Expression Of Survivin And PEDF In Melanoma And The Role Of Them In The Apoptosis Of Melanoma A375 Cells Induced By EGCG

Part I Expression of Survivin and PEDF in Cutaneous MalignantMelanoma and the Relationship between them.Objective To investigate the expression of survivin and PEDF (pigment epithelium derived factor ) and their clinical significance in cutaneous malignant melanoma(CMM).Methods The expression of survivin and PEDF was measured by streptavidin-peroxidase complex immunohistochemical technique in 48 cases of CMM and 23 cases of nevus.Result The positive expression percentage of survivin in CMM was significantly higer than that of nevus, while the positive expression percentage of PEDF in that was similar. The expression of survivin and PEDF in CMM was positively related to pathological histology, Clark's grade, the metastasis to lymph nodes and clinical outcome. The expression of PEDF was inversely correlated with the high expression of survivin in CMM.Conclusions Survivin might play important roles in the carcinogenesis process and progression of CMM and have important implications in the diagnosis and prognostic assessment. PEDF might play negative modulation roles in the progression of CMM. Part II Effect of EGCG on the Biological Behavior of Melanoma A375 cellsObjective TO study the effects of EGCG on the biological behavior of Melanoma A375 cells.Method The melanoma cell line A375 was chosen in this experiment. The inhibitoryeffects of chemotherapeutical drugs on A375 cell line were assayed with MTT test.The EGCG group was divided into different groups by concentration and time.Changes of apoptosis in morphology and chemistry were detected by Phasemicroscope, Electron microscopy and Hochest nuclear staining respectively.Apoptosis was quantitatively determined by TUNEL and detected by Annexin Vstaining. Changes of apoptosis in chemistry were detected by DNA electrophoresis aswell.Results EGCG obviously inhibited the A375 cell growth with dose-andtime-dependent. By Phase microscope, Electron microscopy, Hochest nuclear staining,DNA electrophoresis, Annexin V and TUNEL staining, we all detected the occurrenceof apoptosis. Annexin V and TUNEL staining was in a dose-and time-dependentmanner.Conclusion EGCG can effectively induce apoptosis in A375 cells. Part III The role of Survivin and PEDF in the apoptosis of Melanoma A375 cells induced by EGCGObjective To study the role of Survivin and PEDF in the process of apoptosis, so as to provide the basis for validating the mechanisms of EGCG induce apoptosis. Methods The EGCG group was divided into different groups by concentration and time. The survivin and PEDF protein expression was detected with streptavidin-peroxidase complex immunohistochemical technique and westen blot. Expression of Survivin and PEDF mRNA was detected by RT-PCR. The caspase 3 activities was detected by caspase kit. Results The Survivin protein expression was decreased with the increasing of EGCGconcentration while the expression of PEDF protein was increased by immunohistochemical detection. Western blot showed that the increased expression of Survivin protein and the decreased expression of PEDF in melanoma A375 cells induced by EGCG which is time-dependent. Survivin mRNA was down-regulated and PEDF mRNA was up-regulated with the increase of dose of EGCG The ratio of Survivin and β-actin was negatively associated with the inhibitory rate of cell proliferation and the rate of apoptosis (p<0.01). The ratio of PEDF and β-actin was positively associated with the inhibitory rate of cell proliferation and the rate of apoptosis (p<0.01). The change of caspase-3 activity was in a dose-and time-dependent manner. The increase of of caspase-3 activity positively associated with the decreased expression of Survivin and the decreased expression of PEDF. Conclusion Survivin and PEDF gene expression may play a critical role in the apoptosis of melanoma A375 cells induced by EGCG which throught modulation the caspase-3 activity.