The Effects And Mechanism Of MMP-13 On The Regulation Of Melanoma Blood Supply Pattern

Objective1.The expression levels of MMP-13 in human melanoma tissue samples were detected to evaluate the relationship between MMP-13 expression and the clinicopathological parameters of melanoma.Besides,the impacts of MMP-13 in the malignat behaviour of melanoma such as metastasis and patient prognosis were also elucidated to demonstrate the related clinicopathological significance.The effects of MMP-13 on melanoma blood supply pattern were revealed via the observation of the relationship between MMP-13 and Vasculogenic Mimicry,mosaic vessles as well endothelium dependent vessles.Also,the impacts of MMP-13 on tube formation of both melanoma cells and endothelium cells were observed.2.The possible molecular mechanisms of MMP-13 on vasculogenic mimicry formation were also explored.The cleavage fragments of laminin-5 by MMP-13 were detected by western blotting.Furthermore,the relation of MMP-13 and tumor blood supply pattern was revealed via the establishment of mouse bearing melanoma model.The effects of these cleavage fragments on the related functions of melanoma cells were observed to analyze the influences of the laminin-5 fragments by MMP-13 on the tube formation of melanoma cells.Besides,as a Vasculogenic Mimicry marker,the expression of VE-cadherin in both human melanoma tissue samples as well as melanoma cell lines was also detected to find the possible molecular mechanism and signal transduction pathway by which MMP-13 influences the formation of vasculogenic mimicry.The effects of MMP-13 on the migration of HUVEC were also evaluated to explore the possible mechanism of MMP-13 related promotion of endothelium dependent vessels.3.The expression of VE-cadherin and 0-catenin were evaluated to analyse the relationship and clinical significance between MMP-13 expression and VE-cadherin.Methods1.Immunohistochemisty methods were used to detect the expression of MMP-13 in human malignant melanoma tissue samples(n=79).The Vasculogenic Mimicry number,mosaic vessel number and MVD in the melanoma tissues were also counted.The correlation between the expression levels of MMP-13 in melanoma cells and clinicopathologic parameters were analyzed statistically.The expression of VE-cadherin and ?-catenin was also detected via immunohistochemistry staining methods.The correlation between the MMP-13 expression and the expression of VE-cadherin and ?-catenin was evaluated and the related significance was analyzed.2.A375 and B16-F10 melanoma cells were culture in vitro.The melanoma cells stably transfected with pcDNA-3-MMP-13 or pRNAT-MMP-13 siRNA was used to observe the effects of different MMP-13 expression levels on the migration,invasion and tube formation ability of melanoma cells.The human umbilical vein endothelial cells(HUVEC)were also cultured to evaluate the effects MMP-13 in the culture medium on the invasion and tube formation ability of HUVEC.3.Melanoma bearing mouse model was established to observe and analyse the correlation of MMP-13 expression and tumor blood supply pattern in the different stages of melanoma growth.4.Western-blotting was used to detect the cleavage fragments of MMP-13 on laminin-5.Three dimensional culture assays were used for observing the effects of the laminin-5 cleavage fragments by MMP-13 on the migration,invasion and tube formation ability.Besides,western blotting was used for detecting the expression of VE-cadherin under different MMP-13 expression levels.Immunofluorescence technique was used to observe the effects of exogenous addition of MMP-13 on the expression and localization of(3-catenin in melanoma cells.Results1.Of the 79 malignant melanomas,36 melanomas have higher expression of MMP-13 while the other 43 melanomas have lower expression.61%occurs metastasis of the melanomas with higher expression of MMP-13(n=36).However,as to the melanoma tissues with lower expression of MMP-13(n=43),only 26%have metastasis.Kaplan-Meier survival analysis showed that the patients whose tumors have higher expression levels of MMP-13 have lower overall survival rates.2.The melanoma tissues which have higher expression levels of MMP-13 have lower Vasculogenic Mimicry number while the melanoma tissues which have lower expression of MMP-13 have higher Vasculogenic Mimicry number.In the cultured melanoma cells,the cells with up-regulated MMP-13 expression levels have decreased ability of tube formation on Matrigel.However,an increased ability of tube formation on Matrigel can be observed after the MMP-13 expression levels were down-regulated.Besides,MMP-13 significantly increased the tubes formed on the Matrigel by HUVEC(P<0.05).3.The expression of MMP-13 in the implanted melanoma increased with tumor growth and inversely correlated with Vasculogenic Mimicry number while negatively related with MVD(P<0.05).4.Different from the tube promotion ability of tumor cells by MMP-2,MMP-13 cleaved laminin-5 into cleavage fragments with smaller fragments which inhibit the tube-forming ability of melanoma cells.5.The human melanoma tissues with higher expression levels of MMP-13 have lower levels of VE-cadherin.Lower membrane positive rates as well as higher cytoplasm and nucleus positive rates can also be found in the melanoma tissues with higher expression of MMP-13.The melanoma cells with up-regulated expression levels of MMP-13 have lower levels of VE-cadherin.Furthermore,higher positive expression of ?-catenin in the cell cytoplasm and nucleus can be found after exogenous addition of MMP-13.6.MMP-13 promoted the invasion ability of HUVEC on Matrigel and which is related with the cleavage of Ln-5 by MMP-13.Conclusions1.The higher expression of MMP-13 in melanoma tissues was correlated with metastasis.2.MMP-13 negatively regulated Vasculogenic Mimicry formation while positively regulated endothelium dependent vessels.3.MMP-13 may play in the replacement of Vasculogenic Mimicry by endothelium dependent vessels to be main blood supply pattern.4.MMP-13 inhibits and destroys Vasculogenic Mimicry formation via the degradation of Ln-5 into smaller cleavage fragments.Besides,the cleavage of VE-cadherin by MMP-13 also plays roles in the MMP-13 related inhibition of Vasculogenic Mimicry formation.5.The degradation of VE-cadherin by MMP-13 resulted in the release of ?-catenin from the VE-cadherin-?-catenin complex,which helps to promote melanoma metastasis.6.MMP-13 promoted the migration ability of endothelium cells which resulted in promotion of endothelium dependent angiogenesis.