Initial Study On Caveolin-1 Regulating Angiogenesis In Hepatocellular Carcinoma

BACKGROUNDS and OBJECTIVESHepatocellular carcinoma (HCC) is one of the most common solid tumors throughout the world. Though surgical ablation and liver transplantation are the effective measures for treating HCC, the effect is dissatisfactory. Metastasis and recurrence of HCC are the maximal barrier influencing therapeutic effect. As HCC is characterized by hypervascularization, angiogenesis is involved in its invasion and migration through the interaction between tumor cells and vascular endothelial cells.Caveolin-1, an essential constituent of the plasma membrane invaginations named as caveolae, is known to be involved in cancer invasion and angiogenesis. Numerous studies indicated that the role of caveolin-1 expression depends on the cell types and cancer stages. Whereas in some cases caveolin-1 acts as a tumor suppressor, several more recent studies found that caveolin-1 overexpression was involved in invasion and angiogenesis. During study on HCC, it showed that down-regulation of caveolin-1 reduced lymphangiogenic factor variables, vascular endothelial growth factor (VEGF) expression and prevent lymphatic metastasis in mouse hepatocarcinoma cells. On our early research, we found that caveolin-1 is one of the upregulated genes by SuperArray microarrays on screening of HCC metastasis related genes. However, the connection between the expression of caveolin-1 and cancer invasion and angiogenesis in HCC remains a puzzle and its mechanism is still not clear. The majority of studies have revealed that VEGF was one of the key positive regulators during angiogenesis in HCC. However, the relevance of caveolin-1 to VEGF induced angiogenesis was mainly focused on endothelial cells. The connection between the expression of caveolin-1 in tumor cell and angiogenesis induced by VEGF remains uncertain. Based on these previous findings, we supposed that caveolin-1 could induce VEGF expression in HCC, enhanceing tumor angiogenesis and promoting the invasion of HCC. METHODSTo confirm our hypothesis, we proposed following experiments. Firstly, the caveolin-1 protein and mRNA expression in tissue specimens of HCC and human hepatocarcinoma cell lines were determined by using immunohistochemistry, Western blot, and quantitative Real-Time PCR (qRT-PCR). The levels of caveolin-1 were correlated with the clinicopathologic variables, VEGF, microvessel density (MVD) and unpaired artery (UA). Secondly, used the lentiviral expression vector of human caveolin-1 with RNA interference approach, down-regulation of caveolin-1 was correlated with VEGF expression, invasiveness and migration in SMMC7721 by qRT-PCR, ELISA, invasion assay and wound healing scratch assay. Lastly, the effect of conditioned medium from supernatant of SMMC7721 treated with caveolin-1 RNAi on eNOS expression, proliferation and capillary-like tubule formation of human umbilical vein endothelial cells (HUVEC) was screened by Western blot, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and angiogenesis two-dimensional gel model in vitro.RESULTSPart 1: The characteristics of caveolin-1 expression in HCC and its relationship with the clinicopathologic variables and angiogenesis1. In 26 tissue specimens of HCC with intrahepatic metastases, caveolin-1 expression of tumor tissues was significantly higher than that of nomal (P=0.046), cirrhosis (P=0.021) or adjacent tumor tissues (P=0.039). In all 75 tissue specimens of HCC, the caveolin-1 expression intensity (P=0.001), proportion (P=0.002) or score (P=0.001) was significantly correlated with metastasis. Levels of caveolin-1 correlated positively with VEGF expression (r=0.293, P=0.011), MVD (r=0.361, P=0.001) and UA (r=0.388, P=0.001). A Kaplan-Meier survival curve showed that the survival rate of patients with higher caveolin-1 expression was considerably worse than that of patients with lower caveolin-1 expression. Univariate analyses identified tumor size, portal vein tumor thrombus, metastasis, caveolin-1 intensity, caveolin-1 score, caveolin-1 expression, VEGF expression, MVD and UA as significant prognostic factors for cancer-specific survival. Multivariate analyses indicated that only tumor size and VEGF levels were independent prognostic factors for cancer-specific survival.2. The caveolin-1 protein and mRNA were loss in HepG2 with low invasive ability, and then significantly increasing in SMMC7721 with high invasive ability (P<0.01). Moreover, the expression of caveolin-1 in SMMC7721 was significantly associated with VEGF (r=0.888, P<0.01).Part 2: Construction of recombinant lentivirol expression vector carring human caveolin-1 with RNA interference and its correlation with VEGF expression, invasiveness and migration of SMMC77211. Construction of recombinant lentivirol expression vector carring human caveolin-1 with RNA interference could express in SMMC7721 presistandtly and stablely. The transduction efficiency of SMMC7721 was (96.0±1.2)%. The caveolin-1 mRNA level was knocked down as 90%. The protein expression of caveolin-1 was significantly declined.2. Caveolin-1 silenced in SMMC7721 resulted in the mRNA level of VEGF in SMMC7721 declining about 50% compared with negative infection (P<0.05). Meanwhile, the secreted VEGF expression were decreased about 20% by ELISA (P<0.05).3. After caveolin-1 was knocked down, the invasiveness and migration of SMMC7721 were prevented at every phase (P<0.01), and dramaticly restored again by supplemented with recombinant human VEGF (P<0.05). The results implied that the invasion of SMMC7721 were positively regulated by caveolin-1 expression through inducing VEGF expression.Part 3: The effect of conditioned medium from SMMC7721 with caveolin-1 silenced on eNOS expression, proliferation and capillary-like tubule formation of HUVEC1. The cell purity was maintained and variation was reduced by primary cultured HUVEC. It indicated that conditioned medium from SMMC7721 with caveolin-1 silenced could significantly hampered HUVEC proliferation (P<0.01).2. Capillary-like tubules developed in conditioned medium from SMMC7721 with caveolin-1 silenced were reduced (P<0.05), and dramatically reorganized by supplemented with recombinant human VEGF (P<0.05). MVD by Immunocytochemistry (CD34 labled) was performed at the twenty-fourth hour to further confirm the results.3. Conditioned medium from SMMC7721 with caveolin-1 silenced could significantly reduce the phosphorylation of eNOS on the serine 1177 (P<0.05). However, the total protein of eNOS was unchanged. CONCLUSI ON1. Overexpression of caveolin-1 in HCC was positively correlated with metastasis, poor prognosis and angiogenesis. The caveolin-1 expression in human hepatocarcinoma cell lines SMMC7721 with high invasiveness was increased, correlating positively with VEGF. Caveolin-1 was one of hallmarker for predicting the invasion and prognosis of HCC.2. Construction of recombinant lentivirol expression vector carring human caveolin-1 with RNA interference could express in SMMC7721 presistandtly and stablely. Caveolin-1 silenced in SMMC7721 reduced the mRNA level of VEGF in SMMC7721 and the secreted VEGF expression in supernatant. Following by caveolin-1 down-regulated, the invasiveness and migration of SMMC7721 were prevented, and dramaticly restored again by supplemented with recombinant human VEGF. The results implied that the invasion of SMMC7721 were positively regulated by caveolin-1 expression through inducing VEGF expression.3. Conditioned medium from SMMC7721 with caveolin-1 silenced could significantly hampered HUVEC proliferation. Capillary-like tubules developed in conditioned medium from SMMC7721 with caveolin-1 silenced were reduced, and dramatically reorganized by supplemented with recombinant human VEGF. The activation of eNOS was prevented too. It indicated that VEGF down-regulation induced by caveolin-1 silenced hampered capillary-like tubule formation of HUVEC through reducing the activation of eNOS induced by VEGF during angiogenesis. These data highlights the fact that overexpression of caveolin-1 in HCC promotes invasion and metastasis in HCC through up-regulating VEGF induced angiogenesis. It may open new insights about the possibility of novel therapeutic strategies able to block the tumor's blood supply and to decrease invasion and metastasis in HCC.