The Relationship Between Fas/Fas Ligand, PCNA And Apoptosis With Human Malignant Melanoma

Malignant melanoma (MM) is one kind of malignant tumor origin from neural crest melanocytes, and it often occurs in the skin and the mucous membranes. MM is quite often secret with highly malignant degree, and it is a high invasion and easily transfusion into blood vessel and the lymph node. The recurrence rate of MM is high, it develops very quickly and worse prognosis. MM is insensitivity to many conventional therapy methods such as radiotherapy, chemotherapy, operation and immunotherapy. However, the pathogenesis of malignant melanoma and metastasis and apoptosis mechanisms of tumor cells are still not clearly. Constantly, many scientists devote themselves to study human malignant melanoma from many aspects, and they gradually pay attention to Fas/FasL system.Fas, Apo-1/CD95, is a member of the tumor necrosis factor receptor (TNFR) superfamily, which induces cell death in sensitive cells when ligated by its physiological ligand Fas ligand (FasL), and it plays an important role in the regulation of general tissue homeostasis. The Fas/FasL system has closely relation with the development of malignant melanoma. In the normal condition, the Fas/FasL system mediates apoptosis and immune monitoring. However, biology effective of the Fas/FasL system is not very clearly in tumor. At present, it is seldom about the expression and the role of Fas/FasL in malignant melanoma. In the Fas/FasL system, there are another two very important members, soluble Fas (sFas) and soluble FasL (sFasL). Soluble Fas is the protein product of Fas mRNA variation splicing, and sFasL is its ligand. During the apoptosis mediation, sFas is an inhibitor to inhibit the complex formation of Fas/FasL. A further complicating aspect in the immune escape of FasL-expressing tumors is the role of soluble FasL. Therefore, many people believed that there were possible relation between soluble Fas and worse prognosis in metastatic MM. But the studies about sFas are different and there exists argue in malignant tumor.The occurrence and development of malignant melanoma are not only closely related with cell infinite multiplication, but also with apoptosis. At present, the expression and the role of Fas/FasL are not completely comprehended in malignant melanoma. The Fas/FasL system induces apoptosis as a pair of membrane proteins. The disable and abnormal of Fas/FasL expressions cause the signals of Fas/FasL destruction and incompetence. Therefore, abnormal cells escape immunologic surveillance, and they are survival and developed. In order to recognize the mechanism of MM development, we research the relation between Fas/FasL and MM to provide the theory basis and prognostic evaluation of malignant melanoma.The tumor occurrence is a multiple factor and the multi-stage process, its mechanism is complex. In recent years, many studies have proven the balance between the cell apoptosis and cellular proliferation are broken, which caused the tumor occurrence. Along with malignant melanoma occurrence and the malignant degree, the apoptosis mediated by Fas system is suppressed, while cell proliferation is very active. Poliferating cell nuclear antigen (PCNA) is an index to evaluate cell proliferation in malignant tumor. There is important significance to examine the PCNA of malignant tumor since it judge the tumor malignant degree, the clinical instruction treats and improves the prognosis. And apoptosis precisely respond by TdT-mediated dUTP nick end labeling method (TUNEL).The objective of this research is to study the expression of Fas, Fas-ligand (Fas/FasL), sFas and proliferating cell nuclear antigen (PCNA) in malignant melanoma, melanocytic nevus and normal skin, and apoptosis in malignant melanoma, illuminate its relationship. We provide the clue of MM therapy and prognosis and deeply understand the mechanisms of genesis and development of MM. We observe these indexes by HE dying, immunohistochemistry, immunofluorescence, TUNEL and the ABC-ELISA.1. The difference expression of Fas and FasL by immunohistochemistry in different group tissues: the positive staining of Fas was mainly located in the cytoplasma, while cell membrane was minority staining. The positive staining of FasL was mainly located in the cytoplasma and cell membrane. The expression rates of Fas were respectively 31.25%(5/16),57.89%(11/19),76.19%(16/21),92.85%(26/28), while the expression rates of FasL were respectively 75.0% (12/16),52.63%(10/19),19.04%(4/21),3.57%(1/28) in MM with lymph node metastasis, MM without lymph node metastasis, melanocytic nevus and normal skin groups. The expressions of Fas were obviously difference in different groups (χ~2=525.53,v=3,P<0.01), and the expressions of FasL were obviously difference in different groups(χ~2=1275.77,v=3,P<0.01).2. The difference fluorescence intensity of Fas and FasL in different group tissues by immunofluorescence: The fluorescence intensity of Fas were respectively 59.80±91.76,178.20±163.54,410.52±239.24,581.25±164.58, and fluorescence intensity of FasL were respectively 581.72±353.03,295.82±289.62,88.09±186.28,5.62±29.73 in MM with lymph node metastasis, MM without lymph node metastasis, melanocytic nevus and normal skin groups. The fluorescence intensity of Fas were obviously difference in different groups (P<0.01), and the fluorescence intensity of FasL were obviously difference in different groups (P<0.01).3. The difference apoptosis indexes by TUNEL in different group tissues: apoptosis cells were suffusion distributed in melanocytic nevus group, while scatter in MM with lymph node metastasis group. Apoptotic indexes of melanocytic nevus were significantly higher than other groups (P<0.05), and those of MM with lymph node metastasis group were lower than those of MM without lymph node metastasis group (P<0.05).4. The difference expression of PCNA by immunohistochemistry in different group tissues: PCNA indexes were respectively 3.99±1.96%,6.21±3.03%,54.96±14.82% in MM, melanocytic nevus and normal groups. There were significantly statistics difference (P<0.01). And PI of MM was higher than that of normal group and melanocytic nevus group (P<0.01), while there was no statistics difference between posterior two groups(P>0.05).5. The difference expression of sFas by ABC-ELISA in different group tissues: The concentration of sFas were respectively 4.18±1.88,7.33±3.78,36.32±11.57 ng/ml in normal group,melanocytic nevus group and MM. There were significantly statistics difference in there groups(F=37.49,v=2,18,P<0.0001), while there was no statistics difference between normal group and melanocytic nevus group (P>0.05).In this research we proved through the experiment these conclusions as follows. 1. There are negative correlation between the Fas expression and the MM malignant degree, that is to say, the malignant degree of MM is higher following the decreased of fas expression. 2. There are positive correlation between the FasL expression and the MM malignant degree. The malignant degree of MM is higher following the increased of fas expression. 3. The expressions of Fas/FasL have relationship with apotosis of tumor cell. The expression of Fas and FasL are balance in benign melanin, so these induce to active apotosis and the increase of AI. However, following the increase of malignant degree, they are unbalance in malignant melanoma so it is not benefits to active apotosis and AI is deduce. 4. The expressions of PCNA and sFas reflect the malignant degree of MM, and the more expression of PCNA, the more malignant degree of melanoma. PCNA might become an candidate index to evaluate prognosis of MM. 5. The densities of sFas in patient serum have relation with malignant melanoma malignant, and it have diagnostic value for MM.