Preclinical evaluation of 4-methylthiobutyl isothiocyanate on liver cancer and cancer stem cells with different p53 status.

Isothiocyanates from plants of the order Brassicales are considered promising cancer chemotherapeutic phytochemicals. However, their selective cytotoxicity on liver cancer has been barely researched. Therefore, in the present study, we systematically studied the chemotherapeutic potency of 4-methylthiobutyl isothiocyanate (MTBITC). Selective toxicity was investigated by comparing its effect on liver cancer cells and their chemoresistant subpopulations to normal primary hepatocytes and liver tissue slices. Additionally, in a first assessment, the in vivo tolerability of MTBITC was investigated in mice. Growth arrest at G2/M and apoptosis induction was evident in all in vitro cancer models treated with MTBITC, including populations with cancer initiating characteristics. This was found independent from TP53; however cell death was delayed in p53 compromised cells as compared to wt-p53 cells which was probably due to differential BH3 only gene regulation i. e. Noxa and its antagonist A1. In normal hepatocytes, no apoptosis or necrosis could be detected after repeated administration of up to 50 µM MTBITC. In mice, orally applied MTBITC was well tolerated over 18 days of treatment for up to 50 mg/kg/day, the highest dose tested. In conclusion, we could show here that the killing effect of MTBITC has a definite selectivity for cancer cells over normal liver cells and its cytotoxicity even applies for chemoresistant cancer initiating cells. Our study could serve for a better understanding of the chemotherapeutic properties of isothiocyanates on human liver-derived cancer cells.

Modification of plant Rac/Rop GTPase signalling using bacterial toxin transgenes.

Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co-expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive-like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co-expressed constitutively active Rop4, blocking the hypersensitive response caused by over-expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop-dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop-dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.

In vitro cytotoxicity of cyclodextrin-bonded birch bark extract.

Triterpenoids from birch bark, like betulin, seem to have an anticancer potential which needs to be further investigated. Aim of this study was first to explore whether a cyclodextrin-solubilised triterpenoid extract (STE) from birch bark induces selective cytotoxic effects in primary liver cancer cells compared to healthy human hepatocytes. Second, selective cytotoxicity against several tumour cell lines should be analysed. For this purpose, human liver cancer cells derived from mouse xenografts (LIXF 575), healthy human hepatocytes, and 42 different human tumour cell lines were incubated with different concentrations of STE corresponding to 4.3 µM - 137.5 µM betulin (BE). Cytotoxicity was tested with the WST-1 cell proliferation assay, apoptosis with caspase 3/7-activity, and necrosis was determined by the propidiumiodid uptake assay. The pathway of cytotoxic effects was further investigated by immunoblotting of apoptosis inducing factor (AIF) and p53. The monolayer assay was used to analyse selectivity of STE towards different tumour cell lines. STE significantly (p < 0.001) reduced viability and induced apoptosis of LIXF cells in low concentrations corresponding to 8.6 µM BE, while human hepatocytes were affected only in concentrations ≥ 68.8 µM. Cell death occurred in a p53 independent manner, and AIF was not involved. The mean IC50 in the 42 tumour cell lines corresponded to 4.3 µM BE and ranged from 2.05 µM to 8.95 µM BE content. Selectivity was, therefore, rather low. In conclusion, STE exhibits in low concentrations cytotoxicity in a broad spectrum of primary cancer cells and cancer cell lines, which is, at least in LIXF cells, induced by caspase 3/7 mediated apoptosis. STE is far less toxic in hepatocytes. The anticancer potential of STE should be further characterised and also investigated in animal models.

Primary resistance to cetuximab in a panel of patient-derived tumour xenograft models: activation of MET as one mechanism for drug resistance.

Cetuximab (Erbitux®) targets the epidermal growth factor receptor (EGFR) and is approved for treatment of colorectal and head and neck cancer. Despite wide expression of EGFR, only a subgroup of cancer patients responds to cetuximab therapy. In the present study we assessed the cetuximab response in vivo of 79 human patient-derived xenografts originating from five tumour histotypes. We analysed basic tumour characteristics including EGFR expression and activation, mutational status of KRAS, BRAF and NRAS, the expression of EGFR ligands and the activation of HER3 (ErbB3) and the hepatocyte growth factor receptor MET. Based on these results, a cetuximab response score including positive and negative factors affecting therapeutic response is proposed. Positive factors are high expression and activation of EGFR and its ligands epiregulin or amphiregulin, negative factors are markers for downstream pathway activation independent of EGFR. In cetuximab resistant NSCL adenocarcinoma LXFA 526 and LXFA 1647, overexpression due to gene amplification and strong activation of MET was identified. Knock-down of MET by siRNA in the corresponding cell lines showed that anchorage-independent growth and migration are dependent on MET. MET knock down sensitized LXFA 526L and LXFA 1647L to EGF. Combined treatments of a MET inhibitor and cetuximab were additive. Therefore, combination therapy of cetuximab and a MET inhibitor in selected lung cancer patients could be of high clinical significance.

Autocatalytic processing of Clostridium difficile toxin B. Binding of inositol hexakisphosphate.

Clostridium difficile toxins A and B are major virulence factors responsible for induction of pseudomembranous colitis and antibiotic-associated diarrhea in men. The toxins possess a multidomain structure and only the N-terminal glucosyltransferase domain, which inactivates Rho GTPases by glucosylation, is translocated into the cytosol of target cells. Processing of the toxin occurs by autocatalytic cleavage and is activated by inositol hexakisphosphate (InsP6). Here we studied the inherent protease activity in fragments of toxin B and determined the site of toxin B that interacts with InsP6. We report that a fragment of toxin B, comprised of residues 1-955, is cleaved in the presence of InsP6. In contrast, mutants of the catalytic triad of the putative cysteine protease domain did not cleave this fragment. [3H]InsP6 bound to holotoxin B and to the fragment 1-955, but not to a fragment comprising residues 900-2366 or the glucosyltransferase domain (residues 1-544). Binding to the putative cysteine protease domain (residues 544-955) was also observed. InsP6-binding was specific and saturable. Isothermal titration calorimetry revealed a Kd value of 2.4 microm for binding of InsP6 to toxin fragment 544-955 with a stoichiometry factor of 0.86. Lysine 600 of toxin B was identified as essential amino acid for InsP6 binding and for InsP6-dependent activation of the protease activity.

Processing of Clostridium difficile toxins.

The pathogenicity of Clostridium difficile depends on the large clostridial glucosylating toxins A and B (TcdA and TcdB). The proteins accomplish their own uptake by a modular structure comprising a catalytic and a binding/translocation domain. Based on a proteolytic processing step solely the catalytic domain reaches the cytosol. Within the cells, the glucosyltransferases inactivate small GTPases by mono-O-glucosylation. Here, a short overview is given regarding latest insights into the intramolecular processing, which is mediated by an intrinsic protease activity.

Human alpha-defensins inhibit Clostridium difficile toxin B.

BACKGROUND & AIMS: Clostridium difficile toxins A and B are major virulence factors implicated in pseudomembranous colitis and antibiotic-associated diarrhea. The toxins are glucosyltransferases, which inactivate Rho proteins involved in cellular signaling. Human alpha-defensins as part of the innate immune system inactivate various microbial pathogens as well as specific bacterial exotoxins. Here, we studied the effects of alpha-defensins human neutrophil protein (HNP)-1, HNP-3, and enteric human defensin (HD)-5 on the activity of C difficile toxins A and B. METHODS: Inactivation of C difficile toxins by alpha-defensins in vivo was monitored by microscopy, determination of the transepithelial resistance of CaCo-2 cell monolayers, and analysis of the glucosylation of Rac1 in toxin-treated cells. In vitro glucosylation was used to determine K(m) and median inhibitory concentration (IC(50)) values. Formation of defensin-toxin complexes was analyzed by precipitation and turbidity studies. RESULTS: Treatment of cells with human alpha-defensins caused loss of cytotoxicity of toxin B, but not of toxin A. Only alpha-defensins, but not beta-defensin-1 or cathelicidin LL-37, inhibited toxin B-catalyzed in vitro glucosylation of Rho guanosine triphosphatases in a competitive manner, increasing K(m) values for uridine 5'-diphosphate-glucose up to 10-fold. The IC(50) values for inhibition of toxin B-catalyzed glucosylation by the alpha-defensins were 0.6-1.5 micromol/L. At high concentrations, defensins (HNP-1 > or = 2 micromol/L) caused high-molecular-mass aggregates, comparable to Bacillus anthracis protective antigen and lethal factor. CONCLUSION: Our data indicate that toxin B interacts with high affinity with alpha-defensins and suggest that defensins may provide a defense mechanism against some types of clostridial glucosylating cytotoxins.

Clostridium difficile glucosyltransferase toxin B-essential amino acids for substrate binding.

Recently the crystal structure of the catalytic domain of Clostridium difficile toxin B was solved ( Reinert, D. J., Jank, T., Aktories, K., and Schulz, G. E. (2005) J. Mol. Biol. 351, 973-981 ). On the basis of this structure, we studied the functional role of several amino acids located in the catalytic center of toxin B. Besides the (286)DXD(288) motif and Trp(102), which were shown to be necessary for Mn(2+) and UDP binding, respectively, we identified by alanine scanning Asp(270), Arg(273), Tyr(284), Asn(384), and Trp(520) as being important for enzyme activity. The amino acids Arg(455), Asp(461), Lys(463), and Glu(472) and residues of helix alpha17 (e.g. Glu(449)) of toxin B are essential for enzyme-protein substrate recognition. Introduction of helix alpha17 of toxin B into Clostridium sordellii lethal toxin inhibited modification of Ras subfamily proteins but enabled glucosylation of RhoA, indicating that helix alpha17 is involved in RhoA recognition by toxin B. The data allow the design of a model of the interaction of the glucosyltransferase domain of toxin B with its protein substrate RhoA.

Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity.

The action of Clostridium difficile toxins A and B depends on processing and translocation of the catalytic glucosyltransferase domain into the cytosol of target cells where Rho GTPases are modified. Here we studied the processing of the toxins. Dithiothreitol and beta-mercaptoethanol induced auto-cleavage of purified native toxin A and toxin B into approximately 250/210- and approximately 63-kDa fragments. The 63-kDa fragment was identified by mass spectrometric analysis as the N-terminal glucosyltransferase domain. This cleavage was blocked by N-ethylmaleimide or iodoacetamide. Exchange of cysteine 698, histidine 653, or aspartate 587 of toxin B prevented cleavage of full-length recombinant toxin B and of an N-terminal fragment covering residues 1-955 and inhibited cytotoxicity of full-length toxin B. Dithiothreitol synergistically increased the effect of myo-inositol hexakisphosphate, which has been reported to facilitate auto-cleavage of toxin B (Reineke, J., Tenzer, S., Rupnik, M., Koschinski, A., Hasselmayer, O., Schrattenholz, A., Schild, H., and Von Eichel-Streiber, C. (2007) Nature 446, 415-419). N-Ethylmaleimide blocked auto-cleavage induced by the addition of myo-inositol hexakisphosphate, suggesting that cysteine residues are essential for the processing of clostridial glucosylating toxins. Our data indicate that clostridial glucosylating cytotoxins possess an inherent cysteine protease activity related to the cysteine protease of Vibrio cholerae RTX toxin, which is responsible for auto-cleavage of glucosylating toxins.

Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function.

Clostridium difficile causes pseudomembranous colitis and is responsible for many cases of nosocomial antibiotic-associated diarrhea. Major virulence factors of C. difficile are the glucosylating exotoxins A and B. Both toxins enter target cells in a pH- dependent manner from endosomes by forming pores. They translocate the N-terminal catalytic domains into the cytosol of host cells and inactivate Rho guanosine triphosphatases by glucosylation. The crystal structure of the catalytic domain of toxin B was solved in a complex with uridine diphosphate, glucose, and manganese ion, exhibiting a folding of type A family glycosyltransferases. Crystallization of fragments of the C-terminus of toxin A, which is characterized by polypeptide repeats, revealed a solenoid-like structure often found in bacterial cell surface proteins. These studies, which provide new insights into structure, uptake, and function of the family of clostridial glucosylating toxins, are reviewed.

Insulin and insulin-like growth factor-1 promote mast cell survival via activation of the phosphatidylinositol-3-kinase pathway.

OBJECTIVE: Mast cells (MCs) play central roles for the onset and development of immediate-type and inflammatory allergic reactions. Since the inverse relationship between atopic disorders and diabetes mellitus has been observed in animals and humans, we investigated the effects of insulin (Ins) on MC signaling and biological function. METHODS: In bone marrow-derived MCs (BMMCs) from wild-type as well as SHIP-deficient mice Ins as well as insulin-like growth factor-1 (IGF-I)-triggered intracellular signaling events and MC effector functions were studied. RESULTS: We found that the addition of either Ins or IGF-1 to BMMCs triggers the phosphorylation of protein kinase B (PKB) and p38 kinase but not extracellular signal-regulated kinase (Erk). We also found that Ins/IGF-1 stimulates the tyrosine phosphorylation of SHIP1 and, in keeping with this, Ins/IGF-1-induced PKB phosphorylation is higher in SHIP1-/- BMMCs and is inhibited in SHIP+/+ as well as SHIP1-/- BMMCs with inhibitors of phosphatidylinositol-3-kinase (PI3K). Ins/IGF-1, like antigen (Ag), also stimulates the Rac-dependent activation of PAK as well as the production of hydrogen peroxide (H2O2). To elucidate the role of Ins and IGF-1 in MC biology, we studied their effects on Ag-mediated degranulation and MC survival. Although both only slightly enhanced Ag-mediated degranulation, they significantly promoted MC survival in the absence of IL-3 in a PI3K-dependent manner. CONCLUSION: The promotion of BMMC survival by induction of Ins/IGF-1 signaling may, in part, be responsible for the inverse correlation observed between atopic disorders and diabetes mellitus.

Exchange of a single amino acid switches the substrate properties of RhoA and RhoD toward glucosylating and transglutaminating toxins.

Rho GTPases are the preferred targets of various bacterial cytotoxins, including Clostridium difficile toxins A and B, Clostridium sordellii lethal toxin, the cytotoxic necrotizing factors (CNF1) from Escherichia coli, and the dermonecrotizing toxin (DNT) from Bordetella species. The toxins inactivate or activate specific sets of Rho GTPases by mono-O-glucosylation and deamidation/transglutamination, respectively. Here we studied the structural basis of the recognition of RhoA, which is modified by toxin B, CNF1, and DNT, in comparison with RhoD, which is solely a substrate for lethal toxin. We found that a single amino acid residue in RhoA and RhoD defines the substrate specificity for toxin B and lethal toxin. Change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin. Accordingly, change of the equivalently positioned phenylalanine 85 in RhoD with serine allowed glucosylation by toxin B. Comparable results were achieved with the Rho-activating and transglutaminating enzymes CNF1 and DNT. Here, amino acid glutamate 64 of RhoA and the equivalent aspartate 76 of RhoD define substrate specificity for CNF1 and DNT, respectively. These data indicate that single amino acid residues located in the switch II region of Rho proteins determine enzyme specificity for diverse bacterial toxins.

Cholesterol-dependent pore formation of Clostridium difficile toxin A.

The large clostridial cytotoxins toxin A and toxin B from Clostridium difficile are major virulence factors known to cause antibiotic-associated diarrhea and pseudomembranous colitis. Both toxins mono-glucosylate and thereby inactivate small GTPases of the Rho family. Recently, it was reported that toxin B, but not toxin A, induces pore formation in membranes of target cells under acidic conditions. Here, we reassessed data on pore formation of toxin A in cells derived from human colon carcinoma. Treatment of 86Rb+-loaded cells with native or recombinant toxin A resulted in an increased efflux of radioactive cations induced by an acidic pulse. The efficacy of pore formation was dependent on membrane cholesterol, since cholesterol depletion of membranes with methyl-beta-cyclodextrin inhibited 86Rb+ efflux, and cholesterol repletion reconstituted pore-forming activity of toxin A. Similar results were obtained with toxin B. Consistently, methyl-beta-cyclodextrin treatment delayed intoxication of cells in a concentration-dependent manner. In black lipid membranes, toxin A induced ion-permeable pores only in cholesterol containing bilayers and at low pH. In contrast, release of glycosylphosphatidylinositol-anchored structures by phosphatidylinositol specific phospholipase C treatment did not reduce cell sensitivity toward toxins A and B. These data indicate that in colonic cells toxin A induces pore formation in an acidic environment (e.g. endosomes) similar to that reported for toxin B and suggest that pore formation by clostridial glucosylating toxins depends on the presence of cholesterol.

Change of the donor substrate specificity of Clostridium difficile toxin B by site-directed mutagenesis.

The large cytotoxins of Clostridia species glycosylate and thereby inactivate small GTPases of the Rho family. Clostridium difficile toxins A and B and Clostridium sordellii lethal toxin use UDP-glucose as the donor for glucosylation of Rho/Ras GTPases. In contrast, alpha-toxin from Clostridium novyi N-acetylglucosaminylates Rho GTPases by using UDP-N-acetylglucosamine as a donor substrate. Based on the crystal structure of C. difficile toxin B, we studied the sugar donor specificity of the toxins by site-directed mutagenesis. The changing of Ile-383 and Gln-385 in toxin B to serine and alanine, respectively, largely increased the acceptance of UDP-N-acetylglucosamine as a sugar donor for modification of RhoA. The K(m) value was reduced from 960 to 26 mum for the double mutant. Accordingly, the potential of the double mutant of toxin B to hydrolyze UDP-N-acetylglucosamine was higher than that for UDP-glucose. The changing of Ile-383 and Gln-385 in the lethal toxin of C. sordellii allowed modification of Ras in the presence of UDP-N-acetyl-glucosamine and reduced the acceptance of UDP-glucose as a donor for glycosylation. Vice versa, the changing of the equivalent residues in C. novyi alpha-toxin from Ser-385 and Ala-387 to isoleucine and glutamine, respectively, reversed the donor specificity of the toxin from UDP-N-acetylglucosamine to UDP-glucose. These data demonstrate that two amino acid residues are crucial for the co-substrate specificity of clostridial glycosylating toxins.

Complex formation between the postsynaptic scaffolding protein gephyrin, profilin, and Mena: a possible link to the microfilament system.

Gephyrin is an essential component of the postsynaptic cortical protein network of inhibitory synapses. Gephyrin-based scaffolds participate in the assembly as well as the dynamics of receptor clusters by connecting the cytoplasmic domains of glycine and GABA(A) receptor polypeptides to two cytoskeletal systems, microtubules and microfilaments. Although there is evidence for a physical linkage between gephyrin and microtubules, the interaction between gephyrin and microfilaments is not well understood so far. Here, we show that neuronal gephyrin interacts directly with key regulators of microfilament dynamics, profilin I and neuronal profilin IIa, and with microfilament adaptors of the mammalian enabled (Mena)/vasodilator stimulated phosphoprotein (VASP) family, including neuronal Mena. Profilin and Mena/VASP coprecipitate with gephyrin from tissue and cells, and complex formation requires the E-domain of gephyrin, not the proline-rich central domain. Consequently, gephyrin is not a ligand for the proline-binding motif of profilins, as suspected previously. Instead, it competes with G-actin and phospholipids for the same binding site on profilin. Gephyrin, profilin, and Mena/VASP colocalize at synapses of rat spinal cord and cultivated neurons and in gephyrin clusters expressed in transfected cells. Thus, Mena/VASP and profilin can contribute to the postulated linkage between receptors, gephyrin scaffolds, and the microfilament system and may regulate the microfilament-dependent receptor packing density and dynamics at inhibitory synapses.