ABSTRACT
BACKGROUND/AIMS: Deoxycholic acid (DCA), a secondary bile acid, has been implicated to promote colon cancer growth and progression. However, its molecular mechanisms are largely unknown. In this study, we investigated the effects of DCA on proliferation, migration, and invasiveness of colon cancer cells (HT-29). METHODS: HT-29 cells were incubated with either medium (control) only or DCA for 24-48 hours. Time courses of RT-PCR for vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1alpha mRNA expression, Western blotting for VEGF and matrix metalloproteinase (MMP)-9, zymography for MMP-9 activation, and wound-migration assay were determined after various concentrations of DCA (0-80mum) treatment. Moreover, these experiments were reassessed after pretreatments (2-6 hours) with specific inhibitors of various signal pathways. RESULTS: DCA enhanced HIF-1alpha mRNA expression, VEGF mRNA and VEGF protein expression, MMP-9 protein expression/activation, and cell migration ability in a dose-related manner. DCA-induced VEGF protein expression was inhibited by pretreatment with NS-398 (COX-2 inhibitor), PDTC (NF-kappaB inhibitor), or tauroursodeoxycholic acid (TUDC). DCA-induced cell migration ability was inhibited by pretreatment of GF109203X, a protein kinase C inhibitor. DCA-induced MMP-9 protein expression/activation was inhibited by pretreatment with SB203580, U0126, or PDTC. CONCLUSIONS: DCA significantly upregulates invasive and angiogenic potentials of human colon cancer cells through multiple signal transduction pathways.
Subject(s)
Humans , Cell Movement/drug effects , Colonic Neoplasms/metabolism , Deoxycholic Acid/pharmacology , HT29 Cells , Hypoxia-Inducible Factor 1/metabolism , Matrix Metalloproteinase 9/metabolism , Neoplasm Invasiveness , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Vascular Endothelial Growth Factor A/metabolismABSTRACT
BACKGROUND: The incidence of atherosclerosis is well correlated with the progression of type 2 diabetes mellitus. High plasma glucose in uncontrolled diabetic patients evokes many vascular complications such as atherosclerosis. Specifically, high glucose was reported to induce thrombospondin-1 (TSP-1), which activates matrix metalloproteinase-2 (MMP-2) and leads to the invasion of vascular smooth muscle cells (VSMCs) into the intima. Catechins with antioxidant effects are known to inhibit MMP-2 activity. Therefore, this study was aimed at revealing the effect of epicatechin, one of catechins, on high glucose-induced TSP-1 and the invasiveness of VSMCs. METHODS: VSMCs were primarily isolated from Sprague-Dawley rat aorta. The VSMCs were incubated with different doses (30, 100 and 300 micrometer) of epicatechin under high glucose concentration (30 mM). The TSP-1 protein and mRNA expressions were analyzed by performing Western blotting and Northern blot analyses, respectively. RT-PCR was performed to observe the MMP-2 mRNA expression. Gelatin zymography was performed for the measurement of MMP-2 activity. Invasion assays were performed to evaluate the invasiveness of VSMCs. RESULTS: Epicatechin inhibited the high glucose-induced TSP-1 expression and the MMP-2 activity in a dose-dependent manner. Also, epicatechin inhibited the high glucose-induced invasiveness of VSMCs across the matrix barrier in a dose-dependent fashion. CONCLUSION: Collectively, epicatechin may prevent the high glucose-induced proliferation and invasion of VSMCs by inhibiting the TSP-1 expression and the MMP-2 activity. Therefore, epicatechin appears to play a protective role in the development of atherosclerosis.
Subject(s)
Animals , Humans , Rats , Antioxidants , Aorta , Atherosclerosis , Blood Glucose , Blotting, Northern , Blotting, Western , Catechin , Diabetes Mellitus, Type 2 , Gelatin , Glucose , Incidence , Matrix Metalloproteinase 2 , Muscle, Smooth, Vascular , Rats, Sprague-Dawley , RNA, Messenger , Thrombospondin 1ABSTRACT
BACKGROUND: The incidence of atherosclerosis is well correlated with the progression of type 2 diabetes mellitus. High plasma glucose in uncontrolled diabetic patients evokes many vascular complications such as atherosclerosis. Specifically, high glucose was reported to induce thrombospondin-1 (TSP-1), which activates matrix metalloproteinase-2 (MMP-2) and leads to the invasion of vascular smooth muscle cells (VSMCs) into the intima. Catechins with antioxidant effects are known to inhibit MMP-2 activity. Therefore, this study was aimed at revealing the effect of epicatechin, one of catechins, on high glucose-induced TSP-1 and the invasiveness of VSMCs. METHODS: VSMCs were primarily isolated from Sprague-Dawley rat aorta. The VSMCs were incubated with different doses (30, 100 and 300 micrometer) of epicatechin under high glucose concentration (30 mM). The TSP-1 protein and mRNA expressions were analyzed by performing Western blotting and Northern blot analyses, respectively. RT-PCR was performed to observe the MMP-2 mRNA expression. Gelatin zymography was performed for the measurement of MMP-2 activity. Invasion assays were performed to evaluate the invasiveness of VSMCs. RESULTS: Epicatechin inhibited the high glucose-induced TSP-1 expression and the MMP-2 activity in a dose-dependent manner. Also, epicatechin inhibited the high glucose-induced invasiveness of VSMCs across the matrix barrier in a dose-dependent fashion. CONCLUSION: Collectively, epicatechin may prevent the high glucose-induced proliferation and invasion of VSMCs by inhibiting the TSP-1 expression and the MMP-2 activity. Therefore, epicatechin appears to play a protective role in the development of atherosclerosis.
Subject(s)
Animals , Humans , Rats , Antioxidants , Aorta , Atherosclerosis , Blood Glucose , Blotting, Northern , Blotting, Western , Catechin , Diabetes Mellitus, Type 2 , Gelatin , Glucose , Incidence , Matrix Metalloproteinase 2 , Muscle, Smooth, Vascular , Rats, Sprague-Dawley , RNA, Messenger , Thrombospondin 1ABSTRACT
CD63, which belongs to the tetraspanin membrane proteins, has been proposed to play an important role in inhibiting melanoma metastasis. To determine whether reduction of CD63 expression, which frequently occurs in the malignant progression of human melanoma, is responsible for metastasis promotion, we transfected the antisense CD63 cDNA into MelJuso melanoma cells having endogenous CD63 expression. The antisense CD63 transfectant clones showing decreased CD63 expression displayed increased cell motility, matrix-degrading activity, and invasiveness in vitro when compared with the control transfectant cells. The antisense CD63 cDNA-transfected cells also exhibited altered adhesiveness to extracellular matrix. The results suggest that reduced CD63 expression contributes to the invasive and metastatic ability of human melanoma cells.