Induction of Apoptosis by Acetylated Black Tea Polyphenol through

Chapter 29

Downloaded by IOWA STATE UNIV on October 19, 2014 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch029

Induction of Apoptosis by Acetylated Black Tea Polyphenol through Reactive Oxygen Species Production, Cytochrome c Release, and Caspases Activation in Human Leukemia HL-60 Cells 1

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Min-Hsiung Pan , Hui-Jun Kang , Wei-Jen Chen , Chih-Yu Lo , Shiming L i , Shengmin Sang , and Chi-Tang Ho 3

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Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung, Taiwan Department of Biomedical Sciences, Chung Shan Medical University, No, 110, Section 1, Chien-Kuo N. Road, Taichung 402, Taiwan Department of Food Science, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901 Department of Chemistry Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854 2

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The growth inhibitory effects of black tea polyphenol, theaflavin (TF-1) and its peracetylated derivative (ATF-1) in human leukemia cancer cells were examined. TF-1 and ATF-1 displayed strong growth inhibitory effects against human leukemia HL-60 cells. ATF-1 was more potent against the growth of HL-60 cells and induction of apoptosis through modulation of mitochondrial functions regulated by reactive oxygen species (ROS). ROS generation occurs in the early stages of ATF-1-induced apoptosis, preceding cytochrome c release, caspase activation, and DNA fragmentation. The molecular mechanism of ATF-1-induced apoptosis was also investigated.

© 2008 American Chemical Society

In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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346 Tea (Camellia sinensis) is the most popular beverage, after water, consumed worldwide; the major tea beverage is black tea, especially in the western nations. Black tea leaves are produced through extensive enzymatic oxidation of polyphenols to polymerized products, such as theaflavines and thearubigins. The major theaflavins in black tea are theaflavin, theaflavin-3-gallate, theaflavin-3'gallate, and theaflavin-3,3'-digallate (7). Green tea polyphenols such as (-) epigallocatechin-3-gallate (EGCG) have been demonstrated to have several inhibitory properties on the growth of tumor cell lines (2). The molecular mechanism of antitumor growth might operate through blocking the signal transduction pathway (3). However, other tea constituents such as theaflavins from black tea also have anti-proliferative or anti-carcinogenic activities (4). Recently, Lu et al. (5) reported that black tea significantly inhibited proliferation and enhances apoptosis in mouse skin tumor models. Haider and Bhaduri (6) reported that theaflavins and thearubigins have antioxidative properties on human red blood cells. Among black tea components, theaflavins are generally considered to be the more effective components for the inhibition of carcinogenesis, but it is unclear which of these theaflavins is the most effective one. Chemoprevention, a promising strategy to prevent cancer is the use of either natural or synthetic substances or their combination to block, reverse or retard the process of carcinogenesis (7). Apoptosis plays a fundamental role in the maintenance of tissues and organ systems by providing a controlled cell deletion to balanced cell proliferation. A common feature of cancer cells is their ability to evade apoptosis as a result of alterations that block cell death signaling pathways (8). It is now apparent that many dietary chemopreventive agents with promise for human consumption can also preferentially inhibit the growth of tumor cells by targeting one or more signaling intermediates leading to induction of apoptosis (9). In the current study, was first examined the antiproliferative effects of TF-1 and ATF-1 on human leukemia cells. The results clearly demonstrate that ATF-1 can induce apoptosis in a dose-dependent manner in HL-60 cells. The molecular mechanisms of the apoptotic effects induced by ATF-1 were further evaluated. It is suggested that ATF-1 modulates the production of ROS, Bcl-2 family proteins, the release of cytochrome c, and the activation of caspases in ATF-1 induced apoptosis. The results of the present study will provide molecular basis for designing compounds and understanding the healthy effect of black tea.

Materials and Methods Cell Culture Human promyelocyte leukemia (HL-60) cells obtained from American Type Culture Collection (Rockville, MD) were grown in 90% RPMI 1640 and


347 10% fetal bovine serum (GIBCO BRL, Grand Island, NY), supplemented with 2 mM glutamine (GIBCO BRL), 1% penicillin/streptomycin (10000 units of penicillin/mL and 10 mg streptomycin/mL). Medium was normally changed to phenol red-free RPMI 1640 before polyphenol treatment. Propidium iodide was obtained from Sigma Chemical Co. (St. Louis, MO).


Materials Anhydrous pyridine, acetic anhydride, 4-(dimethylamino)-pyridine, (4DMAP), 99+% Sephadex LH-20 gel, and HPLC sorbent (C-18) were purchased from Sigma-Aldrich Chemical Co (St. Louis, MO). Ethyl acetate (EtOAc), methanol and water (HPLC grade) were purchased from Fisher Scientific (Springfield, NJ). Black tea extract was obtained from WellGen, Inc (New Brunswick, NJ). Ethyl acetatefractionof theaflavin extract was subjected to a Sephadex LH20 column and eluted with acetone solution (40%, v/v). During the elution process, their unique benzotropolone structure of theaflavin with orange-red color can be easily monitored. Crude theflavin (TF-1) was collected. Crude TF-1 was further purified by a self pack C-18 column in a 20% methanol isocratic method. Fractions were checked by TLC and pure TF-1fractionswere collected. TF-1 was ready for further acetylation reaction.

Procedure for the Synthesis of TF-1 and Its Peracetylated Derivative TF-1 (0.21 mmol) was dissolved in 4 mL EtOAc and the 4-DMAP (1.05 mmol) was dissolved in anhydrous pyridine (0.86 mL). The 4-DMAP/pyridine and acetic anhydride (10.63 mmol) was subsequently added dropwise to TF1/EtOAc which had been stirred and placed in ice bath. The mixture was refluxed for 24 hours. The mixture was refluxed for 6 h. Then, the reaction mixture was cooled to room temperature and quenched with water. The mixture was then extracted with EtOAc, washed with brine and dried over sodium sulfate. After removing the solvent, the mixture was dissolved in 1.00 mL acetone and applied to LH-20 column which was eluted with 50% acetone solution. Their purities as determined by HPLC were at least greater than 95% and 93% for ATF-1 (HPLC assay). AFT-1 showed positive ESI-MS m/z 943 [M+H] . +

Determination of Cell Viability Cell viability was determined for indicated compounds based on the trypan blue exclusion method. Briefly, HL-60 cells were plated at a density of lxlO 5


348 cells/mL into 24-well plates. After overnight growth, cells were pretreated with a series of concentrations of TF-1 and ATF-1 for 24 h. The final concentrations of dimethyl sulfoxide (DMSO) in the culture medium were

Induction of Apoptosis by Acetylated Black Tea Polyphenol through

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