Targeting neurokinin-3 receptor: a novel anti-angiogenesis strategy for cancer treatment

Abstract

Angiogenesis is essential for tumor growth and metastasis, controlling angiogenesis is a promising strategy in cancer treatment. However, thus farther severe side effects of anti-angiogenic drugs have been rather demonstrated, stimulating interest in seeking novel targets of anti-angiogenesis. Neurokinin receptors, also known as tachykinin receptors, are usually considered as drug targets due to diverse physiological functions and their tractability. Although Neurokinin B, the selective natural agonist of neurokinin-3 receptor, have been shown to exhibit anti-angiogenesis activity, the effect and mechanism of neurokinin-3 receptor-mediated angiogenesis still remains unclear. In the present study, we demonstrated that [Mephe7]NKB, an analogue of NKB, possess significant anti-angiogenic effect on CAM. Furthermore, by introducing the tumor angiogenesis homing sequence (NGR), we designed and synthesized two novel agonist analogues of NK3R, NK3R-A1 and NK3R-A2. Both of the two analogues exhibit more efficient anti-migration effect on HUVECs by activating NK3R in vitro, and showed potent antitumor activities with no significant side effects in vivo. Taken together, our results illuminated that NK3R might be a potential novel target for the anti-angiogenesis therapy. Notably, NK3R-A1 might be used as a template for the development of the anti-tumor drugs on the basis of the anti-angiogenesis strategy.

Keywords: anti-angiogenesis; antitumor therapy; neurokinin 3 receptor; neurokinin B.

Figure 1. The anti-angiogenic effect of NKB mediated by NK3R on CAM

(A) Egg CAM was assayed in chick embryos as described in Materials and Methods. PBS, [Gly⁶]NKB[–10], NKB or a combination of [Gly⁶]NKB[–10] was applied to the CAM of day 8 chicken embryos. After 48 h, CAMs were dissected out and the representative areas were photographed (n = 10). The column charts displayed the relative rate of vascular density (B, D) and area (C, E) treated with NKB or co-treated with NKB and [Gly⁶]NKB[–10]. Data are mean ± SEM from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2. NK3R selective agonist analogues exert anti-angiogenic property on CAM through NK3R

(A) Chick CAM assay of angiogenesis. [MePhe⁷]NKB, NK3R-A1, NK3R-A2, or a combination of [Gly⁶]NKB[–10], respectively, was applied to the CAM of day 8 chicken embryos. The representative photos are taken after 48 h (n = 10). The columncharts displayed the relative rate of vascular density (B, D) and area (C, E) of [MePhe⁷]NKB, NK3R-A1 and NK3R-A2 treated group, respectively, or the relative rate of vascular density (F) and area (G) co-treated with NK3R selective agonist analogues and [Gly⁶]NKB[–10], respectively. (H) The gelatin sponge-CAM assay of neovascularization at microscopic level on histological sections. Representative photos are shown (400 ×). Data are mean ± SEM from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3. NK3R-A1, NK3R-A2 and [MePhe⁷]NKB could suppress the migration of HUVECs in vitro

(A–B) Wound healing assay. HUVECs were treated with PBS, NK3R-A1, NK3R-A2 or [MePhe⁷]NKB. During 24 h healing period, the cells migrating into the wound area were visualized and the digital images were captured for every 6 h, and representative photos were shown (200 ×) (A). (B) The histograms showed the relative width of the wound area within 24 h treated with the optimal concentration of NK3R-A1, NK3R-A2 and [MePhe⁷]NKB, respectively. (C–E) Transwell migration assay. HUVECs treated with NK3R-A1, NK3R-A2 and [MePhe⁷]NKB were seeded as indicated in the upper chamber. After 12 h incubation, the migrated cells were stained with hematoxylin and photographed (C) and representative images were shown (200 ×). The histograms reflected the number of migrated HUVECs treated with a range of concentration of NK3R-A1 (D) or NK3R-A2 (E) in 12 h. Data are mean ± SEM from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4. NK3R antagonist [Gly6]NKB[–10] could antagonize the anti-angiogenic effect induced by NK3R-A1, NK3R-A2 and [MePhe7]NKB in vitro

(A–D) Competitive antagonism was measured by using [Gly⁶]NKB[–10] in Wound healing assay. (A) HUVECs were treated with PBS, NK3R-A1, NK3R-A2 or [MePhe⁷]NKB alone, or co-treated with [Gly⁶]NKB[–10]. During 24 h healing period, the cells migrating into the wound area were photographed for every 6hand representative photos were shown (200 ×). The analysis of the relative width of wound area was displayed in column chart (B–D). (E–H) Competitive antagonism was measured by using [Gly⁶]NKB[–10] in Transwell migration assay. The representative images are shown (200 ×). (E) The histograms indicated the numbers of migrated HUVECs co-treated with [Gly⁶]NKB[–10] and NK3R-A1 (F), NK3R-A2 (G) or [MePhe⁷]NKB (H), respectively, in 12 h. Data are mean ± SEM from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5. Effects of analogues, NK3R-A1, NK3R-A2 and [MePhe⁷]NKB, on tumor growth in S180 sarcoma-bearing BALB/c mice

(A) Mice treatment protocol: BALB/c micewere subcutaneously implanted with 2 × 10⁶ cells/mouse on the right flank (day 0). Seventy-two hours after inoculation, forty mice with S180 cells were randomly divided into eight groups. Peptides were continuously administrated intravenously by tails for 14 days (0.1 ml/10 g, once a day): group 1 with NS (negative control), group 2 with cyclophosphamide (CTX, positive control), groups 3 and 4 were injected with peptide [MePhe⁷]NKB (0.2 and 1 mg/kg, respectively), groups 5 and 6 were injected with peptide NK3R-A1 (0.2 and 1 mg/kg, respectively), groups 7 and 8 were injected with peptide NK3R-A2 (0.2 and 1 mg/kg, respectively). All mice were sacrificed after 24 h of the last administration, and the tumor tissues were excised and weighed. (B) The growth curves of the tumor were recorded with a caliper and the tumor volume was measured every two day according to the equation: tumor volume = length × width2/2. (C) The body weight changes curve of each group of BABL/c mice during the intravenous administration of peptides for 14 days. (D–E) At the end of the experiment (day 15), the mice were sacrificed, and the tumors were isolated, photographed and weighed. (F–G) CD31 staining. Tumor tissues from each group were stained using immunohistochemistry for CD31 and representative photos were shown (200 ×) (F), the evaluation of immunostaining for microvasculature density (MVD) in tumor tissues was displayed in bar graph (G). Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.