DHA-mediated enhancement of TRAIL-induced apoptosis in colon cancer cells is associated with engagement of mitochondria and specific alterations in sphingolipid metabolism

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid present in fish oil, may exert cytotoxic and/or cytostatic effects on colon cancer cells when applied individually or in combination with some anticancer drugs. Here we demonstrate a selective ability of subtoxic doses of DHA to enhance antiproliferative and apoptotic effects of clinically useful cytokine TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in cancer but not normal human colon cells. DHA-mediated stimulation of TRAIL-induced apoptosis was associated with extensive engagement of mitochondrial pathway (Bax/Bak activation, drop of mitochondrial membrane potential, cytochrome c release), activation of endoplasmic reticulum stress response (CHOP upregulation, changes in PERK level), decrease of cellular inhibitor of apoptosis protein (XIAP, cIAP1) levels and significant changes in sphingolipid metabolism (intracellular levels of ceramides, hexosyl ceramides, sphingomyelines, sphingosines; HPLC/MS/MS). Interestingly, we found significant differences in representation of various classes of ceramides (especially C16:0, C24:1) between the cancer and normal colon cells treated with DHA and TRAIL, and suggested their potential role in the regulation of the cell response to the drug combination. These study outcomes highlight the potential of DHA for a new combination therapy with TRAIL for selective elimination of colon cancer cells via simultaneous targeting of multiple steps in apoptotic pathways.     

Activation-associated alterations in neutrophil pyridine nucleotide levels: a potential regulatory role for calcium and calmodulin

The concentration of NADP + NADPH in resting human neutrophils has been measured to be 24.0 +/- 2.7 X 10(-18) mol/cell. Upon activation with opsonized zymosan A, phorbol myristic acetate or N-formylmethionyl-leucylphenylalanine, neutrophil NADP + NADPH pools increase to 80.5, 84.0, and 54.0 X 10(-18) mol/cell, respectively. These increases in pyridine nucleotide concentration are blocked by the addition of the calcium antagonist 8-(N,N-dimethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride, while calcium ionophore A23187, in the presence of calcium, will trigger the increase in the absence of other stimuli. Calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide also inhibit stimulus-induced increases in the NADP + NADPH pool. These studies are interpreted as suggesting a role for calcium and calmodulin, and possibly protein kinase C in the regulation of pyridine nucleotide concentration in the activated neutrophil.     

Systems biology and its potential role in radiobiology

About a century ago, Conrad Röentgen discovered X-rays, and Henri Becquerel discovered a new phenomenon, which Marie and Pierre Curie later coined as radio-activity. Since their seminal work, we have learned much about the physical properties of radiation and its effects on living matter. Alas, the more we discover, the more we appreciate the complexity of the biological processes that are triggered by radiation exposure and eventually lead (or do not lead) to disease. Equipped with modern biological methods of high-throughput experimentation, imaging, and vastly increased computational prowess, we are now entering an era where we can piece some of the multifold aspects of radiation exposure and its sequelae together, and develop a more systemic understanding of radiogenic effects such as radio-carcinogenesis than has been possible in the past. It is evident from the complexity of even the known processes that such an understanding can only be gained if it is supported by mathematical models. At this point, the construction of comprehensive models is hampered both by technical inadequacies and a paucity of appropriate data. Nonetheless, some initial steps have been taken already and the generally increased interest in systems biology may be expected to speed up future progress. In this context, we discuss in this article examples of relatively small, yet very useful models that elucidate selected aspects of the effects of exposure to ionizing radiation and may shine a light on the path before us.     

PAF-mediated pulmonary edema: a new role for acid sphingomyelinase and ceramide

Platelet-activating factor (PAF) induces pulmonary edema and has a key role in acute lung injury (ALI). Here we show that PAF induces pulmonary edema through two mechanisms: acid sphingomyelinase (ASM)-dependent production of ceramide, and activation of the cyclooxygenase pathway. Agents that interfere with PAF-induced ceramide synthesis, such as steroids or the xanthogenate D609, attenuate pulmonary edema formation induced by PAF, endotoxin or acid instillation. Our results identify acid sphingomyelinase and ceramide as possible therapeutic targets in acute lung injury.     

Pertussis toxin-induced lymphocytosis is associated with alterations in thymocyte subpopulations.

Pertussis toxin (Ptx), an important adjuvant for inducing certain organ-specific autoimmune diseases in mice, exerts multiple effects upon the immune system. In addition to its adjuvant effects, which include enhancement of delayed-type hypersensitivity and increased antibody production. Ptx elicits a marked lymphocytosis with a concomitant decrease in thymic weight. In vitro studies indicate that Ptx acts directly on thymocytes and that both susceptible and resistant populations exist. It is believed that these susceptible cells are released into the circulation and account, in part, for the T cell component of the lymphocytosis. We have used flow cytometry to analyze the CD4, CD8, and Thy-1 phenotypes of thymic and peripheral T cells from Ptx-treated mice. In the thymus, there is a dramatic decrease in the number of CD4+CD8+ (double positive) cells at all doses tested (0.25, 0.50, and 1.0 microgram) by day 4 after Ptx treatment. The double negative and single positive populations remain relatively constant. Analysis of Thy-1 expression reveals a significant reduction in Thy-1hi thymocytes, with little change in the Thy-1lo population. Thus Ptx primarily affects and depletes, in a dose-dependent fashion, thymic T cells with an immature phenotype. These results mimic those of corticosteroids, although neither prior adrenalectomy nor treatment with the antiglucocorticoid RU486 are able to prevent the effects of Ptx. In the periphery of Ptx-treated animals, the relative increase in the number of CD4+ T cells is more than that of CD8+ T cells. Double positive and Thy-1hi cells cannot be detected in appreciable numbers. These results are consistent with the concept that Ptx may drive immature thymocytes through accelerated maturation for release into the periphery as single positive, predominantly CD4+, Thy-1lo cells. Increased numbers of such cells may in part account for the immunopotentiating effects of Ptx, particularly as they relate to the induction of organ-specific autoimmune disease. Treatment with purified Ptx beta-oligomer fails to elicit any of the responses described above, indicating that the holotoxin is required for such activities.     

A potential role for zinc alterations in the pathogenesis of Alzheimer's disease

Alzheimer's disease (AD), one of the major causes of disability and mortality in Western societies, is a progressive age-related neurodegenerative disorder. Increasing evidence suggests that the etiology of AD may involve disruptions of zinc (Zn) homeostasis. This review discusses current evidence supporting a potential role of Zn and zinc transporters (ZnTs) in processing of the amyloid beta protein precursor (APP) and amyloid beta (Aβ) peptide generation and aggregation.     

Chromosomal terminal methylation status is associated with gut microbiotic alterations

Molecular and Cellular Biochemistry - We explored the association of fecal bacterial species and somatic telomere changes in patients with chronic disease. The results showed that the length of the...     

Developmental biology: a growing role for computer simulations

Keeping cells separated in well-defined domains is essential for development. A new computational-experimental study elucidates the physical mechanisms that establish and maintain the dorsal-ventral compartment boundary in the Drosophila wing disc and demonstrates the increasing value of computer simulations in developmental biology.     

Substantial sulfatide deficiency and ceramide elevation in very early Alzheimer's disease: potential role in disease pathogenesis

In addition to pathology in the gray matter, there are also abnormalities in the white matter in Alzheimer's disease (AD). Sulfatide species are a class of myelin-specific sphingolipids and are involved in certain diseases of the central nervous system. To assess whether sulfatide content in gray and white matter in human subjects is associated with both the presence of Alzheimer's disease (AD) pathology as well as the stage of dementia, we analyzed the sulfatide content of brain tissue lipid extracts by electrospray ionization mass spectrometry from 22 subjects whose cognitive status at time of death varied from no dementia to very severe dementia. All subjects with dementia had AD pathology. The results demonstrate that: (i) sulfatides were depleted up to 93% in gray matter and up to 58% in white matter from all examined brain regions from AD subjects with very mild dementia, whereas all other major classes of lipid (except plasmalogen) in these subjects were not altered in comparison to those from age-matched subjects with no dementia; (ii) there was no apparent deficiency in the biosynthesis of sulfatides in very mild AD subjects as characterized by the examination of galactocerebroside sulfotransferase activities in post-mortem brain tissues; (iii) the content of ceramides (a class of potential degradation products of sulfatides) was elevated more than three-fold in white matter and peaked at the stage of very mild dementia. The findings demonstrate that a marked decrease in sulfatides is associated with AD pathology even in subjects with very mild dementia and that these changes may be linked with early events in the pathological process of AD.     

Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome

We have investigated the interrelationship between diet, gut microbial ecology, and energy balance using a mouse model of obesity produced by consumption of a prototypic Western diet. Diet-induced obesity (DIO) produced a bloom in a single uncultured clade within the Mollicutes class of the Firmicutes, which was diminished by subsequent dietary manipulations that limit weight gain. Microbiota transplantation from mice with DIO to lean germ-free recipients promoted greater fat deposition than transplants from lean donors. Metagenomic and biochemical analysis of the gut microbiome together with sequencing and metabolic reconstructions of a related human gut-associated Mollicute (Eubacterium dolichum) revealed features that may provide a competitive advantage to members of the bloom in the Western diet nutrient milieu, including import and processing of simple sugars. Our study illustrates how combining comparative metagenomics with gnotobiotic mouse models and specific dietary manipulations can disclose the niches of previously uncharacterized members of the gut microbiota.     

Conditions for a theoretical biology: is biology truly an autonomous science?

In a line of a previous paper, the conditions for a theoretical biology were discussed and it was pointed out that the primary condition is that biology is an autonomous science. This statement is connected to the problem of reductionism. A discussion of the autonomy of biology shows that reductionism cannot be maintained, although particularly in physiology often physics and mathematics are used. Development, organization and evolution of biological systems are typical areas of autonomous biological researches and of autonomous theoretical developments. A sort of reduction to history seems today a nonsensical attempt to reduce the area of free theoretical biological activity.     

Manganese accumulates in iron-deficient rat brain regions in a heterogeneous fashion and is associated with neurochemical alterations

Previous studies have shown that iron deficiency (ID) increases brain manganese (Mn), but specific regional changes have not been addressed. Weanling rats were fed one of three semipurified diets: control (CN), iron deficient (ID), or iron deficient/manganese fortified (IDMn+). Seven brain regions were analyzed for Mn concentration and amino acid (glutamate, glutamine, taurine, γ-aminobutyric acid) concentrations. Both ID and IDMn+ diets caused significant (p<0.05) increases in Mn concentration across brain regions compared to CN. The hippocampus was the only brain region in which the IDMn+ group accumulated significantly more Mn than both the CN and ID groups. ID significantly decreased GABA concentration in hippocampus, caudate putamen, and globus pallidus compared to CN rats. Taurine was significantly increased in the substantia nigra of the IDMn+ group compared to both ID and CN. ID also altered glutamate and glutamine concentrations in cortex, caudate putamen, and thalamus compared to CN. In the substantia nigra, Mn concentration positively correlated with increased taurine concentration, whereas in caudate putamen, Mn concentration negatively correlated with decreased GABA. These data show that ID is a significant risk factor for central nervous system Mn accumulation and that some of the neurochemical alterations associated with ID are specifically attributable to Mn accumulation.     

Selective phosphotyrosine phosphatase inhibition and increased ceramide formation is associated with B-cell death by apoptosis

Bis(maltolato)oxovanadium(IV) (BMOV), a protein phosphotyrosine phosphatase inhibitor, selectively induced apoptosis (as quantitated by TUNEL staining) in a B-cell line (Ramos) but not in a T-cell line (Jurkat). The pattern of BMOV-induced protein tyrosine phosphorylation was different in B-cells versus T-cells. Further, BMOV induced a 2-fold increase in ceramide levels in B-cells but not in T-cells and this resembled the ceramide increase following activation of the B-cell antigen receptor. A 2-fold increase in the ratio of ceramide to sphingomyelin in B-cells treated with BMOV suggested that sphingomyelinase activation was the result of the sustained tyrosine phosphorylation of specific proteins and activated the cell death pathway.     

Visceral adipose inflammation in obesity is associated with critical alterations in tregulatory cell numbers

Background

The development of insulin resistance (IR) in mouse models of obesity and type 2 diabetes mellitus (DM) is characterized by progressive accumulation of inflammatory macrophages and subpopulations of T cells in the visceral adipose. Regulatory T cells (Tregs) may play a critical role in modulating tissue inflammation via their interactions with both adaptive and innate immune mechanisms. We hypothesized that an imbalance in Tregs is a critical determinant of adipose inflammation and investigated the role of Tregs in IR/obesity through coordinated studies in mice and humans.

Methods and Findings

Foxp3-green fluorescent protein (GFP) “knock-in” mice were randomized to a high-fat diet intervention for a duration of 12 weeks to induce DIO/IR. Morbidly obese humans without overt type 2 DM (n = 13) and lean controls (n = 7) were recruited prospectively for assessment of visceral adipose inflammation. DIO resulted in increased CD3⁺CD4⁺, and CD3⁺CD8⁺ cells in visceral adipose with a striking decrease in visceral adipose Tregs. Treg numbers in visceral adipose inversely correlated with CD11b⁺CD11c⁺ adipose tissue macrophages (ATMs). Splenic Treg numbers were increased with up-regulation of homing receptors CXCR3 and CCR7 and marker of activation CD44. In-vitro differentiation assays showed an inhibition of Treg differentiation in response to conditioned media from inflammatory macrophages. Human visceral adipose in morbid obesity was characterized by an increase in CD11c⁺ ATMs and a decrease in foxp3 expression.

Conclusions

Our experiments indicate that obesity in mice and humans results in adipose Treg depletion. These changes appear to occur via reduced local differentiation rather than impaired homing. Our findings implicate a role for Tregs as determinants of adipose inflammation.     

Cell cycle arrest of hematopoietic cell lines after treatment with ceramide is commonly associated with retinoblastoma activation

BACKGROUND: Leukaemia cells differ from their normal counterparts in that their ability to properly regulate survival, proliferation, differentiation, and apoptosis is aberrant. Understanding the molecular mechanisms controlling cell proliferation and developing therapeutic strategies to correct nonfunctional regulatory mechanisms are emerging areas of medical research. Ceramide, a metabolite of membrane sphingomyelin hydrolysis, has recently emerged as a key regulator of cellular proliferation, differentiation, and apoptosis in leukaemia cells. METHODS: Leukaemia cell lines were treated with a biologically active analogue of ceramide, C(2)-ceramide. Cell cycle status was assessed flow cytometrically using propidium iodide. Induction of apoptosis was confirmed by annexin V staining of externalised phosphatidylserine and retinoblastoma activation was determined by Western blotting. RESULTS: C(2)-ceramide induced activation of retinoblastoma tumour suppressor protein, G(0)/G(1) cell cycle arrest, or apoptosis in leukaemia cell lines. In addition, these effects differed depending upon cell type, thus confirming the pleiotropic nature of the ceramide signalling pathway. Most cells studied responded to exogenous C(2)-ceramide by entering growth arrest, evidently resulting from activation of retinoblastoma protein, and by displaying some degree of apoptosis. CONCLUSIONS: Taken together, these findings suggest that signalling via ceramide has novel therapeutic applications for treatment of leukaemia.     

Phenotypic changes in 3T3 cells associated with the change of sphingolipid synthesis by a ceramide analog, 2-decanoylamino-3-morpholino-1-phenylpropanol (compound RV538)

A culture of BALB/c 3T3 cells grown in the presence of 40 microM of the ceramide analog compound RV538 (2-decanolyamino-3-morpholino-1-phenylpropanol) for several passages caused a substantial decrease in the level of all glycosphingolipids and an accumulation of ceramide and sphingomyelin. Associated with these chemical changes of sphingolipid composition and metabolism, the following phenotypic changes were observed: (i) loss of the cobblestone appearance at cell density saturation and development of fibroblastic appearance with partial overlapping of cells; (ii) reduction of cell growth rate; (iii) enhanced production of lactic acid; (iv) enhanced rate of glucose transport; and (v) higher incidence of large colony formation with infiltrating appearance in soft agar. Cell morphology changes, lactate production, and enhanced sugar uptake were reversed by co-culturing cells with gangliosides, particularly trisialogangliosides. Thus, these phenotypic changes mimicking those of oncogenically transformed cells are closely related to the blocked synthesis of glycolipids in these cells, whereas other changes may be caused by an accumulation of ceramide and sphingomyelin.     

Labeled chemical biology tools for investigating sphingolipid metabolism,trafficking and interaction with lipids and proteins

The unraveling of sphingolipid metabolism and function in the last 40 years relied on the extensive study of inherited human disease and specifically-tailored mouse models. However, only few of the achievements made so far would have been possible without chemical biology tools, such as fluorescent and/or radio-labeled and other artificial substrates, (mechanism-based) enzyme inhibitors, cross-linking probes or artificial membrane models. In this review we provide an overview over chemical biology tools that have been used to gain more insight into the molecular basis of sphingolipid-related biology. Many of these tools are still of high relevance for the investigation of current sphingolipid-related questions, others may stimulate the tailoring of novel probes suitable to address recent and future issues in the field. This article is part of a Special Issue entitled Tools to study lipid functions.