14K prolactin derived 14-mer antiangiogenic peptide targets bradykinin-/nitric oxide-cGMP-dependent angiogenesis

Abstract

Over the past few decades, VEGF-targeted antiangiogenic therapy for cancers has gained increasing attention. Nevertheless, there are still several limitations such as the potential resistance mechanisms arising in cancer cells against these therapies and their potential adverse effects. These limitations highlight the need for novel anti-angiogenesis molecules and better understanding of the mechanisms of tumor angiogenesis. In the present study, we investigated the antiangiogenic properties of a novel 14-mer antiangiogenic peptide (14-MAP) derived from N-terminal 14 kDa buffalo prolactin and characterized its mode of action. 14-MAP at the picomolar concentration inhibited VEGF- and bradykinin (an autacoid peptide expressed in vascular tissues in pathophysiology, BK)-stimulated endothelial nitric oxide (eNO) production, cell migration, and proliferation in endothelial cells and vessel development in the chick embryo. Although this peptide inhibited both VEGF- and BK-dependent angiogenic processes, its action was more pronounced in the latter. Moreover, the interference of 14-MAP with the eNO synthase (eNOS)-cyclic GMP pathway was also identified. A combination of a low dose of Avastin, a widely used drug targeting VEGF-dependent angiogenesis, and 14-MAP significantly reduced tumor size in an in vivo model of human colon cancer. Taken together, our results suggest that 14-MAP, a BK- and eNOS-dependent antiangiogenic peptide, might be useful for overcoming the limitation of VEGF-targeted antiangiogenic therapy in cancer patients. However, further studies will be required to further characterize its mode of action and therapeutic potential.

Keywords: antiangiogenic peptide; anti‐angiogenesis; bradykinin; endothelial nitric oxide synthase; prolactin.

Fig. 1

Inhibition of VEGF‐ and BK‐induced endothelial cell functions by 14‐MAP. (A, B) Cell migration analysis by wound healing assay. Cell migration analysis by wound healing assay of 14‐MAP on VEGF pathway, scale 100 μm (C) and BK pathway (D). (E) Cell proliferation analysis, and (F, G) (scale 50 μm) Angiogenic vessel growth analysis by CAM assay of 14‐MAP on VEGF‐ and BK‐pathways, respectively. 14‐MAP and Scr, 10 pg·mL⁻¹ (approx. 7 pm); VEGF, 10 ng·mL⁻¹ (approx. 0.3 nm); BK, 1.24 μg·mL⁻¹ (approx. 1.24 μm). Cntl, control; Scr, scramble (GSQCAAGTMNLKIF). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Arrows indicate the deteriorating (14‐MAP, 14‐MAP+BK, and 14‐MAP+VEGF) and ameliorating (VEGF) vascular network. Statistical analysis was conducted using Tukey's or Dunnett's test in conjunction with the one‐way ANOVA test or Dunnett's T3 test in conjunction with Welch's one‐way ANOVA. All mean data were represented with standard error (SEM). Each experiment represents three independent biological experiments.

Fig. 2

Inhibitory effect of 14‐MAP in eNOS pathway. (A) NO production analysis of 14‐MAP on VEGF pathway in EC. (B) NO production analysis of 14‐MAP on eNOS/NO/sGC/cGMP pathway in EC. (C) Cell migration analysis of 14‐MAP on eNOS/NO/sGC/cGMP pathway in EC. (D) Diagram of eNOS/NO/sGC/cGMP pathway correlated with 14‐MAP. Cntl, control; Scr, scramble (GSQCAAGTMNKIF). **P < 0.01; ***P < 0.001; ****P < 0.0001. Statistical analysis was conducted using Tukey's or Dunnett's test in conjunction with the one‐way ANOVA test or Dunnett's T3 test in conjunction with Welch's one‐way ANOVA. All mean data were represented with standard error (SEM). Each experiment represents three independent biological experiments.

Fig. 3

Interference of BK‐BKRs pathway by 14‐MAP. (A) EC cell migration. (B) EC proliferation. (C) NO production. 14, 14‐MAP; BK, B2R agonist; Cntl, control; L8, [Leu8]Des‐Arg9‐BK/B1R antagonist; R9, Des‐Arg9‐BK/B1R agonist; Scr, scramble. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Statistical analysis was conducted using Tukey's or Dunnett's test in conjunction with the one‐way ANOVA test or Dunnett's T3 test in conjunction with Welch's one‐way ANOVA. All mean data were represented with standard error (SEM). Each experiment represents three independent biological experiments.

Fig. 4

14‐MAP effects on cancer. (A) Tumor growth monitoring in HT29‐xenografted Balb/c nude mice. (B) Time‐dependent body weight monitoring of the mice. (C) HT29 cell migration analysis. (D) HT29 cell proliferation analysis. Cntl, control (PBS). Arrows indicated I.V. injection (A, B). Avastin (M.W., 149 kDa) and 14‐MAP (M.W., 1.4 kDa) were treated as 0.5 mg·kg⁻¹ and 0.5 μg·kg⁻¹, respectively (A, B), and 5 ng·mL⁻¹ (approx. 34 pm) and 50 pg·mL⁻¹ (approx. 35 pm), respectively (C, D). *P < 0.05; **P < 0.01 vs control. Statistical analysis was conducted using the Student's t‐test and nonparametric Kruskal–Wallis test. All mean data were represented with standard error (SEM). Five mice per group were used in the experiment.