Difference in F-Actin Depolymerization Induced by Toxin B from the Clostridium difficile Strain VPI 10463 and Toxin B from the Variant Clostridium difficile Serotype F Strain 1470

Clostridium difficile toxin A (TcdA) and toxin B (TcdB) are the causative agent of the C. difficile-associated diarrhea (CDAD) and its severe form, the pseudomembranous colitis (PMC). TcdB from the C. difficile strain VPI10463 mono-glucosylates (thereby inactivates) the small GTPases Rho, Rac, and Cdc42, while Toxin B from the variant C. difficile strain serotype F 1470 (TcdBF) specifically mono-glucosylates Rac but not Rho(A/B/C). TcdBF is related to lethal toxin from C. sordellii (TcsL) that glucosylates Rac1 but not Rho(A/B/C). In this study, the effects of Rho-inactivating toxins on the concentrations of cellular F-actin were investigated using the rhodamine-phalloidin-based F-actin ELISA. TcdB induces F-actin depolymerization comparable to the RhoA-inactivating exoenzyme C3 from C. limosum (C3-lim). In contrast, the Rac-glucosylating toxins TcdBF and TcsL did not cause F-actin depolymerization. These observations led to the conclusion that F-actin depolymerization depends on the toxin’s capability of glucosylating RhoA. Furthermore, the integrity of focal adhesions (FAs) was analyzed using paxillin and p21-activated kinase (PAK) as FA marker proteins. Paxillin dephosphorylation was observed upon treatment of cells with TcdB, TcdBF, or C3-lim. In conclusion, the Rho-inactivating toxins induce loss of cell shape by either F-actin depolymerization (upon RhoA inactivation) or the disassembly of FAs (upon Rac1 inactivation).     

Ethanol selectively impairs clathrin-mediated internalization in polarized hepatic cells

Although alcoholic liver disease is clinically well-described, the molecular basis for alcohol-induced hepatotoxicity is not well understood. Previously, we determined that the clathrin-mediated internalization of asialoglycoprotein receptor was impaired in ethanol-treated WIF-B cells whereas the internalization of a glycophosphatidylinositol-anchored protein thought to be endocytosed via a caveolae/raft-mediated pathway was not changed suggesting that clathrin-mediated endocytosis is selectively impaired by ethanol. To test this possibility, we examined the internalization of a panel of proteins and compounds internalized by different mechanisms in control and ethanol-treated WIF-B cells. We determined that the internalization of markers known to be internalized via clathrin-mediated mechanisms was impaired. In contrast, the internalization of markers for caveolae/raft-mediated endocytosis, fluid phase internalization or non-vesicle-mediated uptake was not impaired in ethanol-treated cells. We further determined that clathrin heavy chain accumulated at the basolateral surface in small puncta in ethanol-treated cells while there was decreased dynamin-2 membrane association. Interestingly, the internalization of resident apical proteins that lack any known internalization signals was also disrupted by ethanol suggesting that these proteins are internalized via clathrin-mediated mechanisms. This conclusion is consistent with our findings that dominant negative dynamin-2 overexpression impaired internalization of known clathrin markers and single spanning apical residents, but not of markers of fluid phase or raft-mediated internalization. Together these results indicate that ethanol exposure selectively impairs hepatic clathrin-mediated internalization by preventing vesicle fission from the plasma membrane.     

Inhibition of insulin-stimulated glucose transport in 3T3-L1 cells by Clostridium difficile toxin B, Clostridium sordellii lethal toxin, and Clostridium botulinum C2 toxin

The role of the actin cytoskeleton and/or GTPases of the Rho/Rac-family in glucose transport regulation was investigated in 3T3-L1 cells with clostridial toxins which depolymerize actin by inactivation of Rho/Rac (Clostridium difficile toxin B and Clostridium sordellii lethal toxin (LT)) or by direct ADP-ribosylation (Clostridium botulinum C2 toxin). Toxin B and C2 reduced insulin-stimulated, but not basal, 2-deoxyglucose (2-DOG) uptake rates in 3T3-L1 fibroblasts. In parallel, the toxins produced morphological alterations of the cells reflecting disruption of the actin cytoskeleton. Both toxins reduced the maximum response to insulin but failed to alter the half-maximally stimulating concentrations of insulin. In 3T3-L1 adipocytes, the lethal toxin reduced the effect of insulin on 2-DOG uptake, whereas toxin B and C2 failed to affect glucose transport or cell morphology. When cells were exposed to the toxins after treatment with insulin, both toxin B and the lethal toxin, in contrast to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, failed to reduce the 2-DOG uptake rates. Thus, both translocation to the plasma membrane and internalization of glucose transporters were inhibited by the toxins, whereas the PI 3-kinase inhibitor selectively affects translocation. The data suggest that the effects of the clostridial toxins on trafficking of glucose transporters are mediated by the depolymerization of the actin cytoskeleton and are an indirect consequence of Rho or Rac inactivation. It is suggested that pathways signalling through Rac or Rho may play a modulatory role in glucose transport regulation through their effects on the actin network. Received: 28 October 1997 / Accepted: 19 January 1998     

Antiviral Activity of Hederasaponin B from Hedera helix against Enterovirus 71 Subgenotypes C3 and C4a

Enterovirus 71 (EV71) is the predominant cause of hand, foot and mouth disease (HFMD). The antiviral activity of hederasaponin B from Hedera helix against EV71 subgenotypes C3 and C4a was evaluated in vero cells. In the current study, the antiviral activity of hederasaponin B against EV71 C3 and C4a was determined by cytopathic effect (CPE) reduction method and western blot assay. Our results demonstrated that hederasaponin B and 30% ethanol extract of Hedera helix containing hederasaponin B showed significant antiviral activity against EV71 subgenotypes C3 and C4a by reducing the formation of a visible CPE. Hederasaponin B also inhibited the viral VP2 protein expression, suggesting the inhibition of viral capsid protein synthesis.These results suggest that hederasaponin B and Hedera helix extract containing hederasaponin B can be novel drug candidates with broad-spectrum antiviral activity against various subgenotypes of EV71.     

The NFL-TBS.40-63 anti-glioblastoma peptide enters selectively in glioma cells by endocytosis

Glioblastoma are the most frequent and aggressive tumour of the nervous system despite surgical resection associated with chemotherapy and radiotherapy. Recently, we showed that the NFL-TBS.40-63 peptide corresponding to the sequence of a tubulin-binding site of neurofilaments, enters selectively in glioblastoma cells where it blocks microtubule polymerization, inhibits their proliferation, and reduces tumour development in rats bearing glioblastoma (0050 and 0040). Here, we characterized the molecular mechanism responsible for the uptake of NFL-TBS.40-63 peptide by glioblastoma cells. Unlike other cell penetrating peptides (CPPs), which use a balance between endocytosis and direct translocation, the NFL-TBS.40-63 peptide is unable to translocate directly through the membrane when incubated with giant plasma membrane vesicles. Then, using a panel of markers and inhibitors, flow cytometry and confocal microscopy investigations showed that the uptake occurs mainly through endocytosis. Moreover, glycosaminoglycans and αVβ3 integrins are not involved in the NFL-TBS.40-63 peptide recognition and internalization by glioblastoma cells. Finally, the signalling of tyrosine kinase receptors is involved in the peptide uptake, especially via EGFR overexpressed in tumour cells, indicating that the uptake of NFL-TBS.40-63 peptide by glioblastoma cells is related to their abnormally high proliferative activity.     

Brown spider venom toxins interact with cell surface and are endocytosed by rabbit endothelial cells

Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations. Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. The aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom. After treating endothelial cells with venom toxins, we observed that the venom interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates. When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells. The venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L. intermedia venom on endothelial cells is not mediated by venom internalization.     

Fluopsin C induces oncosis of human breast adenocarcinoma cells

Aim:

Fluopsin C, an antibiotic isolated from Pseudomonas jinanesis, has shown antitumor effects on several cancer cell lines. In the current study, the oncotic cell death induced by fluopsin C was investigated in human breast adenocarcinoma cells in vitro.

Methods:

Human breast adenocarcinoma cell lines MCF-7 and MD-MBA-231 were used. The cytotoxicity was evaluated using MTT assay. Time-lapse microscopy and transmission electron microscopy were used to observe the morphological changes. Cell membrane integrity was assessed with propidium iodide (PI) uptake and lactate dehydrogenase (LDH) assay. Flow cytometry was used to measure reactive oxygen species (ROS) level and mitochondrial membrane potential (Δψm). A multimode microplate reader was used to analyze the intracellular ATP level. The changes in cytoskeletal system were investigated with Western blotting and immunostaining.

Results:

Fluopsin C (0.5-8 μmol/L) reduced the cell viability in dose- and time-dependent manners. Its IC₅₀ values in MCF-7 and MD-MBA-231 cells at 24 h were 0.9 and 1.03 μmol/L, respectively. Fluopsin C (2 μmol/L) induced oncosis in both the breast adenocarcinoma cells characterized by membrane blebbing and swelling, which was blocked by pretreatment with the pan-caspase inhibitor Z-VAD-fmk. In MCF-7 cells, fluopsin C caused PI uptake into the cells, significantly increased LDH release, induced cytoskeletal system degradation and ROS accumulation, decreased the intracellular ATP level and Δψm. Noticeably, fluopsin C exerted comparable cytotoxicity against the normal human hepatocytes (HL7702) and human mammary epithelial cells with the IC₅₀ values at 24 h of 2.7 and 2.4 μmol/L, respectively.

Conclusion:

Oncotic cell death was involved in the anticancer effects of fluopsin C on human breast adenocarcinoma cells in vitro. The hepatoxicity of fluopsin C should not be ignored.     

Binding Studies on Isolated Porcine Small Intestinal Mucosa and in vitro Toxicity Studies Reveal Lack of Effect of C. perfringens Beta-Toxin on the Porcine Intestinal Epithelium

Beta-toxin (CPB) is the essential virulence factor of C. perfringens type C causing necrotizing enteritis (NE) in different hosts. Using a pig infection model, we showed that CPB targets small intestinal endothelial cells. Its effect on the porcine intestinal epithelium, however, could not be adequately investigated by this approach. Using porcine neonatal jejunal explants and cryosections, we performed in situ binding studies with CPB. We confirmed binding of CPB to endothelial but could not detect binding to epithelial cells. In contrast, the intact epithelial layer inhibited CPB penetration into deeper intestinal layers. CPB failed to induce cytopathic effects in cultured polarized porcine intestinal epithelial cells (IPEC-J2) and primary jejunal epithelial cells. C. perfringens type C culture supernatants were toxic for cell cultures. This, however, was not inhibited by CPB neutralization. Our results show that, in the porcine small intestine, CPB primarily targets endothelial cells and does not bind to epithelial cells. An intact intestinal epithelial layer prevents CPB diffusion into underlying tissue and CPB alone does not cause direct damage to intestinal epithelial cells. Additional factors might be involved in the early epithelial damage which is needed for CPB diffusion towards its endothelial targets in the small intestine.     

A probiotic strain of Escherichia coli,Nissle 1917, given orally exerts local and systemic anti-inflammatory effects in lipopolysaccharide-induced sepsis in mice

Background and purpose:

Escherichia coli Nissle 1917 is a probiotic strain used in the treatment of intestinal immune diseases, including ulcerative colitis. The aim of the present study was to test if this probiotic bacterium can also show systemic immunomodulatory properties after oral administration.

Experimental approach:

The probiotic strain was administered to rats or mice for 2 weeks before its assay in two experimental models of altered immune response, the trinitrobenzenesulphonic acid (TNBS) model of rat colitis, localized in the colon, and the lipopolysaccharide (LPS) model of systemic septic shock in mice. Inflammatory status was evaluated both macroscopically and biochemically after 1 week in the TNBS model or after 24 h in the LPS shock model. In addition, splenocytes were obtained from mice and stimulated, ex vivo, with concanavalin A or LPS to activate T or B cells, respectively, and cytokine production (IL-2, IL-5 and IL-10) by T cells and IgG secretion by B cells measured.

Key results:

E. coli Nissle 1917 was anti-inflammatory in both models of altered immune response. This included a reduction in the pro-inflammatory cytokine tumour necrosis factor-α both in the intestine from colitic rats, and in plasma and lungs in mice treated with LPS. The systemic beneficial effect was associated with inhibited production of the T cell cytokines and by down-regulation of IgG release from splenocyte-derived B cells.

Conclusions and implications:

The anti-inflammatory effects of E. coli Nissle 1917 given orally were not restricted to the gastrointestinal tract.     

Sertraline inhibits the transport of PAT1 substrates in vivo and in vitro

Background and Purpose

Intestinal nutrient transporters may mediate the uptake of drugs. The aim of this study was to investigate whether sertraline interacts with the intestinal proton-coupled amino acid transporter 1 PAT1 (SLC36A1).

Experimental Approach

In vitro investigations of interactions between sertraline and human (h)PAT1, hSGLT1 (sodium-glucose linked transporter 1) and hPepT1 (proton-coupled di-/tri-peptide transporter 1) were conducted in Caco-2 cells using radiolabelled substrates. In vivo pharmacokinetic investigations were conducted in male Sprague–Dawley rats using gaboxadol (10 mg·kg⁻¹, p.o.) as a PAT1 substrate and sertraline (0–30.6 mg·kg⁻¹). Gaboxadol was quantified by hydrophilic interaction chromatography followed by MS/MS detection.

Key Results

Sertraline inhibited hPAT1-mediated L-[³H]-Pro uptake in Caco-2 cells. This interaction between sertraline and PAT1 appeared to be non-competitive. The uptake of the hSGLT1 substrate [¹⁴C]-α–methyl-D-glycopyranoside and the hPepT1 substrate [¹⁴C]-Gly-Sar in Caco-2 cells was also decreased in the presence of 0.3 mM sertraline. In rats, the administration of sertraline (0.1–10 mM, corresponding to 0.3–30.6 mg·kg⁻¹, p.o.) significantly reduced the maximal gaboxadol plasma concentration and AUC after its administration p.o.

Conclusions and Implications

Sertraline is an apparent non-competitive inhibitor of hPAT1-mediated transport in vitro. This inhibitory effect of sertraline is not specific to hPAT1 as substrate transport via hPepT1 and hSGLT1 was also reduced in the presence of sertraline. In vivo, sertraline reduced the amount of gaboxadol absorbed, suggesting that the inhibitory effect of sertraline on PAT1 occurs both in vitro and in vivo. Hence, sertraline could alter the bioavailability of drugs absorbed via PAT1.     

Predominant contribution of OATP1B3 to the hepatic uptake of telmisartan, an angiotensin II receptor antagonist, in humans.

Telmisartan, a nonpeptide angiotensin II receptor antagonist, is selectively distributed to liver. In the present study, we have characterized the contribution of organic anion transporting polypeptide (OATP) isoforms to the hepatic uptake of telmisartan by isolated rat hepatocytes, human cryopreserved hepatocytes, and human transporter-expressing cells. Because it is difficult to evaluate the transport activity of telmisartan because of its extensive adsorption to cells and culture materials, we performed the uptake study in the presence of human serum albumin. The saturable uptake of telmisartan into isolated rat hepatocytes took place in a Na(+)-independent manner and was inhibited by pravastatin, taurocholate, and digoxin, which are Oatp substrates and inhibitors, but not by organic cation, tetraethylammonium, indicating the involvement of Oatp isoforms in its uptake into rat hepatocytes. To identify which human OATP transporters are important for the hepatic uptake of telmisartan, the uptake assay was carried out using OATP1B1- and OATP1B3-expressing human embryonic kidney 293 cells and cryopreserved human hepatocytes. The uptake of telmisartan by OATP1B3-expressing cells was saturable (K(m) = 0.81 microM) and significantly higher than that by vector-transfected cells. In contrast, no significant uptake was observed in OATP1B1-expressing cells. We also observed the saturable uptake of telmisartan by human hepatocytes. Thirty micromolar estrone-3-sulfate, which can selectively inhibit OATP1B1-mediated uptake compared with OATP1B3, did not inhibit the uptake of telmisartan in human hepatocytes, whereas it could inhibit the uptake of estradiol 17beta-d-glucuronide mediated by OATP1B1. These results suggest that OATP1B3 is predominantly involved in the hepatic uptake of telmisartan in humans.     

The toxicity of liposomal chloramphenicol for Tetrahymena pyriformis

The toxicity of suspensions of anionic and neutral liposomes containing chloramphenicol was compared with the toxicity of free drug solution for the protozoan, Tetrahymena pyriformis. The liposomal form of the drug was found to be markedly more toxic, anionic and neutral liposomes being equally effective in killing the cells. Cellular uptake and inhibition of uptake by cytochalasin B of diformazan liposomes and polystyrene beads was found to be similar. Uptake of [³H] cholesterol-labelled liposomes was decreased in old cells and absent in killed cells. Quantitative aspects of the internalization of CAP in liposome form are discussed.     

Astragalus polysaccharide stimulates glucose uptake in L6 myotubes through AMPK activation and AS160/TBC1D4 phosphorylation

Aim:

To establish the mechanism responsible for the stimulation of glucose uptake by Astragalus polysaccharide (APS), extracted from Astragalus membranaceus Bunge, in L6 myotubes in vitro.

Methods:

APS-stimulated glucose uptake in L6 myotubes was measured using the 2-deoxy-[³H]-D-glucose method. The adenine nucleotide contents in the cells were measured by HPLC. The phosphorylation of AMP-activated protein kinase (AMPK) and Akt substrate of 160 kDa (AS160) was examined using Western blot analysis. The cells transfected with 4P mutant AS160 (AS160-4P) were constructed using gene transfer approach.

Results:

Treatment of L6 myotubes with APS (100−1600 μg/mL) significantly increased glucose uptake in time- and concentration-dependent manners. The maximal glucose uptake was reached in the cells treated with APS (400 μg/mL) for 36 h. The APS-stimulated glucose uptake was significantly attenuated by pretreatment with Compound C, a selective AMPK inhibitor or in the cells overexpressing AS160-4P. Treatment of L6 myotubes with APS strongly promoted the activation of AMPK. We further demonstrated that either Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ) or liver kinase B1 (LKB1) mediated APS-induced activation of AMPK in L6 myotubes, and the increased cellular AMP: ATP ratio was also involved. Treatment of L6 myotubes with APS robustly enhanced the phosphorylation of AS160, which was significantly attenuated by pretreatment with Compound C.

Conclusion:

Our results demonstrate that APS stimulates glucose uptake in L6 myotubes through the AMP-AMPK-AS160 pathway, which may contribute to its hypoglycemic effect.     

Behavioral deficits and cellular damage following developmental ethanol exposure in rats are attenuated by CP-101,606, an NMDAR antagonist with unique NR2B specificity

NMDAR-mediated excitotoxicity has been implicated in some of the impairments following fetal ethanol exposure. Previous studies suggest that both neuronal cell death and some of the behavioral deficits can be reduced by NMDAR antagonism during withdrawal, including antagonism of a subpopulation of receptors containing NR2B subunits. To further investigate NR2B involvement, we selected a compound, CP-101,606 (CP) which binds selectively to NR2B/2B stoichiometries, for both in vitro and in vivo analyses. For the in vitro study, hippocampal explants were exposed to ethanol for 10 days and then 24 h following removal of ethanol, cellular damage was quantified via propidium iodide fluorescence. In vitro ethanol withdrawal-associated neurotoxicity was prevented by CP (10 and 25 nM). In vivo ethanol exposure was administered on PNDs 1-7 with CP administered 21 h following cessation. Activity (PNDs 20-21), motor skills (PNDs 31-33), and maze navigation (PNDs 43-44) were all susceptible to ethanol insult; treatment with CP (15 mg/kg) rescued these deficits. Our findings show that CP-101,606, a drug that blocks the NR2B/2B receptor, can reduce some of the damaging effects of “3rd trimester” alcohol exposure in our rodent model. Further work is clearly warranted on the neuroprotective potential of this drug in the developing brain.     

In vitro anti-cancer activity of chamaejasmenin B and neochamaejasmin C isolated from the root of Stellera chamaejasme L

Aim:

To examine the anti-cancer effects of chamaejasmenin B and neochamaejasmin C, two biflavonones isolated from the root of Stellera chamaejasme L (known as the traditional Chinese herb Rui Xiang Lang Du) in vitro.

Methods:

Human liver carcinoma cell lines (HepG2 and SMMC-7721), a human non-small cell lung cancer cell line (A549), human osteosarcoma cell lines (MG63, U2OS, and KHOS), a human colon cancer cell line (HCT-116) and a human cervical cancer cell line (HeLa) were used. The anti-proliferative effects of the compounds were measured using SRB cytotoxicity assay. DNA damage was detected by immunofluorescence and Western blotting. Apoptosis and cell cycle distribution were assessed using flow cytometry analysis. The expression of the related proteins was examined with Western blotting analysis.

Results:

Both chamaejasmenin B and neochamaejasmin C exerted potent anti-proliferative effects in the 8 human solid tumor cell lines. Chamaejasmenin B (the IC₅₀ values ranged from 1.08 to 10.8 μmol/L) was slightly more potent than neochamaejasmin C (the IC₅₀ values ranged from 3.07 to 15.97 μmol/L). In the most sensitive A549 and KHOS cells, the mechanisms underlying the anti-proliferative effects were characterized. The two compounds induced prominent expression of the DNA damage marker γ-H2AX as well as apoptosis. Furthermore, treatment of the cells with the two compounds caused prominent G₀/G₁ phase arrest.

Conclusion:

Chamaejasmenin B and neochamaejasmin C are potential anti-proliferative agents in 8 human solid tumor cell lines in vitro via inducing cell cycle arrest, apoptosis and DNA damage.     

Renal toxicity of ethylene glycol results from internalization of calcium oxalate crystals by proximal tubule cells

Ethylene glycol exposure can lead to the development of renal failure due to the metabolic formation of calcium oxalate monohydrate (COM) crystals. The renal damage is closely linked to the degree of COM accumulation in the kidney and most likely results from a COM-induced injury to proximal tubule (PT) cells. The present studies have measured the binding and internalization of COM by primary cultures of normal PT cells from humans and from Wistar and Fischer-344 rats in order to examine the roles of these uptake processes in the resulting cytotoxicity. Internalization was determined by incubation of cells with [¹⁴C]-COM at 37 °C, removal of bound COM with an EDTA incubation, followed by solubilization of cells, as well as by transmission electron microscopy of COM-exposed cells. COM crystals were internalized by PT cells in time- and concentration-dependent manners. COM crystals were bound to and internalized by rat cells about five times more than by human cells. Binding and internalization values were similar between PT cells from Wistar and Fischer-344 rats, indicating that a differential uptake of COM does not explain the known strain difference in sensitivity to ethylene glycol renal toxicity. Internalization of COM correlated highly with the degree of cell death, which is greater in rat cells than in human cells. Thus, surface binding and internalization of COM by cells play critical roles in cytotoxicity and explain why rat cells are more sensitive to COM crystals. At the same level of COM accumulation after ethylene glycol exposure or hyperoxaluria in vivo, rats would be more susceptible than humans to COM-induced damage.