylori infection and the subsequent influx of inflammatory cells causes a massive release of reactive oxygen species (ROS) during the inflammatory reaction. The ROS then cause the pathologic changes seen in the infected tissues. In this study, human gastric adenocarcinoma cell line ATCC 1739 (a cell line not previously evaluated) was examined for its production of interleukin-1β, -6, -8, and TNF-α when cocultured in a ratio of 10:1 H. pylori to adenocarcinoma cells, to determine its value as a model to demonstrate the inflammatory response. Results from this study indicated that ATCC 1739 cells only reliably produced IL-8 when cocultured with H. pylori and stimulated with TNF-α. The production of IL-1β, IL-6, and TNF-α by the ATCC 1739 cells was no different in H. pylori-exposed cells than non-exposed cells. It was concluded that the ATCC 1739 cell line is not suitable to study the effects of coculture with H. pylori on cytokine production.     

Cellular immune responses to autologous chronic myelogenous leukaemia cells in vitro

 Using a modification of the autologous mixed lymphocyte/tumour cell culture (MLTC), it is demonstrated here that lymphocytes from chronic-phase myelogenous leukaemia (CML) patients (n = 58), but not from their HLA-identical siblings, proliferated upon coculture with autologous tumour cells. However, in most cases, the level of proliferation measured was low (stimulation index <3, n = 37). This was most likely related to the amount of interleukin-10 (IL-10) released into the culture medium by the CML cells, because addition of neutralizing anti-IL-10 serum to MLTC markedly enhanced proliferative responses. In addition, supplementation of media with IL-1α further enhanced proliferative responses and a combination of anti-IL-10 serum and IL-1α was more effective than either agent alone. Only HLA-DR-matched CML cells, but not HLA-DR-mismatched CML cells or matched or mismatched PBMC restimulated proliferation of IL-2-dependent T cell lines derived from MLTC supplemented with IL-1α and anti-IL-10 serum. The responding cells under these conditions were predominantly CD4⁺ and secreted IL-2, and interferon γ; some secreted IL-4, but none secreted IL-10. These data therefore suggest the existence of an HLA-DR-restricted DTH/Th1-type of tumour-specific immunity in CML patients, which may be down-regulated in vitro by excessive secretion of IL-10 together with depressed secretion of IL-1. Received: 9 November 1995 / Accepted: 8 February 1996     

Partial purification and characterization of a novel human factor that augments the expression of class I MHC antigens on tumour cells

A cytokine which augments the expression of major histocompatibility complex (MHC) I antigens on K562 and gastric carcinoma tumour (HR) cells, has been isolated from the culture supernatant of Concanavalin-A (Con-A) activated human peripheral blood mononuclear cells. The factor, termed MHC augmenting factor (MHC- AF) has been partially purified by Sephadex G- 100 column chromatography, preparative isoelectric focusing and HPLC with ion- exchange as well as sizing columns. MHC-AF activity is associated with a 35 kDa molecule which has pI of 6.0. Interferon (IFN)-α, \, tumour necrosis factor (TNF), Interleukin (IL)-2, IL-4, IL-5 and IL-7 had no significant effect in MHC- AF bioassay, but IFN-γ had significant MHC-AF activity. Antibodies to IFN-α, IFN-\ and TNF-α did not block the activity of MHC-AF, but anti-IFN-y antibodies could partially neutralize the activity. However, unlike IFN-γ, MHC-AF activity was resistant to pH 2.0 treatment. Purified MHC-AF preparations did not have any activity in WISH cell/encephalo myocarditis virus (EMC) IFN bioassays. In addition, anti-IFN-y affinity column did not retain MHC-AF activity. These results indicate that a MHC-AF distinct from IFN-γ, is produced by activated human mononuclear cells.     

Voltage-dependent calcium channels in young and old human red cells

Presence of subtypes of voltage-dependent Ca channels was investigated in young and old human red cells, employing immunological and flux-kinetics methods. Western blots showed specific reaction toward polyclonal rabbit antibodies raised against a highly conserved residue of α_1C, subunit of high-voltage activated Ca channels (pan α₁) and against conserved residues of α_1C and α_1E subunits. No specific reaction was detected with antibodies against conserved residues of α_1A, α_1B, or α_1D subunits. Only a single band (approx 260 kDa) was revealed on anti-pan α_1A or anti-α_1E blots, whereas two bands (200 and 230 kDa) were detected by α_1C exposure, Blots from old cells always showed diminished band intensity. Channel activity was assessed by studying the effect of voltage-dependent Ca channels blockers' under conditions likely to alter the red cell membrane potential, through incubation in media of different composition. In a 150 mM NaCl+5 mM KCl medium, blockers of L-, R-, and Q-type caused a 15–50% reductions of ⁴⁵Ca influx into cells, which had the Ca pump inactivated by either exhaustive adenosine triphosphate depletion or presence of vanadate plus substrates. Additionally, some P/Q-and N-type blockers also reduced Ca influx to various extents (25–60%). Old cells were generally insensitive to L-type but not to non-L-type, blockers. Raising external K to about 70–80 mM reduced by 50–100% inhibition by L-type blockers. Incubation in a gluconate medium containing 150 mM Na+5 mM K practically abolished the action of L-type blockers, but only slightly reducing that by non-L-type. The results, clearly demonstrate presence of L- and R-type Ca channels, apparently occurring in different functional states in young and old cells. Other non-L-type channels were also demonstrated only by pharmacological means. A possible physiological role for these channels is discussed.     

Volume-regulatory Amino Acid Release from the Protozoan Parasite Crithidia luciliae

The unicellular protozoan parasite, Crithidia luciliae, responded to osmotic swelling by undergoing a regulatory volume decrease. This process was accompanied by the efflux of amino acids (predominantly alanine, proline and glycine). The relative loss of the electroneutral amino acids proline, valine, alanine and glycine was greater than that for the anionic amino acid, glutamate; there was negligible loss of the cationic amino acids, lysine, arginine and ornithine. The characteristics of amino acid release were investigated using a radiolabeled form of the nonmetabolized alanine analogue α-aminoisobutyrate. α-Aminoisobutyrate efflux was activated within a few seconds of a reduction of the osmolality, and inactivated rapidly (again within a few seconds) on restoration of isotonicity. The initial rate of efflux of α-aminoisobutyrate from cells in hypotonic medium was unaffected by the extracellular amino acid concentration. Hypotonically activated α-aminoisobutyrate efflux (as well as the associated regulatory volume decrease) was inhibited by the sulfhydryl reagent N-ethylmaleimide but was not inhibited by a range of anion transport blockers. As in the efflux experiments, unidirectional influx rates for α-aminoisobutyrate increased markedly following reduction of the osmolality, consistent with the swelling-activated amino acid release mechanism allowing the flux of solutes in both directions. Hypotonically activated α-aminoisobutyrate influx showed no tendency to saturate up to an extracellular concentration of 50 mm. The functional characteristics of the amino acid release mechanism are those of a channel, with a preference for electroneutral and anionic amino acids over cationic amino acids. However, the pharmacology of the system differs from that of the anion-selective channels that are thought to mediate the volume-regulatory efflux of organic osmolytes from vertebrate cells. Received: 13 May 1996/Revised: 9 July 1996     

Functional genomics of PPAR-γ in human immunomodulatory cells

Keeping in view the fact that peroxisome-proliferators activated receptors-PPARs (α,γ) play a crucial role in atherogenic inflammation, the present study was addressed to explore as to how selective and specific PPAR-γ gene silencing within human mononuclear cells affects genes involved in lipid metabolism and innate immune process. Such a study revealed that with respect to control cells, the PPAR-γ knock-out cells exhibited significant reduction in the expression of genes coding for PPAR- α, CD-36, LDL-R as well as significant increase in the expression of genes coding for IL-4, IL-8, IFN-γ, CX3CR1, hTERT. However, the expression of genes coding for LXR-α and Receptor-C _k could not be detected in PPAR-γ knock-out cells. Based on these results, we propose that PPAR-γ gene has the inherent capacity to influence the lipid mediated inflammation process in atherosclerotic lesions.     

Cytotoxicity in feline leukemia virus subgroup-c infected fibroblasts is mediated by adherent bone marrow mononuclear cells

Summary The pathogenesis of retrovirus-induced erythroid aplasia in cats is unknown. In studies to define mechanisms of cytotoxicity associated with retroviral infections, bone marrow mononuclear cells (BMMC) from healthy specific pathogenfree cats were co-cultured with uninfected feline embryonic fibroblasts (FEA cells) and FEA cells infected with feline leukemia virus (FeLV) of subgroup A (FEA-A) or subgroup C (FEA-C). Moderate to marked cytotoxicity (CPE) developed in co-cultures of BMMC and FEA-C cells on Days 5 to 7 of incubation but not in co-cultures of BMMC and FEA-A or BMMC and uninfected cells (FEA-CT). Cytotoxicity was associated with adherent cells of light density (1.056) from bone marrow and peripheral blood, which were positive for alpha naphthyl butyrate esterase activity. Stimulation of adherent cells with phorbol ester or addition of recombinant human tumor necrosis factor-alpha (rhTNF-α) caused similar CPE in FEA-CT cells. The TNF-α concentrations in the culture supernatants of BMMC+FEA-C were higher than those of BMMC+FEA-A or BMMC+FEA-CT, and addition of anti-TNF antibodies to the cultures blocked the CPE. These data support the hypothesis that macrophages exposed to FeLV-C cause CPE in co-cultures of BMMC and FEA cells by a mechanism involving TNF-α. It is suggested that TNF-α may be involved in the suppression of hematopoiesis in cats which develop FeLV-C induced erythroid aplasia.     

Heparan sulfate expression is affected by inflammatory stimuli in primary human endothelial cells

In diabetes the endothelium is either chronically or transiently exposed to hyperglycemic conditions. In addition, endothelial dysfunction in diabetes is related to changes in the inflammatory response and the turnover of extracellular matrix. This study was undertaken to study the effects of inflammatory stimuli on one particular matrix component, the heparan sulfate (HS) proteoglycans (PGs) synthesized by primary human umbilical cord vein endothelial cells (HUVEC). Such cells were cultured in vitro in 5 mM and 25 mM glucose. The latter concentration was used to mimic hyperglycemic conditions in short-term experiments. HUVEC were also cultured in the presence of the inflammatory agents tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), interleukin 1β (IL-1β) and transforming growth factor β (TGF-β). The cells were labeled with ³⁵S-sulfate and ³⁵S-PGs were recovered for further analyses. The major part of the ³⁵S-PGs was secreted to the medium, irrespective of type of stimuli. Secreted ³⁵S-PGs were therefore isolated and subjected to further analyses. TNF-α and IL-1α slightly increased the release of ³⁵S-PGs to the culture medium, whereas IL-1β treatment gave a significant increase. The different treatments neither changed the ratio of ³⁵S-HS and ³⁵S-chondroitin sulfate (CS) nor the macromolecular properties of the ³⁵S-PGs. However, the ³⁵S-HS chains were slightly increased in size after TNF-α treatment, and slightly decreased after TGF-β treatment, but not affected by the other treatments. Compositional analysis of labeled disaccharides showed changes in the amount of 6-O-sulfated glucosamine residues after treatment with TNF-α, IL-1α and IL-1β. Western immunoblotting showed that major HSPGs recovered from these cells were collagen XVIII, perlecan and agrin, and that secretion of these distinct PGs was increased after IL-1β stimulation. Hence, short term inflammatory stimuli increased the release of HSPGs in HUVEC and affected both the size and sulfation pattern of HS, depending on type of stimuli.     

Target-cell-induced anergy in natural killer cells: suppression of cytotoxic function

 Our earlier studies have demonstrated that natural killer (NK) cells are the effectors that participate during the spontaneous regression of AK-5 tumour in syngeneic hosts. We have shown that the tumour cells are killed by necrosis and apoptosis. In this study, we have examined the induction of functional anergy in NK cells following coculture with fixed AK-5 tumour cells at high ratio. NK cells, upon coculture with fixed AK-5 cells (1:1 ratio), showed loss of cytotoxic function against both AK-5 (antibody-dependent cell cytotoxicity) as well as YAC-1 targets. The response of these cells to the activation by recombinant interleukin-2 and recombinant interferon γ was poor. Induction of tumour necrosis factor α (TNFα) secretion was observed after coculture of NK cells with fixed AK-5 cells. The cocultured cell supernatant inhibited the cytotoxic activity of NK cells, which was partially restored with anti-TNFα antibody. In addition, NK cells, after treatment with fixed tumour cells showed overexpression of the Fas receptor. We have also observed induction of apoptosis in cocultured NK cells. These studies suggest that the fixed tumour cells (antigen) at high ratio are able to suppress NK cell function as well as induce death in NK cells. Received: 16 September 1999 / Accepted: 13 January 2000     

Signaling mechanisms in tumor necrosis factor alpha-induced death of microvascular endothelial cells of the corpus luteum

The microvasculature of the corpus luteum (CL), which comprises greater than 50% of the total number of cells in the CL, is thought to be the first structure to undergo degeneration via apoptosis during luteolysis. These studies compared the apoptotic potential of various cytokines (tumor necrosis factor α, TNFα; interferon gamma, IFNγ; soluble Fas ligand, sFasL), a FAS activating antibody (FasAb), and the luteolytic hormone prostaglandin F_2α (PGF_2α) on CL-derived endothelial (CLENDO) cells. Neither sFasL, FasAb nor PGF_2α had any effect on CLENDO cell viability. Utilizing morphological and biochemical parameters it was evident that TNFα and IFNγ initiated apoptosis in long-term cultures. However, TNFα was the most potent stimulus for CLENDO cell apoptosis at early time points. Unlike many other studies described in non-reproductive cell types, TNFα induced apoptosis of CLENDO cells occurs in the absence of inhibitors of protein synthesis. TNFα-induced death is typically associated with acute activation of distinct intracellular signaling pathways (e.g. MAPK and sphingomyelin pathways). Treatment with TNFα for 5–30 min activated MAPKs (ERK, p38, and JNK), and increased ceramide accumulation. Ceramide, a product of sphingomyelin hydrolysis, can serve as an upstream activator of members of the MAPK family independently in numerous cell types, and is a well-established pro-apoptotic second messenger. Like TNFα, treatment of CLENDO cells with exogenous ceramide significantly induced endothelial apoptosis. Ceramide also activated the JNK pathway, but had no effect on ERK and p38 MAPKs. Pretreatment of CLENDO cells with glutathione (GSH), an intracellular reducing agent and known inhibitor of reactive oxygen species (ROS) or TNFα-induced apoptosis, significantly attenuated TNFα-induced apoptosis. It is hypothesized that TNFα kills CLENDO cells through elevation of reactive oxygen species, and intracellular signals that promote apoptosis.     

Induction of endonuclease G-mediated apopotosis in human oral squamous cell carcinoma cells by protein kinase C inhibitor safingol

PKC inhibitor safingol suppressed the growth of human oral squamous cell carcinoma (SCC) cells significantly at concentrations that inhibit PKC isoforms. Safingol inhibited the translocation of PKC following treatment with 12-o-tetradecanoylphorbol 13-acetate (TPA) in PKC α-EGFP-transfected cells, but not in PKC β-EGFP- transfected cells, indicating selective inhibition for PKC α in oral SCC cells. Flow cytometric analysis and DNA analysis by agarose gel electrophoresis revealed an increase in the proportion of sub-G₁ cells and DNA fragmentation in safingol-treated cells. Mitochondrial membrane potential was decreased, and cytochrome c was released from mitochondria. However, the safingol-induced cell death was not accompanied by activation of caspase 3 and cleavage of poly (ADP-ribose) polymerase (PARP). The broad-spectrum caspase inhibitor BD-fmk failed to prevent safingol-induced cell death. Another apoptogenic factor endonuclease G, but not apoptosis-inducing factor (AIF), was also released from mitochondria and translocated to the nucleus. These results suggest that PKC α inhibitor safingol induces an endonuclease G- mediated apoptosis in a caspase-independent manner.     

Lysosomal storage of oligosaccharide and glycosphingolipid in imino sugar treated cells

Sandhoff and Tay-Sachs disease are autosomal recessive GM2 gangliosidoses where a deficiency of lysosomal β-hexosaminidase results in storage of glycoconjugates. Imino sugar (2-acetamido-1,4-imino-1,2,4-trideoxy-L-arabinitol) inhibition of β-hexosaminidase in murine RAW264.7 macrophage-like cells led to lysosomal storage of glycoconjugates that were characterised structurally using fluorescence labelling of the free or glycolipid-derived oligosaccharides followed by HPLC and mass spectrometry. Stored glycoconjugates were confirmed as containing non-reducing GlcNAc or GalNAc residues resulting from the incomplete degradation of N-linked glycoprotein oligosaccharide and glycolipids, respectively. When substrate reduction therapeutics N-butyl-deoxynojirimycin (NB-DNJ) or N-butyldeoxygalactonojirimycin (NB-DGJ) were applied to the storage phenotype cells, an increase in glucosylated and galactosylated oligosaccharide species was observed due to endoplasmic reticulum α-glucosidases and lysosomal β-galactosidase inhibition, respectively. Hexosaminidase inhibition triggered a tightly regulated cytokine-mediated inflammatory response that was normalised using imino sugars NB-DNJ and NB-DGJ, which restored the GM2 ganglioside storage burden but failed to reduce the levels of GA2 glycolipid or glycoprotein-derived N-linked oligosaccharides. Using a chemically induced gangliosidosis phenotype that can be modulated with substrate lowering drugs, the critical role of GM2 ganglioside in the progression of inflammatory disease is also demonstrated.     

Immunostimulatory effect by aqueous extract of <Emphasis Type="Italic">Hizikia fusiforme</Emphasis> in RAW 264.7 macrophage and whole spleen cells

Hizikia fusiforme is a commonly used food that possesses potent anti-bacterial, anti-fungal, and anti-inflammatory activities. The immunostimulatory activities of aqueous extract of Hizikia fusiforme (HFAE) in RAW 264.7 macrophages and whole spleen cells were investigated. HFAE activated RAW 264.7 macrophages to produce cytokines such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in a dose-dependent manner. In addition, HFAE induced the mRNA expression of TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophages. Moreover, HFAE stimulated proliferation of whole spleen cells and reference mitogen. Taken together, the results demonstrate that HFAE potently activates the immune function by regulating NO, TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophage and promoting spleen cell proliferation.