Epigallocatechin gallate (EGCG) is the predominant tea polyphenol and it exhibits a hydrophilic character. The lipophilized EGCG derivative (LEGCG) was synthesized by enzymatic esterification of EGCG with lauric acid to enhance its bioactivity. The tetralauroyl EGCG was confirmed by high-performance liquid chromatography-tandem mass spectrometry and further identified as 3 , 5 , 3 , 5 -4-O-lauroyl EGCG by 1 H and 13 C nuclear magnetic resonance. The anti-proliferation effect of LEGCG on DU145 human prostate carcinoma cells was evaluated by MTT assay. In addition, the underlying molecular mechanism by which LEGCG exerts anti-proliferation efficacy was elucidated by flow cytometry and immunoblot analysis. Results suggested that LEGCG exhibited a dose-dependent anti-proliferation effect on DU145 cells by G0/G1 phase arrest and induction of apoptosis. LEGCG induced cell cycle arrest via p53/p21 activation, which down-regulated the cyclin D1 and CDK4 expression. In addition, LEGCG induced apoptosis by increasing the Bax/Bcl-2 ratio, the cytochrome c release, and the caspases cleavage on DU145 cells. The results provide theoretical support to prevent prostate cancer with LEGCG.Nutrients 2020, 12, 92 2 of 10 cancer affects [16]. Therefore, we hypothesized that LEGCG has the potential to prevent prostate cancer. In the present study, enzymatic esterification of EGCG was implemented. The high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and 1 H and 13 C nuclear magnetic resonance (NMR) were used to determine the chemical structures of LEGCG.The inhibitory effect and the underlying mechanism of LEGCG on DU145 cells were investigated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, flow cytometry, and immunoblot analysis.
Materials and Methods
MaterialsEGCG, MTT, and propidium iodide (PI) were purchased from Sigma-Aldrich Co. (St. Louis, MO., USA). Annexin V-FITC/PI kit was from Biolegend (San Diego, CA, USA). Cyclin D1, CDK 4, p21, p27, p53, Bax, Bcl-2, cytochrome c, cleaved caspase-9, cleaved caspase-3, and GAPDH antibodies were from Cell Signaling Technology (Danvers, MA, USA).
Preparation of Crude EGCG DerivativeLEGCG was prepared according to the literature [17]. Briefly, EGCG and lauric acid were mixed at a mole ratio of 1:1 in ethanol, and then 5% of Lipozyme TLIM (5% w/w) was added. The mixture was heated at 45 • C for 12 h in a screw-capped glass bottle. The reaction was terminated by the removal of the enzyme through filter paper, and the product was concentrated to get crude LEGCG.