To Study The Correlation Of Caspase 8,9 And EMT Regulating Proteins In The Early Stage Of Tumor Microcirculation Of The Tumor Cells Around Lineraly Patterned Programmed Cell Necrosis And Vasculogenic Mimicry

Object ive:To study the correlation of Caspase8,9 and EMT regulating proteins in the early stage of tumor microcirculation of the tumor cells around linearly patterned programmed cell necrosis and vasculogenic mimicry. Though increasing hypoxic and ischemic microenvironment of tumor cells by inhibiting LPPCN information and VM information after inhibiting Capase8 which inhibited dearth receptor-dependent pathway and 9 which inhibited mitochondrial pathway-dependent pathway, the expression of EMT associated proteins were identified of the tumor cells from layer I to IV of LPPCN and VM, and investigate the important role of the tumor cells undergoing EMT in the information of VM.Methods:1. Building the mouse modelsThe C57 mouse melanoma transplant tumor models:The B16 melanoma tissues which were stored in liquid nitrogen were incubated for 20-30sec in a 43℃water bath and centrifuged at 1000rpm for 10 min, and then the supernatant was abandoned. The tumor tissues were cut up and diluted with 0.9%sodium to ensure a final cell density of 0.2x10'cells/ml. The mouse groin was sanitized with an alcohol swab and 0.2ml single tumor cell suspension was injected subcutaneously. After tumors appeared, the mice were randomly divided into the Caspase8 inhibitor group, the Caspase9 inhibitor group, the Caspase8 and 9 inhibitors group, and the control group. The mice were killed at 15d after giving Caspase8,9 inhibitors. After killing the mice, the engrafted animal melanoma tissues were removed, fined with formalin and embedded in paraffin. The 4-μm paraffin-embedded tumor tissues were mounted onto APES-coated slides.2. The expressions of EMT associated proteins of the tumor cells from layer I to IV of LPPCN and VMThe formation of LPPCN and VM was compared in the Caspase8 inhibitor group, the Caspase9 inhibitor group, the Caspase8 and 9 inhibitors group, and the control group. The expressions of EMT associated proteins of the tumor cells surrounding LPPCN and VM especially of the tumor cells from layer I to IV of LPPCN and VM were compared in the Caspase8 inhibitor group, the Caspase9 inhibitor group, the Caspase8 and 9 inhibitors group, and the control group. To investigate the tumor cells from layerⅠtoⅣof LPPCN might play an important role in the formation of VM. The tumor cells from layerⅠtoⅣof LPPCN were the tumor cells from first to forth layer of LPPCN respectively; the tumor cells from layerⅠtoⅣof VM were the tumor cells from first to forth layer of VM respectively.Results:1. The LPPCN binds in every groupThe LPPCN binds in the Caspase8 group (11.30±1.930), Caspase9 group (22.23±4.133), Caspae8 and 9 group (19.60±2.579), and the control group (31.57±5.352) had statistical significance (F=5.310, P=0.002). Especially of the Caspase8 inhibitor group (q=20.271, P=0.007), the Caspase8 and 9 inhibitors group (q=11.971, p=0.021) were decreased obviously compared with the control group.2. The quantity of VM in every groupThe quantity of VM in the Caspase8 group (1.90±0.232), Caspase9 group (2.00±0.328), Caspae8 and 9 group (2.03±0.222), and the control group (2.61±0.246) did not have statistical significance (F=1.558, P=0.204).3. The expression of EMT associated proteins of tumor cells from layerⅠtoⅣof LPPCN and VMThe Twistl nuclear expression of the tumor cells from layerⅠtoⅣof LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group had statistical significance(Caspase8 inhibitor group F=9.029,P=0.000; Caspase9 inhibitor group F=5.035,P=0.006; Caspase8 and 9 inhibitors group F=6.356, P=0.002; the control group F=15.259, P=0.000).The Twistl nuclear expression of the tumor cells in the same layer of LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group had statistical significance(theⅠlayer F=5.894,P=0.003; theⅡlayer F=6.715,P=0.001; theⅢlayer F=4.892,p=0.007; theⅣlayer F=5.732,p=0.003). The Twistl nuclear expression of the tumor cells from layerⅠtoⅣof VM in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not have statistical significance(Caspase8 inhibitor group F=0.906,P=0.449; Caspase9 inhibitor group F=0.189,P=0.905; Caspase8 and 9 inhibitors group F=0.711,P=0.553; the control group P=1.406,P=0.262).The Twistl nuclear expression of the tumor cells in the same layer of VM in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not had statistical significance(theⅠlayer F=0.233,P=0.873; theⅡlayer F=0.275,p=0.843; theⅢlayer F=0.426,P=0.736; theⅣlayer F=0.608, P=0.645).The Twistl cytoplasm expression of the tumor cells from layerⅠtoⅣof LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group had statistical significance(Caspase8 inhibitor group F=0.096,P=0.962; Caspase9 inhibitor groupF=0.054,p=0.983; Caspase8 and 9 inhibitors group F=1.309,P=0.277; the control group F=0.226,P=0.878).The Twistl cytoplasm expression of the tumor cells in the same layer of LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group had statistical significance(theⅠlayer F=2.488,P=0.066; theⅡlayer F=2.292,p=0.084; theⅢlayer F=0.062,p=2.357; theⅣlayer F=2.262,P=0.087). The Twistl cytoplasm expression of the tumor cells from layerⅠtoⅣof VM in Caspase8 inhibitor group, Caspase9 inhibitor group, CaspaseS and 9 inhibitors group, and the control group did not have statistical significance(Caspase8 inhibitor group F=0.350,P=0.789; Caspase9 inhibitor group F=0.755,P=0.522; Caspase8 and 9 inhibitors group F=0.571,p=0.636; the control group F=1.005,P=0.395).The Twistl cytoplasm expression of the tumor cells in the same layer of VM in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not had statistical significance(theⅠlayer F=2.186, P=0.096; theⅡlayer F=1.756, P=0.162; theⅢlayer F=1.120,p=0.346; theⅣlayer F=2.105, P=0.106).The Snail expression of the tumor cells from layerⅠtoⅣof LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group had statistical significance(Caspase8 inhibitor group F=3.359,P=0.021; Caspase9 inhibitor group F=1.115,P=0.347; Caspase8 and 9 inhibitors group F= 1.191,p=0.317; the control group F=0.493,P=0.688).The Snail expression of the tumor cells in the same layer of LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group had statistical significance(theⅠlayer F=6.810,p=0.000; theⅡlayer F= 13.132,p=0.000; theⅢlayer F= 14.571,p=0.000; theⅣlayer F=13.612,p=0.OOO). The Snail expression of the tumor cells from layer I to IV of VM in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not have statistical significance(Caspase8 inhibitor group F=1.590,p=0.196; Caspase9 inhibitor group F=1.386,P=0.251; Caspase8 and 9 inhibitors group F=2.267,P=0.084; the control group F=1.997,P=0.119).The Snail of the tumor cells in the same layer of VM in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not had statistical significance(theⅠlayer F=1.446,P=0.443; theⅡlayer F=3.183,P=0.027; theⅢlayer F=0.920P=0.433; theⅣlayer F=2.371,P=0.074).The Slug expression of the tumor cells from layerⅠtoⅣof LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group had statistical significance(Caspase8 inhibitor group F=6.446,P=0.000; Caspase9 inhibitor group F=31.339,P=0.000; Caspase8 and 9 inhibitors group F=2.659,P=0.052; the control group F=3.785,P=O.O13).The Slug expression of the tumor cells in the same layer of LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group had statistical significance(theⅠlayer F=4.888,p=0.003; theⅡlayer F=4.221,P=0.007; theⅢlayer F=4.777vP=0.004; theⅣlayer F=6.642,P=0.000). The Slug expression of the tumor cells from layer I toⅣof VM in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not have statistical significance(Caspase8 inhibitor group F=0.574,P=0.633; Caspase9 inhibitor group F=2.009,P=0.117; Caspase8 and 9 inhibitors group F=2.391,P=0.071; the control group F=0.776,.P=0.510).The Slug of the tumor cells in the same layer of VM in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not had statistical significance(theⅠlayer F=0.559,P=0.643; theⅡlayer F=0.995,P=0.398; theⅢlayer F=1.312,P=0.274; theⅣlayer F=0.766,P=0.515).The E-cadherin expression of the tumor cells from layerⅠtoⅣof LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not have statistical significance(Caspase8 inhibitor group F=0.277,P=0.842; Caspase9 inhibitor group F=0.020,P=0.996; Caspase8 and 9 inhibitors group F=0.087,P=0.993; the control group F=0.031,p=0.993).The E-cadherin expression of the tumor cells in the same layer of LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not have statistical significance(theⅠlayer F=0.419,P=0.740; theⅡlayer F=0.177,P=0.912; theⅢlayer F=0.177,P=0.912; theⅣlayer F=0.322,P=0.809). The E-cadherin expression of the tumor cells from layerⅠtoⅣof VM in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not have statistical significance(Caspase8 inhibitor group F=0.676,P=0.569; Caspase9 inhibitor group F=0.730,p=0.536; Caspase8 and 9 inhibitors group F=0.478,P=0.698; the control group F=0.261,P=0.854).The E-cadherin of the tumor cells in the same layer of VM in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not had statistical significance(theⅠlayer F=0.967,P=0.411; theⅡlayer F=1.188,P=0.317; theⅢlayer F=0.633,P=0.595; the IV layer F=0.632,p=0.667).The Vimentin expression of the tumor cells from layer I to IV of LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not have statistical significance(Caspase8 inhibitor group F=1.573,P=0.200; Caspase9 inhibitor group F=0.560,P=0.642; Caspase8 and 9 inhibitors group F=2.644,P=0.051; the control group F=1.910,P=0.132).The Vimentin expression of the tumor cells in the same layer of LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not have statistical significance(theⅠlayer F=2.064,P=0.109; theⅡlayer F=3.679vP=0.014; theⅢlayer F=3.898,P=0.011; theⅣlayer F=1.852,P=0.142). The Vimentin expression of the tumor cells from layerⅠtoⅣof VM in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not have statistical significance(Caspase8 inhibitor group F=1.982,P=0.121; Caspase9 inhibitor group F=2.36O,p=0.075; Caspase8 and 9 inhibitors group F=1.399,P=0.247; the control group F=1.399,P=0.128).The Vimentin of the tumor cells in the same layer of VM in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not had statistical significance(the I layer F=1.310,P=0.274; theⅡlayer F=0.665,p=0.575; theⅢlayer F=1.329,P=0.268; theⅣlayer F=2.405,P=0.071).The Fibronectin expression of the tumor cells from layerⅠtoⅣof LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not have statistical significance(Caspase8 inhibitor group F=0.611,P=0.609; Caspase9 inhibitor group F=2.144,P=0.099; Caspase8 and 9 inhibitors group F=0.768,p=0.541; the control group F=2.678,P=0.074).The Fibronectin expression of the tumor cells in the same layer of LPPCN in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not have statistical significance(theⅠlayer F=1.071,P=0.364; theⅡlayer F=0.870,P=0.459; theⅢlayer F=0.937,p=0.427; theⅣlayer F=0.655,P=0.582). The Fibronectin expression of the tumor cells from layerⅠtoⅣ of VM in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not have statistical significance(Caspase8 inhibitor group F=l.053,P=0.372; Caspase9 inhibitor group F=0733,P=0.543; Caspase8 and 9 inhibitors group F=0.538,P=0.657; the control group F=2.620,P=0.054).The Fibronectin of the tumor cells in the same layer of VM in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, and the control group did not had statistical significance(theⅠlayer F=0.611,P=0.609; theⅡlayer F=1.102,P=0.390; theⅢlayer F=2.459,P=0.066; theⅣlayer F=1.262,P=0.291).Conclusion:1. LPPCN exists in mouse melanomaLinearly or network-like LPPCN cells exist in C57 B16 melanoma. LPPCN is a new kind of tumor cell death model and is different from the typical cell death model: apoptosis and necrosis. The molecular mechanism underling LPPCN may be similar to the mitochondria-dependent apoptosis-signaling pathway.2. The LPPCN binds in every groupCompared with the control group, the LPPCN binds were decreased significantly in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group, especially in the Caspase8 inhibitor group and the Caspase8 and 9 inhibitors group. LPPCN is a caspase-dependent, new kind of tumor cell death model.3. The quantity of VM in every groupCompared with the control group, the quantity of VM was not changed in Caspase8 inhibitor group, Caspase9 inhibitor group, Caspase8 and 9 inhibitors group. The formation of VM may not directly have relationship with Caspase8,9.4. The tumor cells from l ayerⅠtoⅣof LPPCN and VM express EMT assoc i ated proteinsThe tumor cells form layerⅠtoⅣof LPPCN and VM express EMT associated proteins Twistl, Snail, Slug, Vimentin and Fronection but almost not express E-cadherin. The tumor cells form layer I to IV of LPPCN and VM undergo EMT.5. The expression of EMT associated proteins of the tumor cells from layer I to IV of LPPCN and VM after inhibiting Caspase8,9With LPPCN information deceased by inhibiting Caspase8,9, the expressions of Twistl nuclear, Snail and Slug decreased by layers, while the changes of Twistl cytoplasm, E-cadherin, Vimentin and Fronectin not obvious of the tumor cells from layerⅠtoⅣof LPPCN. The expression of Twistl nuclear, Twist1 cytoplasm, Snail, Slug, E-cadherin, Vimentin and Fronectin not decreased by layers of the tumor cells from layerⅠtoⅣof VM.6. The tumor cells from layerⅠtoⅣof LPPCN might be the materials foundation of VM formationEMT is a process, which epithelial cells loss polarity and homeostasis, but acquire the behavior of motile and migratory. EMT is an important process in embryogenesis, the formation of tissue, the healing of wound, the formation of various fibrotic diseases, and the development and the invasion and metastasis of the tumor. EMT was reported as a process by which epithelial cells lose their characteristics, such as cell-cell adhesion, polarity, and lack of motility, and acquired mesenchymal features, including motility, invasion, and resistance to apoptosis. The tumor cells from layersⅠtoⅣof LPPCN undergoing EMT under comparative hypoxia not only undergo morphologic changes, but could also autocrine many kinds of cytokines including TGF-β,TNF-α,MCP-1,IGF,FGF, autocrine matrix metalloproteinases to break down extracellular matrix, and autocrineⅣcollagen and PAS positive material. Therefore, tumor cells undergoing EMT might be the materials foundation for VM formation.