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Abstract
Epigallocatechin gallate (EGCG), or epigallocatechin 3-gallate, is the ester of epigallocatechin and gallic acid, and is a type of catechin. EGCG may be therapeutic for many disorders including diabetics and some types of cancer. However it is unknown whether EGCG can induce transdifferentiation of pancreatic cells in pancreatitis. The aim of this study was to investigate the effects of EGCG on the expression of pancreatic regenerating related markers in pancreatic AR42J cells, a model of pancreatic progenitor cells. AR42J cells, differentiated with betacellulin and activin A, were cultured with/without EGCG in a time-dependent manner. Cell growth rate, levels of mRNA, and protein expression were examined with the MTT assay, quantitative PCR, and Western blots, respectively. The results showed that AR42J cell growth rates were inhibited by EGCG in a dose-dependent manner. mRNA and protein expression of amylase, insulin and neurogenin 3 (ngn 3) increased in AR42J cells treated with EGCG. Additionally, we demonstrated that the signal transduction pathway of mitogen-activated protein (MAP) kinase is active in EGCG-treated AR42J cells. ERK and JNK phosphorylation decreased in cells treated with EGCG but not p38 phosphorylation. Activation of the p38 MAP kinase pathway was confirmed by specific MAP kinase pathways inhibitors: U0126 for ERK, SP600126 for JNK, and SB203580 for p38. Activated p38 phosphorylation was inhibited by the specific p38 inhibitor SB203580 but p38 phosphorylation was inhibited with increased EGCG treatment. The ERK and JNK MAP kinase pathways were not affected by EGCG treatment. Although further studies are needed, these results suggest that EGCG affects the induction of pancreatic cell regeneration by increasing the ngn 3 protein and mRNA expression and activating the p38 MAP kinase pathway.
Figures and Tables
Fig. 1
The inhibition of rat pancreatic tumor cell line, AR42J cells after treatment with EGCG. Cells were plated at 1 × 105 cells per 60 mm culture plate, and incubated for 24 hr. The cells were treated with various concentrations of EGCG for 48 hr. The growth inhibition was measured by the metabolic-dye-based MTT assay. Results are expressed as the means ± S.E. of three independent experiments.
Fig. 2
Effects of EGCG treatment on the levels on neurogenin 3, α-amylase and insulin mRNA and protein in AR42J cells. A, C, E: after 24, 48 hr incubation with EGCG total RNAs were isolated and revers-transcribed. The resulting cDNAs were subjected to PCR with the incubated primers and reaction products were subjected to electophoresis in a 1% agarose gel and visualized by EtBr staining. GAPDH was used as internal control. B, D, F: The cells were lysed and then cellular proteins were separated by SDS-ployacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with proper antibodies. Proteins were visualized using an ECL detection system. β-actin was used as internal control.
Fig. 3
Expression levels of MAP Kinase signaling pathway, pERK, pp38 and pJNK in AR42J cells treated with EGCG for 24, 48 hrs. A, B, C: after 24, 48 hr incubation with/without EGCG, total proteins were lysed and then cellular proteins were separated by SDS-ployacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with proper antibodies. Proteins were visualized using an ECL detection system. β-actin was used as internal control. Band intensities in the immunoblots were quantified by densitometry using L Process (Version 2.01, Fujifilm, Stanford) and Multi Gauge software (Version 2.02, Fujifilm). Band intensities were normalized relative to the internal control and background. Multiple experiments were combined and data was expressed as % of the control (set as 100%). Statistical Analysis was performed using Student's T-test; significance was set at p < 0.05. D: after cells were incubated with/without EGCG for 48 hrs, U0126, SB203580, SP600126 inhibiters were treated for 15 mins. Effects of MAP Kinase pathway were monitored by use of proper antibodies. Proteins were visualized using an ECL detection system. β-actin was used as internal control.
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