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PubMed2006

Snail silencing effectively suppresses tumour growth and invasiveness.

The transcription factor Snail has been recently proposed as an important mediator of tumour invasion because of its role in downregulation of E-cadherin and induction of epithelial-mesenchymal transitions (EMT). This behaviour has led to the consideration of Snail as a potential therapeutic target to block tumour progression. In this report, we provide evidence for this hypothesis. We show that silencing of Snail by stable RNA interference in MDCK-Snail cells induces a complete mesenchymal to epithelial transition (MET), associated to the upregulation of E-cadherin, downregulation of mesenchymal markers and inhibition of invasion. More importantly, stable interference of endogenous Snail in two independent carcinoma cell lines leads to a dramatic reduction of in vivo tumour growth, accompanied by increased tumour differentiation and a significant decrease in the expression of MMP-9 and angiogenic markers and invasiveness. These results indicate that use of RNA interference can be an effective tool for blocking Snail function, opening the way for its application in new antiinvasive therapies.

Animals↗

PubMed2005

Second intermediate host land snails and definitive host animals of Brachylaima cribbi in southern Australia.

This study of infection of southern Australian land snails with Brachylaima cribbi metacercariae has shown that all commonly encountered native and introduced snails are susceptible second intermediate hosts. The range of infected snails is extensive with metacercariae-infected snails being present in all districts across southern Australia. C. virgata has the highest average natural metacercarial infection intensity of 6.1 metacercariae per infected snail. The susceptibility of birds, mammals and reptiles to B. cribbi infection was studied in South Australia by capturing, dissecting and examining the intestinal tract contents of animals which commonly eat land snails as a food source. Indigenous Australian little ravens (Corvus mellori), which are a common scavenger bird, and two other passeriform birds, the black bird (Turdus merula) and the starling (Sturnus vulgaris), which are both introduced European birds, were found to have the highest infection rates of all animals examined. Other birds found infected with B. cribbi were an emu (Dromaius novaehollandiae), chickens (Gallus gallus) and a pigeon (Columba livia). Natural infections were also detected in field mice (Mus domesticus) and shingleback lizards (Tiliqua rugosa) although the intensity of infection was lower than that observed in birds. Susceptibility studies of laboratory mice, rats and ducks showed that mice developed patent infections which persisted for several weeks, rats developed a short-lived infection of three weeks' duration and ducks did not support infection. This study has shown for the first time that a brachylaimid can infect a wide host range of birds, mammals and reptiles in nature.

Animals↗

PubMed1997

Influence of egg cannibalism on growth, survival and feeding in hatchlings of the land snail Helix aspersa Müller (Gastropoda, Pulmonata, Stylommatophora).

Under controlled conditions, growth, survivorship and several nutritional parameters (ingestion, egestion and assimilation) were measured weekly in hatchlings that either ate a conspecific egg after birth (cannibalistic) or not (non-cannibalistic) and in food-deprived individuals (control group). Subsequently, the snails were fed on Taraxacum officinale. After 4 days, cannibalistic snails were 1.3 times heavier than food-deprived snails and 100% survived (75.8% in the control group and 40% of the non-cannibalistic individuals). Mortality, particularly in smaller snails, might be a consequence of food deprivation. Nutritional and energetic gains of oophagy increased both future survivorship and growth. After 11 weeks, cannibalistic snails were 1.4 times heavier and had higher survival rates than food-deprived ones although ingestion, egestion rates and assimilation efficiency were similar in both groups. The larger wet weight of cannibalistic snails after 4 days induced a higher food consumption and thus a higher growth rate. The influence of oophagy on life-history traits is discussed in relation to costs and benefits.

Animals↗

PubMed2002

Harmful effect of immunotherapy in children with combined snail and mite allergy.

BACKGROUND: With respect to allergy, the possibility of cross-reactivity between snail and mite is well recognized, and anecdotal reports suggesting that allergen immunotherapy with mite extract can worsen snail-induced allergy exist. OBJECTIVE: We describe the effect of immunotherapy in 4 children with snail-mite allergy. METHODS: Four children (1 boy and 3 girls; 9-13 years of age) had consistent clinical histories (mild immediate respiratory symptoms after ingestion) and positive skin reactions for allergy to snail. They also had mite-induced asthma and were therefore prescribed subcutaneous specific immunotherapy and subsequently followed. RESULTS: Several months (8-25) after starting immunotherapy, all children experienced life-threatening reactions, anaphylaxis, and respiratory failure after inadvertent ingestion of snail. Skin reactivity to the fresh food increased in all patients. CONCLUSIONS: This observation confirms that in patients with combined mite-snail allergy, immunotherapy should be avoided.

Adolescent↗

PubMed2005

E-cadherin and vitamin D receptor regulation by SNAIL and ZEB1 in colon cancer: clinicopathological correlations.

E-cadherin (CDH1) gene expression is strictly regulated. The transcriptional factors SNAIL and ZEB1 are involved in its repression, whereas activation of vitamin D receptor (VDR) by vitamin D induces its transcription. We study the expression and functional correlation of SNAIL, CDH1, VDR and ZEB1 genes and examine their possible involvement in colon cancer. The expression of these four genes was measured by real time-PCR in 114 patients with colorectal cancer, and tumor characteristics were analyzed in each patient. SNAIL expression was associated with downregulation of CDH1 (P < 0.001) and VDR (P < 0.001) gene products. We also found a positive correlation between CDH1 and VDR expressions. However, the association between SNAIL and CDH1 was not found in patients with high expression of ZEB1. We observed a correlation between downregulation of: a) ZEB1 and presence of polyps in surgical resections; b) VDR and poor differentiation and c) CDH1 and poor differentiation, vascular invasion, presence of lymph node metastases and advanced stages; as well as a trend toward a correlation between SNAIL expression in tumors and vascular invasion. The correlations between SNAIL, CDH1, VDR and ZEB1 and the association between reduced expression of CDH1 and VDR and aggressive tumor characteristics emphasize the value of analyzing these genes in colon cancer patients for prognostic purposes and for predicting response to possible therapies with vitamin D or its analogs.

Adult↗

PubMed2000

Intrinsic metabolic depression in cells isolated from the hepatopancreas of estivating snails.

Many animals across the phylogenetic scale are routinely capable of depressing their metabolic rate to 5-15% of that at rest, remaining in this state sometimes for years. However, despite its widespread occurrence, the biochemical processes associated with metabolic depression remain obscure. We demonstrate here the development of an isolated cell model for the study of metabolic depression. The isolated cells from the hepatopancreas (digestive gland) of the land snail (Helix aspersa) are oxygen conformers; i.e., their rate of respiration depends on pO(2). Cells isolated from estivating snails show a stable metabolic depression to 30% of control (despite the long and invasive process of cell isolation) when metabolic rate at the physiological pH and pO(2) of the hemolymph of estivating snails is compared with metabolic rate at the physiological pH and pO(2) of the hemolymph of control snails. When the extrinsic effects of pH and pO(2) are excluded, the intrinsic metabolic depression of the cells from estivating snails is still to below 50% of control snails. The in vitro effect of pO(2) on metabolic rate is independent of pH and state (awake or estivating), but the effects of pH and state significantly interact. This suggests that pH and state change affect metabolic depression by similar mechanisms but that the metabolic depression by hypoxia involves a separate mechanism.

Animals↗

PubMed2006

Parasites alter host phenotype and may create a new ecological niche for snail hosts.

By modifying the behaviour and morphology of hosts, parasites may strongly impact host individuals, populations and communities. We examined the effects of a common trematode parasite on its snail host, Batillaria cumingi (Batillariidae). This widespread snail is usually the most abundant invertebrate in salt marshes and mudflats of the northeastern coast of Asia. More than half (52.6%, n=1360) of the snails in our study were infected. We found that snails living in the lower intertidal zone were markedly larger and exhibited different shell morphology than those in the upper intertidal zone. The large morphotypes in the lower tidal zone were all infected by the trematode, Cercaria batillariae (Heterophyidae). We used a transplant experiment, a mark-and-recapture experiment and stable carbon isotope ratios to reveal that snails infected by the trematode move to the lower intertidal zone, resume growth after maturation and consume different resources. By simultaneously changing the morphology and behaviour of individual hosts, this parasite alters the demographics and potentially modifies resource use of the snail population. Since trematodes are common and often abundant in marine and freshwater habitats throughout the world, their effects potentially alter food webs in many systems.

Animals↗

PubMed2006

Caspase-like activity is essential for long-term synaptic plasticity in the terrestrial snail Helix.

Although caspase activity in the nervous system of mollusks has not been described before, we suggested that these cysteine proteases might be involved in the phenomena of neuroplasticity in mollusks. We directly measured caspase-3 (DEVDase) activity in the Helix lucorum central nervous system (CNS) using a fluorometrical approach and showed that the caspase-3-like immunoreactivity is present in the central neurons of Helix. Western blots revealed the presence of caspase-3-immunoreactive proteins with a molecular mass of 29 kDa. Staurosporin application, routinely used to induce apoptosis in mammalian neurons through the activating cleavage of caspase-3, did not result in the appearance of a smaller subunit corresponding to the active caspase in the snail. However, it did increase the enzyme activity in the snail CNS. This suggests differences in the regulation of caspase-3 activity in mammals and snails. In the snail CNS, the caspase homolog seems to possess an active center without activating cleavage typical for mammals. In electrophysiological experiments with identified snail neurons, selective blockade of the caspase-3 with the irreversible and cell-permeable inhibitor of caspase-3 N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp-(OMe)-fluoro-methylketone prevented development of the long-term stage of synaptic input sensitization, suggesting that caspase is necessary for normal synaptic plasticity in snails. The results of our study give the first direct evidence that the caspase-3-like activity is essential for long-term plasticity in the invertebrate neurons. This activity is presumably involved in removing inhibitory constraints on the storage of long-term memory.

Animals↗

PubMed1995

Antioxidant defenses and metabolic depression in a pulmonate land snail.

During arousal from estivation oxygen consumption by land snails (Otala lactea) increases severalfold. To determine whether snails prepared for an accompanying rise in the rates of oxyradical generation by altering their antioxidant defense mechanisms, changes in the activities of antioxidant enzymes and lipid peroxidation products were quantified in foot and hepatopancreas of control, 30-day estivating, and aroused snails. Compared with controls, estivating O. lactea showed significant increases in the activities of foot muscle superoxide dismutase (SOD) (increasing by 56-67%), catalase (51-72%), and glutathione S-transferase (79-108%), whereas, in hepatopancreas, SOD (57-78%) and glutathione peroxidase (93-144%) increased. Within 40 min after arousal began, hepatopancreas glutathione peroxidase activity had returned to control values, but SOD showed a further 70% increase in activity but then returned to control levels by 80 min. Estivation had no effect on total glutathione (GSH + 2 GSSG) concentrations in tissues, but GSSG content had increased about twofold in both organs of 30-day dormant snails. Lipid peoxidation (quantified as thiobarbituric acid reactive substances) was significantly enhanced at the onset of arousal from dormancy, indicating that oxidative stress and tissue damage occurred at this time. The data suggest that antioxidant defenses in snail organs are increased while snails are in the hypometabolic state as a preparation for oxidative stress during arousal.

Animals↗

PubMed2005

The transcription factor snail induces tumor cell invasion through modulation of the epithelial cell differentiation program.

Abberant activation of the process of epithelial-mesenchymal transition in cancer cells is a late event in tumor progression. A key inducer of this transition is the transcription factor Snail, which represses E-cadherin. We report that conditional expression of the human transcriptional repressor Snail in colorectal cancer cells induces an epithelial dedifferentiation program that coincides with a drastic change in cell morphology. Snail target genes control the establishment of several junctional complexes, intermediate filament networks, and the actin cytoskeleton. Modulation of the expression of these genes is associated with loss of cell aggregation and induction of invasion. Chromatin immunoprecipitation experiments showed that repression of selected target genes is associated with increased binding of Snail to their promoters, which contain consensus Snail-binding sites. Thus, Snail constitutes a master switch that directly represses the epithelial phenotype, resulting in malignant carcinoma cells.

Actins↗

PubMed2002

Cloning, isolation, and IgE-binding properties of Helix aspersa (brown garden snail) tropomyosin.

BACKGROUND: Gastropod consumption is quite frequent in the Mediterranean countries and cross-reactivities with crustaceans have been described, but the mechanism of this allergenic cross-reactivity has not been studied in detail. This study aimed to produce recombinant Helix aspersa (brown garden snail) tropomyosin and investigate its implication for cross-reactivity among invertebrates. METHODS: A tropomyosin-specific cDNA encoding H. aspersa tropomyosin was synthetized, and recombinant allergen was overexpressed in Escherichia coli as nonfusion protein. IgE-binding reactivity was studied by immunoblotting and immunoblot inhibition experiments with sera from snail-allergic patients. RESULTS: Cloned brown garden snail tropomyosin shares high homology with other edible mollusk tropomyosins (84-69% identity) as well as with those from arthropods (65-62%), and less homology with vertebrate ones (56% identity). Tropomyosin reacted with 18% of the sera from patients with snail allergy. Inhibition experiments, using natural and recombinant tropomyosins, showed different degrees of cross-reactivity between invertebrate tropomyosins. Sera from snail-allergic subjects recognized tropomyosins in both mollusks and crustacean extracts. CONCLUSIONS: Tropomyosin represents a minor allergen in snail extracts, but it is clearly involved in invertebrate cross-reactivity.

Amino Acid Sequence↗

PubMed2006

Transcription factor snail modulates hormone expression in established endocrine pancreatic cell lines.

The development of differentiated cells from undifferentiated progenitor cells is one of the central tenets of developmental biology. However, under conditions of tissue morphogenesis, regeneration, and cancer, this process of development is reversed and fully differentiated cells transition to an undifferentiated phenotype. Here we present evidence that the zinc-finger transcription factor Snail modulates this transition in differentiated pancreatic endocrine cell lines. During passage and growth of these cell lines, Snail expression is induced in a subset of cells within the culture, concomitant with a decrease in insulin and/or glucagon expression. As the cells cluster and exit the cell division cycle, nuclear levels of Snail are reduced and hormone expression is resumed. Snail represses proinsulin and proglucagon gene transcription, and reduction of Snail levels by small interfering RNA treatment increases proinsulin gene expression. We propose that Snail modulates the dynamic balance between differentiated and dedifferentiated cells allowing their migration and proliferation. These findings may be relevant to providing approaches for the enhancement of beta-cell growth in individuals with diabetes mellitus.

Cell Differentiation↗

PubMed2004

Snail and E47 repressors of E-cadherin induce distinct invasive and angiogenic properties in vivo.

The transcription factors Snail and E47 are direct repressors of E-cadherin, with both inducing a full epithelial-mesenchymal transition and invasive behaviour in vitro when expressed in the prototypic epithelial MDCK cell line. The role of these repressors in the invasive process and in other tumorigenic properties is, nevertheless, still poorly understood. However, organotypic cultures and in vivo transplantation assays indicate that cells expressing MDCK-Snail and MDCK-E47 exhibit significant differences. MDCK-Snail cells have a higher infiltrative potential than MDCK-E47 cells. Interestingly, both cell types induce angiogenesis of the host stromal tissue in transplantation assays, but this property is greatly enhanced in transplants of MDCK-E47 cells. Xenografted tumours induced in nude mice also show signs of strong angiogenic potential, again markedly increased in tumours induced by MDCK-E47 which exhibit a higher vessel density and proliferation rate than those induced by MDCK-Snail cells. These results suggest differential roles for Snail and E47 E-cadherin repressors in tumour progression where Snail is implicated in promoting the initial invasion and E47 plays an active role in tumour cell growth by promoting angiogenesis.

Actins↗

PubMed1982

Isolation and properties of vitellogenic ferritin from snails.

The iron storage protein ferritin is the principal yolk protein in oocytes of the snails Planorbarius corneus L. and Lymnaea stagnalis L. This report gives an account of the isolation procedure and of some properties of snail ferritins. The isolation procedure includes a heat-denaturation step, gel filtration on Sepharose 6B and two ultracentrifugation steps followed by electrophoresis. Ferritins from both snails are highly reminiscent of vertebrate and plant ferritins in terms of heat stability, absorption spectrum and ultrastructure, and both share common antigen determinants with horse spleen ferritin. In different electrophoresis systems snail ferritins display considerable heterogeneity and microheterogeneity. Electrophoresis in the presence of sodium dodecyl sulphate (SDS) yields two major polypeptides with molecular weights of 19 000 and 24 000, which are interpreted to be authentic subunits of the ferritin molecule. Different organs and tissues of the snails differ in subunit composition. Midgut gland ferritin consists predominantly of the 19 000 Mr polypeptide, while in embryos only the 24 000 Mr band was found. No carbohydrates or lipids could be detected by staining acrylamide gels. Results from SDS/acrylamide electrophoresis, electrophoresis under non-denaturing conditions on gradient gels and from isoelectric focusing indicate that the ferritins of both snails are composed of at least two different types of ferritin that are tissue-specific. One ferritin is typical of somatic tissue (midgut gland) and is most probably a homopolymer of the 19 000 Mr subunit. The other ferritin is typical of oocytes, but since it is an exogenous protein it is also encountered in the midgut gland (the presumed site of yolk synthesis) and the haemolymph. Vitellogenic ferritin is either a homopolymer of the 24 000 Mr subunit or is predominantly composed of it. So far, there is no evidence for a precursor-product relationship between the two subunits.

Animals↗

PubMed2005

Intraspecific variation of venom injected by fish-hunting Conus snails.

Venom peptides from two species of fish-hunting cone snails (Conus striatus and Conus catus) were characterized using microbore liquid chromatography coupled with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and electrospray ionization-ion trap-mass spectrometry. Both crude venom isolated from the venom duct and injected venom obtained by milking were studied. Based on analysis of injected venom samples from individual snails, significant intraspecific variation (i.e. between individuals) in the peptide complement is observed. The mixture of peptides in injected venom is simpler than that in the crude duct venom from the same snail, and the composition of crude venom is more consistent from snail to snail. While there is animal-to-animal variation in the peptides present in the injected venom, the composition of any individual's injected venom remains relatively constant over time in captivity. Most of the Conus striatus individuals tested injected predominantly a combination of two neuroexcitatory peptides (s4a and s4b), while a few individuals had unique injected-venom profiles consisting of a combination of peptides, including several previously characterized from the venom duct of this species. Seven novel peptides were also putatively identified based on matches of their empirically derived masses to those predicted by published cDNA sequences. Profiling injected venom of Conus catus individuals using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry demonstrates that intraspecific variation in the mixture of peptides extends to other species of piscivorous cone snails. The results of this study imply that novel regulatory mechanisms exist to select specific venom peptides for injection into prey.

Animals↗

PubMed2006

Suppression of Na+/K+-ATPase activity during estivation in the land snail Otala lactea.

Entry into the hypometabolic state of estivation requires a coordinated suppression of the rate of cellular ATP turnover, including both ATP-generating and ATP-consuming reactions. As one of the largest consumers of cellular ATP, the plasma membrane Na+/K+-ATPase is a potentially key target for regulation during estivation. Na+/K+-ATPase was investigated in foot muscle and hepatopancreas of the land snail Otala lactea, comparing active and estivating states. In both tissues enzyme properties changed significantly during estivation: maximal activity was reduced by about one-third, affinity for Mg.ATP was reduced (Km was 40% higher), and activation energy (derived from Arrhenius plots) was increased by approximately 45%. Foot muscle Na+/K+-ATPase from estivated snails also showed an 80% increase in Km Na+ and a 60% increase in Ka Mg2+ as compared with active snails, whereas hepatopancreas Na+/K+-ATPase showed a 70% increase in I50 K+ during estivation. Western blotting with antibodies recognizing the alpha subunit of Na+/K+-ATPase showed no change in the amount of enzyme protein during estivation. Instead, the estivation-responsive change in Na+/K+-ATPase activity was linked to posttranslational modification. In vitro incubations manipulating endogenous kinase and phosphatase activities indicated that Na+/K+-ATPase from estivating snails was a high phosphate, low activity form, whereas dephosphorylation returned the enzyme to a high activity state characteristic of active snails. Treatment with protein kinases A, C or G could all mediate changes in enzyme properties in vitro that mimicked the effect of estivation, whereas treatments with protein phosphatase 1 or 2A had the opposite effect. Reversible phosphorylation control of Na+/K+-ATPase can provide the means of coordinating ATP use by this ion pump with the rates of ATP generation by catabolic pathways in estivating snails.

Adenosine Triphosphate↗

PubMed2004

COX-2, MMP-9, and Noguchi classification provide additional prognostic information about adenocarcinoma of the lung. A study of 117 patients from Brazil.

We report immunohistochemical staining results for cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 in primary tumors of 117 patients with resected adenocarcinoma of the lung (median follow-up, 20 months). For COX-2, we graded the degree of tumor staining according to the sum of staining intensity and the proportion of cells staining. For MMP-9, we used morphometry to quantify cytoplasmic staining. We used the Cox proportional hazards model to analyze overall survival. With only 29 patients censored at last follow-up, after controlling for the effect of pathologic stage, staining for COX-2 and MMP-9 and subtype of tumor were related significantly to survival (P < 6 x 10(-5)). The effects of COX-2 and MMP-9 were opposite. Whereas any staining for COX-2 decreased the hazard and increased survival time, increased staining for MMP-9 increased the hazard and decreased survival time. The results also suggested that staining for COX-2 decreases with dedifferentiation. Our results suggest that staining for the combination of COX-2 and MMP-9 and categorizing tumors into papillary and nonpapillary types may provide important prognostic information for patients with resected adenocarcinoma of the lung; it is possible that these 3 variables could aid decisions about postoperative adjuvant treatment.

Adenocarcinoma↗

PubMed2006

Snail-dependent and -independent epithelial-mesenchymal transition in oral squamous carcinoma cells.

Disappearance of E-cadherin is a milestone for epithelial-mesenchymal transition (EMT), found both in carcinomas and in some fibrotic diseases. We have studied the mechanisms of EMT in oral squamous cell carcinoma (SCC) cells isolated from primary tumor (43A) and its recurrent tumor (43B). Whereas the cells from primary carcinoma displayed a typical phenotype of squamous epithelial cells including E-cadherin and laminin-332 (laminin-5), cells from recurrent tumor expressed characteristics of dedifferentiated, EMT-experienced tumors. 43B cells expressed E-cadherin repressors ZEB-1/deltaEF1 and especially ZEB-2/SIP1, which therefore appear as candidates for endogenous EMT in these cells. Differences between endogenous and exogenous EMT were assessed by transfecting 43A cells with SNAIL cDNA. SNAIL-transfected cells showed complete EMT phenotype with fibroblastoid appearance, vimentin filaments, E-cadherin/N-cadherin switch, lack of hemidesmosomes and, as a new feature of EMT, lack of laminin-332 synthesis. Upregulation of ZEB-1 and ZEB-2 was evident in these cells, suggesting that SNAIL can regulate these E-cadherin repressors. New monoclonal antibodies against SNAIL showed nuclear immunoreactivity not only in the SNAIL-transfected cells but also in carcinoma cells lacking production of Lm-332 and showing signs of EMT. These results suggest that changes in the epithelial cell differentiation program and EMT in SCC cells can result from the interplay among several E-cadherin repressors; however, SNAIL alone is able to accomplish a complete EMT.

Aged↗