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1.
Anthrax toxin, the major virulence factor of Bacillus anthracis, consists of three polypeptides: protective antigen (PrAg), lethal factor (LF) and oedema factor (EF). To intoxicate mammalian cells, PrAg binds to its cellular receptors and is subsequently activated via proteolysis, yielding a carboxyl-terminal fragment which coordinately assembles to form heptamers that bind and translocate LF and EF into the cytosol to exert their cytotoxic effects. Substantial progress has been made in recent years towards the characterisation of the structure and function of anthrax toxin, and this has greatly facilitated rational drug design of antianthrax agents. There is also emerging evidence that toxins can be manipulated for cancer therapy. LF can efficiently inactivate several mitogen-activated protein kinase kinases (MAPKKs) via cleavage of their amino-terminal sequences. Consequently, antitumour effects of wild type lethal toxin were observed after treatment of mitogen-activated protein kinase (MAPK)-dependent tumours such as human melanomas. Modification of the toxin's proteolytic activation site limits its cytotoxicity to certain cell types and creates a versatile method of treatment. One approach that has successfully achieved specific tumour targeting is the alteration of the furin cleavage of PrAg so that it is not activated by furin, but, alternatively, by proteases that are highly expressed by tumour tissues, including matrix metalloproteases and urokinase. 相似文献
2.
Newman ZL Sirianni N Mawhinney C Lee MS Leppla SH Moayeri M Johansen LM 《Antimicrobial agents and chemotherapy》2011,55(3):1028-1035
Anthrax lethal toxin (LT) is the major virulence factor for Bacillus anthracis. The lethal factor (LF) component of this bipartite toxin is a protease which, when transported into the cellular cytoplasm, cleaves mitogen-activated protein kinase kinase (MEK) family proteins and induces rapid toxicity in mouse macrophages through activation of the Nlrp1b inflammasome. A high-throughput screen was performed to identify synergistic LT-inhibitory drug combinations from within a library of approved drugs and molecular probes. From this screen we discovered that auranofin, an organogold compound with anti-inflammatory activity, strongly inhibited LT-mediated toxicity in mouse macrophages. Auranofin did not inhibit toxin transport into cells or MEK cleavage but inhibited both LT-mediated caspase-1 activation and caspase-1 catalytic activity. Thus, auranofin inhibited LT-mediated toxicity by preventing activation of the Nlrp1b inflammasome and the downstream actions that occur in response to the toxin. Idebenone, an analog of coenzyme Q, synergized with auranofin to increase its protective effect. We found that idebenone functions as an inhibitor of voltage-gated potassium channels and thus likely mediates synergy through inhibition of the potassium fluxes which have been shown to be required for Nlrp1b inflammasome activation. 相似文献
3.
Alfano RW Leppla SH Liu S Bugge TH Herlyn M Smalley KS Bromberg-White JL Duesbery NS Frankel AE 《Molecular cancer therapeutics》2008,7(5):1218-1226
Anthrax lethal toxin (LeTx) shows potent mitogen-activated protein kinase pathway inhibition and apoptosis in melanoma cells that harbor the activating V600E B-RAF mutation. LeTx is composed of two proteins, protective antigen and lethal factor. Uptake of the toxin into cells is dependent on proteolytic activation of protective antigen by the ubiquitously expressed furin or furin-like proteases. To circumvent nonspecific LeTx activation, a substrate preferably cleaved by gelatinases was substituted for the furin LeTx activation site. Here, we have shown that the toxicity of this matrix metalloproteinase (MMP)-activated LeTx is dependent on host cell surface MMP-2 and MMP-9 activity as well as the presence of the activating V600E B-RAF mutation, making this toxin dual specific. This additional layer of tumor cell specificity would potentially decrease systemic toxicity from the reduction of nonspecific toxin activation while retaining antitumor efficacy in patients with V600E B-RAF melanomas. Moreover, our results indicate that cell surface-associated gelatinase expression can be used to predict sensitivity among V600E B-RAF melanomas. This finding will aid in the better selection of patients that will potentially respond to MMP-activated LeTx therapy. 相似文献
4.
5.
Moayeri M Wiggins JF Lindeman RE Leppla SH 《Antimicrobial agents and chemotherapy》2006,50(8):2658-2665
Bacillus anthracis lethal toxin (LT) produces symptoms of anthrax in mice and induces rapid lysis of macrophages derived from certain inbred strains. LT is comprised of a receptor binding component, protective antigen (PA), which delivers the enzymatic component, lethal factor (LF), into cells. We found that mouse macrophages were protected from toxin by the antitumor drug cis-diammineplatinum (II) dichloride (cisplatin). Cisplatin was shown to inhibit LT-mediated cleavage of cellular mitogen-activated protein kinases (MEKs) without inhibiting LF's in vitro proteolytic activity. Cisplatin-treated PA lost 100% of its ability to function in toxicity assays when paired with untreated LF, despite maintaining the ability to bind to cells. Cisplatin-treated PA was unable to form heptameric oligomers required for LF binding and translocation. The drug was shown to modify PA in a reversible noncovalent manner. Not surprisingly, cisplatin also blocked the actions of anthrax edema toxin and of LF-Pseudomonas aeruginosa exotoxin A fusion peptide (FP59), both of which require PA for translocation. Treatment of BALB/cJ mice or Fischer F344 rats with cisplatin at biologically relevant concentrations completely protected the animals from a coadministered lethal dose of LT. However, treatment with cisplatin 2 hours before or after animals received a lethal bolus of toxin did not protect them. 相似文献
6.
Protection from anthrax toxin-mediated killing of macrophages by the combined effects of furin inhibitors and chloroquine 总被引:1,自引:0,他引:1
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Cell surface proteolytic processing of anthrax protective antigen by furin or other furin-related proteases is required for its oligomerization, endocytosis, and function as a translocon for anthrax lethal and edema factors. Countering toxin lethality is essential to developing effective chemotherapies for anthrax infections that have proceeded beyond the stage at which antibiotics are effective. The primary target for toxin is the macrophage, which can be killed by lethal factor via both necrotic and apoptotic pathways. Here we show that three high-affinity inhibitors of furin efficiently blocked killing of murine J774A.1 macrophages by recombinant protective antigen plus lethal factor: RRD-eglin and RRDG-eglin, developed by engineering the protein protease inhibitor eglin c, and the peptide boronic acid inhibitor acetyl-Arg-Glu-Lys-boroArg pinanediol. Inhibition of killing was dose dependent and correlated with prevention of protective antigen processing. Previous studies have shown that weak bases, such as chloroquine, which neutralize acidic compartments, also interfere with toxin-dependent killing. Here we show that combining furin inhibitors and chloroquine strongly augments the inhibition of toxin-dependent killing, suggesting that combined use of antifurin drugs and chloroquine might provide enhanced therapeutic benefits. Reversible furin inhibitors protected against anthrax toxin killing for at least 5 h, but by 8 h, toxin-dependent killing resumed even though furin inhibitors were still active. An irreversible chloromethylketone inhibitor did not exhibit this loss of protection. 相似文献
7.
Abi-Habib RJ Urieto JO Liu S Leppla SH Duesbery NS Frankel AE 《Molecular cancer therapeutics》2005,4(9):1303-1310
Anthrax lethal toxin, composed of protective antigen and lethal factor, was tested for cytotoxicity to human melanoma cell lines and normal human cells. Eleven of 18 melanoma cell lines were sensitive to anthrax lethal toxin (IC(50) < 400 pmol/L) and 10 of these 11 sensitive cell lines carried the V599E BRAF mutation. Most normal cell types (10 of 15) were not sensitive to anthrax lethal toxin and only 5 of 15 normal human cell types were sensitive to anthrax lethal toxin (IC(50) < 400 pmol/L). These cells included monocytes and a subset of endothelial cells. In both melanoma cell lines and normal cells, anthrax toxin receptor expression levels did not correlate with anthrax lethal toxin cytotoxicity. Furthermore, an anthrax toxin receptor-deficient cell line (PR230) did not show any enhanced sensitivity to anthrax lethal toxin when transfected with anthrax toxin receptor. Anthrax lethal toxin toxicity correlated with elevated phosphorylation levels of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 in both melanoma cell lines and normal cells. Anthrax lethal toxin-sensitive melanoma cell lines and normal cells had higher phospho-MEK1/2 levels than anthrax lethal toxin-resistant melanoma cell lines and normal tissue types. U0126, a specific MEK1/2 inhibitor, was not toxic to anthrax lethal toxin-resistant melanoma cell lines but was toxic to 8 of 11 anthrax lethal toxin-sensitive cell lines. These results show that anthrax lethal toxin toxicity correlates with elevated levels of active MEK1/2 pathway but not with anthrax toxin receptor expression levels in both normal and malignant tissues. Anthrax lethal toxin may be a useful therapeutic for melanoma patients, especially those carrying the V599E BRAF mutation with constitutive activation of the mitogen-activated protein kinase pathway. 相似文献
8.
9.
Anthrax toxin blocks priming of neutrophils by lipopolysaccharide and by muramyl dipeptide 总被引:5,自引:0,他引:5
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We studied the pretreatment of human polymorphonuclear neutrophils (PMN) with purified preparations of the anthrax toxin components--protective antigen (PA), edema factor (EF), and lethal factor (LF)--and their effects on release of superoxide anion (O-2) after stimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). PMN isolated in the absence of lipopolysaccharide (LPS) (less than 0.1 ng/ml) released only small amounts of O-2 after FMLP stimulation; pretreatment with anthrax toxin had little effect. The release of O-2 was increased fivefold by prior treatment with 3 ng/ml LPS for 1 h at 37 degrees C, an effect referred to as priming. PMN were primed to an equivalent extent by treatment with 100 ng/ml N-acetyl-muramyl-L-alanyl-D-isoglutamine (muramyl dipeptide [MDP]). Pretreatment of PMN with anthrax toxin components PA plus EF or PA plus LF inhibited priming by LPS or MDP, as shown by the reduction in the release of O-2 up to 90% relative to controls not treated with toxin; single toxin components were inactive. The inhibition was markedly reduced when priming with LPS or MDP was carried out before exposure to toxin. O-2 release after stimulation by phorbol myristate acetate was not increased by priming, and pretreatment with toxin did not inhibit O-2 release after this stimulus. Evidently, anthrax toxin inhibits the priming that is normally induced in PMN by bacterial products and is necessary for the full expression of antimicrobial effects. 相似文献
10.
Ding Y Boguslawski EA Berghuis BD Young JJ Zhang Z Hardy K Furge K Kort E Frankel AE Hay RV Resau JH Duesbery NS 《Molecular cancer therapeutics》2008,7(3):648-658
We hypothesized that signaling through multiple mitogen-activated protein kinase (MAPK) kinase (MKK) pathways is essential for the growth and vascularization of soft-tissue sarcomas, which are malignant tumors derived from mesenchymal tissues. We tested this using HT-1080, NCI, and Shac fibrosarcoma-derived cell lines and anthrax lethal toxin (LeTx), a bacterial toxin that inactivates MKKs. Western blots confirmed that LeTx treatment reduced the levels of phosphorylated extracellular signal-regulated kinase and p38 MAPK in vitro. Although short treatments with LeTx only modestly affected cell proliferation, sustained treatment markedly reduced cell numbers. LeTx also substantially inhibited the extracellular release of angioproliferative factors including vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor. Similar results were obtained with cell lines derived from malignant fibrous histiocytomas, leiomyosarcomas, and liposarcomas. In vivo, LeTx decreased MAPK activity and blocked fibrosarcoma growth. Growth inhibition correlated with decreased cellular proliferation and extensive necrosis, and it was accompanied by a decrease in tumor mean vessel density as well as a reduction in serum expression of angioproliferative cytokines. Vital imaging using high-resolution ultrasound enhanced with contrast microbubbles revealed that the effects of LeTx on tumor perfusion were remarkably rapid (<24 h) and resulted in a marked reduction of perfusion within the tumor but not in nontumor tissues. These results are consistent with our initial hypothesis and lead us to propose that MKK inhibition by LeTx is a broadly effective strategy for targeting neovascularization in fibrosarcomas and other similar proliferative lesions. 相似文献
11.
TNF-α mediated suppression of tissue type plasminogen activator expression in vascular endothelial cells is NF-κB- and p38 MAPK-dependent 总被引:1,自引:0,他引:1
E. ULFHAMMER P. LARSSON L. KARLSSON T. HRAFNKELSDÓTTIR M. BOKAREWA† A. TARKOWSKI† S. JERN 《Journal of thrombosis and haemostasis》2006,4(8):1781-1789
12.
Abboud N Chow SK Saylor C Janda A Ravetch JV Scharff MD Casadevall A 《The Journal of experimental medicine》2010,207(11):2395-2405
One important function of humoral immunity is toxin neutralization. The current view posits that neutralization results from antibody-mediated interference with the binding of toxins to their targets, a phenomenon viewed as dependent only on antibody specificity. To investigate the role of antibody constant region function in toxin neutralization, we generated IgG2a and IgG2b variants of the Bacillus anthracis protective antigen-binding IgG1 monoclonal antibody (mAb) 19D9. These antibodies express identical variable regions and display the same specificity. The efficacy of antibody-mediated neutralization was IgG2a > IgG2b > IgG1, and neutralization activity required competent Fcγ receptor (FcγR). The IgG2a mAb prevented lethal toxin cell killing and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase cleavage more efficiently than the IgG1 mAb. Passive immunization with IgG1 and IgG2a mAb protected wild-type mice, but not FcγR-deficient mice, against B. anthracis infection. These results establish that constant region isotype influences toxin neutralization efficacy of certain antibodies through a mechanism that requires engagement of FcγR. These findings highlight a new parameter for evaluating vaccine responses and the possibility of harnessing optimal FcγR interactions in the design of passive immunization strategies. 相似文献
13.
目的探讨尿激酶型纤溶酶原激活物(urokinaseplasminogenactivator,uPA)在子宫内膜异位症发病机制中的作用。方法应用免疫组织化学方法检测40例子宫内膜异位症患者异位内膜及在位内膜、30例正常子宫内膜组织(对照组)中uPA蛋白表达水平。结果uPA蛋白主要定位于细胞质,异位内膜、在位内膜、对照组腺上皮细胞及间质细胞均表达uPA蛋白,且在血管内皮细胞呈阳性表达;异位内膜间质细胞uPA阳性表达率为85.0%,高于异位内膜腺上皮细胞(60.0%)(P〈0.05),在位内膜间质细胞(57.5%)(P〈0.05)及正常内膜间质细胞(40.0%)(P〈0.05);uPA蛋白在异位内膜I~Ⅱ期与Ⅲ~Ⅳ期的表达差异无统计学意义(P〉0.05)。结论uPA在异位内膜间质细胞的高表达促使其转移、黏附、侵袭、生长,导致子宫内膜异位症的发生、发展。 相似文献
14.
Gaobing Wu Yuzhi Hong Aizhen Guo Chunfang Feng Sha Cao Cheng-Cai Zhang Ruiping Shi Yadi Tan Ziduo Liu 《Antimicrobial agents and chemotherapy》2010,54(11):4750-4757
Effective measures for the prophylaxis and treatment of anthrax are still required for counteracting the threat posed by inhalation anthrax. In this study, we first demonstrated that the chimeric protein LFn-PA, created by fusing the protective antigen (PA)-binding domain of lethal factor (LFn) to PA, retained the functions of the respective molecules. On the basis of this observation, we attempted to develop an antitoxin that targets the binding of lethal factor (LF) and/or edema factor (EF) to PA and the transportation of LF/EF. Therefore, we replaced PA in LFn-PA with a dominant-negative inhibitory PA (DPA), i.e., PAF427D. In in vitro models of anthrax intoxication, the LFn-DPA chimera showed 3-fold and 2-fold higher potencies than DPA in protecting sensitive cells against anthrax lethal toxin (LeTx) and edema toxin (EdTx), respectively. In animal models, LFn-DPA exhibited strong potency in rescuing mice from lethal challenge with LeTx. We also evaluated the immunogenicity and immunoprotective efficacy of LFn-DPA as an anthrax vaccine candidate. In comparison with recombinant PA, LFn-DPA induced significantly higher levels of the anti-PA immune response. Moreover, LFn-DPA elicited an anti-LF antibody response that could cross-react with EF. Mice immunized with LFn-DPA tolerated a LeTx challenge that was 5 times its 50% lethal dose. Thus, LFn-DPA represents a highly effective trivalent vaccine candidate for both preexposure and postexposure vaccination. Overall, we have developed a novel and dually functional reagent for the prophylaxis and treatment of anthrax.Inhalational anthrax, caused by inhalation of the adversity-resistant spores, is a fatal disease, with a mortality rate approaching 80% (30). Although the naturally occurring inhalational form of anthrax is rare, malicious release of anthrax spores, particularly as weaponized anthrax spores, in a bioterrorism event kills civilians as well as creates great panic. This has stimulated the search for effective methods for the therapy and prevention of anthrax.The principal virulence factors of Bacillus anthracis consist of an antiphagocytic capsule composed of poly-d-glutamic acid (PGA) and a secreted bacterial toxin. The former is encoded by genes located on plasmid pXO1, and the latter is encoded by plasmid pXO2 (47). The anthrax toxin, which is predominantly responsible for the etiology of anthrax, belongs to the family of bacterial binary AB-type toxins, which consist of a receptor-binding B subunit known as the protective antigen (PA) and two catalytic A subunits, i.e., the lethal factor (LF) and edema factor (EF). PA combines with either LF or EF to form the lethal toxin (LeTx) and edema toxin (EdTx), respectively (47). Currently, the standard approach for anthrax therapy is to kill the germinating bacilli by administering aggressive antibiotics. However, antibiotic therapy is ineffective once systematic anthrax symptoms appear because by that time, fatal concentrations of the anthrax toxin have accumulated in the patient''s body (41). Moreover, the emergence of antibiotic-resistant strains as a result of natural evolution or intentional modification by genetic engineering also poses a new challenge to traditional antibiotic treatment (13, 14). Therefore, the development of an antitoxin for combined use with antibiotic therapy is of high priority.At present, the process by which anthrax toxins enter cells and act is relatively well understood. Initially, the B subunit, i.e., the 83-kDa PA (PA83), binds to specific cell surface receptors through its C-terminal binding domain, and this is then proteolytically cleaved by furin or furin-like protease into a 20-kDa N-terminal fragment (PA20) and an active 63-kDa C-terminal fragment (PA63) (5, 15, 19, 28). After dissociation of PA20, cell-bound PA63 self-assembles into a ring-shaped homo-oligomer (heptamer or octamer) termed a prepore (18, 52). Simultaneously, the prepore competitively binds up to three molecules of LF and/or EF to form toxin complexes (9, 23, 33). These complexes are then internalized into the cells by receptor-mediated endocytosis and delivered to an endosome, where the acidic pH triggers the conformational transition of the prepore to generate the pore (31). Ultimately, LF and EF are translocated through the pore into the cytosol, where they exert their respective catalytic effects, leading to the manifestation of the anthrax symptoms (32).The elucidation of the molecular mechanism of anthrax toxin action has provided us with new strategies for developing antitoxins for anthrax treatment. To date, several potential antitoxins that target different steps of anthrax toxin intoxication are under development (37). The PA-binding domain of LF (LFn) or LFn-based fusion proteins is sufficient for binding to the PA63 formed prepore and can inhibit the anthrax toxin by competitively inhibiting the binding of LF to the prepore (1, 3, 20, 34). Another powerful antitoxin is the dominant-negative mutant of PA (DPA), which can be proteolytically activated to form dominant-negative inhibitory PA63 (DPA63). DPA63 coassembles with wild-type PA63 and blocks its ability to translate LF and EF (42, 46). On the basis of these findings, we decided to combine the competitive inhibitory activity of LFn and the dominant-negative inhibitory activity of DPA into a single-component reagent. Toward this end, we first constructed the chimera LFn-PA and demonstrated that this chimera could be proteolytically activated to produce an active PA63 moiety and a functional LFn-PA20 part. We then further replaced the PA moiety with a dominant-negative mutation, PAF427D, which has been described in sufficient detail elsewhere, to generate LFn-DPA (6, 21, 46). We next investigated the antitoxin potency of LFn-DPA in vitro and in vivo.Vaccination with the anthrax vaccine is the best strategy for preexposure prevention and postexposure prophylaxis (45). Now the only licensed anthrax vaccine for human use in the United States is the anthrax vaccine absorbed (AVA), also known as BioThrax. AVA has several disadvantages, such as batch-to-batch variation, an ill-defined composition, side effects, and a lengthy vaccination schedule (47). A second generation of vaccines based on highly purified recombinant PA (rPA) is under development. Although well-defined and homogeneous, rPA-based vaccines have immunogenicity and potency similar to those of AVA (17). This means that vaccination with rPA-based vaccines may be burdensome, just like vaccination with AVA. This underscores the need for developing new and more effective anthrax vaccines. Therefore, we evaluated the potential of LFn-DPA in anthrax prophylaxis. 相似文献
15.
Clinical significance of urokinase-type plasminogen activator receptor (uPAR) expression in cancer 总被引:9,自引:0,他引:9
The involvement of the urokinase-type plasminogen activator (uPA) system in particular has been extensively studied in the pathogenesis of cancer. The molecular role of the uPA receptor (uPAR) is well characterized with its participation in cell migration and extracellular matrix (ECM) degradation. Over-expression of uPAR in cancer has been demonstrated in many studies and is considered an attractive target for anticancer agents. We and others have down-regulated uPAR expression in an attempt to inhibit cancer metastasis based on its molecular role. Uniquely, uPAR which is a glycosyl phosphatidylinositol anchored protein is not only bound to the cell surface but also has a soluble form, suPAR. There is now accumulated clinical and experimental evidence supporting the significant role of uPAR and its soluble counterpart in a number of solid cancers. The expression of uPAR can be associated with tumor cells or stromal cells or both. Differences observed in the expression of uPAR using immunohistochemistry (IHC) are likely explained by the use of different antibodies and techniques rather than true cellular differences and are reviewed here. This review summarizes the clinical relevance of uPAR and its soluble form in the prognosis and diagnosis of different cancers. 相似文献
16.
Pertussis toxin-sensitive G proteins as mediators of the signal transduction pathways activated by cytomegalovirus infection of smooth muscle cells. 总被引:5,自引:0,他引:5
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T Shibutani T M Johnson Z X Yu V J Ferrans J Moss S E Epstein 《The Journal of clinical investigation》1997,100(8):2054-2061
We demonstrated recently that the arachidonic acid (AA) cascade is involved in cytomegalovirus (CMV)-induced generation of reactive oxygen species (ROS) and the activation of nuclear factor (NF)-kappaB in human smooth muscle cells (SMCs). Since AA release from neutrophils is mediated by pertussis toxin (PTx)-sensitive guanine nucleotide-binding (G) proteins, we hypothesized by analogy that CMV stimulates ROS generation in SMCs and ultimately activates NF-kappaB via a PTx-sensitive G protein-coupled pathway. Our first test of this hypothesis demonstrated that PTx blocked AA release induced by CMV infection of SMCs, as well as blocked the terminal products of this reaction, ROS generation and NF-kappaB activation. More proximal components of the pathway were then examined. CMV infection increased phosphorylation and activity of cytosolic phospholipase A2 (cPLA2), an enzyme causing AA release; these effects were inhibited by PTx. CMV infection activated mitogen-activated protein (MAP) kinase, a key enzyme for cPLA2 phosphorylation, an effect also inhibited by PTx. Finally, inhibition of MAP kinase kinase (MAPKK), which phosphorylates and thereby activates MAP kinase, inhibited CMV-induced ROS generation. These data demonstrate that a PTx-sensitive G protein-dependent signaling pathway mediates cellular effects of CMV infection of SMCs. The downstream events include phosphorylation and activation of MAP kinase by MAPKK and subsequent phosphorylation and activation of cPLA2 (with its translocation to cell membranes), followed by stimulation of the AA cascade, which generates intracellular ROS and thereby activates NF-kappaB. 相似文献
17.
Inhibitors of protein kinases: CGP 41251, a protein kinase inhibitor with potential as an anticancer agent. 总被引:4,自引:0,他引:4
D Fabbro E Buchdunger J Wood J Mestan F Hofmann S Ferrari H Mett T O'Reilly T Meyer 《Pharmacology & therapeutics》1999,82(2-3):293-301
CGP 41251 was originally identified as an inhibitor of protein kinase C (PKC), inhibiting mainly the conventional PKC subtypes, and subsequently shown to inhibit the vascular endothelial growth factor (VEGF) receptor kinase insert domain-containing receptor, which is involved in angiogenesis. CGP 41251 inhibits reversibly intracellular PKC activity, induction of c-fos and the corresponding activation of the mitogen-activated protein kinase induced by either tumor promoting phorbol esters, platelet-derived growth factor, or basic fibroblast growth factor, but not by the epidermal growth factor. CGP 41251 inhibited the ligand-induced autophosphorylation of the receptors for platelet-derived growth factor, stem cell factor, and VEGF (kinase insert domain-containing receptor) that correlated with the inhibition of the mitogen-activated protein kinase activation, but did not affect the ligand-induced autophosphorylation of the receptors for insulin, insulin-like growth factor-I, or epidermal growth factor. CGP 41251 showed broad antiproliferative activity against various tumor and normal cell lines in vitro, and is able to reverse the p-glycoprotein-mediated multidrug resistance of tumor cells in vitro. CGP 41251 showed in vivo antitumor activity as single agent and inhibited angiogenesis in vivo. Thus, CGP 41251 may suppress tumor growth by inhibiting tumor angiogenesis (via its effects on the VEGF receptor tyrosine kinases) in addition to directly inhibiting tumor cell proliferation (via its effects on PKCs). 相似文献
18.
Induction of potent TRAIL-mediated tumoricidal activity by hFLEX/Furin/TRAIL recombinant DNA construct. 总被引:1,自引:0,他引:1
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to exert selectively cytotoxic activity against many tumor cells but not normal cells. On the other hand, the ligand for the receptor tyrosine kinase Fms-like tyrosine kinase 3 (Flt3L) is a growth factor for hematopoietic progenitors and is a potent stimulating factor for dendritic and NK cells. Previously, we have demonstrated that it is possible to inhibit the outgrowth of primary tumors by the administration of an hFlex (the extracellular domain of the Flt3L) and TRAIL (amino acid residues 95-281) secreted fusion protein. Here, we report that by the insertion of a linker sequence encoding the cleavage site for the Golgi-expressed endoprotease furin between the DNA sequences encoding hFlex and TRAIL, the tumoricidal activity of the cleaved TRAIL protein generated was greatly enhanced in comparison to the hFlex/TRAIL fusion protein. Furthermore, we demonstrate that intratumoral injection of the hFlex/furin/TRAIL DNA, in conjunction with cationic liposomes, significantly suppressed the outgrowth of the human CNE-2 nasopharyngeal tumor xenografts in SCID mice. In situ histological examinations confirmed the expression of TRAIL in the treated tumor nodules and the induction of apoptosis was also evidenced by the presence of numerous pyknotic nuclei. 相似文献
19.
Te-Hui Liu Jon Oscherwitz Bruce Schnepp Jana Jacobs Fen Yu Kemp B Cease Philip R Johnson 《Molecular therapy》2009,17(2):373-379
Bacillus anthracis represents a formidable bioterrorism and biowarfare threat for which new vaccines are needed with improved safety and efficacy over current options. Toward this end, we created recombinant adeno-associated virus type 1 (rAAV1) vectors containing synthetic genes derived from the protective antigen (PA) or lethal factor (LF) of anthrax lethal toxin (LeTx) and tested them for immunogenicity and induction of toxin-neutralizing antibodies in rabbits. Codon-optimized segments encoding activated PA (PA63), or LF, were synthesized and cloned into optimized rAAV1 vectors containing a human cytomegalovirus (hCMV) promoter and synthetic optimized leader. Serum from rabbits immunized intramuscularly with rAAV1/PA (monovalent), rAAV1/LF (monovalent), rAAV1/PA + rAAV1/LF (bivalent), or rAAV1/enhanced green fluorescent protein (control) exhibited substantial PA- and LF-specific antibody responses at 4 weeks by both western blot (> 1:10,000 dilution) and enzyme-linked immunosorbent assay (ELISA) (mean end-point titer: 32,000–260,000), and contained anthrax LeTx–neutralizing activity in vitro, with peak titers approximating those of a rabbit hyperimmune antisera raised against soluble PA and LF. Compared to the monovalent groups (rAAV1/PA or rAAV1/LF), the bivalent group (rAAV1/PA + rAAV1/LF) exhibited marginally higher ELISA and neutralization activity with dual specificity for both PA and LF. The finding of robust neutralizing antibody responses after a single injection of these rAAV1-based vectors supports their further development as candidate anthrax vaccines. 相似文献
20.
Djouder N Aneiros E Cavalié A Aktories K 《The Journal of pharmacology and experimental therapeutics》2003,304(3):1243-1250
Using Rho GTPases-inhibiting clostridial cytotoxins, we showed recently in RBL cells that the GTPase Rac is involved in FcepsilonRI (high-affinity receptor for IgE) signaling and receptor-mediated calcium mobilization, including influx via calcium release-activated calcium channels. Here, we studied the role of Rho GTPases in muscarinic M1 receptor signaling in RBL 2H3-hm1 cells. Clostridium difficile toxin B, which inactivates Rho, Rac, and Cdc42, and Clostridium sordellii lethal toxin, which inhibits Rac but not Rho, blocked M1-mediated exocytosis, indicating that Rac but not Rho is involved in the regulation of receptor-mediated exocytosis. Although antigen-induced FcepsilonRI stimulation caused tyrosine phosphorylation of the Rac guanine nucleotide exchange factor Vav, M1 stimulation by carbachol activated Rac independently of Vav. The Rac-inactivating toxins blocked M1 receptor-induced membrane translocation of the pleckstrin homology domain of protein kinase B, which is a phosphoinositide 3-kinase effector. The M1-induced calcium release from internal stores was not affected by toxin B; however, the subsequent calcium influx from the extracellular space was inhibited. The data suggest that besides capacitative calcium entry, the M1 signaling pathway activates further calcium entry channels with mechanisms that are not affected by the inhibition of Rac. 相似文献