Evaluation of anti-cancer efficiency of resveratrol analogue on prostate cancer cells in vitro

Prostate cancer (PCa) is the second most common cancer type in men in developing countries. PCa has a clinically heterogeneous course, which initiates slowly and progresses rapidly, and may be fatal. Carcinoma progression is associated with mesenchymal fenotype achievement and epithelial feature loss, which has been known as epithelial-mesenchymal transition (EMT). Therefore, metastatic characteristics of cancer cells increase by cell migration and invasion ability achievement via EMT. Resveratrol is a potential chemo-preventive and chemotherapeutic agent that is found in red grape. Resveratrol has anti-tumorigenic effect in various cancer types, and it has been revealed that it may lead to EMT inhibition. DMU-212, one of the synthesized resveratrol analogue, has high selectivity and activity against various cancer types, and it has stronger anti-tumor activity than resveratrol. In recent years, the relevance on studies including inhibitors obtained from plants has been increasing on behalf of therapeutic strategy development against cancer, because of high death incidence in prostate cancer, despite of developments in early diagnosis and treatment. Thus, in this thesis, it was aimed to determine the mechanisms that may prevent invasion and metastasis in PCa cells by considering EMT as a therapeutic target and investigating the effects of DMU-212, a new stilbene derivative, on EMT, which has not been studied in PCa cells so far. Cytotoxic effects of DMU-212 active ingredient was determined by WST-8 test and colony formation experiments in LNCaP, PC-3 and DU-145 cells. Apoptotic effects of identified IC50 doses of DMU-212 were evaluated by Annexine V method, whereas its effect on cell cycle was assessed by propidium iodide (PI) analysis. The effects of IC50 doses on cell migration was studied by wound healing, on tumor invasion was analyzed by cell invasion analyses, and its effects on EMT was investigated by qRT-PCR in order to observe the changes in EMT-related gene expression levels. The alterations of E-cadherin and vimentin, which are EMT indicators, were determined by immunofluorescence labeling method. Expression profiles of cyclin B1, cyclin D1, E-cadherin, and β-catenin proteins were evaluated by western blot analysis. The changes in transcription factor NFkB activity was investigated by dual luciferase activity. It was determined that low level of DMU-212 has high cytotoxic effect on LNCaP, PC-3, and DU-145 cell lines, and it led to G2/M arrest by inducing apoptosis in those cell lines. It was also found that DMU-212 inhibited migration and invasion in prostate cancer cells. This mechanism may be the result of increased expression of TFP1-2 gene. We also found that this effect may occur by inhibition of transcriptional β-catenin activity via attenuation of Wnt-β catenin signal pathway in PC-3 cells. In DU-145 cells, gene expression results showed that DMU-212 inhibited EMT-related TGF-β signaling. It was found that DMU-212 increased E-cadherin and decreased vimentin in prostate cancer cells. Furthermore, it was determined that NFkB transcription factor had high activity on LNCaP and PC-3 cells. In conclusion, DMU-212 may be a promising agent in order to provide new opportunities devoted for metastatic prostate cancer prevention with its strong anti-cancer activity.