The Function Of Vasohibin 1 In Inhibition Of Colon Cancer Cells And Angiogenesis

Background and aimsColorectal cancer (CRC) is a major cause of cancer death in the world. Colon cancer remains a major global public health problem. There are 100,000 new cases of colon cancer and 50,00 cases estimated death a year. The incidence of colon cancer in all tumor is 10%-15% in China. The therapy of colon cancer includes surgery, radiation therapy, chemotherapy, Targeted cancer therapies. Much more need to be done for further research to develop new therapeutic strategies however there was still high recurrence rate in5 years after curative resection and adjuvant chemotherapy. Angiogenesis is an important factor in the development of tumor so it is a hot area that how to inhibit angiogenesis in cancer. Angiogenesis is controlled by the homeostasis between angiogenesis stimulators and inhibitors.Recently Vasohibinl was found as new angiogenesis inhibitor which can inhibit angiogenesis in cancer tissue. Vasohibin family has two members VASH1 and VASH2. VASH1 selectively express on the endothelial cells which is induced by angiogenesis stimulators such as VEGF and FGF-2. Human VASH1 have two isoforms:one is full length VASH1-A and another is the spliced variant VASH1-B. VASH2 as a homology of VASH1 is expressed preferentially in mononuclear cells (MNCs). VASH1 is expressed in endothelial cells (ECs) at the termination zone halting angiogenesis and VASH2 is expressed at the sprouting front promoting angiogenesis. VASH1 can inhibit migration and proliferation of endothelial cells.VASH1 as an angiogenesis inhibitor take an important role in tumor introduced angiogenesis such as breast cancer, urothelial carcinoma, hepatocellular carcinoma (HCC) and lung cancer correlating with a clinically relevant predictor of tumor patient prognosis. However there is no study focusing on the expression of VASH1 on colon cancer cells and the role of VASH1 in colon cancer. VASH1 can also express in the cancer cells however the function of VASH1 in cancer cells is still unclear.In this study we we retrospectively analyzed the VASH1 expression of tumor tissue and the correlation between VASH1 and clinicopathology was evaluated. Based on this result overexpression VASH1-A and VASH1-B in HT29 cells and knockdown VASH1 in HCT116 cells tests if VASH1 can influence growth, migration and adhesion by apoptosis or senescence. Using flank tumor model and metastasis model further test the function of VASH1 in tumor growth and metastasis. To confirm the role of VASH1 in colon cancer and find new therapy for colon cancer.Methods1.Immunohistochemical staining for the expression of VASH1 in colon cancer stroma.Samples of paraffin-embedded tissue sections and clinicopathological features were obtained from 79 colon cancer at Ji nan central hospital affiliated Shandong University. We carried out immunohistochemical staining the primary antibody: VASH1, VEGF-A, CD34 (as a marker for vascular endothelial cells), VEGF-C and D2-40 (as a lymphatic vessel marker). The correlation between stroma VASH1 and CD34, D2-40, VEGF-A, VEGF-C was analyzed. The correlation between stroma VASH1, CD34, VEGF-A, D2-40, VEGF-C and clinicopathologic characteristics was also confirmed. Tube formation assay was preformed for VASH1 function as an inhibitor in angiogenesis.2. Immunohistochemical staining for the expression of VASH1 in colon cancer cells.Colon cancer tissue slides were stained by the antibodies of VASH1. Immunohistochemistry result was analyzed. We examined the correlation VASH1 in colon cancer cells status with clinicopathologic characteristics.3. The expression of VASH1 in different cancer cell lines were tested by Real Time RT-PCR and Western Blot to confirm the levels of VASH1-Aand VASH1-B.4. HT29 cells were overexpressed VASH1-A or VASH1-B. Growth curve, the [3H]-thymidine incorporation assays, plate colony formation were performed. Apoptosis and senescence were tested by SA-β-gal staining and FACS.5. Test the function of VASH1 in angiogenesis using the HUVEC tube formation assay6. Knockdown VASH1 in HCT116 and Growth curve, the [3H]-thymidine incorporation assays, plate colony formation were performed to make sure the ability of cell growth. Trans-well migration assay and wound closure assay were carried out for migration. Adhesion assay also was performed for the ability of adhesion.7. HT29 (5x106) with VASH1-A, VASH1-B or vector were injected into lateral sublethal. The tumor size were measured every there days by Vernier calipers after 5-6 days. When the tumor grow nearly 2mm all the mice were sacrificed. And tumor size were calculated by the formula 1/2 (length2 xwidth). Tumor weight were measured.KI-67,Cleaved Caspase-3 and CD34 were tested by IHC. SA-β-gel were performed for senescence.8. Growth of the colon cancer cells in vivo:HCT116 (4x106) with VASH1 shRNA or control shRNA were injected into lateral sublethal. The tumor size, tumor weight and KI-67 were measured.9. HCT116(2x106) with VASH1 shRNA or with control shRNA were injected into lateral tail vein. The animals were sacrificed after 6 weeks and their lungs and livers were harvested. Count the tumor nodes by anatomical microscope. The lung and liver tissue were embedded in OCT and HE staining was preformed.Results1. VASHl localize in the endothelial cell of colon cancer vasculature which can inhibit the tube formation (p<0.01). We first evaluated the presence of VASH1 in human colon cancer stroma. The study include 75 patients. VASH1 express in the blood endothelial cells. The expression of VASH1 in colon cancer stroma is higher than paracancerous tissue (p<0.001). The expression of VEGF-A、CD34、 VEGF-C、 D2-40 in colon cancer tissue is also higher than paracancerous tissue (p<0.01). There is a positive correlation between VASH1 and MVD and no correlation with VEGF-A, VEGF-C, LVD. The stroma VASH1 negative correlates with tumor size (p=0.021124), TNM stage (p=0.0069078) and Distant metastasis (p=0.0031225). Transfection of VASH1-A and VASH1-B in HUVECs decreased the tube formation by the reduced numbers of branch points.2. We can found that VASH1 also express in the colon cancer cells. There is not correlation between VASH1 and gender, age, pathologic types, tumor size, TNM stage, tumor differentiation, lymph node metastasis, overall survival, disease-free survival, distant metastasis. However VASH1 is significant predictor of other organs metastasis(p=0.01613).3. The expression levels of VASH1-A and VASH1-B in different cancer cells were determined using Real Time PCR. In the present project we analyzed VASH1-A(the majorVASH1 isoform) and VASH1-B(the alternative splicing isoform). We found all the cell lines express VASH1-A, VASH1-B.The expression of VASH1-A, VASH1-B in HT29 is lowest than other cell lines and HCT116 express higher than other colon cancer cell lines. The expression of VASH1-A, VASH1-B in breast cancer cell lines MCF7 is highest than other cell lines.4. We found that overexpression VASH1 A and VASH1 B can inhibit the proliferation of HT29 by growth curve, the [3H]-thymidine incorporation assays and the colony formation assay (p<0.05). We find VASH1-B can induce apoptosis (p<0.01) and VASH1-A can lead senescence in HT29 cells (p<0.05).5. VASH1 knockdown significantly promote the proliferation of HCT116 (P<0.01) by grow curve, the [3H]-thymidine incorporation assays and the colony formation The adhesion assay shows that adhesion of HCT116 is increased after transfected shRNA. The trans-well migration assay and wound closure assay support that HCT116-shRNA promotes cancer cells’migration(P<0.05).6. Overexpression of VASH1 in HT29 cells inhibited tumor growth (p=0.025) and tumor weight(p<0.01). Transfection of VASH1-A or VASH1-B decreased KI-67 (p<0.05). Transfection of VASH1-A (p<0.05) or VASH1-B (p<0.01) increased Cleaved Caspase-3. Transfection of VASH1-A increase SA-β-Gal positive cell populations in the tumor tissues (p<0.01).7. When injected subcutaneously, we found that HCT116 with shRNA promoted tumor growth (p<0.01). Noticeably, the tumor weights, which we obtained on days 33, showed that HCT116 with shRNA can promote tumor growth(p<0.05). HCT116 with shRNA increased KI-67 (p<0.01).8. To investigate knockdown VASH1 roles in vivo metastasis, we inject HCT116 with shRNA or control shRNA into the mouse by tail vein. Mice were killed after 6 weeks. Examination of the lungs revealed metastasis nodules in the HCT116 with shRNA group was higher than the HCT116 with control shRNA group (p<0.05) which was consistent with the livers result (p<0.01). Conclusion1. VASH1 express in the blood vessel endothelial cells and can inhibit tube formation.There is negative correlation between stroma VASH1 and tumor size, TNM stage, distant metastasis. However the expression of VASH1 in colon cancer cells positively correlates with distant metastasis. There maybe different ways between VASH1 in colon cancer tissue and in colon cancer cells.2. The expression of VASH1 in colon cancer cells can inhibit proliferation, adhesion, migration. We find VASH1-B can induce apoptosis and VASH1-A can lead senescence in colon cancer cells. There are different inhibition function of VASH1-A and VASHl-B in colon cancer cells.3. Transfection of VASH1 decreased tumor growth and tumorigenesis and increased apoptosis and senescence in vivo.In the tumor model knockdown VASH1 can promote tumor growth. The tumor size and tumor weight in shRNA group were higher than control shRNA group; the tumor nodes in shRNA group is higher than control shRNA group. It is same with the vitro result that the expression of VASH1 in colon cancer cells can inhibit tumor growth and metastasis in vivo.4. In conclusion VASH1 can inhibit colon cancer by angiogenesis and cancer cell growth, adhesion, and migration so VASH1 can be a new target for colon cancer therapy.