VEGF-D-enhanced lymph node metastasis of ovarian cancer is reversed by vesicular stomatitis virus matrix protein

Abstract

Lymphatic metastasis is a poor prognostic factor in ovarian cancer, which correlates to the majority of cancer deaths. Matrix protein (MP) of vesicular stomatitis virus (VSV) exhibits potent antitumor and antiangiogenic activities through inducing apoptosis and inhibiting angiogenesis. In this study, the antitumor and antimetastatic effects of MP were further investigated. Wild-type SKOV3 (WT-SK) cells were successfully transfected with empty vector pcDNA3.1 plasmid, or pcDNA3.1-VEGF-D recombinant plasmid to construct cell lines named EV-SK, and VEGFD-SK, respectively. Inhibition of VEGFD-SK cell migration and invasion was detected by Transwell and wound healing assay. Then, lymphogenous metastatic model of ovarian cancer was established by injecting VEGFD-SK cells subcutaneously into the left hindlimb claw pad of nude mice. The inducted apoptotic effect of MP on VEGFD-SK cells were assessed by flow analysis and Hoechst-33258 staining, respectively, in vitro. The in vivo antitumor and antiangiogenic activities of MP gene were evaluated with lymphogenous metastatic model of ovarian cancer. Tumor volume and lymphatic metastasis rates were measured. Lymphatic vessels were delineated using Evan's blue and LYVE-1 staining. Expression of VEGF-D and MMP-2 were evaluated by immunostaining. Apoptosis of tumor cells was analyzed by Hoechst-33258 staining. Mice bearing VEGFD-SK tumor cells displayed more rapid tumorigenesis, higher lymphogenous metastatic tendency and increased lymphatic vessel density compared with the mice bearing WT-SK or EV-SK cells. However, VEGF-D-enhanced metastasis was evidently reversed by MP. MP significantly reduced the invasion of VEGFD-SK cells, tumor volume, lymphatic metastasis rates and lymphatic vessel density compared with control groups (P<0.05), accompanied with down-expression of VEGF-D and MMP-2 and increased apoptosis. Our data indicate that MP has strong antitumor and antimetastatic abilities, and it may be a promising therapeutic strategy against the lymphatic metastasis of human ovarian cancer.

Figure 1

RT-PCR and apoptotic effect on VEGFD-SK cells. (A) RT-PCR analysis of expression of MP in vitro. High expression of MP in VEGFD-SK cells transfected with pVAX-MP:lip was detected (1,036 bp). GAPDH was used for the internal standard. The displaying panel of electrophoretic image presents the following: DNA ladder marker (a), WT-SK (b), EV-SK (c), VEGFD-SK (d), pVAX:lip (e), and pVAX-MP:lip (f), respectively. (B) Overexpression of VEGF-D using RT-PCR could be detected in VEGFD-SK cells, but not in WT-SK and EV-SK cells (1,077 bp). However, the VEGF-D expression was suppressed when VEGFD-SK cells suffered from pVAX-MP:lip. The overexpression of VEGF-D did not decreased obviously when VEGFD-SK cells were treated with pVA. (C) VEGF-D expression in vivo was analysis by RT-PCR. (D) The expression of VEGF-D in vivo was determined with immunohistochemistry using VEGF-D antibody: VEGFD-SK tumors were detected with much stronger staining. Whereas, the VEGFD-SK tumors in pVAX-MP:lip group present nearly negative staining. Tumors in pVAX:lip group also exhibited strong staining. Very weak VEGF-D protein was observed in WT-SK and EV-SK tumors. (E) Hoechst-33258 stained fluorescence microscopy (x200) in vitro: apoptosis cells were observed apparently when the VEGFD-SK cells were treated with pVAX-MP:lip (arrow). (F) A quantitative comparison of apoptotic cells was carried out by flow analysis stained with Annexin V-FITC and propidium iodide: pVAX-MP significantly increased VEGFD-SK cells apoptosis compared with the controls.

Figure 2

Inhibition of cell invasion and migration induced by pVAX-MP:lip in vitro. (A) Wound healing analysis of migration. The cell motility was enhanced in the VEGFD-SK group. However, the cell motility was decreased in the pVAX-MP:lip group: the wound widths in the pVAX-MP:lip group was wider than in VEGFD-SK or pVAX:lip group. (B) Transwell assay was performed to evaluate cell invasion. Migratory capacity was increased in VEGFD-SK cells (x100). The number of invading cells in the VEGFD-SK group was increased compared with other groups. While the numbers of invading cells in the pVAX-MP:lip groups were obviously lower than in the pVAX:lip group (x100). (C) VEGFD-SK cells had much high migration ability, but pVAX-MP:lip group had the lowest migration tendency in the wound healing assay (^*P<0.05 WT-SK group versus VEGFD-SK group; ^**P<0.05 pVAX-lip group versus pVAX:lip group).

Figure 3

Tumor growth in nude mice. The graphs indicate that tumor volume in VEGFD-SK group was larger than it in WT-SK and EV-SK groups. However, the tumor growth in pVAXMP:lip group is extremely suppressed after systemic treatment (P<0.05 versus any other group). The pVAX:lip group also exhibited faster tumor growth.

Figure 4

Qualitative and quantitative analyses of LVD and metastasis rate of LNs. (A) Top panel, qualitative LV was evaluated by Even's blue lymphangiogram. There are dilated LVs around tumors in VEGFD-SK and pVAX:lip groups (arrow, blue lymphatic vessels). Nearly no visible LVs were seen when VEGFD-SK tumors were treated with pVAX-MP:lip. A few of LVs appeared in the WT-SK and EV-SK groups. Middle panel, LYVE-1 staining shows that rich nascent LVs present in VEGFD-SK and pVAX:lip group. However, very few LVs appear in pVAX-MP:lip group. Sparse LVs in WT-SK and EV-SK groups were detected. Bottom panel, microscopic images show that clumpy deposits of metastatic growth have occupied almost entire the LNs tissues in VEGFD-SK and pVAX:lip groups, but very few metastasis was established in pVAX-MP:lip group. (B) Quantitative analyses of LVD were calculated by LYVE-1 staining. Tumor in VEGFD-SK and pVAX:lip groups displayed increased LVD compared with tumor in WT-SK and EV-SK groups. However, pVAX-MP:lip group show obviously reductions in LVD (^*P<0.05 versus WT-SK and EV-SK groups respectively). (C) The comparison of metastasis rates in tumor-draining LNs show that VEGFD-SK and pVAX:lip groups have a much higher metastasis tendency than other groups (arrow, tumor cells in lymph node). The VEGF-D-enhanced metastases to multilevel LNs were evidently reversed after the pVAX-MP:lip administration (^*P<0.05 versus other groups).

Figure 5

Expression of MMP-2 and comparisons of apoptotic indexes among different groups. Top panel, there was much stronger MMP-2 staining in tumor tissue of VEGFD-SK group compared with the weak staining in WT-SK group and EV-SK group. The pVAX:lip group also exhibited deep MMP-2 staining; whereas the pVAX-MP:lip group showed negative MMP-2 staining. Middle panel, microscopic fluorescence images show apoptotic cells of tumor tissues of different groups by Hoechst-33258 staining. VEGFD-SK tumor tissue manifested a few apoptosis of tumor cells; whereas pVAX-MP:lip group tumor tissue revealed a significant enhancement in apoptosis of tumor cells (arrow, apoptotic cell). However, no appreciable difference was observed between WT-SK group and EV-SK group. Bottom panel, VEGFD-SK group tumor tissue manifested a significant suppression of apoptotic tumor cells in contrast to other groups. Whereas higher apoptosis index was observed after the pVAX-MP:lip administration (^*P<0.05 versus other groups).

Figure 6

Mouse weight. No significant differences in mouse weight was found among the five groups (P>0.05).