Antisense oligonucleotide targeting midkine suppresses in vivo angiogenesis

Abstract

Aim: To evaluate the effect of antisense oligonucleotide targeting midkine (MK-AS) on angiogenesis in chick chorioallantoic membrane (CAM) and in situ human hepatocellular carcinoma (HCC).

Methods: An in situ human hepatocellular carcinoma (HCC) model and CAM assay were used in this experiment. The effect of MK-AS on angiogenesis was evaluated by cell proliferation assay and hematoxylin-eosin (HE) staining.

Results: MK-AS significantly inhibited human umbilical vein endothelial cells (HUVEC) and in situ human HCC growth. At the same time, MK-AS suppressed the angiogenesis both in human hepatocellular carcinoma cell line (HEPG2)-induced CAM and in situ human HCC tissues.

Conclusion: MK-AS is an effective antiangiogenesis agent in vivo.

Figure 1

RT-PCR (A) and Western-blotting analysis (B) of MK expression in human umbilical vein endothelial cells (HUVEC) after transfection with 0.4 μmol/L MK-AS (lane 4), MK-SEN (lane 3), Lipofectin alone (lane 2) for 24 h. Lane 1 represents cells without transfection. GAPDH and β-actin were used as control respectively.

Figure 2

Effect of MK on growth of HUVEC. Cells after transfection with 0.4 μmol/L MK-AS or MK-SEN for 24 h were analyzed by MTS assay. Data were expressed as mean ± SD from four independent experiments.

Figure 3

Angiogenesis in normal chick CAM (A), HepG2-induced CAM (B), and HepG2-induced CAM treated with 0.4 μmol/L MK-AS (C). Chick CAM assays were used to assess the impact of MK-AS on angiogenesis in vivo.

Figure 4

Immunohistochemical analysis of HCC xenograft angiogenesis after treatment with MK-SEN (A), 25 mg/kg of MK-AS per day (B), 50 mg/kg of MK-AS per day (C), and 100 mg/kg of MK-AS per day (D). Representative views of anti-CD34 antibody-stained vascular endothelium (brown) (х 200).

Figure 5

Tumors resected from mice at the experimental end point. Resected tumors were sectioned and stained as described in the text. Microvessel densities (MVD) were determined by counting CD34-positive endothelial cells in the sections and presented as mean ± SE positive cells/field from the three “hot-spot” fields. ^aP < 0.05, ^bP < 0.01 vs control group.