INTRODUCTION: Osteopontin (OPN) mediates metastasis and invasion of hepatocellular carcinoma (HCC). Epigallocatechin-3-gallate (EGCG), found in green tea, suppresses HCC tumor growth in vitro. We sought to investigate the role of EGCG in modulating OPN in cell lines of metastatic HCC. METHODS: Experimental HCC cell lines included HepG2 and MHCC-97H HCC cells, which express high levels of OPN, and the Hep3B cells, which express lesser levels of OPN. Cells were treated with EGCG (0.02-20 μg/mL) before measurement of OPN with enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. Scratch assay measured cell migration. Binding of the OPN promoter to RNA pol II was evaluated by the use of Chromatin-IP assay after EGCG treatment. Transcriptional regulation of OPN was investigated with luciferase reporter plasmids containing various deletion fragments of the human OPN promoter. Measurement of the half-life of OPN mRNA was conducted using actinomycin D. RESULTS: Treatment of MHCC-97H and HepG2 cells with 2 μg/mL and 20 μg/mL EGCG caused a ∼6-fold and ∼90-fold decrease in secreted protein levels of OPN (All P < .001). OPN mRNA was decreased with EGCG concentrations of 0.2-20 μg/ml (All P < .001). The 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (ie, MTT) assay revealed that differences in OPN expression were not due to viability of the HCC cell lines. Promoter assay and chromatin immunoprecipitation analysis revealed no effect of EGCG on the transcriptional regulation of OPN. Posttranscriptionally, EGCG decreased the half-life of OPN mRNA from 16.8 hours (95% confidence interval 9.0-125.1) to 2.5 hours (95% confidence interval 2.1-3.2) (P < .001). Migration was decreased in EGCG treated cells at 24 hours (8.0 ± 2.4% vs 21.2 ± 10.8%, P < .01) and at 48 hours (13.2 ± 3.6% vs 53.5 ± 19.8%, P < .001). CONCLUSION: We provide evidence that EGCG decreases OPN mRNA and secreted OPN protein levels by decreasing the half-life of OPN mRNA in MHCC-97H cells. The translatability of EGCG for patients with HCC is promising, because EGCG is an inexpensive, easily accessible chemical with an extensive history of safety.
INTRODUCTION:Osteopontin (OPN) mediates metastasis and invasion of hepatocellular carcinoma (HCC). Epigallocatechin-3-gallate (EGCG), found in green tea, suppresses HCC tumor growth in vitro. We sought to investigate the role of EGCG in modulating OPN in cell lines of metastatic HCC. METHODS: Experimental HCC cell lines included HepG2 and MHCC-97H HCC cells, which express high levels of OPN, and the Hep3B cells, which express lesser levels of OPN. Cells were treated with EGCG (0.02-20 μg/mL) before measurement of OPN with enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. Scratch assay measured cell migration. Binding of the OPN promoter to RNA pol II was evaluated by the use of Chromatin-IP assay after EGCG treatment. Transcriptional regulation of OPN was investigated with luciferase reporter plasmids containing various deletion fragments of the humanOPN promoter. Measurement of the half-life of OPN mRNA was conducted using actinomycin D. RESULTS: Treatment of MHCC-97H and HepG2 cells with 2 μg/mL and 20 μg/mL EGCG caused a ∼6-fold and ∼90-fold decrease in secreted protein levels of OPN (All P < .001). OPN mRNA was decreased with EGCG concentrations of 0.2-20 μg/ml (All P < .001). The 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (ie, MTT) assay revealed that differences in OPN expression were not due to viability of the HCC cell lines. Promoter assay and chromatin immunoprecipitation analysis revealed no effect of EGCG on the transcriptional regulation of OPN. Posttranscriptionally, EGCG decreased the half-life of OPN mRNA from 16.8 hours (95% confidence interval 9.0-125.1) to 2.5 hours (95% confidence interval 2.1-3.2) (P < .001). Migration was decreased in EGCG treated cells at 24 hours (8.0 ± 2.4% vs 21.2 ± 10.8%, P < .01) and at 48 hours (13.2 ± 3.6% vs 53.5 ± 19.8%, P < .001). CONCLUSION: We provide evidence that EGCG decreases OPN mRNA and secreted OPN protein levels by decreasing the half-life of OPN mRNA in MHCC-97H cells. The translatability of EGCG for patients with HCC is promising, because EGCG is an inexpensive, easily accessible chemical with an extensive history of safety.
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