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To analysis biological functions of the VEGF produced and secreted by each transfected cell lines, the endothelial tube formation assay was carried out using the Millipore In Vitro Angiogenesis Assay Kit which provided a simple model of angiogenesis in which the induction or inhibition of tube formation by exogenous signals can be easily monitored. The human umbilical vein endothelial cells (HUVEC) were seeded onto the surface of the polymerized ECMatrix with the conditioned medium from each transfectant cells. After 6 hours incubation at 37o_{C, cells or tubes were visualized by}

provided dye as shown in Figure 5.5 and the cell networking structures were quantified according to the scoring system explained in Charpter 2, Section 2.2.10.3. The average numerical values of three individual experiments for each transfected cell line were shown in Figure 5.6.

The HUVEC tube formation ability was strongly promoted by the conditioned medium from positive control (human VEGF, 10ng/ml) and LNCaP-WT. The HUVEC cells were able to form well-assembled and complex mesh like structures in the presents of conditioned medium from positive control and LNCaP-WT (Figure 5.5). For positive control and LNCaP-WT group, the average numerical value associated with tube formation pattern was 5 and 4.25±0.83 respectively. In contrast, the HUVEC cells remained randomly separated without any signs of organization and unable to complete tube formation when applied with the culture medium only (negative control). For negative control, the average numerical value associated with tube formation pattern Page | 167

was 1.25±0.83. The HUVEC cells were begin to migrate and align themselves and some of them were able to formed capillary tubes in presence of the conditioned medium from LNCaP-R109A, LNCaP-R109/129A and LNCaP-V but no closed polygons pattern were observed. For LNCaP-R109A, LNCaP-R109/129A and LNCaP-V group, the average numerical value associated with tube formation pattern was 2.75±0.44, 2.25±0.83 and 2.5±0.50 respectively. With the treatment of conditioned medium from LNCaP-WT, HUVEC cells tube formation ability were significantly increased by 1.5-, 1.9-, 1.7- and 3.4-fold compared to the effect of LNCaP-R109A, LNCaP-R109/129A, LNCaP-V and negative control respectively (Students t-test P<0.05). Differences of HUVEC cells tube formation ability either between LNCaP-R109A and LNCaP-V or between LNCaP-R109/129A and LNCaP-V were not significant (Students t-test P>0.05). It has also been observed that the number of branch points formed in LNCaP-WT group was higher than that formed in LNCaP-R109A, LNCaP-R109/129A, LNCaP-V and negative control group.

Figure 5.5 HUVEC cell network formations on ECMatrix with conditioned medium from different transfected cell lines

Images of HUVEC cell network structure were taken after 6 hours incubation in endothelia cell growth media with conditioned medium from different cell lines. Positive control (A), negative control (B), LNCaP-WT (C), LNCaP-R109A (D), LNCaP-R109/129A (E), and LNCaP-V (F).

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B

C

D

Figure 5.6 Cell network formation values of HUVEC cells with conditioned medium from different transfected cell lines

The effect of conditioned medium from different transfected cell lines was assessed and the cell network formation values were quantified according to the cell network pattern. The values from five random fields per well were averaged and three individual assays were performed (mean±SD). Positive control (P), LNCaP-WT (1), LNCaP-R109A (2), LNCaP-R109/129A (3), LNCaP-V (4) and negative control (N).

In order to determine whether the biological activities of VEGF played an important role in tube formation, HUVEC cells in conditioned medium from different cell lines were treated with anti-VEGF in the angiogenesis assay. After 6 hours incubation at 37o_{C, cells were stained with reagent from Millipore In Vitro Angiogenesis Assay Kit as}

shown in Figure 5.8 and the cell network patterns were analyzed as described previously. With the treatment of anti-VEGF, HUVEC cells in LNCaP-WT conditioned medium were unable to form polygons pattern and most of them were randomly separated without any signs of organization. For LNCaP-WT group, the average numerical value associated with tube formation pattern was 1.75±1.26 which is significantly reduced by 2.4-fold compared to samples without anti-VEGF (Students t-test P<0.05). The average numerical values for the rest of the groups including the negative control stayed as same level as samples without anti-VEGF (Students t-test P>0.05). No statistical difference was found between the average numerical value of LNCaP-WT group and that of LNCaP-V group in presence of anti-VEGF (Students t-test P>0.05).

Figure 5.8 Inhibition of cell network formation by anti-VEGF antibody

(A) Images of HUVEC cell network structure were taken after 6 hours incubation with treatment of anti-VEGF antibody in conditioned medium from LNCaP-WT (1) and LNCaP-V (2). (B) The cell network formation values were quantified according to the cell network pattern: LNCaP-WT (1) LNCaP-R109A (2), LNCaP-R109/129A (3) and LNCaP-V (4). The values from five random fields per well were averaged and three individual assays were performed (mean±SD).

A

B

5.3 Discussion

The relationship between the tumourigenicity-promoting function of C-FABP and its ability of binding to and transporting fatty acids were examined and showed that fatty acid-binding and transporting ability is essential for C-FABP to promote tumor growth and expansion. However, whether the increased expression of wild type and mutant C-FABPs are associated with the level of putative target VEGF is still not clear.

In the first part of this chapter, the levels of VEGF expression both in transfected cell lines and in their conditioned medium were measured by Western blot and ELISA. These studies demonstrated that VEGF expression in LNCaP cells transfected with C-FABP was significantly increased compared to VEGF level in cells transfected with mutant C-FABPs or the vector. These results suggested that increase expression of C-FABP can elevate the expression level of VEGF in LNCaP cell modle. Our previous studies results on Rama 37 cell model and prostate cancer cell PC-3M model as mentioned previously also supported these findings [158, 159]. VEGF, as the most proangiogenic factor, plays a crucial role on solid tumor development and expansion. It was reported that the order of VEGF expression in different prostate cancer cell lines as judged by intensity of immunohistochemical staining was PC-3 (a parental cells of PC-3M) > Du145 > LNCaP [182]. The malignancy ranking of these prostate cancer cell lines is in similar sequence as VEGF expression levels. In clinical research, the higher levels of VEGF expression in plasma were observed in prostate cancer patients compared with healthy controls. Based on these findings, it has been indicated that VEGF may facilitate to prostate cancer malignant progression possibly by stimulate angiogenesis.

In the second part of Chapter 5, the angiogenic function of VEGF produced by different transfected cell lines was examined. Immunohistochemistry was performed to investigate the relationship between C-FABP, VEGF and angiogenesis in tumors produced by nude mice inoculated with different transfected cell lines. The tumors from LNCaP-WT group, strong expression of VEGF has been detected and microvessel densities in these tumors were increased 2.8-, 3.5- and 7.0-fold respectively in comparison to those developed from LNCaP-R109A, LNCaP-R109/129A and LNCaP-V. These results suggested that higher expression of C-FABP can stimulate angiogenesis through upregulation of VEGF. The tube formation assay was also carried out using the Millipore In Vitro Angiogenesis Assay Kit in which the angiogenic function of VEGF in conditioned medium from each transfected cell lines can be qualitatively and quantitatively measured. The results showed that tube formation ability of HUVEC cells with the treatment of conditioned medium from LNCaP-WT cells were significantly increased by 1.5-, 1.9-, 1.7- and 3.4-fold compared to the effect of LNCaP-R109A, LNCaP-R109/129A, LNCaP-V and negative control respectively. On the other hand, the angiogenesis ability of HUVEC cells in LNCaP-WT conditioned medium was suppressed when co-cultured with anti-VEGF antibody. These results combined with those reported previously [158, 159] confirmed that the angiogenesis was indeed produced by VEGF, rather than other possible angiogenesis factors.