Determination of the effects of TGF-β1 on adamts8 gene expression in a colon cancer model

ADAMTS-8 is a member of the family of intercellular matrix proteases called ADAMTS (A Disintegrin-like And Metalloproteinase with Thrombospondin Motifs). ADAMTS-1 and ADAMTS-8 are potent angioinhibitors that inhibit new vessel formation induced by vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). While TGF-β cytokine has tumor suppressor functions in the early phase of tumorigenesis, it is known to lead to tumor progression and metastasis in the late phase of the tumor. No study has been found that demonstrates the function of ADAMTS-8 in colorectal cancer with TGF-β stimulation. This thesis study aims to determine the contribution of TGF-β to the transcriptional regulation of ADAMTS-8 in a colorectal cancer model (SW480). As a result of this thesis, it was determined by the MTT test that TGF-β had a proliferative effect at a dose of 20 ng/mL on SW480 cells. Then, it was determined by Real Time PCR and Western Blot studies that TGF-β cytokine caused an increase in the expression of ADAMTS-8 at mRNA and protein levels at different time intervals. TGF-β-mediated transcriptional activity of the ADAMTS-8 promoter was investigated by transient- transfection of five different lengths of promoter fragments into SW480 cells. As a result of transfection, it was determined that the most active promoter parts were pMet_TS-8[-223/+323], pMet_TS-8[-662/+323] and pMet_TS-8[-851/+323]. As a result of pathway inhibition studies, it was determined that the regulation of ADAMTS-8 induced by TGF-β was via p38 MAPK, MEK, JNK, PI3K and SMAD-3 intracellular signaling pathways at the mRNA and protein level. It was determined by transfection study that the increase in transcriptional activity was regulated via p38 MAPK, MEK, JNK, PI3K and SMAD-3 pathways. With administration of TGF-β in SW480 cells, the functional binding of the SMAD2/3 transcription factor to certain regions in the ADAMTS-8 promoter was confirmed by Chromatin Immunoprecipitation Assay (ChIP) analysis.