The Effects Of Interferon-gamma On Vasculogenic Mimicry Of Melanoma Cells

Objective To investigate the effect of interferon-gamma(IFN-γ) on migration,invasion and vasculogenic mimicry(VM)formation ability of human melanoma cell line MUM-2B.Methods Human melanoma cell line MUM-2B cultured in vitro were divided into three groups:cells in control group were cultured in DMEM medium with 10% FBS; cells in treatment groups were cultured in DMEM medium with 10% FBS, and 10 μg/L IFN-γ and 100 μg/L IFN-γ was added in the medium respectively; Part 1: To investigate the effect of IFN-γ on VM formation of MUM-2B cells in vitro 1. Wound healing assay was performed to investigate the effect of IFN-γ on the migration ability of MUM-2B cells; 2. Transwell invasion assay was performed to investigate the effect of IFN-γ on the invasion ability of MUM-2B cells; 3. Three-dimensional culture was performed to investigate the i effect of IFN-γ on the ability of VM tube-like structure formation of MUM-2B cells; Part 2: To discuss the mechanism of the effect of IFN-γ on the ability of VM formation of MUM-2B cells 4. Western blot was performed to analyze effect of IFN-γ on protein level of p-STAT1 of MUM-2B cells; 5. RT-PCR was performed to analyze the effect of IFN-γ on m RNA level of EMT markers of MUM-2B cells; 6. Western blot was performed to analyze the effect of IFN-γ on protein level of EMT markers of MUM-2B cells; 7. Western blot was performed to analyze the effect of IFN-γ on protein level ofEMT associated regulators of MUM-2B cells; 8. Western blot was performed to analyze the effect of IFN-γ on protein level of VEGF of MUM-2B cells. 9. Gelatin zymography assay was performed to detected the effect of IFN-γ on the activity of MMP-2 and MMP-9 of MUM-2B cells;Results 1. The result of wound-healing assay showed that the migration distance of cells in treatment groups was decreased compared with the control group,and the migration distance of cells in 100 μg/L IFN-γ treatment group was decreased compared with the 10 μg/L IFN-γ treatment group(P<0.05); 2. The result of matrigel invasion assay showed that the number of cells invaded through the matrigel in treatment groups was decreased compared with the control group, and the number of cells invaded through the matrigel in 100 μg/L IFN-γ treatment group was decreased compared with the 10 μg/L IFN-γ treatment group(P<0.05); 3. The result of 3-D culture showed that cells in the control group can form typical VM tube-like structures, whereas cells in treatment groups cannot; 4. The result of Western blot showed that the protein expression of p-STAT1 of cells in treatment groups was increased compared with the control group, and the protein expression of p-STAT1 of cells in 100μg/L IFN-γ treatment group was increased compared with the 10 μg/L IFN-γ treatment group(P<0.05). 5. The result of RT-PCR showed that the m RNA expression of VE-cadherin and Vimentin in treatment groups was decreased compared with the control group, the m RNA expression of VE-cadherin and Vimentin in 100 μg/L IFN-γ treatment group was decreased compared with the 10 μg/L IFN-γ treatment group(P<0.05); the m RNA expression of E-cadherin in treatment groups was increased comparedwith the control group, the m RNA expression of E-cadherin in 100 μg/L IFN-γ treatment group was increased compared with the 10 μg/L IFN-γ treatment group(P<0.05); 6. The result of Western blot showed that the protein expression of VE-cadherin and Vimentin in treatment groups was decreased compared with the control group, the protein expression of VE-cadherin and Vimentin in 100 μg/L IFN-γ treatment group was decreased compared with the 10 μg/L IFN-γ treatment group(P<0.05); the protein expression of E-cadherin in treatment groups was increased compared with the control group, the protein expression of E-cadherin in 100 μg/L IFN-γ treatment group was increased compared with the 10 μg/L IFN-γ treatment group(P<0.05); 7. The result of Western blot showed that the expression of EMT associated regulator Twist1 and Slug of cells in treatment groups was decreased compared with the control group, and the protein expression of Twist1 and Slug of cells in 100μg/L IFN-γ treatment group was decreased compared with the 10 μg/L IFN-γ treatment group(P<0.05). 8. The result of Western blot showed that the protein expression of VEGF of cells in treatment groups was decreased compared with the control group, and the protein expression of VEGF of cells in 100μg/L IFN-γ treatment group was decreased compared with the 10 μg/L IFN-γ treatment group(P<0.05). 9. The result of gelatin zymography assay showed that the activity of MMP-2 and MMP-9 of cells in treatment groups was decreased compared with the control group, the activity of MMP-2 and MMP-9 of cells in 100 μg/L IFN-γ treatment group was decreased compared with the 10 μg/L IFN-γ treatment group(P<0.05);Conclusion 1. The result of wound-healing assay, matrigel invasion assay and 3-D culture showed that the migration distance, the number of cells invaded through thematrigel and the VM formation ability of MUM-2B cells in IFN-γ treatment groups were all decreased compared with the control group. These data suggest that IFN-γ inhibits the migration, invasion and VM formation ability of MUM-2B cells in vitro. 2. The result of RT-PCR and Western Blot showed that IFN-γ promoted the expression of p-STAT1 and E-cadherin of MUM-2B cells,and inhibited the expression of VE-cadherin,Vimentin, Twist1 and Slug of MUM-2B cells. These data suggest that IFN-γ inhibits the migration, invasion and VM formation ability of MUM-2B cells through suppressing EMT which depends on the activation of STAT1. 3. The result of Western Blot and gelatin zymography assay showed that IFN-γ inhibited the expression of VEGF and the activity of MMP-2 and MMP-9 of MUM-2B cells. These data suggest that IFN-γ inhibits the VM formation ability of MUM-2B cells through suppressing the expression of VEGF,and also through suppressing the activity of MMP-2 and MMP-9 which could provide space for VM formation by ECM degredation.