The Anti-angiogenesis Activity Study Of Novel Designed Peptide Analogues Of Neurokinin 3 Receptor Agonist

Research backgroundAngiogenesis is the fundamental process by which new blood vessels are formed.Extensive research has shown that this event play an essential role for tumorigenesis.The study of tumor angiogenesis therefore represents a promising area of research for development of anti-cancer therapeutics.Of note,in recent years several angiogenesis inhibitors which have been certificated by Food and Drug Administration of America(FDA),especially vascular endothelial growth factor antibodies have been found the severe side effects of these drugs at preclinical or clinical level.Therefore,it is crucial for oncology to look for novel targets of anti-angiogenesis therapy.Although the roles of neurokinin-3 receptor(NK3R),which is a significant member of tachykinin receptors family,in various physiological processes have been well established,the effects and mechanism of it in angiogenesis remain unclear.Disruption of endogenous Neurokinin B(NKB),which preferentially binds with NK3 R,targeting of endothelium via a multi-component mechanism to oppose vascular remodeling could function as an endogenous angiogenesis inhibitor in previous studies,suggesting that the inhibition of NK3 R pathway can effectively block neovascularization.We designed two analogues based on an agonist of NK3 R,[MePhe7]NKB,named NK3R-A1 and NK3R-A2.Furthermore,motifs NGR and CNGRC were introduced into our designed analogues to augment the anti-tumor effect of them.This study was undertaken to verify the anti-angiogenic properties of three agonist analogues of NK3 R contained [MePhe7]NKB,NK3R-A1 and NK3R-A2 in vitro and in vivo.Additionally,a selective antagonist of NK3 R was used to clarify the anti-angiogenic mechanism of peptides above-mentioned.Research MethodsOn the basis of our previous work,we designed two analogues which termed with NK3R-A1 and NK3R-A2 based on the modified structure of an agonist ofNK3 R,[MePhe7]NKB.Afterwards,[MePhe7]NKB,NK3R-A1,NK3R-A2 and a selective antagonist of NK3 R [Gly6]NKB[3-10] were synthesized by Fmoc synthesis protocol and purified by RP-HPLC subsequently,and the mass spectrum was used to identify the Molecular Weight(MW)of synthesized peptides.Furthermore,the anti-angiogenic effect of [MePhe7]NKB,NK3R-A1 and NK3R-A2 were first performed by gelatin sponge chick embryo chorioallantoic membrane(gelatin sponge-CAM),and [Gly6]NKB[3-10],an antagonist of NK3 R was used in the corresponding competitive binding assay to examine the mechanism of peptides.Meanwhile,the in vitro response to drugs was investigated by wound healing assay and Transwell migration assay using the Human Umbilical Vein Endothelial Cells(HUVECs)and the corresponding competitive assays for [MePhe7]NKB,NK3R-A1 and NK3R-A2 have also been tested by the [Gly6]NKB[3-10].To eliminate the potent influence of drugs on HUVECs,MTT proliferation assay was used before the migration assays.Additionally,the in vivo anti-tumor effects of peptides were analyzed in 40 BALB/c mice which were successfully transplanted S180 sarcoma cells and i mmunohistochemical(IHC)staining assay was performed to evaluate the anti-tumor angiogenic effect of peptides on microvasculars of tumors after the last administration.Research ResultsAll peptides including three agonist analogues of NK3 R contained[MePhe7]NKB(Asp-Met-His-Asp-Phe-Phe-MePhe-Gly-Leu-Met-NH2),NK3R-A1(Asn-Gly-Arg-Gly-Gly-Asp-Phe-Phe-MePhe-Gly-Leu-Met-NH2),NK3R-A2(Cys-Asn-Gly-Arg-Cys-Gly-Gly-Asp-Phe-Phe-MePhe-Gly-Leu-Met-NH2)and a selective antagonist of NK3 R [Gly6]NKB[3-10](His-Asp-Phe-Gly-Val-Gly-Leu-Met-NH2)were successfully chemically synthesized following the protocol of Fmoc solid phase.RP-HPLC was utilized to purify the peptides synthesized above and then identified with mass spectrum to confirm that we obtained the expected peptides.Moreover,[MePhe7]NKB,NK3R-A1 and NK3R-A2 all showed a significant anti-angiogenic effect on gelatin sponge-CAM assay,nevertheless,the anti-angiogenic effects were eliminated when the selective antagonist of NK3 R,[Gly6]NKB[3-10] had been administrated in the correspondingcompetitive binding assays.The similar results could be found in vitro assays.All three detected peptides[MePhe7]NKB,NK3R-A1 and NK3R-A2 did not exhibit obvious anti-proliferation effect on HUVECs in 24 h according to the results in MTT assay,which suggest that it would be reasonable to control the drug processing time in 24 h in the following assays.Three agonist analogues of NK3 R [MePhe7]NKB,NK3R-A1 and NK3R-A2 were found to be good anti-migration inhibitor at the concentration of 50μM,25μM and 25μM,respectively,in wound healing assay.And the inhibition ratios of these peptides reached to 20.0%,73.2% and 53.7%,respectively.Similar results could be seen in Transwell migration assay,the migrated cells treated with 5μM[MePhe7]NKB,NK3R-A1 and NK3R-A2,respectively,were 2.3-,5.0-and 7.0-fold,respectively,less than the negative control treated with serum-free RPMI 1640 medium.Of note,the response of HUVECs to peptides NK3R-A1 and NK3R-A2 were superior to the others in migration assays,suggesting that they held the anti-angiogenic function of NKB successfully.The anti-angiogenic effects of agonist analogues of NK3 R were suppressed after the preprocessing of[Gly6]NKB[3-10],demonstrating that the anti-angiogenic effects of them are mediated by the NK3 R pathway.The relative healing rate reached to 96.3% and97.6%,respectively,in the groups of NK3R-A1 and NK3R-A2 which after the treatment of [Gly6]NKB[3-10],and the relative healing rate of the group[MePhe7]NKB combined with [Gly6]NKB[3-10] was nearly equal to the rate of negative control,suggesting that the anti-angiogenic effects of them in vitro were almost completely antagonized by [Gly6]NKB[3-10] in wound healing competitive binding assay.The same with the wound healing assay,anti-angiogenic effects of three peptides were antagonized by [Gly6]NKB[3-10] to some extent in Transwell migration competitive binding assay,the migrated cells of antagomistic groups of[MePhe7]NKB,NK3R-A1 and NK3R-A2,respectively were 3.9-,2.8-and 3.3-fold more than the groups treated with the peptides above alone.Comparing with the negative group,the tumor volumes were dramatically decreased in the drug processing period,which less than a half of the negative group.Tumors were extracted and weighed at day 15,and tumor weights of groupsdisposed with the drugs were significantly decreased,especially for the high concentration group of [MePhe7]NKB and low concentration group of NK3R-A1.And the anti-tumor rate of these two groups reached to ~60%.In addition,the results of Immunohistochemical staining(IHC)showed that the microvessel density(MVD)treated with peptides of stained tissue showed a conspicuous depletion in comparison with the group of control administrated with the normal saline(NS).As expected,the MVDs treated with NK3R-A1 and NK3R-A2,respectively,were both less than the group of [MePhe7]NKB,indicating that the NK3R-A1 and NK3R-A2 exhibited the property of anti-tumor angiogenesis.ConclusionIn summary,our investigation for examining the anti-angiogenic effect and the mechanism of these agonist analogues of NK3 R,[MePhe7]NKB,NK3R-A1 and NK3R-A2 demonstrated that the NK3 R might be a potential novel target for the anti-angiogenesis therapy.In addition,we proposed a kind of anti-angiogenic drug templates: NK3R-A1,which might promote the development of the anti-tumor therapy.