FS145, the first flea-derived disintegrin, inhibits angiogenesis through specifically binding integrin αvß3.

FS145, a protein containing a WGD motif, was previously described from the salivary transcriptome of the flea Xenopsylla cheopis. Nevertheless, its biological function and complete structure are still uncertain. Herein, FS145 was confirmed to adopt a common αßß structure with the WGD motif exposed on its surface and located right at the top of a loop composed of residues 72-81. Furthermore, FS145 dose-dependently inhibited the proliferation, adhesion, migration, and tube formation of HUVECs by not only binding to integrin αvß3 but also by subsequently inactivating the FAK/Src/MAPK pathway along with the reduction of the expression of MMP-2, MMP-9, VEGFA, bFGF, Ang2, Tie2, HIF-1α, and FAK. Moreover, FS145 also inhibited aortic vessel sprout and showed strong anti-angiogenic activities as assessed ex vivo, by employing the rat aortic ring assay, chick embryo chorioallantoic membrane, and zebrafish embryo models. Altogether, our results suggest that FS145 suppresses angiogenesis ex vivo and in vitro by blocking integrin αvß3. The current study reveals the first anti-angiogenesis disintegrin with WGD motif from invertebrates and provides a beneficial pharmacological activity to inhibit abnormal angiogenesis.

The in vitro anticancer effects of FS48 from salivary glands of Xenopsylla cheopis on NCI-H460 cells via its blockage of voltage-gated K+ channels.

Voltage-gated K+ (Kv) channels play a role in the cellular processes of various cancer cells, including lung cancer cells. We previously identified and reported a salivary protein from the Xenopsylla cheopis, FS48, which exhibited inhibitory activity against Kv1.1-1.3 channels when assayed in HEK 293T cells. However, whether FS48 has an inhibitory effect on cancer cells expressing Kv channels is unclear. The present study aims to reveal the effects of FS48 on the Kv channels and the NCI-H460 human lung cancer cells through patch clamp, MTT, wound healing, transwell, gelatinase zymography, qRT-PCR and WB assays. The results demonstrated that FS48 can be effective in suppressing the Kv currents, migration, and invasion of NCI-H460 cells in a dose-dependent manner, despite the failure to inhibit the proliferation. Moreover, the expression of Kv1.1 and Kv1.3 mRNA and protein were found to be significantly reduced. Finally, FS48 decreases the mRNA level of MMP-9 while increasing TIMP-1 mRNA level. The present study highlights for the first time that blood-sucking arthropod saliva-derived protein can inhibit the physiological activities of tumour cells via the Kv channels. Furthermore, FS48 can be taken as a hit compound against the tumour cells expressing Kv channels.

The toxin mimic FS48 from the salivary gland of Xenopsylla cheopis functions as a Kv1.3 channel-blocking immunomodulator of T cell activation.

The Kv1.3 channel has been widely demonstrated to play crucial roles in the activation and proliferation of T cells, which suggests that selective blockers could serve as potential therapeutics for autoimmune diseases mediated by T cells. We previously described that the toxin mimic FS48 from salivary gland of Xenopsylla cheopis downregulates the secretion of proinflammatory factors by Raw 264.7 cells by blocking the Kv1.3 channel and the subsequent inactivation of the proinflammatory MAPK/NF-κB pathways. However, the effects of FS48 on human T cells and autoimmune diseases are unclear. Here, we described its immunomodulatory effects on human T cells derived from suppression of Kv1.3 channel. Kv1.3 currents in Jurkat T cells were recorded by whole-cell patch-clamp, and Ca2+ influx, cell proliferation, and TNF-α and IL-2 secretion were measured using Fluo-4, CCK-8, and ELISA assays, respectively. The in vivo immunosuppressive activity of FS48 was evaluated with a rat DTH model. We found that FS48 reduced Kv1.3 currents in Jurkat T cells in a concentration-dependent manner with an IC50 value of about 1.42 µM. FS48 also significantly suppressed Kv1.3 protein expression, Ca2+ influx, MAPK/NF-κB/NFATc1 pathway activation, and TNF-α and IL-2 production in activated Jurkat T cells. Finally, we show that FS48 relieved the DTH response in rats. We therefore conclude that FS48 can block the Kv1.3 channel and inhibit human T cell activation, which most likely contributes to its immunomodulatory actions and highlights the great potential of this evolutionary-guided peptide as a drug template in future studies.

Anti-inflammatory effects of FS48, the first potassium channel inhibitor from the salivary glands of the flea Xenopsylla cheopis.

The voltage-gated potassium (Kv) 1.3 channel plays a crucial role in the immune responsiveness of T-lymphocytes and macrophages, presenting a potential target for treatment of immune- and inflammation related-diseases. FS48, a protein from the rodent flea Xenopsylla cheopis, shares the three disulfide bond feature of scorpion toxins. However, its three-dimensional structure and biological function are still unclear. In the present study, the structure of FS48 was evaluated by circular dichroism and homology modeling. We also described its in vitro ion channel activity using patch clamp recording and investigated its anti-inflammatory activity in LPS-induced Raw 264.7 macrophage cells and carrageenan-induced paw edema in mice. FS48 was found to adopt a common αßß structure and contain an atypical dyad motif. It dose-dependently exhibited the Kv1.3 channel in Raw 264.7 and HEK 293T cells, and its ability to block the channel pore was demonstrated by the kinetics of activation and competition binding with tetraethylammonium. FS48 also downregulated the secretion of proinflammatory molecules NO, IL-1ß, TNF-α, and IL-6 by Raw 264.7 cells in a manner dependent on Kv1.3 channel blockage and the subsequent inactivation of the MAPK/NF-κB pathways. Finally, we observed that FS48 inhibited the paw edema formation, tissue myeloperoxidase activity, and inflammatory cell infiltrations in carrageenan-treated mice. We therefore conclude that FS48 identified from the flea saliva is a novel potassium channel inhibitor displaying anti-inflammatory activity. This discovery will promote understanding of the bloodsucking mechanism of the flea and provide a new template molecule for the design of Kv1.3 channel blockers.

Tablysin-15 inhibits osteoclastogenesis and LPS-induced bone loss via attenuating the integrin αvß3 pathway.

Excessive osteoclast leads to the imbalance in bone reconstruction and results in osteolytic diseases, such as osteoporosis and rheumatic arthritis. Integrin αvß3 abundantly expresses on osteoclast and plays a critical role in the formation and function of osteoclast, therefore, blockage of αvß3 has become an attractive therapeutic option for osteolytic diseases. In this study, we find that Tablysin-15, a RGD motif containing disintegrin, concentration-dependently suppresses RANKL-induced osteoclastogenesis, F-actin ring formation and bone resorption without affecting the cell viabilities. Tablysin-15 binds to integrin αvß3 and inhibits the activation of FAK-associated signaling pathways. Tablysin-15 also suppresses the activation of NF-кB, MAPK, and Akt-NFATc1 signaling pathways, which are crucial transcription factors during osteoclast differentiation. Moreover, Tablysin-15 decreases the osteoclastogenesis marker gene expression, including MMP-9, TRAP, CTSK, and c-Src. Finally, Tablysin-15 significantly inhibits LPS-induced bone loss in a mouse model. Taken together, our results indicate that Tablysin-15 significantly suppresses osteoclastogenesis in vitro and in vivo, thus it might be a excellent candidate for treating osteolytic-related diseases.

Disintegrin Tablysin-15 Suppresses Cancer Hallmarks in Melanoma Cells by Blocking FAK/Akt/ERK and NF-κB Signaling.

BACKGROUND: Integrins are crucial anti-cancer therapy targets. We previously showed that tablysin-15 is an integrin antagonist with its Arg-Gly-Asp motif in a novel structural context. OBJECTIVE: Here we investigated the anti-cancer effects and mechanisms of action of tablysin-15 in melanoma cells. METHODS: Cell adhesion, competitive binding, cell viability, and ATP chemiluminescence assays were used to analyze the binding of tablysin-15 to αvß3 integrin and its phenotypic effects. Wound healing, transwells, and zymography were performed to detect motility and matrix metalloproteinase- 2/-9 activities. PARP and caspase-3 cleavage were used as apoptosis assays, while LDH release and flow cytometry were used for necrosis and cell cycle analysis. The expression of mRNAs and proteins of target molecules was measured by qRT-PCR and western blotting, respectively. RESULTS: Tablysin-15 dose-dependently inhibited the proliferation, migration, and invasion of M21 cells through integrin αvß3. The proliferation inhibition caused by tablysin-15 was attributable to G0/G1 phase arrest rather than apoptosis or necrosis. Furthermore, tablysin-15 suppressed MMP-2/- 9 activities and the mRNA expression of MMP-2/-9 and COX-2 but was upregulated TIMP-1 in M21 cells. Meanwhile, tablysin-15 suppressed the expression of cyclin D1/E and CDK 2/6, the phosphorylation of FAK, Akt, and ERK, and nuclear translocation of NF-κB, while increasing the expression of the CDK inhibitor p21waf1/C1. Taken together, tablysin-15 might inhibit melanoma cell metastasis and proliferation by competing with αvß3 integrin, thereby blocking FAK-associated signaling pathways and nuclear translocation of NF-κB. CONCLUSION: Tablysin-15 has reliable anti-cancer effects against M21 melanoma cells, suggesting tablysin-15 is a promising anti-tumor drug.

The anticancer properties and mechanism of action of tablysin-15, the RGD-containing disintegrin, in breast cancer cells.

αvß3 integrin expressed on cancer cell surfaces is associated with important cancer hallmarks including survival and metastasis and is thus a potential anticancer drug target. Tablysin-15 contains the RGD motif and is a high-affinity αvß3 integrin antagonist. The aim of this study was to investigate the antitumor effect and mechanism of action of tablysin-15 against αvß3 integrin high-expressing breast cancer cell lines in vitro and in vivo. Tablysin-15 dose-dependently inhibited the proliferation, migration, and invasion of two breast cancer cell lines via the αvß3 integrin in vitro. Proliferation inhibition was attributable to G0/G1 phase cell cycle arrest rather than apoptosis or necrosis. Furthermore, tablysin-15 downregulated the activity and mRNA expression of MMP-2/-9, VEGF, and COX-2 but upregulated TIMP-1/-2 mRNA in both cell lines. Further, tablysin-15 suppressed the expression of CDK2, CDK6, cyclin D1, and cyclin E, the phosphorylation of FAK, Akt, GSK-3ß, and ERK, and the nuclear translocation of NF-κB while increasing the expression of the CDK inhibitor p21waf1/C1. Lastly, tablysin-15 provided effective antitumor protection in vivo. Thus, tablysin-15 inhibits the metastasis and proliferation of breast cancer cells through binding αvß3 integrin and blocking FAK-associated signaling pathways as well as nuclear translocation of NF-κB.

Functional Characterization of a Novel Lipopolysaccharide-Binding Antimicrobial and Anti-Inflammatory Peptide in Vitro and in Vivo.

Antimicrobial peptides (AMPs) are key components of host immune defense of vertebrates against microbial invasions. Here, we report a new AMP (esculentin-1GN) characterized from the skin of the frog Hylarana guentheri. Esculentin-1GN (GLFSKKGGKGGKSWIKGVFKGIKGIGKEVGGDVIRTGIEIAACKIKGEC) with high amphipathic α-helical structure in membrane-mimetic environments has the microbial-killing activity by destruction of the cell membrane. Moreover, esculentin-1GN inhibits LPS-induced expression of proinflammatory nitric oxide, interleukin-1ß, interleukin-6, and tumor necrosis factor while it enhances expression of interleukin-10. Furthermore, esculentin-1GN can bind to d-(+)-galacturonic acid and LPS. Meanwhile, esculentin-1GN suppresses the activation of inflammatory response pathway induced by LPS. In addition, esculentin-1GN significantly reduces acute inflammation in carrageenan-induced mice paw. Taken together, the novel LPS-binding esculentin-1GN with antimicrobial and anti-inflammatory activities will be an excellent temple for designing new antibiotic formulations.