Small peptides are emerging and promising agents in developing new therapeutics for different diseases (Saladin et al., 2009
; Rosca et al., 2011
). Peptide agents have significant merits compared to conventional proteins and large synthetic molecules. The advantages of peptides as drugs over proteins are high specificity, low immunogenicity and toxicity, better solubility in water and stable product quality between batches (Sulochana and Ge, 2007
). One disadvantage is short half-life in vivo, which could be overcome by peptide modification, conjugation to a macromolecule, or combination with a delivery vehicle (Rosca et al., 2011
; Bhise et al., 2011
; Grdisa, 2011
Previously several anti-angiogenic peptides were identified, derived from endogenous proteins such as thrombospondin-1, collagen, laminin, decorin, platelet factor-4, kininostatin, and VEGFR. Among them a number of anti-angiogenic peptides are in clinical trials (Sulochana and Ge, 2007
; Rosca et al., 2011
). Although many trials have been conducted for anti-angiogenic peptides, peptide agents for inhibiting lymphangiogenesis have not been reported. In this study we investigated the anti-lymphangiogenic effects of small somatotropin peptides. The peptides from IL-17 receptor C (SP5001) and placental lactogen (SP5033) potently inhibited LEC proliferation (). Four peptides derived from brush border myosin-1 (SP5028), chorionic somatomammotropin (SP5030), transmembrane protein 45A (SP5031) and chorionic somatomammotropin-like 1 (SP5032) strongly inhibited LEC migration and adhesion (, ). SP5030, SP5031 and SP5032 were strong inhibitors of the LEC capillary-like tube formation. Among them SP5031 was the most active LEC tube formation inhibitor ().
In the present study we could identify two classes of lymphangiogenesis inhibitors. The first class which includes SP5001 and SP5033 inhibit lymphangiogenesis by inhibiting proliferation of lymphatic endothelial cells and the second class which includes SP5030, SP5031 and SP5032 inhibit lymphangiogenesis by mainly inhibiting lymphatic endothelial cell migration and adhesion (). SP5033 showed significant apoptotic activity on HUVEC, LEC and MEC exhibiting caspase-3/7 activity (). Interestingly the amino acid sequence of SP5033 (LLRISLLLIESWLE) was very similar to the previously studied 14-mer tilted peptide (LLRISLLLIQSWLE) from 16-kDa fragments of prolactin (PRL) (Nguyen et al., 2006
) with a difference only in the 10th
amino acid. The glutamic acid (Glu, E) in SP5033 is replaced with the glutamine (Gln, Q) in the tilted PRL peptide. It has been reported that the tilted PRL peptide led to cell apoptosis (Nguyen et al., 2006
). Also it was shown that PRL protein induced apoptosis in a prostate cancer model (Giuffrida et al., 2010
). These results with prolactin (PRL) peptide and protein suggest that the PRL protein has active domains which may include the sequence of SP5033 exhibiting apoptotic activity. Also the apoptotic property of SP5033 may be associated with its structure which may be conserved regardless of the 10th
amino acid replacement. However SP5001, the other peptide in this class, was not associated with caspase-3/7 activity even though it showed potent proliferation inhibitory effect as seen by increased LDH levels (). This suggests that SP5001 has anti-proliferative activity through other pathways such as arresting the cell cycle or other cytotoxic pathways that are different from the caspase-3/7 dependent apoptosis pathway. The second class peptides comprising SP5030, SP5031 and SP5032 inhibited LEC migration and adhesion and were potent lymphatic tube formation inhibitors compared to the first class of peptides (, and ). These two classes of peptides allow one to manipulate endothelial cell proliferation and migration separately via independent pathways. The receptors and signaling pathways targeted by the two classes of peptides resulting in the control of cell proliferation and migration remain to be identified.
Classification of somatotropin peptides
We have identified several anti-lymphangiogenic sequences derived from various proteins including IL-17 receptor C, brush border myosin-1, neuropeptide FF receptor 2, transmembrane protein 45A, which are not associated with angiogenesis or lymphangiogenesis. These finding may contribute to the understanding of the poorly understood physiological roles of these proteins. For example our present study of SP5001, the peptide derived from IL-17 receptor C protein, could lead us to hypothesize that there might be lymphangiogenesis-related physiological roles of the IL-17 receptor C protein which is known as a receptor for IL-17A and F. Recently it was reported that a ligand IL-17 promotes the expression of VEGFC in non-small-cell lung carcinomas inducing lymphangiogenesis and inflammation (Chen et al., 2010
). We had not expected this correlation between IL-17 and lymphangiogenesis before we identified SP5001. We hypothesize that IL-17 receptor C might work as an endogenous scavenger of IL-17 and the active site for anti-lymphangiogenic and anti-inflammatory activity could be closely related to our peptide sequence; further studies are required to validate this hypothesis.
In this study we identified that SP5031 is the best inhibitor of lymphangiogenesis by blocking LEC migration, adhesion and tube formation potently. Surprisingly we found that SP5031 significantly inhibited the migration of the highly metastatic MDA-MB-231 triple-negative breast cancer cells as well. SP5031 blocked the migration of MDA-MB-231 induced by a full complement of media which includes 10% FBS by 83% at a concentration of 50μM (). These dual inhibition results suggest that SP5031 may have both anti-tumorigenic and anti-lymphangiogenic activity.
There are three important consequences of inhibiting migration of MDA-MB-231 cells in addition to blocking lymphangiogenesis. The first is that SP5031 could delay the transport of tumor cells into the lymph nodes by decreasing motility of the cancer cells. Previous data suggest that the lymph node metastasis is greatly facilitated by migration of tumor cells into the lymph nodes via chemotactic agents (Shields et al., 2007
). Secondly, inhibition of MDA-MB-231 migration by SP5031 suggests that SP5031 may have direct anti-tumorigenic activity. Cancer cell migration is cooperative with tumor microenvironment for further tumorigenesis (Hanahan and Weinberg, 2011
). Thus the inhibition of cancer cell migration can be a phenotype of anti-tumorigenesis. Finally anti-lymphangiogenic activity of SP5031 will decrease the density of lymphatic vessels around the tumor by inhibiting new lymphatic vessel formation, which could help to limit tumor metastasis. Additional studies are needed to characterize anti-tumor and anti-metastatic activity of SP5031 in vivo in different cancer models.
When it comes to anti-angiogenic efficacy of the peptides, we have identified three leading peptides: peptides derived from brush border myosin-1 (SP5028), chorionic somatomammotropin (SP5030) and chorionic somatomammotropin-like 1 (SP5032). Interestingly both SP5030 and SP5032 are derived from chorionic somatomammotropin related proteins. Their amino acid sequences are very similar: 10 of 13–14 amino acids are identical between the two sequences. These anti-angiogenic peptides could be applied to the treatment of different angiogenesis dependent diseases such as cancer, age-related macular degeneration (AMD), or rheumatoid arthritis.
Angiogenesis and lymphangiogenesis are both important processes in health and diseases. Somatotropin peptides are active in both inhibiting lymphangiogenesis and angiogenesis (). These properties are expected be useful for modulating those phenomena in diseases. In particular these peptides would be applied to prevent the deadly spread of cancer cells by inhibiting not only tumor growth but also peritumoral lymphatics. Alternatively, a single compound that targets both tumor-associated blood vessels and tumor-associated lymphatic vessels may decrease tumor size and decrease the incidence of local and distant metastases. Thus these peptides should be further tested in vivo for their simultaneous inhibition of tumor growth and metastasis and other angiogenesis-dependent diseases.