The Study Of Mechanism Of Linearly Patterned Programmed Cell Necrosis In Human Melanoma

Objective:1) To identify if VM and LPPCN existing in human melanoma through a retrospective study with paraffin tissue samples. To explore the correlation between LPPCN, VM and clinicopathologic factors, clinical prognosis in melanoma'patients. Based on these, to investigate the clinical and pathological significance of VM and LPPCN in melanoma'patients.2) To disclose the role of apoptosis-associated proteins and analyze relevant molecular mechanism in LPPCN formation.3) To investigate the expression of Twist1, MMP-2, MMP-9 in melanoma and analyze their role in LPPCN and VM formation.Methods:1) 70 melanoma cases undergoing surgery in Tumor Hospital affiliated of Tianjin Medical University from January 1996 to November 2007 were collected in this study. The information of samples including age, gender, tumor size, location, recurrence and metastasis were collected and analyzed the clinicopathologic character of patients.2) CD31/PAS double staining and HE staining was performed on the slides to confirm the existence of VM and LPPCN. Besides, to count the VM, LPPCN, MVD and analyze the relation between them in melanoma. Based on these, to explore the correlation between LPPCN,VM and clinicopathologic parameter,clinical prognosis in melanoma'patients and investigate the association of LPPCN and VM, endothelium-dependent vessels.3) Immunohistochemistry method was used to detect the expression of apoptosis-associated proteins in scatter patterned programmed cell necrosis cells, Linearly patterned programmed cell necrosis cells and their circum-tumors cells and the expression of invasive-associated proteins in LPPCN-positive group and LPPCN-negative group, VM-positive group and VM-negative group. Staining index was used to evaluate the difference of these proteins expressed and analyzing their role in VM and LPPCN formation.Results:1 VM and LPPCN exists in melanoma1.1 The morphological observed results in melanomaCD31/PAS double staining and HE staining shows 33 cases existed VM and 38 cases existed LPPCN in 70 cases melanoma. Besides,22 cases existed SPPCN and LPPCN.1.2 The relation between LPPCN,VM and clinicopathologic data in melanomaThe occurrence of VM and LPPCN was not correlated with age, gender, location, melanin pigment recurrence and metastasis (P>0.05).The melanoma was prone to generate VM and LPPCN when its volume exceed 20.41cm3(P<0.05) Kaplan-Meier survive plot shows the survive time of VM-positive group was shorter than VM-negative group, the survive time of LPPCN-positive group was shorter than LPPCN-negative group, the survive time of VM and LPPCN-positive group was shorter than VM and LPPCN-negative group (P<0.05)。1.3 The relation between LPPCN,VM and MVD counting in melanomaThe average MVD counting in VM-positive group was higher than that VM-negative group (P<0.05). The average MVD counting in VM and LPPCN-positive group was higher than that VM and LPPCN-negative group (P<0.05)2 The correlation results of every parameterThere is a highly positive correlation between the number of VM and the number of LPPCN, the number of LPPCN cells (r=0.469, r=0.470;P=0.024, P=0.024). There is a highly positive correlation between the number of LPPCN and the number of LPPCN cells (r=0.406, P=0.011).There is a highly positive correlation between the size of tumor and the number of endothelium-dependent vessels(r=0.261, P=0.029).3 The immunohistochemisty staining results of apoptosis-associated proteins3.1 The expression of mitochondrial-mediating apoptosis relative proteins3.1.1 The expression of Bcl-2 and Bax in melanomaThe expression of Bcl-2 in LPPCN-positive group was higher than LPPCN--negative group (P<0.05).The expression of Bcl-2 in SPPCN cells was higher than LPPCN cells (P<0.05). The expression of Bax in SPPCN cells was lower than LPPCN cells (P<0.05). The expression of Bcl-2 in SPPCN circum-tumors cells was lower than LPPCN circum-tumors cells (P<0.05). The expression of Bax in SPPCN circum-tumors cells was higher than LPPCN circum-tumors cells (P<0.05). The expression of Bcl-2 in LPPCN cells was lower than LPPCN circum-tumors cells (P<0.05). The expression of Bax in LPPCN cells was higher than LPPCN circum-tumors cells (P<0.05)。3.1.2 The expression of Caspase9 and Caspase3 in melanomaThe expression of Caspase9 and Caspase3 in LPPCN cells was higher than LPPCN circum-tumors cells(P<0.05). The expression of Caspase9 and Caspase3 in LPPCN-negative group and LPPCN-positive group had not statistical significance (P>0.05).3.2 The expression of tumor necrosis factor family-mediating apoptosis relative-proteins in melanomaThe expression of TNF, TRAIL, Fas, FasL,Caspase8 in LPPCN-negative group and LPPCN-positive group had not statistical significance(.P>0.05). The expression of TNF, TRAIL, Fas, FasL, Caspase8 in LPPCN cells and LPPCN circum-tumors cells had not statistical significance (P>0.05)4 The expression of Twist1,MMP2,MMP-9 in melanomaThe expression of Twistl in LPPCN-positive group was higher than LPPCN-negative group (P<0.05).The expression of Twist1 in VM-positive group was higher than VM-negative group (P<0.05). The expression of Twist1 in LPPCN cells was higher than LPPCN circum-tumors cells (P<0.05).The expression of MMP-2 and MMP-9 in VM-positive group was higher than VM-negative group (P<0.05). The expression of MMP-2 in LPPCN-positive group was higher than LPPCN-negative group (P<0.05)Conclusion:1 VM and LPPCN existed in melanoma and the patients with VM or/and LPPCN has worse clinical prognosis than those without VM and LPPCN. The occurrence of VM and LPPCN was not correlated with age, gender, location, melanin pigment, recurrence and metastasis. The melanoma was prone to generate VM and LPPCN when its volume exceeds 20.41cm3 (diameters about 3 mm).2 LPPCN is a transitive cells'dead way between apoptosis and necrosis. Under the genes control of mitochondrial-mediating apoptosis, some melanoma cells underwent SPPCN by hypoxic condition. SPPCN will develop LPPCN when tumor tissue lacks oxygen severely.3 The proteins of MMP-9,MMP-2,Twist1 expressed by melanoma play a important role in VM formation. The melanoma cells could secrete PAS matter and it participates stromatic remodeling of VM.