Abstract

Angiogenesis, the formation of new blood vessels from the existing vascular bed, has been described as one of the hallmarks of cancer, and plays an essential role in tumor growth, invasion, and metastasis. The development of specific anti-angiogenic agents is an attractive therapeutic approach for the treatment of cancer. Apolipoprotein, Apo(a), is a large glycoprotein with multiple kringle structures, which play an important role in regulating the intermolecular reaction in the body. The evidence shows that Apo(a) has anti-angiogenic activity on vascular endothelial cells and inhibits tumor growth in mice. The 38th kringle on Apo(a) has been found to be homologous to the kringle 5 of plasminogen, a known endogenous anti-angiogenic kringle structure. According to our preliminary study, the 38th kringle of Apo(a) had an inhibitory effect on bovine capillary endothelial (BCE) cells. Seven peptides of the 38th kringle on Apo(a) were synthesized for screening of anti-angiogenic activity. Two synthetic peptides (P4 and P5) based on the 38th kringle structure sequence of Apo(a) were found to inhibit the proliferation of BCE cells. In the current study, we examined the effects of P4 and P5 synthetic peptides on cell proliferation by MTT assay, and VEGF production by ELIAS assay in human prostate cancer cells (C4-2B, LNCap, and PC3), and/or vascular endothelial cells such as human aortic endothelial cells (HAEC), human umbilical vein endothelial cell (HUVEC), and mouse microvascular endothelial cells (MVEC) after 2 days of incubation. The results are shown in the Table 1 and 2 below.

Table 1.

Effect of synthetic peptides on cell proliferation in cancer and vascular endothelial cells (%)

P4P5
Cell proliferation assay was conducted by MTT methos. The % of cell proliferation in peptide treatment groups was compared to the cells without peptide treatment 
HAEC 64.7±16.4 62.4±12.0 
HUVEC 59.4±3.7 71.7±1.2 
MVEC 76.9±6.1 75.8±5.6 
C4-2B 64.1±5.1 60.9±4.3 
LNCap 77.9±2.9 51.8±1.6 
PC3 75.8±3.4 75.4±3.6 
P4P5
Cell proliferation assay was conducted by MTT methos. The % of cell proliferation in peptide treatment groups was compared to the cells without peptide treatment 
HAEC 64.7±16.4 62.4±12.0 
HUVEC 59.4±3.7 71.7±1.2 
MVEC 76.9±6.1 75.8±5.6 
C4-2B 64.1±5.1 60.9±4.3 
LNCap 77.9±2.9 51.8±1.6 
PC3 75.8±3.4 75.4±3.6 
Table 2.

Effect of synthetic peptides on VEGF production in cancer cells (pg/mL)

ControlP4P5
C4-2B 905.0±87.7 694.1±99.1 720.1±106.2 
LNCap 1116.5±191.6 717.9±100.1 794.3±119.7 
PC3 902.6±52.0 743.2±123.7 721.3±92.7 
ControlP4P5
C4-2B 905.0±87.7 694.1±99.1 720.1±106.2 
LNCap 1116.5±191.6 717.9±100.1 794.3±119.7 
PC3 902.6±52.0 743.2±123.7 721.3±92.7 

Our data indicate that these synthetic peptides inhibited the cell proliferation and VEGF production. We also examined the effect of P4 and P5 peptides on angiogenesis in HAEC and MVEC cells after 10h of incubation by Matrigel tube formation assay. The data indicate that angiogenesis declined by 40–50% when P4 or P5 was added to the cell culture. All these data suggest that P4 and P5, synthetic peptides derived from the 38th Kringle of Apo(a) have anti-proliferative and anti-angiogenic effects, which may potentially be applicable for cancer treatment. Further studies are in progress in order to define the mechanisms action of these peptides.

Author notes

Disclosure:Research Funding: Supported by DMCIOAD Research Grant and WSU University Research Grant.