A Pilot Study On The Role Of Caspase Family In The Formation Of Vasculogenic Mimicry In Melanoma

PurposeTo study the role of Capsase3、Capase8,Caspase9and apoptosis mediated by Caspase family in the formation of VM by animal experiment. To further study the role of the Caspase family members in the process of VM formation, the activities of MMP-2and MMP-9was measured by gelatin zymography assay and immunohistochemical methods. To further analyze molecular mechanisms of VM formation, different gene expression of primary tumor cell from Caspase3inhibitor group was assessed by Micro array expression, providing the experimental basis for clinical treatment.Method1) Establish the malignant B16melanoma model in C57BL mice and all the mice were randomly divided into the Caspase3inhibitor group, Caspase8inhibitor combined with Caspase9inhibitor group and the control group. Caspase8inhibitor combined with Caspase9inhibitor every day when the tumor can be touched locally.2) Tumor volumes in differently treated mice were measured every day after therapy. The numbers of Vasculogenic Mimicry in tumor tissues was counted by immunohistochemical and PAS double staining methods. The activities and expressionof MMP-2and MMP-9was measured by gelatin zymography assay and immunohistochemical method.3) The primary B16cell which was treated by Caspase3inhibitor and B16cells were cultured until the cells reached5×106to extract total RNA. The cDNA micro array was hybridized using hybrid membrane with18000clones. After the raw array data were normalize, the differential expression of genes between primary B16and B16was evaluated using fold change(cutoff=1.0). We performed functional enrichment analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) for these differential expressional genes with DAVID (httP://david.abce.neifcrf.gov/)on line tools.4) The capacity of migration of primary B16cell of Caspase3inhibitor group was assessed by wound healing experiments.Result1) Compared with the control group, the tumor in Caspase3inhibitor group and Caspase8inhibitor combined with Caspase9inhibitor group grew much slower (P<0.05) and the numbers of Vasculogenic Mimicry of tumor tissues in Caspase3inhibitor group and Caspase8inhibitor combined with Caspase9inhibitor group were less in the early stage of tumor(P<0.05).2) The activities and expression of MMP-2and MMP-9in Caspase3inhibitor group were lower than the control group (P<0.05), the activity and expression of MMP-9in Caspase8inhibitor combined with Caspase9inhibitor group was lower than the control group (P<0.05).3) Compared with the B16cell, there were1103differentially expressed genes in primary B16cell. The analysis of KEGG pathway showed that these differential expression genes were mainly enriched in more than200pathway including TGF-beta signaling pathway, MAPK signaling pathway, Cell Cycle, Glutathione metabolism, Focal adhesion, Insulin Signaling pathway, Wnt signaling pathway, et al.4) Compared with the B16cell, the migration of primary B16cell of Caspase3inhibitor group decreased.Conclusion1) Caspase family and apoptosis mediated by Caspases play an important role in the formation of VM in malignant melanoma.2) Caspase3inhibitors can depress the formation of the VM by reducing the activity and expression of MMP-2and MMP-9and hindering cell migration in the early stage of tumor.3) Caspase3inhibitors may also hinder the formation of the VM by inhibiting the expression of Idl and Id3.4) Caspase3may not only involved in the process of apoptosis, but also may be involved in the signaling pathway of TGF-β signaling, when the expression of Caspase3was inhibited, many genes of this signaling pathways differentially expressed.