Recombinant Human Arresten and Canstatin Inhibit Angiogenic Behaviors of HUVECs via Inhibiting the PI3K/Akt Signaling Pathway

Abstract

Angiogenetic inhibitors are crucial in tumor therapy, and endogenous angiogenesis inhibitors have attracted considerable attention due to their effectiveness, safety, and multi-targeting ability. Arresten and canstatin, which have anti-angiogenesis effects, are the c-terminal fragments of the α1 and α2 chains of type IV collagen, respectively. In this study, human arresten and canstatin were recombinantly expressed in Escherichia coli (E. coli), and their effects on the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) were evaluated. Regarding the cell cycle distribution test and 5-ethynyl-2'-deoxyuridine (EdU) assays, arresten and canstatin could repress the proliferation of HUVECs at a range of concentrations. Transwell assay indicated that the migration of HUVECs was significantly decreased in the presence of arresten and canstatin, while tube formation assays suggested that the total tube length and junction number of HUVECs were significantly inhibited by these two proteins; moreover, they could also reduce the expression of vascular endothelial growth factor (VEGF) and the phosphorylation levels of PI3K and Akt, which indicated that the activation of the 3-kinase/serine/threonine-kinase (PI3K/Akt) signaling pathway was inhibited. These findings may have important implications for the soluble recombinant expression of human arresten and canstatin, and for the related therapy of cancer.

Keywords: Escherichia coli; angiogenesis; arresten; canstatin.

Figure 1

Construction of recombinant plasmids pRham^TM-arresten and pRham^TM-canstatin, and protein expression and identification. (A) Nucleic acid electrophoresis of re-combinant plasmid. M, DNA marker; Lane 1, pRham^TM-arresten plasmid digestion product; Lane 2, PCR product of arresten; Lane 3, pRham^TM-canstatin plasmid digestion product; Lane 4, PCR product of canstatin. (B) SDS-PAGE detection of the expression of recombinant arresten. M, protein marker; Lane 1, flow through; Lane 2, total protein of recombinant arresten; Lane 3, flow through. Lane 4, purified recombinant arresten. (C) Western blot identification of recombinant arresten and canstatin. M, Western blot molecular weight marker; Lane 1, recombinant arresten; Lane 2, recombinant canstatin. (D) SDS-PAGE detection of the expression of recombinant canstatin. M, protein marker; Lane 1, total protein of recombinant canstatin; Lane 2 and 3, flow through; Lane 4, purified recombinant canstatin. The protein bands for arresten and canstatin are indicated by arrows.

Figure 2

Effect of human arresten and canstatin on the proliferation of HUVECs. (A,B) The proliferation effects of recombinant human arresten and canstatin on HUVECs according to the CCK-8 assay. (C) Determination of cell proliferation using EdU. (D) Effects of recombinant human arresten and canstatin on the cell cycle distribution of HUVECs. Data are expressed as the mean ± standard deviation of triplicate experiments. Scale bars, 100 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate a significant difference compared to the control group.

Figure 3

The influence of recombinant arresten and canstatin on HUVEC migration. (A) Assessment of the migration ability using wound healing test. (B) The Transwell assay indicates that arresten and canstatin significantly inhibited the HUVECs migration and shows the images obtained with a microscope at 100× g magnification. (C,D) The result of wound healing (24 h) and migration (36 h) is represented as the relative cell migration rate. (E) Tube formation assay of HUVECs. (F,G) total length and junction number of HUVEC tube formation after 12 h of recombinant arresten and canstatin treatment. Cells were treated with 20 μg/mL of arresten and canstatin proteins for 24 h, respectively. Scale bars, 200 μm. Data are expressed as the mean ± standard deviation of triplicate experiments. * p < 0.05 and ** p < 0.01 indicate a significant difference compared to the control.

Figure 4

Effect of recombinant human arresten and canstatin on PI3K/Akt signaling pathway. (A) Western blot analysis of VEGF, p-PI3K, PI3K, p-Akt, and Akt proteins in different groups. (B–D) Protein expression levels of VEGF, p-PI3K, and p-Akt. (E) Western blot analysis of p-FAK, FAK, p-ERK, and ERK proteins in different groups. (F,G) Protein expression levels of p-FAK and p-ERK. (H) Schematic diagram of the mechanism by which recombinant human arresten and canstatin could bind to cell surface integrins and inhibit HUVEC proliferation, migration, and tube formation. Recombinant arresten and canstatin reduce VEGF expression and inhibit HUVEC proliferation, migration, and tube formation by suppressing activation of the PI3K/Akt pathway, thereby inhibiting angiogenesis. Data are expressed as the mean ± standard deviation of triplicate experiments. * p < 0.05 indicates a significant difference compared to the control.

Figure 4

Effect of recombinant human arresten and canstatin on PI3K/Akt signaling pathway. (A) Western blot analysis of VEGF, p-PI3K, PI3K, p-Akt, and Akt proteins in different groups. (B–D) Protein expression levels of VEGF, p-PI3K, and p-Akt. (E) Western blot analysis of p-FAK, FAK, p-ERK, and ERK proteins in different groups. (F,G) Protein expression levels of p-FAK and p-ERK. (H) Schematic diagram of the mechanism by which recombinant human arresten and canstatin could bind to cell surface integrins and inhibit HUVEC proliferation, migration, and tube formation. Recombinant arresten and canstatin reduce VEGF expression and inhibit HUVEC proliferation, migration, and tube formation by suppressing activation of the PI3K/Akt pathway, thereby inhibiting angiogenesis. Data are expressed as the mean ± standard deviation of triplicate experiments. * p < 0.05 indicates a significant difference compared to the control.