Angiopoietin-2 (Ang-2) has been identified as an important factor in tumour angiogenesis through its action in blocking the stabilizing actions of Ang-2 and leading to new tumour vessel growth in the presence of vascular endothelial growth factor (VEGF). In the authors’ previous study, over-expression of Ang-2 in Tca8113 tongue tumour cells inhibited growth. The current study aims to clarify the mechanisms of Ang-2-mediated tumour growth inhibition and its role in the regulation of VEGF expression. These studies used tumours derived from Ang-2-transfected Tca8113 cells injected into nude mice. The results showed that Ang-2-transfected tumours displayed aberrant angiogenic vessels with few associated smooth muscle cells. No detectable differences in VEGF expression were observed between Ang-2-transfected and parental tumours. Tumours produced by the Ang-2 transfection also had a higher apoptosis index and lower tumour cell proliferative index than tumours in the control groups. These observations suggest that enhanced expression of Ang-2 has no effect on VEGF expression and results in tumour vessel regression and inhibition of tumour growth.
Growth of solid tumours to a clinically relevant size depends on angiogenic factors forming a vascular network. Many studies have attempted to isolate the molecular mediators of tumour angiogenesis. Amongst the known angiogenic factors, vascular endothelial growth factor (VEGF) has emerged as a central regulator of angiogenesis during physiological and pathological conditions . Angiopoietins (Angs), the ligands for the tyrosine kinase receptor Tie2, are another important family of angiogenic factors. Of the four currently known Angs (Ang-1–4), Ang-2, due to its critical roles in pathological angiogenesis, has naturally become the focus of cancer studies, with many groups examining their expression profiles in various tumour types . Ang-2 is a biological antagonist of Ang-1 and is dramatically expressed at sites of vessel regression and sprouting. It reduces vascular stability and facilitates access of VEGF to endothelial cells . The biological actions of Ang-2 in tumour growth and metastasis have not been fully ascertained. There have been studies showing that in an Ang-2 transgenic gastric animal model, over-expression of Ang-2 stimulated tumour growth and metastasis and these results have been supported by many other experiments .
In the authors’ previous tongue carcinoma study , stably transfected Ang-2 tongue Tca8113 tumour cells were injected subcutaneously into nude mice and tumour development was compared with those derived from cells transfected with the empty vector and parental Tca8113. The results showed that over-expression of Ang-2 in Tca8113 cells prolonged detectable tumour formation and inhibited tumour growth. These phenomena indicated that expression of Ang-2 may have different biologic effects on different tumour tissues and warrants additional investigation.
Angiogenesis is dependent on a tightly regulated balance between angiogenic promoters and inhibitors. Studies suggest that VEGF and Angs play complementary and coordinated roles in the development of the new blood vessels . In this study, the authors investigated how transfection of Ang-2 affected VEGF expression and the mechanism of Ang-2-mediate growth inhibition of tongue carcinoma.
Materials and methods
Transfection and tumour preparation
The pcDNA3.1(−)B/Ang-2 expression plasmid was constructed by subcloning Ang-2 into a pcDNA3.1(−)B vector. The plasmid was transfected into Tca8113 cells using Lipofectamine 2000 (Invitrogen), 3 × 10 6 cells were injected subcutaneously into the back of nude mice. The mice were killed on day 35 after tumour cell inoculation. The tumours were harvested and dissected with half immediately put into nitrogen for real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, and the other half placed in 4% paraformaldehyde for immunohistochemistry .
Quantitative real-time RT-PCR
cDNA was quantified by real-time PCR on the ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). PCR was performed using Sybr Green PCR core reagent (Applied Biosystems) according to the manufacturer’s instructions. PCR amplification of the housekeeping gene, β-actin, was performed for each sample as a control for sample loading and to allow normalization amongst samples. Primer sequences were as follows: VEGF F 5′-AAGGCCCCAAACCAGTAACAAT-3′, VEGF R 5′-GCTGGCAGGGAACGTCTAATAAT-3′; β-actin F 5′-GTGGCCGAGGACTTTGATTG-3′, β-actin R 5′-AGTGGGGTGGCTTTTAGGATG-3′. Each sample was run twice, and each PCR experiment included two non-template control wells.
Monoclonal mouse anti-smooth muscle actin (α-SMA) clone 4A1 and monoclonal mouse anti-proliferating cell nuclear antigen (PCNA) clone PC10 were diluted 1:100 (DAKO Corp., Carpinteria, CA, USA). Immunohistochemical staining of α-SMA was used to assess vessel maturation and PCNA to assess tumour cell proliferation. Blood vessels in paraffin-embedded tissue sections were visualized by staining endothelial cells with polyclonal factor VIII–related antigen (FVIII) diluted 1:500 (DAKO Corp., Carpinteria, CA, USA).
Solid tumours from injected mice were fixed in 4% paraformaldehyde, washed with phosphate buffered saline (PBS), dehydrated through 30%, 70%, 95%, and 100% ethanol and xylene, and embedded in paraffin wax. 3 μm sections were cut and mounted onto slides. Sections were then deparaffinized in xylene and rehydrated in alcohol. They were incubated in 3 ml/l H 2 O 2 to block endogeneous peroxidase activity. Each slide was incubated with normal horse serum for 20 min at room temperature, then α-SMA clone 4A1, PCNA clone PC10, or FVIII was incubated on the tissue section overnight at 4 °C. After incubation in biotinylated mouse anti-goat immunoglobulin G (IgG) at a working dilution of 1:200 for 30 min at room temperature, each slide was rinsed in PBS and incubated in the avidin-biotin peroxidase complex for 40 min at 37 °C. Peroxidase was visualized with 3-3′-diamino-benzidinetetrahydro-chloride solution. PCNA labelling index (LI) = PCNA-positive cells/total cells × 100%.
Terminal dUTP nick-end labelling (TUNEL) staining was performed according to the manufacturer’s protocol (Promega, Madison, WI, USA). Briefly, the sections were fixed with 4% methanol-free paraformaldehyde, washed, permeabilized with 0.2% Triton X-100, washed again, and incubated with the kit’s equilibration buffer. Sections were incubated with a reaction mix containing equilibration buffer, nucleotide mix, and the terminal deoxynucleotidyl transferase enzyme at 37 °C for 1 h and incubated for 15 min at room temperature to stop the terminal deoxynucleotidyl transferase reaction. After washing, sections were stained with 4,6-diamidino-2-phenylindole-2HCl to visualize the nuclei and glass cover slips were applied. TUNEL labelling index (LI) = TUNEL-positive cells/total cells × 100%.
The data were expressed as mean (SD). Differences between experimental groups were analyzed using one-way ANOVA with SPSS for 11.0. All P values presented were two-sided. P < 0.05 was considered significant, P < 0.01 was considered highly significant.
Ang-2 over-expression results in aberrant tumour angiogenesis without affecting VEGF expression. For examination of microvessels in tumour tissue, tumours derived from the parental Tca8113 and those transfected with vector only (Tca8113/pcDNA3.1(−)B) displayed well-defined blood vessels. In tumours derived from Ang-2 transfection (Tca8113/Ang-2), the majority of angiogenic vessels displayed an aberrant structure, characterized by narrow or absent lumina ( Fig. 1 ). Immunohistochemical staining showed there were fewer α-SMA-positive cells around endothelial cells (ECs) from tumours derived from Tca8113/Ang-2 than those from the parental or empty-vector transfected cell tumours. This indicated that the vessels from Ang-2 transfected tumours were immature. Real-time RT-PCR analysis demonstrated that VEGF mRNA expression in Tca8113/Ang-2, Tca8113/pcDNA3.1(−)B and Tca8113 tumours were 4.37 ± 2.73, 4.63 ± 2.96, 5.09 ± 3.94, respectively, which indicated no detectable difference amongst the three groups ( P > 0.05).
Ang-2 over-expression decreased expression of PCNA and induced tumour cell apoptosis. Immunohistochemical staining for PCNA revealed that the number of PCNA-positive cells was less in the tumours from the Tca8113/Ang-2 group, relative to those in the control groups ( P < 0.05; Fig. 2 A) . TUNEL staining of tumour sections from the three groups revealed more TUNEL-positive cells in the tumours from the Tca8113/Ang-2 groups than those in tumours derived from the parental and vector-only transfected groups ( P < 0.05; Fig. 2 B).