4-Acetylantroquinonol B inhibits lipopolysaccharide-induced cytokine release and alleviates sepsis through of MAPK and NFκB suppression.

BACKGROUND: Antrodia cinnamomea is an indigenous medicinal mushroom in Taiwan, commonly used for the treatment of cancers and inflammatory disorders. 4-acetylantroquinonol B (4AAQB) is one of the active component isolated from the mycelium of A. cinnamomea. However, whether 4AAQB exhibits anti-inflammatory effect is not clear. METHODS: The anti-inflammatory activity of 4AAQB was examined by ELISA to measure the pro-inflammatory cytokines production in lipopolysaccharide (LPS)-simulated RAW264.7 cells, peritoneal macrophages and in mice. The effect of 4AAQB for MAPK kinase molecules phosphorylation in LPS-stimulated RAW264.7 macrophage including ERK, JNK and p38 were evaluated. The in vivo efficacy of 4AAQB was also demonstrated. RESULTS: In the present study, we found that 4AAQB exhibits anti-inflammatory effects inhibit tumor necrosis factor-α (TNF-α)/interleukin-6 (IL-6) releasing and LPS-stimulated phagocytes migration without affect cell growth. In addition, the MAPK kinase molecules phosphorylation in LPS-stimulated RAW264.7 macrophage including ERK, JNK and p38 was inhibited by 4AAQB. The phosphorylation of NFκB subunit p65 and IkBα were also decreased after 4AAQB treatment. Furthermore, 4AAQB attenuates the cytokine production in LPS-induced and CLP-induced septic mice. CONCLUSION: These results showed that 4AAQB exhibited anti-inflammatory property both in vitro and in vivo, suggesting that 4AAQB may be a therapeutic candidate which used in inflammatory disorders treatment.

Trowaglerix Venom Polypeptides As a Novel Antithrombotic Agent by Targeting Immunoglobulin-Like Domains of Glycoprotein VI in Platelet.

OBJECTIVE: Currently prescribed antiplatelet drugs have 1 common side effect-an increased risk of hemorrhage and thrombocytopenia. On the contrary, bleeding defects associated with glycoprotein VI (GPVI) expression deficiency are usually slightly prolonged bleeding times. However, GPVI antagonists are lacking in clinic. APPROACH AND RESULTS: Using reverse-phase high-performance liquid chromatography and sequencing, we revealed the partial sequence of trowaglerix α subunit, a potent specific GPVI-targeting snaclec (snake venom C-type lectin protein). Hexapeptide (Troα6 [trowaglerix a chain hexapeptide, CKWMNV]) and decapeptide (Troα10) derived from trowaglerix specifically inhibited collagen-induced platelet aggregation through blocking platelet GPVI receptor. Computational peptide design helped to design a series of Troα6/Troα10 peptides. Protein docking studies on these decapeptides and GPVI suggest that Troα10 was bound at the lower surface of D1 domain and outer surface of D2 domain, which was at the different place of the collagen-binding site and the scFv (single-chain variable fragment) D2-binding site. The newly discovered site was confirmed by inhibitory effects of polyclonal antibodies on collagen-induced platelet aggregation. This indicates that D2 domain of GPVI is a novel and important binding epitope on GPVI-mediated platelet aggregation. Troα6/Troα10 displayed prominent inhibitory effect of thrombus formation in fluorescein sodium-induced platelet thrombus formation of mesenteric venules and ferric chloride-induced carotid artery injury thrombosis model without prolonging the in vivo bleeding time. CONCLUSIONS: We develop a novel antithrombotic peptides derived from trowaglerix that acts through GPVI antagonism with greater safety-no severe bleeding. The binding epitope of polypeptides on GPVI is novel and important. These hexa/decapeptides have therapeutic potential for developing ideal small-mass GPVI antagonists for arterial thrombogenic diseases.

Aggretin Venom Polypeptide as a Novel Anti-angiogenesis Agent by Targeting Integrin alpha2beta1.

VEGF and VEGFR antibodies have been used as a therapeutic strategy to inhibit angiogenesis in many diseases; however, frequent and repeated administration of these antibodies to patients induces immunogenicity. In previous studies, we demonstrated that aggretin, a heterodimeric snake venom C-type lectin, exhibits pro-angiogenic activities via integrin α2ß1 ligation. We hypothesised that small-mass aggretin fragments may bind integrin α2ß1 and act as antagonists of angiogenesis. In this study, the anti-angiogenic efficacy of a synthesised aggretin α-chain C-terminus (AACT, residue 106-136) was evaluated in both in vitro and in vivo angiogenesis models. The AACT demonstrated inhibitory effects on collagen-induced platelet aggregation and HUVEC adhesion to immobilised collagen. These results indicated that AACT may block integrin α2ß1-collagen interaction. AACT also inhibited HUVEC migration and tube formation. Aortic ring sprouting and Matrigel implant models demonstrated that AACT markedly inhibited VEGF-induced neovascularisation. In addition, induction of FAK/PI3K/ERK1/2 tyrosine phosphorylation and talin 1/2 associated with integrin ß1 which are induced by VEGF were blocked by AACT. Similarly, tyrosine phosphorylation of VEFGR2 and ERK1/2 induced by VEGF was diminished in integrin α2-silenced endothelial cells. Our results demonstrate that AACT is a potential therapeutic candidate for angiogenesis related-diseases via integrin α2ß1 blockade.

Improved antithrombotic activity and diminished bleeding side effect of a PEGylated αIIbß3 antagonist, disintegrin.

INTRODUCTION: The applicability of protein drugs is confined by protein degradation and rapid elimination. PEGylation of polypeptides improves protein stability by sterically obstructing the degradation by serum proteases and reduces renal clearance by the increased mass. EXPERIMENTAL APPROACH: We compared the antithrombotic activities of intact rhodostomin (Rn) and PEGylated rhodostomin (PRn) both in vitro and in vivo systems. In addition, the functional half-life in inhibiting platelet aggregation and the tendency in causing bleeding side effect were investigated. RESULTS: Rn and PRn both potently inhibited human and mouse platelet aggregation induced by collagen, thrombin or ADP in vitro with a similar IC50 around 60-100nM. Rotational thromboelastometry assay indicated that PRn was more effective than Rn in preventing clot formation in human whole blood. In platelet-rich plasma from mice injected with Rn or PRn, the inhibitory effects on collagen-induced platelet aggregation were also comparable, but Rn caused higher bleeding tendency. In ferric chloride-induced arterial thrombosis, Rn and PRn significantly prolonged occlusion time at high dosage (0.2µg/g). However, PRn obviously prolonged the occlusion time even given at a lower dosage (0.06µg/g). The functional half-life assay revealed that PEGylation prolonged the in vivo half-life of Rn. CONCLUSIONS: PRn exhibits higher antithrombotic potency and longer half-life in vivo as compared with native Rn on a molar basis. In addition, PRn exhibits a better safety profile at an efficacious antithrombotic dose in vivo. Therefore, PEGylation may be one of the ideal options in modifying disintegrin derivatives as the safe therapeutic agents for integrin-related diseases.

Snake Venom Disintegrin Inhibits the Activation of Toll-Like Receptors and Alleviates Sepsis through Integrin alphaVbeta3 Blockade.

Bacterial infection-induced sepsis is the leading cause of septic inflammatory disease. Rhodostomin (Rn), a snake venom disintegrin, was previously reported to interact with the αVß3 integrin and the TLR4 on phagocyte in attenuating LPS-induced endotoxemia. In this report, we further evaluated the effects of Rn on TLR2-activated monocytes and its in vivo efficacy. Rn effectively suppressed the adhesion, migration, and cytokine release of Pam3CSK4-activated THP-1 cells. Rn specifically bound to integrin αVß3 of TLR2-activated THP-1. Integrin αV and Akt siRNA transfection both restrained Pam3CSK4-elicited cytokine release. Rn decreased the Pam3CSK4-induced phosporylation of MAPKs, degradation of IκB and activation of FAK, Akt, c-Src and Syk. The Pam3CSK4-induced translocation of MyD88, a central adaptor of TLR2, to the cell membrane was also inhibited by Rn treatment. In the polymicrobial inflammatory caecal ligation and puncture model, Rn significantly reduced pro-inflammatory cytokine and chemokine release, alleviated tissue injury and elevated survival rate in vivo. Taken together, in addition to inhibiting the activation of TLR4, Rn exhibits anti-inflammatory activity through antagonizing the activation of phagocytes and interrupting the crosstalk between αVß3 and TLR2-dependent signaling pathways.

4-Acetylantroquinonol B suppresses tumor growth and metastasis of hepatoma cells via blockade of translation-dependent signaling pathway and VEGF production.

Hepatocellular carcinoma (HCC) has become one of most common malignancies and a leading cause of cancer mortality worldwide. Previous study has shown that 4-acetylantroquinonol B (4AAQB) isolated from Antrodia cinnamomea (or niu-chang-chih) was observed to inhibit HepG2 cell proliferation via affecting cell cycle. However, the in vivo effects and antimetastatic activity of 4AAQB have not yet been addressed. This study found that 4AAQB inhibited HepG2 and HuH-7 hepatoma cell growth in both in vitro and in vivo models and exhibited pronounced inhibitory effects on HuH-7 tumor growth in xenograft and orthotopic models. 4AAQB efficiently inhibited the phosphorylation of mTOR and its upstream kinases and the downstream effectors and decreased the production of VEGF and activity of Rho GTPases in HuH-7 cells. Furthermore, 4AAQB inhibited in vitro HuH-7 cell migration and in vivo pulmonary metastasis. The results suggested that 4AAQB is a potential candidate for HCC therapy.

Antirestenosis effect of butein in the neointima formation progression.

The development of restenosis involves migration and hyperproliferation of vascular smooth muscle cells (VSMCs). Platelet-derived growth factor (PDGF) is one of the major factors. Butein modulates inflammatory pathways and affects the proliferation and invasion of the tumor. We investigated the hypothesis that butein might prevent the restenosis process via a similar pathway. Our results demonstrated that butein inhibited PDGF-induced VSMC proliferation and migration as determined by BrdU proliferation and two-dimensional migration scratch assay. Butein also concentration-dependently repressed PDGF-induced phosphorylation of PDGF-receptor ß, mitogen-activated protein kinases, phosphoinositide 3-kinase/Akt, and phopholipase Cγ/c-Src in VSMCs. In addition, in vivo results showed that butein attenuated neointima formation in balloon-injured rat carotid arteries. These results indicate that butein may inhibit PDGF-induced VSMC proliferation and migration, resulting in attenuation of neointima formation after percutaneous transluminal coronary angioplasty. Our study demonstrates for the first time that systemic administration of butein is able to reduce neointima formation after vascular injury.

NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone, a novel antithrombotic agent with dual inhibition of thromboxane A(2) synthesis and calcium entry.

BACKGROUND AND PURPOSE: 1,4-Naphthoquinones exhibit antiplatelet activity both in vivo and in vitro. In the present study, we investigated the antiplatelet effect of a novel naphthoquinone derivative NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone and its mechanism of action. EXPERIMENTAL APPROACH: We measured platelet aggregation, Ca(2+) mobilization, thromboxane B2 formation and P-selectin expression and examined several enzymatic activities. Furthermore, we used the irradiated mesenteric venules in fluorescein sodium-treated mice to monitor the antithrombotic effect of NP-313 in vivo. KEY RESULTS: NP-313 concentration-dependently inhibited human platelet aggregation induced by collagen, arachidonic acid, thapsigargin, thrombin and A23187. NP-313 also inhibited P-selectin expression, thromboxane B(2) formation and [Ca(2+) ](i) elevation in platelets stimulated by thrombin and collagen. NP-313 at 10 µM inhibited cyclooxygenase, thromboxane A(2) synthase, and protein kinase Cα, whereas it did not affect phospholipase A(2) or phospholipase C activity. In the presence of indomethacin and an adenosine 5-diphosphate scavenger, NP-313 concentration-dependently inhibited thrombin- and A23187-induced [Ca(2+)](i) increase through its inhibitory effects on Ca(2+) influx, rather than blocking Ca(2+) release from intracellular stores. NP-313 also inhibited thapsigargin-mediated Ca(2+) influx through store-operated calcium channel but had no effect on Ca(2+) influx through store-independent calcium channel evoked by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol. Nevertheless, it had little effect on cyclic AMP and cyclic GMP levels. Also, intravenously administered NP-313 dose-dependently inhibited the thrombus occlusion of the irradiated mesenteric vessels of fluorescein-pretreated mice. CONCLUSIONS AND IMPLICATIONS: Taken together, these results indicate that NP-313 exerts its antithrombotic activity through dual inhibition of thromboxane A(2) synthesis and Ca(2+) influx through SOCC.

A case of pulmonary aspergilloma and actinomycosis.

Pulmonary aspergilloma and pulmonary actinomycosis are rare pulmonary infectious diseases. Clinical manifestations of pulmonary aspergilloma and pulmonary actinomycosis include chronic cough, fever, chest pain, haemoptysis and other pathologies, but some patients may be asymptomatic. We report a case of a healthy 33-year-old woman without any underlying diseases, who was admitted to Zhongxing Branch of Taipei City Hospital, Taiwan, for intermittent haemoptysis and right upper chest pain, which had persisted for several months. A chest radiograph revealed a focal consolidation in the right upper lobe (RUL) of the lung, which grew in size over time. A sputum study and bronchoscopy revealed no positive findings, although malignancy could not be ruled out. Thus, the patient received a wedge resection of the RUL lesion. Subsequent, pathological examination demonstrated the presence of pulmonary aspergilloma and pulmonary actinomycosis. The patient's symptoms resolved after resection of the RUL lesion.

Pleomorphic hyalinizing angiectatic tumor of soft parts.

Pleomorphic hyalinizing angiectatic tumor (PHAT) of soft parts is a rare, nonmetastasizing tumor of uncertain lineage which was first reported in 1996. Here, we report a case of PHAT and review the literature. A 49-year-old man presented with a soft and progressively enlarging mass over the right buttock for several years. On suspicion that the mass was a right gluteal lipoma, he underwent surgical excision. The excised lesion measured 14 x 6 x 3.5 cm. It had a variegated appearance with a white-tan to yellowish color on the cut surface. Some punctate hemorrhage and vessel thrombosis were seen. Microscopically, the tumor was a PHAT characterized by clusters of ectatic, fibrin-lined, thin-walled vessels, which were surrounded by a mitotically inert, spindled, pleomorphic, neoplastic stroma that contained a variable inflammatory component. Immunohistochemical study showed that the tumor cells were positive for CD34, and negative for S-100, HMB45 and actin. The patient experienced local recurrence 6 months later. The recurrent tumor was widely excised. No evidence of metastasis was found during the 18 months after the second operation. The recurrent lesion had a microscopic appearance that was similar to the initial lesion.

The integrin alpha2beta1 agonist, aggretin, promotes proliferation and migration of VSMC through NF-kB translocation and PDGF production.

BACKGROUND AND PURPOSE: During the development of atherosclerotic plaques, vascular smooth muscle cells (VSMCs) migrate from the media to the intima through the basement membrane and interstitial collagenous matrix, and proliferate to form neointima. Here, we investigate the mechanism of VSMC migration and proliferation caused by aggretin, a snake venom integrin alpha2beta1 agonist. EXPERIMENTAL APPROACH: Cultures of rat and human VSMCs were treated with aggretin and the signal transduction pathways induced by this agonist were examined by Western blotting, immunoprecipitation and electrophoretic mobility shift assay techniques. KEY RESULTS: Aggretin-induced VSMC proliferation was blocked by a monoclonal antibody (mAb) against integrin alpha2 (AII2E10) or against the platelet-derived growth factor receptor (PDGFR)-beta. Proliferation was also blocked by inhibition of the tyrosine kinase Src with PP2, phospholipase C (PLC) with U73122, extracellular signal-regulated kinase (ERK) with PD98059 or nuclear factor-kappa B (NF-kB) activation with pyrrolidine dithiocarbamate (PDTC). VSMC migration towards immobilized aggretin was increased in a modified Boyden chamber and this effect was blocked by alpha2beta1-Src-PLC-MAPK axis inhibitors, but not by PDTC, PDGFR-beta mAb, or a phosphoinositide-3 kinase inhibitor, LY294002. Aggretin stimulated the phosphorylation of PDGFR-beta, Src and ERK in a time-dependent manner. NF-kB translocation and platelet-derived growth factor (PDGF)-BB production were also observed. The ERK activation, NF-kB translocation and PDGF-BB production were blocked by PP2, U73122 and PD98059. CONCLUSIONS AND IMPLICATIONS: Aggretin induces VSMC proliferation and migration mainly through binding to integrin alpha2beta1, and subsequently activates Src, PLC and ERK pathways, inducing NF-kB activation and PDGF production.

Inhibition of tumor formation by snake venom disintegrin.

The metastasis of tumor cells to bone involves migration, invasion and adhesion to bone. Breast and prostate cancer cells have predilection for spreading to bone. Snake venom-derived arginine-glycine-aspartic acid (RGD)-containing disintegrins (e.g. rhodostomin) have been demonstrated to inhibit cell adhesion. Here, we found that rhodostomin inhibited the adhesion of breast and prostate carcinoma cells to both unmineralized and mineralized bone extracellular matrices in a dose-dependent manner, without affecting the viability of tumor cells. In addition, rhodostomin also inhibited the migration and invasion of breast and prostate carcinoma cells. It specifically inhibited the binding of monoclonal antibody (MoAb) 7E3, which recognizes integrin alphavbeta3, to tumor cells, but not those of other MoAbs against other integrin subunits such as alpha2, alpha3, alpha5 and beta1. As breast cancer cells MDA-MB-231 were locally injected into tibia in nude mice, histological examination of the tibia of control group revealed that most of the cancellous bone had been replaced by the breast cancer cells after 28 days' inoculation. In contrast, co-administration of trigramin with cancer cells markedly inhibited tumor growth and bone destruction. Taken together, disintegrins strongly inhibit the adhesion, migration, invasion of tumor cells and also tumor growth of human breast cancer cells in bone as well. Therefore, disintegrins may be developed as alternate therapy for bone metastasis of cancer cells.

Inhibition of neuropathic pain by a potent disintegrin--triflavin.

Injury to peripheral nerves may result in severe and intractable neuropathic pain. Many efforts have been focused on the elucidation of the mechanisms of neuropathic pain. It was found here that integrin plays an important role in the induction of neuropathic pain and treatment of disintegrin is able to attenuate neuropathic pain. The rats were induced hyperalgesia by tightly ligating the L5 spinal nerve and cut just distal to the ligature on one side. Mechanical and thermal stimuli were applied in the middle dermatome of the hind paw. Epidural administration of triflavin (TFV), an arginine-glycine-aspartic acid (RGD) containing disintegrin, inhibited hyperalgesia induced by either mechanical or thermal stimulation. Immunohistochemistry showed that the sprouting of sympathetic nerves into DRG by neuropathic surgery was markedly inhibited by TFV. Beta 1 integrin mRNA of L5 DRG increased immediately 1 day after tight ligation and cut of L5 spinal nerve. However, beta 1 integrin mRNA in uninjured L4 DRG increased later on Day 3 after surgery. On the other hand, alpha-CGRP precursor mRNA decreased in ipsilateral L5 DRG but increased in L4 DRG after neuropathic surgery. Immunohistochemistry shows that beta 3 integrins of L5 as well as L4 increased in response to neuropathic surgery and administration of triflavin antagonized the increasing action. These results suggest that there is interaction between injured and uninjured neurons and the induction of neuropathic pain is related to neuronal sprouting. Disintegrin is able to inhibit neuronal sprouting and the induction of hyperalgesia induced by peripheral nerve injury and may thus be a new category of drugs to be developed for the treatment of neuropathic pain.

Rhodostomin, a disintegrin, inhibits adhesion of neutrophils to fibrinogen and attenuates superoxide production.

Disintegrins are a group of Arg (or Lys)-Gly-Asp-containing snake venom proteins which inhibit platelet aggregation via the blockade of alpha(IIb)beta(3) integrin. Here, we studied the effect of rhodostomin, a disintegrin purified from the venom of Calloselasma rhodostoma, on the functions of neutrophils. By flow cytometric analysis of whole blood, we found that rhodostomin interacted with leukocytes of the myeloid and monocytic lineage as well as with platelets. The binding of rhodostomin to neutrophils could reach saturation in a dose-dependent manner, and its binding was increased in neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) and N-formyl-Met-Leu-Phe. EDTA did not inhibit the binding of rhodostomin. In addition, bound rhodostomin was not internalized. Soluble fibrinogen, a natural ligand of Mac-1 (CD11b/CD18, alpha(M)beta(2)), and the peptide, GRGDS, inhibited the binding of rhodostomin to PMA-activated neutrophils, while 7E3, a monoclonal antibody (mAb) raised against beta(3) integrin, or mAbs raised against alpha(M) and beta(2) integrin did not. Rhodostomin blocked the Mac-1-dependent adhesion of neutrophils to immobilized fibrinogen, in parallel with decreasing the production of superoxide from adherent neutrophils. Taken together, our results indicate that rhodostomin binds to activated neutrophils in an RGD-dependent manner, blocks the adhesion of activated neutrophils to fibrinogen and attenuates superoxide production, suggesting that rhodostomin may have anti-inflammatory properties.

Disintegrin causes proteolysis of beta-catenin and apoptosis of endothelial cells. Involvement of cell-cell and cell-ECM interactions in regulating cell viability.

Disintegrins, the snake venom-derived arginine-glycine-aspartic acid-containing peptides, have been demonstrated to inhibit angiogenesis through induction of endothelial cell apoptosis. However, it is not clear how a disintegrin causes endothelial apoptosis. In this study, we elucidated the action mechanism of disintegrin in causing endothelial apoptosis by using rhodostomin as a tool. We showed that cell detachment was observed at the early stage of rhodostomin treatment. It was initiated through the blockade by integrin alphanubeta3 and was accelerated by a mechanical stretch from neighboring cells. Both rhodostomin and poly(HEME) induced a higher percentage of cells at G2-M phase, the cleavage of beta-catenin and poly(ADP-ribose) polymerase during apoptosis, indicating that cell detachment is a prerequisite for rhodostomin-induced apoptosis. Moreover, pp125(FAK) phosphorylation and actin cytoskeleton were affected upon rhodostomin treatment. The activation of caspase-3 but not that of caspase-9 was detected after rhodostomin treatment. In addition, general caspase inhibitors inhibited the cleavage of beta-catenin and poly(ADP-ribose) polymerase, and DNA fragmentation, whereas they did not prevent cell shape change or detachment. According to these results, we concluded that disintegrin-induced endothelial apoptosis is a complex process, not merely caused by a blockade of endothelial integrin alphanubeta3 but also by an accompanied shape change and mechanical stretches among cells.