Nachweis und Charakterisierung von RANK-Ligand und Osteoprotegerin in humanen Pankreaskarzinomzellen

Summary: The modulation of cellular processes of apoptosis is essential for the normal physiology of cells and of fundamental importance for carcinogenesis. The disturbance of cellular control mechanisms of apoptosis is considered as the critical event in the initiation and the progression of tumours. Reduced apoptosis is further recognised as an important mechanism for the resistance of tumour cells to radio- and chemotherapy. The ductal adenocarcinoma of the pancreas belongs to those tumours which are nearly always resistant to a conventional chemotherapy. Based on actual results of the pathogenesis of pancreatic cancer and the previous findings referring the RANKL/OPG system, it was the aim of this thesis to examine the expression and modulation of OPG mRNA production and OPG protein secretion in human pancreatic cancer cell lines. The following adenocarcinoma cell lines PaTu 8988t, PaTu 8988s, PC-2 und Capan-2 were analysed and the effect of steroid hormones, cytokines and different drugs on the OPG expression was evaluated. The gene expression was determined using northern blot analysis and PCR, the protein secretion by ELISA. Osteoprotegerin (OPG) und Receptor activator of nuclear factor-κB ligand (RANKL) represent specific receptors and ligands of the TNF superfamily, the production of which could be proven up to now in different human tissues. Following the proof of OPG mRNA in pancreatic tissue with the help of a commercial northern blot, OPG could be detected in two of the four examined pancreatic carcinoma cell lines (PaTu 8988t und PC-2). The cell lines PaTu 8988s and Capan-2 on the other hand showed no production of OPG. In addition PaTu 8988t cells expressed RANKL mRNA. In the case of testosterone there was an inhibition dependent on dose and duration of OPG mRNA und protein expression in the pancreatic cancer cell line PaTu 8988t by 70-77%, whereas 5α-DHT and 17β-estradiol showed no effects. The adrenal androgen DHEA impeded the OPG production by 83%. In the case of PC-2 cells on the other hand the OPG expression was lowered by the gonadal androgen 5α-DHT. After the exposition of the cell line PaTu 8988t to the glucocorticoid dexamethasone there was a dose dependent inhibition of OPG mRNA levels and OPG protein production by 23%. Because of the important role of cytokines in the field of tumour immunology and the modulation of apoptosis in pancreatic cancer different cytokines (IL-1β, TNF-α and IL-8) were examined referring their regulation of OPG production. In contrast to many other cell types, these cytokines however showed no influence on OPG expression – except IL-8 –. The treatment with IL-8 resulted in a slight increase in the production of OPG mRNA production in PaTu 8988t cells. The HMG-CoA reductase inhibitors lovastatin and cerivastatin showed an inhibiting effect on the OPG mRNA and OPG protein expression in human pancreatic adenocarcinoma cell lines. Lovastatin as a statin of the first generation reduced the OPG mRNA and protein secretion in PaTu 8988t cells by 90%. In addition OPG mRNA and protein levels were reduced by Cerivastatin in PaTu 8988t cells by 27% and in PC-2 cells by 39%. The reduction through statins war dependent on dose and duration. The inhibition of the OPG production in pancreatic carcinoma cell lines was a further proof of the pleiotropic effects of statins, which should be clinically examined concerning their apoptosis modulation effects on pancreatic carcinoma cells. In conclusion these results in connective view with the results of other groups indicate that the pancreas represents both a site of production and a potential target of OPG, an important osteotropic and apoptosis-modulating cytokine of the TNF receptor superfamily. Future studies should not only investigate the precise interaction of OPG and TRAIL, but also their importance for the creation, progression and metastasis of the pancreatic carcinoma. These findings could be the basis of future clinical studies in which the pinpointed modulation of apoptosis could be implemented as therapeutical principle with patients with pancreatic cancer.