The mechanism of angiogenesis in gastric carcinoma: the role and interaction between interleukin-6 and vascular endothelial growth factor (2/2)

Angiogenesis is currently well known to be essential for the growth, invasion, and metastasis in malignancies. Earlier studies showed that tumor angiogenesis of gastric carcinoma (GC) correlates with disease stage,
lymphatic and distant metastases, recurrence after operation. A wide variety of angiogenic and antiangiogenic factors have been found to be important in this angiogenic process. Among the vast of angiogenic factors, vascular endothelial growth factor (VEGF) is reported to be pivotal for angiogenesis in gastric carcinogenesis. However, no extracellular molecule has yet been
documented as being capable of up-regulating VEGF in GC. Interleukin-6 (IL-6) increases in Helicobacter pylori gastritis and is reported to be associated with the disease status of GC, but the mechanism underlying this association remains unclear. Our earlier research has found a positive association between the serum levels of IL-6 and VEGF in GC patients, which suggested that IL-6 might play a role for angiogenesis in GC via an interaction with VEGF production. Accordingly, we propose a hypothesis that IL-6 simulates tumor angiogenesis in GC via inducing VEGF. In this project, we will verify the association of IL-6, VEGF, and tumor vasculature by the clinical studies on tumor tissues and investigate their relationship by the experimental studies using GC cell lines.
During the first year of this project, we focus on the verification of the clinical association among IL-6, VEGF, and tumor vasculature in GC. GC
samples obtained from surgical specimens at National Taiwan University are collected for immunohistochemical examination of IL-6, VEGF, and tumor microvessels. The immunoreactivities will be graded and then analyzed according to the different clinicopathologic characteristics. We found that there was a positive association among IL-6, VEGF, and tumor vasculature in
different subsets of GC (Table 1, 2). In the experimental studies, we first assessed the effects of dose and duration of IL-6 simulation on VEGF production in different GC cell lines by ELISA (Figure 2a,b). Three signaling pathways, JAK/STAT, Raf/MAPK, and Akt/PI3K, mediate the cellular signals of IL-6 stimulation. Therefore, we next employed firefly luciferase reporter
gene assay to determine the signaling pathway involved in the activation of the promoter of VEGF gene by IL-6 (Figure3). To study the effect of IL-6 on angiogenesis in vitro, the growth and tube formation of human umbilical vein endothelial cells cultured with conditioned media obtained from the GC cells after IL-6 stimulation are to be assessed (Figure 4, 5a-d). In assessment of the effect of IL-6 on angiogenesis in vivo, Matrigel plug assay will be used.
Matrigel plugs were harvested from mice injected subcutaneously with the mixture of Matrigel and conditioned media for gross examination and
determination of hemoglobin densities, which stand for tumor vasculature (Figure 6a-e). We found that IL-6 indeed induced VEGF production in GC cells, which was mediated through JAK/STAT pathway. Meanwhile, we also demonstrated that IL-6 can increase in vitro and in vivo angiogenesis in GC.
During the second year of this project, we have enrolled 250 GC patients and 250 health controls and obtain their WBC DNA to compare the single
nucleotide polymorphisms (SNPs) of IL-6 between these two groups. Our results demonstrated all Taiwanese showed IL-6 -174G/G genotypes. The
SNP of IL-6 at another locus (-634CG) was also analyzed and in progress.
In addition, immunohistoclemical stainings of IL-6 and VEGF have been performed in surgical specimens of GC. The expression of IL-6 and VEGF
will be correlated with each other and related to the clinicopathologic features and microvessel density of GC. These results will further elucidate the role of IL-6 in angiogenesis of GC and shed light on its interaction with VEGF.