Antimicrobial effects of vertebrate TFPI-2 C-terminal peptides2016-10-26
Article from our customer: Kasetty et al. BMC Microbiology (2016) 16:129
Background: In previous studies we reported a novel role of human tissue factor pathway inhibitor-2 (TFPI-2) in innate immunity by serving as a precursor for host defense peptides. Here we further studied the theraputic potential of one selected TFPI-2 derived peptide (mouse) in a murine sepsis model.
Results: A number of selected C-terminal TFPI-2 derived peptides from different species were synthesized and it was found that all of them exert antimicrobial activity against E.coli and P. aeruginosa. The peptide-mediated killing of E. coli was enhanced in human plasma, suggesting an involvement of the classical pathway of the complement.
Conclusion: Our results suggest that the evolutionary conserved C-terminal part of TFPI-2 is an interesting agent for the development of novel antimicrobial therapies.
Fig.6 Treatment of LPS-induced septic shock mice with a mouse TFPI-2 derived peptide. Septic shock in Balb/c mice was induced by i.p.injection of 10 mg/kg E. coli LPS. Thirty minutes after LPS injection, VKG24 (0.5 mg/mouse or ~25 mg/kg body weight) or PBS was administratedi.p. (a)Mouse TFPI-2 derived peptide significantly increased survival of mice. Survival of mice was monitored for 7 days (only the first 96 h is shown in the figure). Statistical comparisons of survival curves were performed using the Mantel-Cox’s test. ***P ≤ 0.0003. (b) Weight was determined daily. (c) Clotting times of aPTT and PT was measured in citrate plasma 24 h after LPS injection (n = 8). (d) The indicated cytokines were analyzed in plasma 24 h after LPS injection (n = 8). Statistical analysis for C-D, one-way ANOVA with Tukey’s multiple comparisons post-test was used. **P ≤ 0.003, ****P ≤ 0.0001
All of the peptides used in this article were synthesized by Ontores (Shanghai, China). The purity (>98 %) of these peptides was confirmed by mass spectral analysis (MALDI-ToF Voyager).
As a leading Biotech company over world, Ontores committed to provide professionally high quality and comprehensive proteomic and genomic industry solution to our global customers. Ontores offers innovative solutions for antibodies, building blocks, oligonucleotides and peptides and carries a broad spectrum of bio-reagents that will meet everyone’s need in their multi-disciplinary research. Ontores also provides cutting edge custom services including antibody production, contract research services and peptide synthesis.
Advances in the Research of TR Peptides Targeting Tumor Cells2016-10-19
Reference：Yang Wang，Autophagy, 126, 949-962
Background: Hydroxychloroquine (HCQ) inhibits autophagy and therefore can sensitize some cancer cells tochemotherapy, but the high doses required limit its clinical use.
Major finding and significance of the present article: loading HCQ into Lip-TR liposomes may increase the effective concentration of the inhibitor in tumor cells, allowing less toxic doses to be used. HCQ/Lip-TR is a leading drug formulation of HCQ with great potential to be optimized further for potency as an autophagy inhibitor. Inaddition, Lip-TR, as a drug delivery carrier, may also be used to deliver other autophagy modulators.
Figure 1. Schematic illustration of autophagy inhibition by HCQ delivered into tumor cells as HCQ/Lip-TR. (1) HCQ/Lip-TR is injected intravenously and targets tumors passively via the EPR effect and actively via binding to ITGAV-ITGB3/integrin avb3 receptors. (2) In the acidic tumor microenvironment, anionic HCQ/Lip-TR becomes cationic, further facilitating HCQ/Lip-TR endocytosis. (3) HCQ/Lip-TR is delivered into cells (I), and HCQ is released (II). The free HCQ enters lysosomes, where it inactivates lysosomalenzymes (III). The late endolysosome fuses with the autophagosome to form an autolysosome (IV), but autophagy is blocked because the lysosomal enzymes have beeninactivated (V).
1.Loading HCQ into liposomes (HCQ/Lip) decorated with a pH-sensitive TH-RGD targeting peptide (HCQ/Lip-TR) can concentrate HCQ in B16F10 tumor cells and lysosomes both invitro and invivo.
Figure 5 Cellular uptake of liposomes. (A) Expression of ITGAV-ITGB3/integrin avb3 receptor on B16F10 and MCF-7 cells. (B-C) Uptake of CFPE-labeled liposomes by (B) B16F10 or (C) MCF-7 cells, measured using confocal laser scanning microscopy. Scale bar: 10 mm. HCQ concentrations (D) within the cell and (E) within lysosomes after 2-h treatment with 100 mM HCQ, HCQ/Lip or HCQ/Lip-TR. Data shown are mean ±SD for 3 replicates per treatment.
Studies in vitro at pH 6.5 showed that the intracellular HCQ concentration was 35.68-fold higher, and lysosomal HCQ concentration 32.22-fold higher, after treating cultures with HCQ/Lip-TR than after treating them with free HCQ.
Figure 6. In vivo targeting ability of liposomes. (A) Representative in vivo (left) and ex vivo (right) images of B16F10 tumors in C57BL/6 mice at 24 h after injection with DiD-labeled liposomes. Quantification of HCQ within (B) tumor cells and (C) lysosomes at 24 h after injection with 60 mg/kg HCQ, HCQ/Lip or HCQ/Lip-TR. Data shown are mean ± SD (5 mice /group, every sample was analyzed 3 times).
The corresponding enhancements observed in mice bearing B16F10 tumors were 15.16-fold within tumor cells and 14.10-fold within lysosomes.
2．HCQ/Lip-TR can more efficiently inhibit tumor growth and prolonged survival time compared with HCQ and HCQ/Lip.
Figure 7. Antitumor efficiency of HCQ/Lip-TR. C57BL/6 mice bearing B16F10 tumors were treated with 60 mg/kg HCQ, HCQ/Lip or HCQ/Lip-TR once every 2 d for 32 d. (A)Changes in animal body weight and (B) tumor growth. (C) Kaplan–Meier survival curves. (D) Western blotting of LC3 and SQSTM1/p62 in tumor extracts after treatment.(E) Representative electron micrographs of tumors after treatment. Arrows indicate autophagic vesicles. Scale bar: 500 nm. The statistical comparison was conductedbetween the PBS group and other groups (HCQ, HCQ/Lip or HCQ/Lip-TR group). *, p < 0.05; **, p < 0.01; and***, p < 0.001.
3.HCQ/Lip-TR can enhance the therapeutic efficacy of DOX ontumor growth both invitro and invivo.
In vitro studies
IC50 of DOX or DOX/Lip at pH 6.5 was significantly lower in the presence of HCQ/Lip-TR (Table 2). This raised the possibility that combining HCQ/Lip-TR with DOX or DOX/Lip would enhance the antitumor efficacy of DOX.
Figure 8. Antitumor efficacy of liposomal DOX when combined with liposomal HCQ. C57BL/6 mice bearing B16F10 tumors were treated with 60 mg/kg HCQ and 2 mg/kg DOX. HCQ treatments were administered once every 2 d for 32 d, while DOX treatments were administered once every 3 d starting on d 8. (A)Changes in animal body weight and (B) tumor growth. (C) Kaplan–Meier survival curves. (D) Representative micrographs of tumor tissue stained by H&E and TUNEL staining. Scale bar: 50 mm. The statistical comparison was conducted between DOX/Lip + HCQ/Lip group and other groups, or between DOX/Lip +HCQ/Lip-TR group and other groups. *, p < 0.05; **, p < 0.01; and***, p < 0.001.
All treatments inhibited tumor growth relative to the growth in the PBS control group(Fig. 8B), with the combination DOX/Lip C HCQ/Lip-TR causing the greatest inhibition. This combination also led tomedian survival > 60 d (Fig. 8C) as well as to more advanced and more extensive apoptosis and necrosis than the other treatments, based on hematoxylin and eosin (H&E) staining and TUNEL staining (Fig. 8D).
4. HCQ/Lip-TR exhibited lower toxicity in vivo compared with free HCQ.
Figure 9. Myelosuppression evaluation. C57BL/6 mice bearing B16F10 tumors were treated with PBS, HCQ, HCQ/Lip or HCQ/Lip-TR (at 240 mg/kg HCQ) by tail-vein injection once every 2 d for 8 d. Numbers of (A) white blood cells (WBC) and (B) platelets (PLT) were determined at various time points. *, p < 0.05; **, p < 0.01; and***, p < 0.001.
Free HCQ caused persistent reductions in both white blood cells (WBC) and platelets(PLT) counts, which were attenuated in the case of liposomal HCQ(Fig. 9). These results suggest that encapsulating HCQ protects against HCQ-induced myelosuppression.
Figure 10. HCQ distribution in vivo. Distribution of HCQ in heart, liver, spleen, kidney, tumor, and small intestine of mice at 24 h after tail-vein injection of HCQ,HCQ/Lip or HCQ/Lip-TR (at 240 mg/kg HCQ; n = 5 animals per condition). *, p <0.05, **, p < 0.01; and***, p < 0.001.
Mice treated with free HCQ were lethargic, and 2 of 10 mice developed bowelobstruction (data not shown), suggesting a high risk of gastrointestinal side effects. This was not observed with liposomal HCQ, which may reflect the fact that encapsulated HCQ showed significantly less accumulation in the intestines than the free inhibitor, and concomitantly greater accumulation in tumor and liver(Fig. 10).
HCQ/Lip-TR was associated with milder anemia and milder myosuppressive reductions in white blood cell and platelet counts than free HCQ, as well as less accumulation in the small intestine, which may reduce risk of intestinal side effects.
5. Mechanism of action: HCQ/Lip-TR blocked autophagic flux in tumor cells.
Figure 5. Autophagy inhibition evaluation of liposomes. B16F10 cells expressing EGFP-LC3 were treated for 2 h with 100 mM HCQ, HCQ/Lip or HCQ/Lip-TR. (A) Representative confocal micrographs showing autophagosomes. Scale bar: 10 mm. (B) Quantification of EGFP puncta using Origin Pro 8.0 software. (C) Western blotting for LC3 and SQSTM1/p62. (D) Quantification of the ratio of LC3-II to LC3-I expression using Image J software. (E) Quantification of levels of SQSTM1/p62 normalized to those in PBS-treated cells using Image J. Data shown are mean ± SD of 3 replicates. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
Transfected cells were treated for 2 h with HCQ, HCQ/Lip or HCQ/Lip-TR, and all cultures showed punctate fluorescence(Fig. 5A), indicating autophagic inhibition. Fluorescence was more obvious with HCQ/Lip-TR than with HCQ or HCQ/Lipat pH 7.4 and 6.5, and fluorescence was significantly higher at the acidic pH only in the case of HCQ/Lip-TR (Fig. 5A-B).
Both of LC3and SQSTM1/p62 play key roles in autophagy and increases in the ratio of LC3-II to LC3-I indicate autophagy inhibition. HCQ/Lip-TR caused a larger increase than HCQ or HCQ/Lip after 2-h treatment at pH 6.5 at a dose of 100 mM HCQ(Fig. 5C-D). At this pH, SQSTM1/p62 expression was higher with HCQ/Lip-TR than the other treatments, confirming stronger autophagy inhibition (Fig. 5C and E).
TR peptide with a terminal cysteine (c[RGDfK]-AGYLLGHINLHHLAHL[Aib]HHIL-Cys) used in this article was synthesized by Zhejiang Ontores Biotechnologies Co. Ltd using standard solid phase methods. As a leading biotech company over the world, Ontores committed to provide professionally high quality and comprehensive proteomic and genomic industry solution to our global customers. Ontores offers innovative solutions for antibodies, building blocks and peptides and carries a broad spectrum of bio-reagents that will meet everyone’s need in their multi-disciplinary research. Ontores also provides cutting edge custom services including antibody production, contract research services and peptide synthesis.
Liquid phase synthesis of peptides2016-10-12
With the development of peptide drugs, research and development of various peptides are of growing interest. Currently available methods of peptide synthesis include liquid-phase peptide synthesis and solid-phase peptide synthesis. The methods of solid-phase peptide synthesis are more commonly used. However, many problems will be encountered during the process of liquid-phase syntheses, such as insolubility, hydrophobicity, and time-consuming when dealing with the intermediates.
Recently, scientists have developed a new solid phase synthesis method based on terminal amide modifications. This method has some advantages, like easy to operate, high yields of crude products, and can synthesize some peptides, which can not be synthesized by commonly used solid-phase peptide synthesis methods.
For example H-Ala-Ala-Gln-Val-Leu-Ile-Ser-Glu-Leu-Ala-Ile-Ala-NH2 Solid-phase peptide synthesis:
HPLC of crude products
liquid-phase peptide synthesis:
HPLC of crude products
HPLC data shows that the purity of products obtained by liquid-phase synthesis method is much higher than that by solid-phase synthesis method. Therefore, we will pay more attention to the development of methods of liquid-phase peptide synthesis.