The Molecular Mechanism Of HGF-induced Expression Of VEGF In MHCC97H Cells

Hepatocellular carcinoma (HCC) is the second cancer in rural, and the third in urban in China. The mortality of cancer is 20.4/100,000 in our country, about 18.8% of mortality rate of total malignant tumors. HCC is a poor prognosis cancer with the biological characteristics of high infiltration of blood vessel and tendency of metastasis, and abundant blood supply. The recurrence and metastasis of liver cancer is a process with multiple steps and elements. Many factors involved in this molecular mechanism, including oncogene, anti-oncogene, transfer related gene, growth factors and its receptors, adhesion molecules, extracellular matrix (ECM), angiogenesis, etc.Scientific researches have focused on angiogenesis in recent years. Studies showed that the growth, metastasis of tumors closely correlated with angiogenesis. The formation of blood vessel of tumors is the basis of growth, metastasis and invasion of malignant tumors. Tumor's continuous growth depended on the neovascularization when the tumor has been developed over 2 mm in diameter. Normally, there are strict space-time regulations on interactions among epithelial-mesenchymal, various cell growth factors and cytokines that can affect angiogenesis. Pathological angiogenesis departed from the normal regulation system and developed out of control during tumorigenesis and its development. Many cytokines that can regulate angiogenesis expressed in various tumors.Vascular endothelial growth factor (VEGF) was considered as one of the most potent angiogenesis factors. The expressed level of VEGF in tumor stood for the level of cell proliferation, migration and blood vessel construction, directly reflecting the speed of tumor growth and the tendency of metastases. VEGF can induced microvessel formation, degradated matrix of vascular endothelial cell, weakens the function of vascular barrier, so that the defect of basal membrane of new vessel will aggravate and the malignant cells will take advantage of it to enter into new vessel. Various factors can affect the expression of VEGF, including cytokines. There are crosstalk among cytokines that involved directly or indirectly in the regulation of transcript and translation of VEGF gene. The regulation of VEGF expression is a very complex process. It can be regulated in different level by various factors, such as regulation in VEGF gene transcript, mRNA stabilization and mRNA transcript. Many angiogenesis factors itself was a potent regulator of VEGF gene expression. These factors probably developed their facilitation of blood vessel through induction and /or magnification of VEGF composition.Hepatocyte Growth Factor (HGF) is a multifunctional growth factor secreted by stroma-cells. HGF exerts paracrine actions in normal or malignant cells through its cell surface tyrosine kinase receptor c-met. HGF can promotes HCC proliferation, motion, angiogenesis, except it play an essential role in normal hepatocyte proliferation. Both of HGF and VEGF can improve the proliferation of vascular epithelial cell and capillary formation. HGF can upregulate potently VEGF expression in variety of cell. HGF has a Synergistic effect with VEGF. It is not clear that the molecular mechanism of the interaction between HGF and VEGF. To understand the mechanism can provide new therapeutic evidence and strategies on controlling over blood vessel formation of HCC.MHCC97H is a human hepatocellular carcinoma cell line with high metastatic potential isolated further from liver cancer cell line MHCC97 established by our institute. This cell line derived from patient with HCC, its biological characteristic closed to clinical status. So, it is an eligible option to study the mechanism of HGF-induced VEGF expression in HCC.Part 1. The effect of HGF on expression of VEGF mRNA and protein and on phosphorylation of transcript factor Sp1 in liver cancer cell line MHCC97HSection 1. HGF-induced expression of VEGF mRNA in MHCC97H: HGF exert its biological function through its cell tyrosine kinase receptor c-met. Different concentration of HGF was given into MHCC97H cell culture. We found the expression of VEGF increasing with the concentration of HGF rising after cultivated for 16 hours. The level of VEGF mRNA elevated after given HGF 20ng/ml and 40ng/ml respectively (1.04±0.16 and 1.20±0. 18). there are no obvious difference between 2 HGF groups and the control group (0.95±0.12), P=0.089及0.17, respectively. The VEGF mRNA of other 2 HGF groups, 60ng/ml and 80ng/ml, was 1.40±0.21及2.68±0.43, P=0. 026 and 0. 0034, respectively.Section 2. Western blot analysis the expression of VEGF protein in MHCC97H cultivated by different concentration of HGF: we can measured VEGF protein in MHCC97H cell without any supernumerary disposal. After cultured with 20ng/ml, 40ng/ml, 60ng/ml, 80ng/ml HGF for 24h, we can see VEGF protein significantly elevated following with arising of the concentration of HGF in MHCC97H cell line. Comparing with the control group, the expression of VEGF protein in group of 20ng/ml, 40ng/ml HGF showed no obvious meaning(P=0.15 and 0.11, respectively) .but it is obviously higher in group of 60ng/ml HGF (1.64±0.16 ) and 80 ng/ml (2.15±0.21) of HGF, P=0.013 and 0.0041, respectively.Section 3. The effect of HGF on Sp1 in MHCC97H cell line: we measured Sp1 protein level in MHCC97H cell line cultivated for 5 min, 10min, 15 min, 30min and 60 min by 60 ng/ml HGF, respectively. The results showed that there are no difference between the group of contrast and groups of HGF cultivated by different time.Section 4. The effect of HGF on phosphorylation of sp1 in MHCC97H Cell line: we further investigated expression patterns of phosphorylation of sp1 in MHCC97H cell line after 60 ng/ml HGF cultivation by 5 min, 10min, 15 min, 30min and 60 min. the results of immunoprecipitation showed that p-Sp1 increased obviously since 15 min cultivated by HGF 60 ng/ml, and there are no further elevated after 15 min.Section 5. Mithramycin, inhibitor of Sp1, suppressed the expression of VEGF mRNA and its following protein in MHCC97H cell line. We incubated MHCC97H cell line for 1 hour by mithramycin before we added 60 ng/ml HGF, and then we compared the level of VEGF mRNA and its following protein with HGF group that without addition of mithramycin. We found that the expression of VEGF mRNA could be inhibited remarkably by mithramycin. Same outcome could be seen for VEGF protein.Part 2. The function of protein kinase C (PKC) in the process of HGF-induced expression of VEGF in MHCC97 cell line.The activation of functional PKC involved in the process of cell response to HGF stimulation. PKC isozymes comprise a family of related serine-threonine kinases that can be grouped into three categories on the basis of their structural and biochemical properties: conventional PKC, novel PKC and atypical PKC. PKC is a very important component of intra-cellular signal pathway. Neovascularization depends on activation of PKC.Section 1: we firstly examined the role of PKC in the process of HGF induced expression of VEGF in MHCC97H cell line by using Ro31-8220, a comprehensive inhibitor of PKC. VEGF protein could be inhibited by Ro31-8220 compared with HGF group.Section 2: we analyzed that the effect of different PKC isoforms on the process of HGF induced expression of VEGF in MHCC97H cell line. Cell were incubated with a selective inhibitor of cPKC, calphostin C (Cal C), and another selective inhibitor of cPKC and nPKC, bisindolymaleimide I(BIM), before stimulation with HGF for 1 hour. Whole cell extracts were prepared and examined for PKCβ, PKCδphosphorylation by Western blot analysis. We found that HGF could attenuated phosphorylation of PKCβand PKCδ. This effect of HGF could be significantly inhibited by Cal C and BIM, respectively.Section 3: the effect of BIM on expression of VEGF. In the BIM group, VEGF mRNA and protein were examined by RT-PCR and Western blot analysis respectively. The results showed that BIM could not block the expression of VEGF mRNA and protein stimulated by HGF although incubated previously with BIM. There are no difference between BIM group and HGF group for the VEGF mRNA and protein. Section 4: the effect of aPKC in HGF-induced VEGF expression. We have excluded the influence of cPKC and nPKC in the process of HGF-induced VEGF expression. We focused on the role of nPKC, especially PKCζin this process. Cell were treated with a selective inhibitor of PKCζ(ζ—PS) for 1 hour before added with HGF. Whole cell extracts were prepared and examined for PKCζphosphorylation by immunoprecipitation using specific antibody that recognize PKCζonly when PKCζwas phosphorylated. And then VEGF protein after 24h incubated by HGF was examined by Western blot analysis. HGF can stimulate activation of PKCζbut not elevate total PKCζlevel.ζ-PS can block effectively the expression of protein stimulated by HGF.Part 3. The role of the pathway of phosphatidylinositol 3 kinase (PI3K) in the process of HGF-induced expression of VEGF in MHCC97H cell line. PI3K existed on a wide variety of cells and could be activated by most of receptor tyrosine kinase, G-protein-coupled receptors and non-receptor tyrosine kinase relative receptor. Akt is one of the downstream signals of PI3K pathway and a serine/threonine kinase as well. To investigate the intracellular events that HGF regulate VEGF expression and Akt activation, we examined the effect of wortmannin and LY294002, specific inhibitors of PI3K, on HGF-stimulated activation of Akt and expression of VEGF mRNA and protein in MHCC97H cell line. Cells were incubated with wortmannin and LY294002 for 1 hour before stimulation with HGF for 15 min, 16 hours and 24 hours respectively. Whole cell extracts at 15 min were prepared and examined for Akt phosphorylation by Western blot analysis using Abs that recognize Akt only when phosphorylated on Ser473 or Thr308. As the result shown, both of inhibitor of PI3K, wortmannin and LY294002 treatment of cells effectively blocked HGF-stimulated phosphorylation of Akt. RT-PCR analysis revealed that wortmannin could hinder the HGF-induced promotion of expression of VEGF mRNA after 16 hours and addition of VEGF protein after 24 hours as well.Part 4. The relationship among of signals in the process of HGF-induced expression of VEGF.Sp1 is a highly glycosylated and phosphorylated protein. Several kinases have been shown to phosphorylate Sp1, including DNA-dependent protein kinase, casein kinase II, protein kinase A, and protein kinase C-ζ. There were studies showed that PKCζwas a downstream effector of PI3K. Our previous researches showed that stimulation of MHCC97H cells with HGF activates multiple signaling cascades, including transcript factor Sp1, PKCζand serine/threonine kinase Akt. To investigate their relationship during the process of HGF-stimulated VEGF expression in MHCC97H cell line, we explored the effect of specific inhibitors of those intracellular signals on each other. MHCC97H cells was added wortmannin andζ-PS for 1 hour, respectively, before stimulation with HGF for 15 min. Whole cell extracts were prepared and examined for phosphorylation of PKC (?), and Sp1 by immunoprecipitation and phosphorylation of Akt by western blot. We found that the inhibitor of PI3K, wortmannin could block HGF-induced phosphorylation of PKCζand Sp1. We investigated the effect of PKCζinhibitor,ζ-PS on the phosphorylation of Akt and Sp1 in MHCC97H cell line simultaneously. The results showed thatζ-PS could depressed phosphorylation of Sp1 effectively but not Akt.Together, those results indicated that the mechanism of HGF-induced VEGF expression in MHCC97H liver cancer cell line implicated pathway of activation of transcript factor Sp1, PKCζand PI3K/Akt, and signals stimulated by HGF passed on from PI3K/Akt to PKCζand then to Sp1. Multiple chemical inhibitors and specific repressible protein of PKCζ,ζ-PS, can block the signal transmission. Those inhibitors and/orζ-PS protein potentially could be developed targets of inhibition of angiogenesis of liver cancer.