Vascular Endothelial Growth factor-C Protein Expression Research Articles

Uses of Vascular Endothelial Growth Factor C as a Lung Adenocarcinoma Prognostic Biomarker.

Lung adenocarcinoma (LUAD) is the most common type of lung cancer and a leading cause of death worldwide. Vascular endothelial growth factor C (VEGF-C) has been identified as a prognosis prediction marker for LUAD. However, VEGF-C protein expression does not appear to significantly relate to LUAD patient survival in several studies. We carried out a bioinformatic analysis to review the effect of VEGF-C mRNA expression on LUAD patient outcomes. GEPIA, UALCAN, TCGAportal, OncoLnc, LCE, GeneMANIA, Metascape, ImmuCellAI, and GSCA online databases were utilized. The expression levels of VEGF-C mRNA between normal tissue and LUAD tissue, overall survival (OS) analysis, function analysis, tumor microenvironment and drug sensitivity were conducted in the current study. We found that the expression level of VEGF-C mRNA was significantly lower in LUAD than normal tissue. Low expression of VEGF-C mRNA was also associated with better OS. VEGF-C expression was correlated with both NF1 and TP53 mutation status. No relationship was observed between VEGF-C and Tr1 or CD4 T-cell infiltrate scores. Additionally, VEGF-C was associated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance. The sensitivity of 5-fluorouracil was positively correlated with VEGF-C, and the sensitivity of TGX221 was negatively correlated with VEGF-C. The activity of BI-2536 and BRD-A94377914 was positively correlated with VEGF-C. Novel LUAD prognostic biomarkers such as VEGF-C mRNA may aid diagnosis and treatment, and may help identify optimal LUAD populations for therapeutic treatments.

VEGFC acts as a double-edged sword in renal cell carcinoma aggressiveness.

Hypoxic zones are common features of metastatic tumors. Due to inactivation of the von Hippel-Lindau gene (VHL), renal cell carcinomas (RCC) show constitutive stabilization of the alpha subunit of the hypoxia-inducible factor (HIF). Thus, RCC represents a model of chronic hypoxia. Development of the lymphatic network is dependent on vascular endothelial growth factor C (VEGFC) and lies at the front line of metastatic spreading. Here, we addressed the role of VEGFC in RCC aggressiveness and the regulation of its expression in hypoxia.Methods: Transcriptional and post transcriptional regulation of VEGFC expression was evaluated by qPCR and with reporter genes. The involvement of HIF was evaluated using a siRNA approach. Experimental RCC were performed with immuno-competent/deficient mice using human and mouse cells knocked-out for the VEGFC gene by a CRISPR/Cas9 method. The VEGFC axis was analyzed with an online available data base (TCGA) and using an independent cohort of patients.Results: Hypoxia induced VEGFC protein expression but down-regulated VEGFC gene transcription and mRNA stability. Increased proliferation, migration, over-activation of the AKT signaling pathway and enhanced expression of mesenchymal markers characterized VEGFC-/- cells. VEGFC-/- cells did not form tumors in immuno-deficient mice but developed aggressive tumors in immuno-competent mice. These tumors showed down-regulation of markers of activated lymphocytes and M1 macrophages, and up-regulation of M2 macrophages markers and programmed death ligand 1 (PDL1). Over-expression of lymphangiogenic genes including VEGFC was linked to increased disease-free and overall survival in patients with non-metastatic tumors, whereas its over-expression correlated with decreased progression-free and overall survival of metastatic patients.Conclusion: Our study revisited the admitted dogma linking VEGFC to tumor aggressiveness. We conclude that targeting VEGFC for therapy must be considered with caution.

MicroRNA-186 regulates the invasion and metastasis of bladder cancer via vascular endothelial growth factor C.

The present study aimed to investigate the expression of microRNA (miRNA or miR)-186 in tumor tissue, blood and urine from patients with bladder cancer. The mechanism by which miR-186 regulates the invasion and metastasis of bladder cancer was also assessed. A total of 76 patients who underwent surgical resection of bladder cancer tissues between August 2012 and January 2016 were included in the present study. Blood and urine samples were also collected from the 76 patients and another 66 healthy subjects. Expression of vascular endothelial growth factor C (VEGF-C) mRNA and miR-186 was measured using reverse transcription-quantitative polymerase chain reaction. Western blot analysis was performed to assess VEGF-C protein expression in tumor tissues. The content of VEGF-C protein in blood and urine samples was measured using an enzyme-linked immunosorbent assay. To identify the direct interaction between miR-186 and VEGF-C mRNA, a dual luciferase reporter assay was performed. The present findings demonstrated that VEGF-C mRNA expression in tumor tissues, blood and urine of bladder cancer patients was upregulated. VEGF-C protein expression in bladder cancer tissues was also enhanced. VEGF-C protein content in blood and urine from bladder cancer patients was elevated, consistent with the results for VEGF-C mRNA. Expression of miR-186 was reduced in tumor tissues, blood and urine. Dual luciferase reporter assay demonstrated that miR-186 regulated the expression of VEGF-C by binding with its 3'-untranslated region. Therefore, the results of the present study indicate that the expression of VEGF-C mRNA and protein is upregulated in tumor tissues, blood and urine from patients with bladder cancer, while that of miR-186 is downregulated in these samples. miR-186 potentially regulates the invasion and metastasis of bladder cancer via VEGF-C, and may become a gene marker for bladder cancer in the future.

Associations between the expression of MTA1 and VEGF-C in esophageal squamous cell carcinoma with lymph angiogenesis and lymph node metastasis.

The aim of the present study was to investigate the association between the expression levels of metastasis-related gene 1 (MTA1) and vascular endothelial growth factor C (VEGF-C) in esophageal squamous cell carcinoma (ESCC) with lymph angiogenesis and lymph node metastasis. The paraffin-embedded tissue samples of 107 cases of ESCC and 56 cases of normal esophageal tissues were collected from the Department of Cardiothoracic Surgery, Suining Central Hospital from March 2013 to January 2014. Immunohistochemical assays were performed to detect the expression levels of MTA1, VEGF-C and D2-40 in ESCC, and the micro-lymphatic vessel density (LVD) was evaluated. Their associations with various clinicopathological parameters were also analyzed. The protein expression levels of MTA1 and VEGF-C in ESCC were significantly higher compared with those in normal esophageal tissues (P<0.05); the high protein expression levels of MTA1 and VEGF-C in ESCC tissues at various tumor-node-metastasis stages exhibited statistically significant differences, as revealed by the Kruskal-Wallis test (P<0.05). The protein expression levels of MTA1 and VEGF-C in ESCC exhibited positive correlations (Spearman's ρ, r=0.512; P=0.000); the LVD level in the group with high expression of MTA1 and VEGF-C was significantly higher compared with in the low expression group (P<0.05). The comparison between MTA1 and VEGF-C protein expression levels in the group with a high rate of lymph node metastasis demonstrated statistically significant differences when compared with in the low lymph node metastasis group (P<0.05). The expression levels of MTA1 and VEGF-C in ESCC exhibited a positive correlation in ESCC, which may co-promote lymph angiogenesis and lymph node metastasis in ESCC; therefore, they may be used as biomarkers for determining the prognosis of ESCC.

Regulation of vascular endothelial growth factor-C by tumor necrosis factor-α in the conjunctiva and pterygium.

Vascular endothelial growth factorC(VEGF-C) plays an important role in the development of a pterygium through lymphangiogenesis. We examined the association between VEGF-C and tumor necrosis factor-α(TNF-α) in the pathogenesis of pterygia. Cultured conjunctival epithelial cells were treated with TNF-α, and the gene expression levels of VEGFC were evaluated by quantitative polymerase chain reaction(qPCR) and VEGF-C protein expression levels were measured using an enzyme-linked immunosorbent assay(ELISA). In addition, using ELISA, we evaluated the VEGF-C protein expression in the supernatants of cultured conjunctival epithelial cells, in which we neutralized TNF-α using anti‑TNF-α antibody. The gene expression of tumor necrosis factor receptor superfamily, member1A(TNFRSF1A), known as TNF receptor1(TNFR1), was confirmed using reverse transcription PCR in cultured conjunctival epithelial cells. Immunofluorescence microscopy was used to examine the localization of VEGF-C and TNFR1 in pterygium tissues and TNFR1 expression in cultured conjunctival epithelial cells. Immunohistochemistry was used to examine the localization of TNFR1 in pterygia and normal conjunctival tissues. VEGFC gene expression increased in cultured conjunctival epithelial cells 24h after the addition of TNF-α. The secretion of VEGF-C protein was significantly increased 48h after the stimulation of cultured conjunctival epithelial cells with TNF-α. Increased VEGF-C protein secretion stimulated by TNF-α was significantly reduced by anti-TNF-α neutralizing antibody treatment. In cultured conjunctival epithelial cells, TNFRSF1A and TNFR1 were expressed. TNFR1 was immunolocalized in normal conjunctival tissues and in human pterygium tissues as well as in VEGF‑C‑positive epithelial cells from human pterygia. Our data demonstrate that TNF-α mediates VEGF-C expression, which plays a critical role in the pathogenesis of pterygia.

Tumor-induced VEGF-C overexpression in retroperitoneal lymph nodes in VX2 carcinoma-bearing rabbits.

ObjectiveTo establish the retroperitoneal lymph node (RLN) metastasis model of cervical carcinoma in rabbits and evaluate the relationship of vascular endothelial growth factor-C (VEGF-C) expression and the lymph node status.MethodsForty-eight rabbits were injected with VX2 cells or RPMI solution at muscular mucosae of the myometrium 0.5 cm away from the cervix. Animals were treated with or without cis-diamminedichloroplatinum(II) (cisplatin: DDP) and sacrificed on days 15, 21, and 27 post-VX2 or RPMI injections. Tumor mass and RLNs were examined histopathologically. Quantitative real-time PCR was used to examine the changes in VEGF-C mRNA expression. Levels of VEGF-C protein expression in tissues were determined using immunohistochemistry staining.ResultsDevelopment of VX2 cervical carcinoma and the RLNs metastasis was confirmed with pathological examination. Significantly increased tumor volume was observed on days 15, 21, and 27 postinjection (P<0.05). The enlargement of RLNs was found on day 21. Expression of VEGF-C was significantly upregulated in peripheral white blood cells, tumor mass, and RLNs in an association with cancer progression. DDP resulted in a suppression of VEGF-C expression, whereas the influences on tumor mass and lymphatic metastasis were insignificant.ConclusionElevated VEGF-C expressions in peripheral white blood cells and RLNs are associated with tumor progression and lymphatic metastasis. DDP treatment inhibits VEGF-C expression and fails to protect against metastatic cervical cancer.

MicroRNA-27b, microRNA-101 and microRNA-128 inhibit angiogenesis by down-regulating vascular endothelial growth factor C expression in gastric cancers.

Vascular Endothelial Growth Factor C (VEGF-C) has critical roles in angiogenesis in human cancers; however, the underlying mechanisms regulating VEGF-C expression remain largely unknown. In the present study, VEGF-C protein expression and the density of blood vessels or lymphatic vessels were determined by immunohistochemistry in 103 cases of gastric cancer tissues. Suppression of VEGF-C by miR-27b, miR-101 and miR-128 was investigated by luciferase assays, Western blot and ELISA. The miRNAs expression levels were detected in human gastric cancers by real-time quantitative PCR. Cell proliferation, migration and invasion assays were performed to assess the effect of miRNAs on gastric cancer cells and human umbilical vascular endothelial cells (HUVECs). Our data showed that high VEGF-C expression was significantly associated with increased tumor size, advanced TNM classification and clinical stage, higher microvessel density (MVD) and lymphatic density (LVD), as well as poor survival in patients with gastric cancer. Furthermore, VEGF-C was found to be a direct target gene of miR-27b, miR-101, and miR-128. The expression levels of the three miRNAs were inversely correlated with MVD. Overexpression of miR-27b, miR-101, or miR-128 suppressed migration, proliferation activity, and tube formation in HUVECs by repressing VEGF-C secretion in gastric cancer cells. We conclude that miR-27b, miR-101 and miR-128 inhibit angiogenesis by down-regulating VEGF-C expression in gastric cancers.

Expression and clinical significance of microRNA-21, maspin and vascular endothelial growth factor-C in bladder cancer.

The present study aimed to explore the expression and clinical significance of microRNA-21 (miR-21), maspin and vascular endothelial growth factor C (VEGF-C) in bladder cancer (BC). A total of 53 BC samples and 12 normal bladder tissue samples were collected. Total messenger RNA (mRNA) was extracted, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to evaluate the expression of miR-21 and maspin in BC and normal bladder tissues. Immunohistochemistry was used for the detection of maspin and VEGF-C protein expression. Furthermore, the correlations between these molecules and certain clinicopathological parameters were investigated, and survival analysis was performed to assess their prognostic significance. miR-21 mRNA expression and VEGF-C protein expression were increased in BC tissues compared with those in normal bladder tissues, whereas maspin mRNA and protein expression levels in BC tissues were significantly decreased (P<0.01). miR-21, maspin and VEGF-C expression were significantly associated with the stage, grade and lymph node metastasis of BC (P<0.05), but not the other clinicopathological features evaluated. There was a marked inverse correlation between the mRNA expression of miR-21 and maspin, with a coefficient of -0.978 (P<0.001). Similarly, there was a significant inverse correlation between the protein expression of maspin and VEGF-C, with a coefficient of -0.589 (P<0.001). Overexpression of miR-21 and VEGF-C, as well as decreased maspin expression, were associated with a poorer prognosis. These results suggested that upregulation of miR-21, decreased maspin expression and enhanced VEGF-C in BC may promote tumor progression. miR-21, maspin and VEGF-C may therefore have significant roles as biomarkers for prognosis and as therapeutic targets of BC.

URG4/URGCP enhances the angiogenic capacity of human hepatocellular carcinoma cells in vitro via activation of the NF-κB signaling pathway.

BackgroundAngiogenesis is essential for tumor growth. Hepatocellular carcinoma (HCC) is characterized by hypervascularity; high levels of angiogenesis are associated with poor prognosis and a highly invasive phenotype in HCC. Up-regulated gene-4 (URG4), also known as upregulator of cell proliferation (URGCP), is overexpressed in multiple tumor types and has been suggested to act as an oncogene. This study aimed to elucidate the effect of URG4/URGCP on the angiogenic capacity of HCC cells in vitro.MethodsExpression of URG4/URGCP in HCC cell lines and normal liver epithelial cell lines was examined by Western blotting and quantitative real-time PCR. URG4/URGCP was stably overexpressed or transiently knocked down using a shRNA in two HCC cell lines. The human umbilical vein endothelial cell (HUVEC) tubule formation and Transwell migration assays and chicken chorioallantoic membrane (CAM) assay were used to examine the angiogenic capacity of conditioned media from URG4/URGCP-overexpressing and knockdown cells. A luciferase reporter assay was used to examine the transcriptional activity of nuclear factor kappa – light – chain - enhancer of activated B cells (NF-κB). NF-κB was inhibited by overexpressing degradation-resistant mutant inhibitor of κB (IκB)-α. Expression of vascular endothelial growth factor C (VEGFC), tumor necrosis factor-α (TNFα), interleukin (IL)-6, IL-8 and v-myc avian myelocytomatosis viral oncogene homolog (MYC) were examined by quantitative real-time PCR; VEGFC protein expression was analyzed using an ELISA.ResultsURG4/URGCP protein and mRNA expression were significantly upregulated in HCC cell lines. Overexpressing URG4/URGCP enhanced - while silencing URG4/URGCP decreased - the capacity of HCC cell conditioned media to induce HUVEC tubule formation and migration and neovascularization in the CAM assay. Furthermore, overexpressing URG4/URGCP increased - whereas knockdown of URG4/URGCP decreased - VEGFC expression, NF-κB transcriptional activity, the levels of phosphorylated (but not total) IκB kinase (IKK) and IκB-α, and expression of TNFα, IL-6, IL-8 and MYC in HCC cells. Additionally, inhibition of NF-κB activity in HCC cells abrogated URG4/URGCP-induced NF-κB activation and angiogenic capacity.ConclusionsThis study suggests that URG4/URGCP plays an important pro-angiogenic role in HCC via a mechanism linked to activation of the NF-κB pathway; URG4/URGCP may represent a potential target for anti-angiogenic therapy in HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1378-7) contains supplementary material, which is available to authorized users.

MiR-101 Suppresses Vascular Endothelial Growth Factor C That Inhibits Migration and Invasion and Enhances Cisplatin Chemosensitivity of Bladder Cancer Cells

BackgroundThe microRNA miR-101 is downregulated in several cancers, including bladder cancer. However, miR-101’s role in the invasion, metastasis, and chemosensitivity of bladder cancer cells remains unclear. This study was conducted to determine miR-101’s role on the lymphangiogenic molecule vascular endothelial growth factor C (VEGF-C) and their effects upon bladder cancer cell migration, invasion, and chemosensitivity to cisplatin.MethodsTwo bladder cancer cell lines (T24 and 5637) and the tool cell line 293T were employed here. Bladder cancer cells were transfected with either a miR-101 overexpression vector or a scrambled-sequence lentivirus, both of which exhibited a high transfection efficiency. Non-transfection was used as a mock negative control. Wound healing and Transwell assays were performed to measure cell migration and invasiveness. A luciferase reporter assay was performed to validate miR-101’s interaction with VEGF-C’s 3′ untranslated region followed by RT-PCR and Western blot confirmation. An MTS assay was used to evaluate the cisplatin sensitivity of the cell lines.ResultsmiR-101 overexpression significantly inhibited the migration and invasiveness while significantly enhancing cisplatin sensitivity. miR-101 negatively regulated VEGF-C protein expression, and VEGF-C overexpression rescued the effects of miR-101 overexpression, indicating that miR-101 negatively regulates VEGF-C protein expression post-transcriptionally. miR-101 and VEGF-C interference independently enhanced cisplatin cytotoxicity in bladder cancer cells.ConclusionsmiR-101 suppresses VEGF-C expression, inhibits cell migration and invasion, and increases cisplatin sensitivity in bladder cancer cells. This study provides new insight into miR-101’s role in bladder cancer and shows miR-101’s promise as a potential molecular target for bladder cancer.

TGF-β1 promotes lymphangiogenesis during peritoneal fibrosis.

Peritoneal fibrosis (PF) causes ultrafiltration failure (UFF) and is a complicating factor in long-term peritoneal dialysis. Lymphatic reabsorption also may contribute to UFF, but little is known about lymphangiogenesis in patients with UFF and peritonitis. We studied the role of the lymphangiogenesis mediator vascular endothelial growth factor-C (VEGF-C) in human dialysate effluents, peritoneal tissues, and peritoneal mesothelial cells (HPMCs). Dialysate VEGF-C concentration correlated positively with the dialysate-to-plasma ratio of creatinine (D/P Cr) and the dialysate TGF-β1 concentration. Peritoneal tissue from patients with UFF expressed higher levels of VEGF-C, lymphatic endothelial hyaluronan receptor-1 (LYVE-1), and podoplanin mRNA and contained more lymphatic vessels than tissue from patients without UFF. Furthermore, mesothelial cell and macrophage expression of VEGF-C increased in the peritoneal membranes of patients with UFF and peritonitis. In cultured mesothelial cells, TGF-β1 upregulated the expression of VEGF-C mRNA and protein, and this upregulation was suppressed by a TGF-β type I receptor (TGFβR-I) inhibitor. TGF-β1-induced upregulation of VEGF-C mRNA expression in cultured HPMCs correlated with the D/P Cr of the patient from whom the HPMCs were derived (P<0.001). Moreover, treatment with a TGFβR-I inhibitor suppressed the enhanced lymphangiogenesis and VEGF-C expression associated with fibrosis in a rat model of PF. These results suggest that lymphangiogenesis associates with fibrosis through the TGF-β-VEGF-C pathway.

Overexpression of MMP-1 and VEGF-C is Associated with a Less Favorable Prognosis in Esophageal Squamous Cell Carcinoma

Background: This study addresses the association of matrix metalloproteinase-1 (MMP-1) and vascular endothelial growth factor-C (VEGF-C) expression in esophageal squamous cell carcinoma (SCC) with clinicopathologic characteristics in the patients. Material and Methods: We profiled the expression of MMP-1 and VEGF-C by cDNA microarray in 4 cases and by reverse transcription-polymerase chain reaction (RT-PCR) in 14 cases of esophageal SCC. Another 90 cases were reviewed by immunohistochemical examination of paraffin-embedded sections. Results: Expression of MMP-1 and VEGF-C mRNA in normal esophageal tissue and tumor tissue was compared. Data were fully consistent with the results of RT-PCR. Immunohistochemistry showed that compared to the normal mucosa MMP-1 and VEGF-C protein expression was upregulated in both esophageal atypical hyperplasia (n = 16) and esophageal SCC. Depth of tumor invasion, lymph node metastasis, and clinical stage were directly associated with prognosis in all cases. Furthermore, median overall survival and disease-free survival were significantly shorter in patients with a higher expression of MMP-1 and VEGF-C than in patients with lower expression levels. Conclusion: We demonstrated that the expression of both MMP-1 and VEGF-C mRNA and protein was upregulated in esophageal SCC tissues. Protein expression was associated with progressive tumor stage and poor prognosis in patients with esophageal SCC.

VEGF-C: An Indicator of Lymph Node Metastasis for Patients that Underwent Esophagectomy

To investigate the expression of vascular endothelial growth factor-C (VEGF-C) and its relationship to lymph node metastasis in esophageal squamous cell cancer after esophagectomy. The expression of VEGF-C protein was detected by immunohistochemistry. Kaplan-Meier method was performed to calculate the survival rate. Cox regression multivariate analysis was performed to determine independent prognostic factors. The difference of VEGF-C protein expression in Tl, T2 and T3 patients was statistically significant (p=0.031). The expression of VEGF-C protein in patients with lymph node metastasis was significantly higher than those without metastasis (p=0.004). VEGF-C protein expression correlated with significantly worsened 5-year survival for all patients as well as those with T3 tumors and N1 nodal status. The result of Cox analysis demonstrated that N stage and VEGF-C protein expression were independent prognostic factors; the multivariate analyses indicated that the N classification is the independent pathological risk factor of VEGF-C expression. VEGF-C expression was found to be significantly associated with T stage and N stage. The patients with VEGF-C protein expression had a significantly lower 5-year survival rate than without VEGF-C protein expression. Lymph node metastasis and VEGF-C protein expression were independent prognostic factors.

Expression and clinical significance of extracellular matrix protein 1 and vascular endothelial growth factor-C in lymphatic metastasis of human breast cancer.

BackgroundExtracellular matrix protein 1 (ECM1) and vascular endothelial growth factor-C (VEGF-C) are secretory glycoproteins that are associated with lymphangiogenesis; these proteins could, therefore, play important roles in the lymphatic dissemination of tumors. However, very little is known about their potential roles in lymphangiogenesis. The aim of this study was to investigate whether correlations exist between ECM1 and VEGF-C in human breast cancer, lymphangiogenesis, and the clinicopathological characteristics of the disease.MethodsECM1 and VEGF-C mRNA and protein expression levels in 41 patients were investigated using real-time reverse transcriptase polymerase chain reaction (RT-PCR), or immunohistochemical (IHC) staining of breast cancer tissue, matched noncancerous breast epithelial tissues, and suspicious metastatic axillary lymph nodes. D2-40 labelled lymph vessels and lymphatic microvessel density (LMVD) were counted. Correlations between ECM1 or VEGF-C protein expression levels, LMVD, and clinicopathological parameters were statistically tested.ResultsThe rate of ECM1 positive staining in breast cancer tissues was higher (31/41, 75.6%) than that in the corresponding epithelial tissues (4/41, 9.8%, P < 0.001) and lymph nodes (13/41, 31.7%, P < 0.001). Similarly, the VEGF-C expression rate in cancer specimens was higher (33/41, 80.5%) than in epithelial tissues (19/41, 46.3%, P < 0.01) or lymph nodes (15/41, 36.6%, P < 0.01). Higher ECM1 and VEGF-C mRNA expression levels were also detected in the tumor tissues, compared to the non-cancerous tissue types or lymph nodes (P < 0.05). ECM1 protein expression was positively correlated with the estrogen receptor status (P < 0.05) and LMVD (P < 0.05). LMVD in the ECM1- and VEGF-C-positive tumor specimens was higher than that in the tissue types with negative staining (P < 0.05).ConclusionsBoth ECM1 and VEGF-C were overexpressed in breast cancer tissue samples. ECM1 expression was positively correlated with estrogen responsiveness and the metastatic properties of breast cancer. We conclude, therefore, that ECM1 and VEGF-C may have a synergistic effect on lymphangiogenesis to facilitate lymphatic metastasis of breast cancer.

Expression of vascular endothelial growth factor C in esophageal carcinoma and its clinical significance

Objective To investigate the expression of vascular endothelial growth factor C (VEGF-C) in human esophageal squamous cell carcinoma (ESCC) and its clinical significance.Methods The expression of VEGF-C in ESCC and its correlation with lymph node metastasis were detected by using immunohistochemistry (IHC) and in situ hybridization (ISH).Three small interfering RNA (siRNA) expression vectors targeting VEGF-C were constructed and transformed into EC9706 cells.The inhibitory effect of siRNA on the expression of VEGF-C protein and mRNA in tranfected cells was analyzed by using Western blotting and reverse transcription-polymerase chain reaction (RT-PCR).Stably transfected cells were transplanted in nude mice later.The expression of VEGF-C in transplant tumor was detected by using IHC and ISH.Results VEGF-C was up-expressed in ESCC.Its expression was correlated significantly with lymph node metastasis.The positive expression rate of VEGF-C in lymph node metastasis group and non-lymph node metastasis group was 90.0％ and 62.1％ respectively (P ＜0.01 ).VEGF-C-targeted siRNA could decrease the expression of VEGF-C mRNA and protein in EC9706 cells effectively.There was significant difference between transfected group (0.32 ± 0.02) and un-transfected group (0.91 ± 0.09,P ＜0.01 ).Correspondingly,the expression of VEGF-C was 9.26 ±0.95,8.94 ±0.81,9.19 ±0.92 and 32.47 ±3.25 respectively.There was significant difference among them (P ＜0.01 ).Conclusion Targeting VEGF-C siRNA can inhibit VEGF-C expression of EC9706 in vivo and in vitro effectively. Key words: Esophageal neoplasm; Vascular endothelial growth factor C

The effects of vascular endothelial growth factor C knockdown in esophageal squamous cell carcinoma

We investigated the role of vascular endothelial growth factor C (VEGF-C) in esophageal squamous cell carcinoma (ESCC) by knocking down VEGF-C expression in the ESCC cell line EC9706. Immunohistochemistry and in situ hybridization techniques were used to detect the expression of VEGF-C expression in ESCC tissues. We also investigated the relationship between VEGF-C expression and lymph node metastasis. We designed a siRNA expression plasmid for VEGF-C and transfected it into EC9706 cells. Stable clones were selected, and VEGF-C expression was analyzed by RT-PCR and western blotting. Cells were inoculated into nude mice. The expression of VEGF-C in the resulting tumors was analyzed by immunohistochemistry and in situ hybridization. VEGF-C is highly expressed in ESCC and correlated with lymph node metastasis, as high levels were observed in patients presenting with lymph node metastases relative to those who did not (P<0.01). Transfection with VEGF-C-siRNA decreased the expression of VEGF-C mRNA and protein. ESCC cells stably transfected with VEGF-C-siRNA expressed very low levels of VEGF-C (P<0.01 compared with control). This knockdown effect persisted when the cells were inoculated into nude mice and allowed to form tumors. The siRNA-targeted knockdown of VEGF-C led to a significant reduction in VEGF-C expression. This siRNA technique could be used for gene therapy in ESCC.

Metastasis-associated protein 1 induces VEGF-C and facilitates lymphangiogenesis in colorectal cancer

To study the correlation between high metastasis-associated protein 1 (MTA1) expression and lymphangiogenesis in colorectal cancer (CRC) and its role in production of vascular endothelial growth factor-C(VEGF-C). Impact of high MTA1 and VEGF-C expression levels on disease progression and lymphovascular density (LVD, D2-40-immunolabeled) in 81 cases of human CRC was evaluated by immunohistochemistry. VEGF-C mRNA and protein expressions in human LoVo and HCT116 cell lines were detected by real-time polymerase chain reaction and Western blotting, respectively, with a stable expression vector or siRNA. The elevated MTA1 and VEGF-C expression levels were correlated with lymph node metastasis and Dukes stages (P < 0.05). Additionally, high MTA1 expression level was correlated with a large tumor size (P < 0.05). A significant correlation was found between MTA1 and VEGF-C protein expressions in tumor cells (r = 0.371, P < 0.05). Similar to the VEGF-C expression level, high MTA1 expression level was correlated with high LVD in CRC (P < 0.05). Furthermore, over-expression of MTA1 significantly enhanced the VEGF-C mRNA and protein expression levels, whereas siRNAs - knocked down MTA1 decreased the VEGF-C expression level. MTA1, as a regulator of tumor-associated lymphangiogenesis, promotes lymphangiogenesis in CRC by mediating the VEGF-C expression.

Heparanase expression correlates with angiogenesis and lymphangiogenesis in human lung cancer.

Heparanase has been thought to be a good molecular marker of tumor, and the heparanase expression level was correlated closely with tumor metastasis. In this study, we investigate the effects of heparanase on angiogenesis and lymphangiogenesis of lung cancer and the relationship between heparanase expression and vascular endothelial growth factor (VEGF), vascular endothelial growth factor-C (VEGF-C). Immunohistochemistry was used to detect the expression of heparanase, VEGF, VEGF-C protein and microvascular density (MVD), lymphatic vessel density (LVD) in 115 cases of non-small cell lung cancer (NSCLC) and 45 cases of adjacent normal tissue samples. Our results showed that heparanase expression was significantly increased in 91 (79.13%) of the 115 cases and correlated with lymph node metastasis (node positive rate 87.0%; node negative rate 36.8%; P=0.003). Heparanase positive expression cases have significantly higher concentration of microvascular density (MVD) and lymphatic vessel density (LVD) as compared with heparanase negative expression cases (P<0.01, P<0.01, respectively), heparanase expression was significantly correlated with VEGF, VEGF-C expression in NSCLC. Heparanase overexpression was associated with angiogenesis and lymphangiogenesis of lung cancer, targeting of heparanase may represent a significant therapeutic potential for lung cancer.

Inhibition of Cyclooxygenase‐2 Suppresses Lymph Node Metastasis via VEGF‐C

Most experimental work addressing cyclooxygenase-2 (COX-2) inhibitor has focused on suppressing hematogenic spread. Little is known about the mechanism by which this inhibitor can also block lymphatic metastasis. Here, the effects of COX-2 inhibitor on vascular endothelial growth factor-C (VEGF-C) expression, lymphangiogenesis and lymph node metastasis were investigated. Utilizing the highly metastatic human lung adenocarcinoma cell line Anip973 and its parental line AGZY83-a, which has a low metastatic capacity, we found elevated VEGF-C and COX-2 immunoreactivity in Anip973 cells compared with AGZY83-a cells. Celecoxib down-regulated expression of VEGF-C mRNA and protein in Anip973 cells while PGE(2) up-regulated expression of VEGF-C mRNA and protein in AGZY83-a cells in a concentration-dependent manner. The expression of COX-2 and VEGF-C was significantly increased in xenografted Anip973 tumors compared with AGZY83-a tumors. The Anip973 tumors showed more lymphatic vessels and lymph node metastasis than the AGZY83-a tumors. In vivo, celecoxib decreased VEGF-C expression in Anip973 tumor-treated mice to a similar level to that in the AGZY83-a tumor-treated mice. Consistent with this decrease in VEGF-C expression, the density of lymphatic vessels and lymph node metastasis in Anip973 tumor-treated mice were suppressed to approximately that found in the AGZY83-a tumor-treated ones. Taken together, our results suggest that the differential expression of COX-2 and VEGF-C might help explain the different metastasis phenotype of lung adenocarcinoma cancer, and that COX-2 inhibitor mediates VEGF-C to block lymphangiogenesis and lymph node metastasis. Thus, COX-2 may be a potential therapeutic target for blocking lymph node metastasis in lung adenocarcinoma.

Relationship between vascular endothelial growth factor C expression level and lymph node metastasis in non small cell lung cancer

To investigate the relationship between vascular endothelial growth factor C (VEGF-C) expression level and lymph node metastasis in non small cell lung cancer (NSCLC). Fifty-two NSCLC patients, 38 males and 14 females, aged (59+/-11) (29-77), were divided into 2 groups based on the pathological examination: lymph node metastasis positive group (n=25) and lymph node metastasis negative group (n=27). RT-PCR and immunohistochemistry were used to detect the mRNA and protein expression of VEGF-C in the tumor tissues and lymph nodes resected during operation. The VEGF-C mRNA expression level in the lung tumor tissue of the lymph node metastasis positive group was 0.273+/-0.179, significantly higher than that in the lymph node metastasis negative group (0.089+/-0.087, P<0.01). The VEGF-C mRNA expression level in the positive lymph node of the lymph node metastasis positive group was 0.207+/-0.174, significantly higher than that in the lymph node metastasis negative group (0.114+/-0.107, P<0.01), but not significantly different from that in the negative lymph nodes of the same group(0.196+/-0.186, P>0.05). The VEGF-C protein positive rate of the lung cancer tissues of the lymph node metastasis positive group was 93.3% (14/15), significantly higher than that of the lymph node metastasis negative group (6.7%, 1/15, P<0.01). The VEGF-C protein positive rate of the metastasis positive lymph noses was 80.4% (37/46), and all 52 metastasis negative lymph nodes were VEGF-C protein negative. VEGF-C mRNA and protein expression levels predict lymph node metastasis and can be useful predictors of lymph node metastasis in NSCLC.