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. 2016 Mar;37(3):734-42.
doi: 10.3892/ijmm.2016.2473. Epub 2016 Jan 29.

Arginine ADP-ribosyltransferase 1 promotes angiogenesis in colorectal cancer via the PI3K/Akt pathway

Lian Yang  1 , Ming Xiao  1 , Xian Li  1 , Yi Tang  1 , Ya-Lan Wang  1
Affiliations

Arginine ADP-ribosyltransferase 1 promotes angiogenesis in colorectal cancer via the PI3K/Akt pathway

Lian Yang et al. Int J Mol Med. 2016 Mar.

Abstract

Arginine adenosine diphosphate (ADP)-ribosyl-transferase 1 (ART1) is known to play an important role in many physiological and pathological processes. Previous studies have demonstrated that ART1 promotes proliferation, invasion and metastasis in colon carcinoma. However, it was unclear whether ART1 is involved in angiogenesis in cases of colorectal cancer (CRC). In the present study, lentiviral vector‑mediated ART1‑cDNA or ART1-shRNA were transfected into LoVo cells, and the LoVo cells transfected with ART1-cDNA or ART1-shRNA were co-cultured with human umbilical vein endothelial cells (HUVECs) to determine the influence of ART1 on HUVECs. The proliferation, migration and angiogenesis of HUVECs were monitored using a cell counting kit-8 assay, a Transwell migration assay and immunohistochemical analysis in intrasplenic allograft tumors, respectively. Hypoxia‑inducible factor 1-α (HIF-1α), total (t-)Akt, phosphorylated (p-)Akt, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) expression levels were detected via western blot analysis. Our results revealed that HUVECs which were co-cultured with ART1-cDNA LoVo cells showed higher proliferation, migration and angiogenic abilities, but a reduction was noted in those cultured with ART1-shRNA LoVo cells; p-Akt, HIF-1α, VEGF and bFGF expression was increased in HUVECs cultured with ART1‑cDNA-transfected LoVo cells, but reduced in ART1-shRNA-transfected LoVo cells. In a mouse xenograft model, we noted that the tumor microvessel density (MVD) was significantly increased in intrasplenic transplanted ART1‑cDNA CT26 tumors but decreased in intrasplenic transplanted ART1‑shRNA tumors. These data suggest that ART1 promoted the expression of HIF-1α via the Akt pathway in tumor cells. It also upregulated VEGF and bFGF and enhanced angiogenesis in HUVECs. Thus, we suggest that ART1 plays an important role in the invasion of CRC cells and the metastasis of CRC.

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Figures

Figure 1
Figure 1
Lentiviral vector-mediated argenine-specific adenosine diphosphate (ADP)-ribosyltransferase 1 (ART1)-shRNA and ART1-cDNA are successfully transfected into LoVo cells. (A) LoVo cells transfected with lentiviral vector-mediated ART1-shRNA and ART1-cDNA. Ten microliters of lentiviral vector was transfected into LoVo cells, and the efficiency was observed under an optical microscope and fluorescence microscope after 96 h, when the infection rate reached 80%. (B) The expression levels of ART1 mRNA and protein in ART1-cDNA LoVo cells were compared with the ART1-shRNA LoVo cells, the lentivirus (LV)-control LoVo cells and the untransfected LoVo cells. *P<0.05 vs. untransfected LoVo cells or LV-control LoVo cells; n=3 experiments.
Figure 2
Figure 2
Proliferation ability of human umbilical vein endothelial cells (HUVECs) co-cultured with supernatant fluid of argenine-specific adenosine diphosphate (ADP)-ribosyltransferase 1 (ART1)-cDNA LoVo cells at ratios of 0, 20, 40, 60, 80 and 100% was compared with those co-cultured with the supernatant fluid of ART1-shRNA LoVo cells, the lentivirus (LV)-control LoVo cells and the untransfected LoVo cells. *P<0.05 vs. untransfected LoVo cells or LV-control LoVo cells; n=3 experiments.
Figure 3
Figure 3
The effect of argenine-specific adenosine diphosphate (ADP)-ribosyltransferase 1 (ART1)-mediated LoVo cells on HUVEC migration. (A) The migratory ability of human umbilical vein endothelial cells (HUVECs) co-cultured with ART1-mediated LoVo cells. (a) HUVECs co-cultured with ART1-shRNA LoVo cells; (b) HUVECs co-cultured with the untransfected LoVo cells; (c) HUVECs co-cultured with LV-control LoVo cells; (d) HUVECs co-cultured with the ART1-cDNA LoVo cells. (B) The comparison of migration of HUVECs co-cultured with ART1-mediated LoVo cells afer 6 and 12 h. *P<0.05 vs. untransfected LoVo cells or lentivirus (LV)-control LoVo cells; n=3 experiments.
Figure 4
Figure 4
The effect of argenine-specific adenosine diphosphate (ADP)-ribosyltransferase 1 (ART1)-mediated LoVo cells on the blood vessel forming ability of human umbilical vein endothelial cells (HUVECs). (A) HUVEC angiogenic abilities when incubated with the supernatant of ART1-mediated LoVo cells. (a) HUVECs cultured with supernatant of ART1-shRNA LoVo cells; (b) HUVECs co-cultured with the untransfected LoVo cells; (c) HUVECs co-cultured with LV-control LoVo cells; (d) HUVECs co-cultured with the ART1-cDNA LoVo cells. (B) The tube length incubated with supernatant of ART1-cDNA LoVo cells was compared with incubated with the supernatant fluid of ART1-shRNA LoVo cells, the lentivirus (LV)-control LoVo cells and the untransfected LoVo cells. *P<0.05 vs. untransfected or LV-control group; n=3 experiments.
Figure 5
Figure 5
Microvessel density (MVD) in mice intrasplenic allograft tumor tissues. (A) The expression of CD34 indicated 'hot spots' and low MVD area in intrasplenic allograft tumors (H&E and immunohistochemical staining; magnification ×400). (B) The comparison of MVD in intrasplenic transplanted argenine-specific adenosine diphosphate (ADP)-ribosyltransferase 1 (ART1)-cDNA CT26 tumors with transplanted ART1-shRNA CT26 cells, LV-control CT26 cells and untransfected CT26 cells. *P<0.05 vs. untransfected or LV-control CT26 cells; n=4 mice in each group.
Figure 6
Figure 6
Arginine-specific adenosine diphosphate (ADP)-ribosyltransferase 1 (ART1) affects total-Akt, p-Akt, HIF-1α, VEGF and bFGF expression in LoVo cells. (A) The expression of total-Akt, p-Akt, hypoxia-inducible factor 1-α (HIF-1-α), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in ART1-mediated LoVo cells. (B) Comparison of Akt, p-Akt, HIF-1α, VEGF and bFGF expression in ART1-cDNA-transfected LoVo cells with that of ART1-shRNA-transfected LoVo cells, LV-control-transfected LoVo cells and untransfected LoVo cells. *P<0.05 vs. untransfected LoVo cells; n=3 experiments.
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