The role of short peptides in tumor angiogenesis

（1. Pharmacology Department，School of Pharmacy，Gannan Medical University；2. Key Laboratory of Ministry of Education for Prevention and Treatment of Cardiovascular and Cerebrovascular Diseases，Gannan Medical University；3. School of Rehabilitation，Gannan Medical University，Ganzhou，Jiangxi 341000；4. School of Health and Related Research，The University of Sheffield，Sheffield，UK S105AD；5. Ganzhou Cancer Precision Medical Engineering Center，Gannan Medical University，Ganzhou，Jiangxi 341000）

Abstract :Because tumor angiogenesis is one of the most prominent mechanisms driving tumor development and progression，anti-angiogenesis is a potential novel anticancer therapy. But more and more studies found conventional antiangiogenic drugs used alone are clinically ineffective in treating advanced solid tumor because of reasons such as indirectly directed against tumor growth，not specific to cancer cells and so forth. With the increasing understanding of short peptides in tumor angiogenesis，the application prospect of short peptides in anti-angiogenesis has attracted attention gradually. This review will contribute to a better understanding of short peptides in the inhibition of tumor angiogenesis.

Key words: Angiogenesis；Cytotoxicity；Growth factors；Short peptides；Tumor

Cancer is a generic term for a large group of diseases that can affect any part of the body；other term used is neoplasms. In 2018，the number of deaths attributed to cancer worldwide was estimated at 9. 6 million［1］. In 1971，Judah Folkman supposed that preventing new blood vessel formation in small cancers could prevent their growth. At the same time，he also introduced the concept of "anti-angiogenesis" as a potential novel anticancer therapy［2］. The first drug to inhibit angiogenesis was approved in 2004［3］. The angiogenesis inhibitors currently available on the market are either，monoclonal antibodies that specifically recognize and bind to vascular endothelial growth factor（VEGF）and prevent it from activating VEGF receptor，or antagonists that bind to VEGF and/or its receptor as well as to other receptors on the surface of endothelial cells or to other proteins in the downstream signaling pathways，blocking their activities. Some angiogenesis inhibitors are immunomodulatory drugsagents that stimulate or suppress the immune system［4-16］. Table 1 summarizes angiogenesis inhibitors approved by the food and drug administration.

Table 1 Food and drug administration approved angiogenic inhibitors

Continue Table 1 Food and drug administration approved angiogenic inhibitors

Conventional antiangiogenic drugs used alone are clinically ineffective in treating advanced solid tumor because of three main reasons：first，they cannot reach and simulate the tumor microenvironment，second，they are indirectly directed against tumor growth because they block the growth of blood vessels rather than blocking the growth of tumor cells themselves，and third，they are not specific to cancer cells thereby，causing side effects in normal cells. Bevacizumab，the most used angiogenic inhibitor，is responsible of bowel perforation［17］，and showed no survival advantage when combined with gemcitabine and erlotinib in phase Ⅱand Ⅲstudies of advanced pancreas cancer bearing patients［18-21］.

Short peptides can be defined as bioactive substances made up of a small number of amino acid residues（20-30 amino acid residues in length）［22］. The overexpression of receptors for peptides in tumor cells，have led to the discovery of novel antitumor drugs with high selectivity and efficacy due to their physicochemical properties，such as small size，ease of synthesis and modification，penetrating ability， and biocompatibility［23］. Many studies reviewed the use of peptides in tumor targeted therapy［23-25］，and it appears that they can be used for different therapeutic purposes（Figure 1）. This review aimed to increase the perspective on the uses of short peptides in the inhibition of tumor angiogenesis. The high-quality articles addressing the activities of short peptides on tumor angiogenesis were reviewed，regardless of the language of publication. Our major exclusion criteria were the journal impact factor（less than 2）and the length of amino acid of the different peptides（more than 30）. The articles reviewed allowed us to understand that short peptides play a fundamental role in tumor angiogenesis because they are derived from angiogenic related molecules. In relation to angiogenesis，short peptides can be derived from growth factors and growth factors receptors，extracellular matrix proteins，type 1 thrombospondin （THBS1） domain，and other angiogenesis related proteins. According to their origins，short peptides can be used as hormone，as vaccine，and as carrier for cytotoxic drug and radionuclide. The majority of them demonstrated potent antitumor activity alone or in association with traditional chemotherapeutic drugs either in-vitro and in-vivo.

In tumor targeted therapy，short peptides can be used as targeting ligands for cytotoxic drug and radionuclide（tumor homing peptides）.

Fig. 1 Treatment options for cancer using short peptides

1 Short peptides derived from the extracellular matrix（ECM）proteins

The ECM is a collection of molecules and fibers that hold the cells of tissues together. There are three major molecules that constitute the ECM；structural proteins（as collagen which provides the matrix with strength），adhesive proteins （as selectins，integrin and cadherin）and，proteoglycans responsible for functioning in cell adhesion. Hence，proteins of ECM have been studied for potential smaller peptides that can interfere with tumor angiogenesis.

1. 1 iRGD PeptidesMany peptides can bind integrin's receptors expressed on tumor cells and possess inhibitory activity［26］， resulting in the inhibition of tumor cells mobility and suppression of metastasis which is related to angiogenesis. Arginineglycine-aspartic acid（RGD）peptide was identified by in vivo phage display in tumor bearing mice in 1997.The RGD cell adhesion sequence peptides have high affinity to the αv （CD51） integrin protein overexpressed in tumor vasculature than in normal vasculature and have inhibitory activity［27］. RGD peptides for scientific research and practical applications have some advantages［28］：（i） RGD is much smaller compared to monoclonal antibodies，and cancer drugs conjugated to internalizing RGD had greater uptake into tumors than unconjugated therapeutics；（ii）the use of RGD minimizes the risk of immune reactivity or pathogen transfer；（iii） the synthesis of RGD peptides is relatively simple and inexpensive. iRGD peptides have anti-metastatic activity through a 3-step process（Figure 2）［29］. CEND-1，an iRGD drug product is actually in phase 1 study in combination with nab-paclitaxel and gemcitabine for metastatic pancreatic cancer［30］. RGD peptides used in metastatic cancer are listed in Table 2［31-39］.

Table 2 Short RGD-peptides used in metastatic cancer

Fig. 2 Schematic representation of the anti-metastatic activity of iRGD peptides

First，their RGD sequence motif mediates binding to αvβ3and αvβ5integrins that are expressed on tumor endothelial cells. Second，upon αVbinding，a protease cleavage event is activated，revealing the cterminal c-end rule （CendR） motif of the peptide.Third，the CendR motif is now able to bind to neuropilin-1 （NRP-1），activating an endocytotic/exocytotic transport pathway.

1. 2 Others ECM-derived peptides

1. 2. 1 Endostatin short peptidesMurine P1（LEQKAASCHNSYIVLCIENSFMTSFSK）is a c-termi nal endostatin peptide that contains 27 amino acid sequences and，three histidine's zinc-binding domain retaining the anti-tumor，anti-migratory activities of endostatin，but not its anti-permeability activity. This peptide demonstrated antitumor activity in BxPC-3 mice xenograft at the dose 7 mg·（kg·d）-1subcutaneous during 28 days［40］.

ES2，IVRRADRAAVP is an eleven amino acid peptide derived from the C-terminus of endostatin.This peptide displayed better anti-angiogenic effect than the complete endostatin sequence［41］.

1. 2. 2 N-cadherin antagonistsCadherins proteins are a super family of cell adhesion molecules（CAM）. Inside the cadherin superfamily，we have the type 1 classical cadherins，which is made up of Ecadherin present in epithelia and N-cadherin found in neural and muscle tissue. Tumor microenvironment overexpress type 1 classical cadherins［42］. N-cadherin is intimately involved in the formation of blood vessels and the maintenance of their integrity，and inhibition of N-cadherin function destabilizes micro-vessels［43］.

ADH-1（ExherinTM）is a short，cyclic pentapeptide vascular-targeting agent with potential antineoplastic and antiangiogenic activities［44］. ADH-1 selectively and competitively binds to and blocks N-cadherin，which may result in disruption of tumor vasculature，inhibition of tumor cell growth，and the induction of tumor cell and endothelial cell apoptosis［45］. ADH-1（50 mg·kg-1）significantly prevents tumor growth and metastasis in a mouse model for pancreatic cancer. ADH-1 prevents tumor cell invasion and metastasis in an orthotopic model for pancreatic cancer using N-cadherin overexpressing BxPC-3 cells［46］.

1. 2. 3 Laminin derived peptidesC16S（DFKLFAVTIKYR） and C16Y （DFKLFAVYIKYR）are antagonists of laminin pro-angiogenic peptides A13（RQVFQVAYIIIKA） and C16 （KAFDITYVRLKF）.C16Y is obtained by replacement of T by Y in shortened amino acid sequence of C16S. C16Y displayed more potent （by ＞70%） in inhibiting attachment to laminin-1 than C16S（by ＜40%）［47］.C16Y also inhibited tube formation at a dose five times lower than that of C16S. In vivo study of angiogenesis using chick chorioallantois membrane （CAM） assay showed that C16S and C16Y at same concentration inhibit angiogenesis induced by C16 at different percentages（63% for C16S and 88% for C16Y）［38］.

2 Short peptides derived from type 1 thrombospondin（THBS1）domain

Thrombospondin 1（THBS1） is an extracellular matrix glycoprotein from platelets and a variety of normal and transformed cells of both mesenchymal and epithelial origin. THBS1 is a potent angiogenic inhibitor that is produced and secreted by many tumor types［48］.The anti-angiogenic activity of TSP1 is localized to three type I repeats in the pro-collagen domain，and several peptides derived from these regions have been identified as angiogenesis inhibitors. This domain directly interacts with CD36 and induces cell death predominantly in endothelial and smooth muscle cells［49］.

3 Short peptides derived from growth factors and growth factor receptors

Growth factors are factors produced by the cell of interest in response to different stimuli or any damage.When cells become hypoxic，they release a factor called vascular endothelial growth factor. This factor can bind to his receptors called vascular endothelial growth factor receptors 1，2 and 3 on the surface of endothelial cells. The binding of VEGF to VEGFRs will trigger intracellular signaling cascade which can result in two types of angiogenesis，sprouting angiogenesis and intussusceptive angiogenesis. Cancer cells overexpress growth factors and their receptors. Table 4 resumes peptides derived from vascular endothelial Growth factor，epidermal growth factor（EGF），fibroblast growth factor（FGF），and transforming growth factor β（TGF-β）and their receptors［53-60］.

Table 3 Short peptides derived from thrombospondin 1 domain

Table 4 Short peptides derived from growth factors and growth factor receptors

4 Other short peptides used for antiangiogenic purpose

LyP-1（CGNKRTRGC）is a homing peptide，that binds the p32 protein found in mitochondrial matrix，nucleus and endoplasmic reticulum of normal and cancer cells［61］. The satisfactory targeting and internalization ability of LyP-1 makes it an attractive homing peptide for ongoing researches in the fields of disease imaging. To date，imaging agents such as fluorescent dyes，radionuclides have been directly labeled with LyP-1，and various nanomaterials including but not limited to Fe3O4NPs，bismuth（Bi），Abraxane，microbubbles（MBs），liposomes，heat shock protein（Hsp）and dendrimers have been selected as platforms for preparing multifunctional Lyp-1-modified complexes.LyP-1 can be employed to produce excellent images of suspected lymph nodes（LNs）before metastasis and lymphatic metastatic tumors， which represents two metastatic states in tumor metastasis into regional LNs［62］.

A7R （Ala-Thr-Trp-Leu-Pro-Pro-Arg）， a new dual targeting ligand for vascular endothelial growth factor receptor 2（VEGFR-2）and neuropilin-1（NRP-1），is a new potent inhibitor of tumor angiogenesis and a targeting ligand for cancer therapy［63］.

SP5-52 peptide（SVSVGMKPSPRP）showed selective recognition of tumor blood vessels，not normal vessels，and was used as a targeting ligand for the treatment of non-small cell lung carcinoma（NSCLC）［64］.

Trebananib is a peptide inhibitor that neutralizes both Ang1 and Ang2 interaction with Tie2 receptor，reducing tumor angiogenesis［65］. It belongs to a new class of therapeutic molecules called peptibodies，consisting of a biologically active peptide that is grafted to the Fc region of an immunoglobulin G（IgG）. Trebananib demonstrated inhibitory activity for both Ang2（IC50=0. 023 nM）and Ang1（IC50=0. 9 nM）interaction with Tie2［66］.

5 Conclusion

Regarding their structure short peptides present many advantages like small size（less than 30 amino acids），their synthesis is cost effective and their degradation products are amino acids which are less immunogenic compared to conventional or traditional cytotoxic drugs［67］. Due to their specificity to cancer cell and cancer cell derived factors，short peptides used alone or in combination constitute a promising alternative to overcome toxicity and common sides effects（primary hypertension and kidney disfunction） seen with already marked drugs targeting tumor angiogenesis. One example is the combination of bevacizumab with trebananib，in first-line treatment of metastatic colorectal cancer［65］. We will even go further by saying that combining antitumor peptide with traditional anti-angiogenic drugs at metronomic dosing could decrease considerably tumor metastasis because at this dosage the bone marrow doesn't have time to produce endothelial progenitor cells，but this still warrants prospective trials.