Fermented Pu-erh Tea Increases In Vitro Anticancer Activities in HT-29 Cells and Has Antiangiogenetic Effects on HUVECs

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1 Journal of Environmental Pathology, Toxicology and Oncology, 32(4): (2013) Fermented Pu-erh Tea Increases In Vitro Anticancer Activities in HT-29 Cells and Has Antiangiogenetic Effects on HUVECs Xin Zhao, 1,2 Jia-Le Song, 1 Jong-Deog Kim, 3 Jong-Suk Lee, 4 Kun Young Park 1 *1 Department of Food Science and Nutrition, Pusan National University, Busan, Korea; 2 Department of Biological and Chemical Engineering, Chongqing University of Education, Chongqing, PR China; 3 Department of Biotechnology, Research Center on Anti-Obesity and Health Care, Chonnam National University, Yosu, Chonnam, Korea; 4 Gyeonggi Biocenter, Gyeonggi Institute of Science and Technology Promotion (GSTEP), Suwon, Gyeonggi-do, Korea * Address all correspondence to: Prof. Kun Young Park, PhD, Department of Food Science and Nutrition, Pusan National University, 30 Jang Jun-dong, Keum Jung-ku, Busan , Korea; Tel.: Tel: (82) ; Fax: (82) ; kunypark@pusan.ac.kr. ABSTRACT: Pu-erh tea is produced in China and known to possess medicinal properties. The anticancer and antiangiogenesis effects of fermented Pu-erh tea on HT-29 colon cancer cells and human umbilical vein endothelial cells, respectively, were examined. Two kinds of unfermented and fermented Pu-erh tea (Seven-son tea cake Pu-erh tea and Xiaguan bowl tea [X]) and green tea were used. An MTT assay showed fermented Pu-erh tea X (85% inhibition) possessed more potent anticancer activities than unfermented Pu-erh tea X (67% inhibition) and green tea (53% inhibition) (P < 0.05). Moreover, fermented Pu-erh tea X increased the number of apoptotic bodies determined through DAPI staining and flow cytometric analysis. Fermented Pu-erh tea X induced apoptosis indicated by increased expression of Bax, caspase-9, and caspase-3 messenger RNA and decreased expression of Bcl-2. Fermented Pu-erh tea X also had an anti-inflammation effect, shown in decreased expression of nuclear factor-κb-p65, inducible nitric oxide synthase, COX-2 messenger RNA and increased expression of IκB-α. Further, fermented Pu-erh teas showed stronger antiangiogenesis effects than the 2 other types of tea. After fermentation, the concentrations of gallic acid, resorcylic acid, quercetin, and kaempferol in Pu-erh tea were increased. These results collectively indicated that fermented and unfermented Pu-erh teas possess stronger anticancer and antiangiogenesis effects than green tea. Furthermore, fermented Pu-erh tea showed stronger functional activities than unfermented Pu-erh tea. KEY WORDS: Camellia sinensis, fermented Pu-erh tea, green tea, anticancer, antiangiogenesis I. INTRODUCTION Tea is a beverage consumed worldwide. Green tea (Camellia sinensis) has been used in traditional Chinese medicine and has gained considerable attention because of its antioxidant, anti-inflammatory, antihypertensive, antidiabetic, and antimutagenic properties. 1 Chinese Pu-erh tea, named after Pu-erh City in the Yunnan province of China, is one of the most famous Chinese teas. 2 This tea is made from the large leaves of C. sinensis O. kuntze var. assamica Kitamura. Pu-erh tea leaves are approximately cm in length, the longest of its kind. These leaves are processed using a special fermentation procedure based on the chemical components of raw or nonfermented Pu-erh tea. With the action of extracellular enzymes that occurs because of the dampness and heat in the tea heap, the tea polyphenols undergo a series of complex and dramatic chemical changes that result in the special flavor and taste of Pu-erh tea. 3 5 Many types of food undergo fermentation processes that increase their medicinal effects. For example, fermented soybeans (doenjang) and fermented cabbage (kimchi) have been shown to exert stronger anticancer effects than raw or unfermented products. 6,7 Microorganisms predominantly found in Yunnan Pu-erh tea undergoing fermentation are Aspergillus niger, Aspergillus gloucu, and species of Penicillium, Rhizopus, Saccharomyces, and Bacterium. A. niger is most predominant, followed by Saccharomyces spp. and then by only a few Bacillus spp. 8,9 Because the quality of Pu-erh /13/$ Begell House, Inc

2 276 Zhao et al. tea is closely related to the postfermentation process, the polyphenol and the γ-aminobutyric acid content of the tea leaves are assessed, along with the free radical scavenging activity, at regular intervals during the fermentation. A particular microorganism (Aspergillus) has been identified as the most beneficial for enhancing the quality of the tea during the fermentation process. 10 Drinking Pu-erh tea for a long period of time can help maintain both mental and physical health. This tea is believed to help reduce high blood pressure and cholesterol levels and to play an important role in preventing heart disease and cancer. 11 Gallic acid is an important component of Pu-erh tea and may have antifungal and antiviral properties. Gallic acid acts as an antioxidant and helps to protect cells from oxidative damage. 12 This compound was found to have cytotoxic activity against cancer cells without harming healthy cells and is used as an astringent to treat internal hemorrhage. 13 Some ointments used to treat psoriasis and external hemorrhoids contain gallic acid. 14 Pu-erh tea can also protect against colon cancer. 15 Various phytochemicals, especially glycoside polyphenols, can be converted into aglycone and other fermented products by beneficial microorganisms. Thus, fermented products can help improve health, including lowering the risk of cancer. The known bioactive components of Pu-erh tea are epicatechin and epigallocatechin. 16 In this study, we investigated the anticancer effects of fermented Pu-erh tea on human colon cancer cells and its antiangiogenic effects on human umbilical vein endothelial cells (HUVECs). These effects were compared with those of unfermented Pu-erh tea and green tea. We also tried to identify active compounds in fermented Pu-erh tea by liquid chromatography mass spectrometry (LC-MS) analysis. II. MATERIALS AND METHODS A. Sources of Pu-erh Teas Seven-son tea cake Pu-erh tea (Pu-erh tea S) (Yunan Province Menghai County Yunhai Tea Factory, Yunan, China) and Xiaguan bowl tea (Pu-erh tea X) (Yunnan Xiaguan Tuocha Co., Ltd., Yunan, China) were purchased in Yunnan, China. Two types of unfermented (U) Pu-erh tea (U-Pu-erh tea S and U-Pu-erh tea X) and 2 types of fermented (F) Pu-erh tea (naturally fermented for 1 year; F-Puerh tea S and F-Pu-erh tea X) also were purchased in Yunnan, China. Green tea (Longjing tea; Hangzhou Longjing Tea Industry Co., Ltd., Zhejiang, China) was used as a control sample. B. Preparation of Tea Extract The Pu-erh tea and green tea were stored at -80 C and freeze-dried to produce a powder. A 20-fold volume of methanol was added to the powdered samples and extracted twice by stirring overnight. The methanol extracts were evaporated using a rotary evaporator (N-1100; Eywla, Tokyo, Japan), concentrated, and then dissolved in dimethylsulfoxide (DMSO) (Amresco, Solon, OH) to adjust to the stock concentration (20% w/v). C. Cancer Cell Preparation HT-29 human colon adenocarcinoma cells were obtained from the Korean Cell Line Bank (Seoul, South Korea). The cancer cells were cultured in RPMI-1640 medium (Welgene Inc., Daegu, South Korea) and supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco Co., Birmingham, MI) at 37 C in a humidified atmosphere containing 5% carbon dioxide (CO 2 ) (model 311; Forma, Waltham, MA). The medium was changed 2 or 3 times each week. D. MTT Assay The anticancer effects of Pu-erh tea was assessed by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The HT-29 human colon carcinoma cells were seeded into a 96-well plate, so that each well contained 180 µl of a cell suspension composed of cells per well. According to the proportion of cells in the suspension, 20 µl of specimen was added to a vessel and cultured for 48 hours at 37 C in a humidified at- Journal of Environmental Pathology, Toxicology and Oncology

3 Pu-erh Tea Increases Anticancer and Antiangiogenesis Effects 277 mosphere containing 5% CO 2. We added 200 µl of MTT solution (5 mg/ml), and the cells were cultured for another 4 hours under the same conditions. After removing the supernatant, 150 µl of DMSO was added to each well and mixed for 30 minutes. Finally, the absorbance of each well was measured by an enzyme-linked immunosorbant assay reader (model 680; Bio-Rad, Hercules, CA) at 540 nm. 17 E. Nuclear Staining with 4,6-Diamidino- 2-Phenylindole (DAPI) Untreated control cells and cells treated with tea extracts (200 µg/ml) were harvested, washed with phosphate-buffered saline (PBS), and fixed with 3.7% paraformaldehyde (Sigma, St. Louis, MO) in PBS for 10 minutes at room temperature. Fixed cells were washed with PBS and stained with a DAPI solution (1 mg/ml) (Sigma) for 10 minutes at room temperature. 18 The cells were washed 2 more times with PBS and examined with a fluorescence microscope (model BX50; Olympus, Tokyo, Japan). F. Flow Cytometry Analysis After treatment with tea samples, the cells were collected by trypsinization; they were washed with cold PBS and resuspended in PBS. The DNA contents of the cells were measured using a DNA staining kit (CycleTESTTM PLUS kit; Becton Dickinson, Heidelberg, Germany). Nuclear fractions stained with propidium iodide were obtained by following the kit protocol. Fluorescence intensity was determined using a FACScan flow cytometer (EPICS XL-MCL; Beckman Coulter KK, Tokyo, Japan) and was analyzed using CellQuest software (Becton Dickinson). 19 G. Reverse Transcriptase Polymerase Chain Reaction of Various Messenger RNAs Total RNA was isolated from HT-29 cells using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer s recommendations. The RNA was digested with RNase-free DNase (Roche, Basel, Switzerland) for 15 minutes at 37 C and purified using an RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer s protocol. Complementary DNA was synthesized from 2 µg of total RNA by incubation at 37 C for l hour with avian myeloblastosis virus reverse transcriptase (GE Healthcare, Little Chalfont, UK) with random hexanucleotides according to the manufacturer s instructions. Primers used to specifically amplify the genes of interest are shown in Table 1. Amplification was performed in a thermal cycler (Eppendorf, Hamburg, Germany) with cycles of denaturation. The polymerase chain reaction (PCR) products were separated in 1.0% agarose gels and visualized with ethidium bromide staining. 20 H. In Vitro Antiangiogenesis Test The HUVECs used in this study were obtained from Young Science Inc. (Seoul, South Korea), with cells from passages 3 to 5. HUVECs were cultured in EBM-2 growth medium (Cambrex, Hopkinton, MA), containing hydrocortisone, epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor-1, vascular endothelial growth factor, ascorbic acid, heparin, and 2% fetal bovine serum at 37 C in a humidified atmosphere containing 5% CO 2. The cells were seeded in culture flasks coated with 2% gelatin (Sigma) and allowed to grow to confluence before the experimental treatment. The formation of tubular structures by HUVECs in Matrigel was used to assess the antiangiogenesis effect of the tea samples. Culture plates (24-well) were coated with 150 µl of Matrigel, which was allowed to solidify at 37 C for 1 hour. HUVECs were seeded on Matrigelcoated wells (25,000 cells/well) and treated with tea samples at a concentration of 50 µg/ml for 24 hours at 37 C in an atmosphere humidified with 5% CO 2. Digital images were captured in randomly selected areas of the wells (5 each) using a digital camera (CoolPix 4500; Nikon, Tokyo, Japan). The formation of tube networks was quantified by summing up the arbitrary length of tubes from Volume 32, Number 4, 2013

4 278 Zhao et al. TABLE 1: Primers of Reverse Transcriptase Polymerase Chain Reaction Gene name Bax Bcl-2 Caspase-9 Caspase-3 NF-κB-p65 IκB-α Inducible nitric oxide synthase COX-2 GAPDH Sequence Forward: 5 -AAG CTG AGC GAG TGT CTC CGG CG-3 Reverse: 5 -CAG ATG CCG GTT CAG GTA CTC AGT C-3 Forward: 5 -CTC GTC GCT ACC GTC GTG ACT TGG-3 Reverse: 5 -CAG ATG CCG GTT CAG GTA CTC AGT C-3 Forward: 5 -GGC CCT TCC TCG CTT CAT CTC-3 Reverse: 5 -GGT CCT TGG GCC TTC CTG GTA T-3 Forward: 5 -CAA ACT TTT TCA GAG GGG ATC G-3 Reverse: 5 -GCA TAC TGT TTC AGC ATG GCA-3 Forward: 5 -CAC TTA TGG ACA ACT ATG AGG TCT CTG G-3 Reverse: 5 -CTG TCT TGT GGA CAA CGC AGT GGA ATT TTA GG-3 Forward: 5 -GCT GAA GAA GGA GCG GCT ACT-3 Reverse: 5 -TCG TAC TCC TCG TCT TTC ATG GA-3 Forward: 5 -AGA GAG ATC GGG TTC ACA-3 Reverse: 5 -CAC AGA ACT GAG GGT ACA-3 Forward: 5 -TTA AAA TGA GAT TGT CCG AA-3 Reverse: 5 -AGA TCA CCT CTG CCT GAG TA-3 Forward: 5 -CGG AGT CAA CGG ATT TGG TC-3 Reverse: 5 -AGC CTT CTC CAT GGT CGT GA-3 each image using the National Institutes of Heath image program. 21 I. LC-MS Analysis The tea extracts were dissolved in DMSO to produce a final concentration of 10 mg/ml and then diluted with 50% methanol to make a final concentration of 2 mg/ml. The diluted sample (5 µl) was analyzed by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). LC- MS/MS was performed using a Finnigan LCQ Advantage MAX ion trap mass spectrometer (Thermo Electron Co., Waltham, MA) equipped with an electrospray ionization source. Separation by high-performance liquid chromatography was performed with a Finnigan Surveyor Modular HPLC System (Thermo Electron Co.) using an Xterra MS C18 column (5 µm, mm; Waters, Milford, MA). Mobile phase A was water, and mobile phase B was acetonitrile; both contained 0.1% formic acid. Gradient elution at a flow rate of 0.2 ml/ min was carried out as follows: 0 5 minutes with 0 40% B (linear gradient), 5 20 minutes with 40 80% B (linear gradient), minutes with % B (linear gradient), and minutes with 100% B (isocratic). Full-scan mass spectra were obtained in the positive and negative ion modes at a range of m/z values of For identifying compound structures, data obtained from the MS/MS analysis were compared with those from an MS/MS spectral library search. 22 J. Statistical Analysis An analysis of variance was performed using data from the experiments for each specimen. The results are presented as the mean ± standard deviation (SD). Differences between the mean of the individual groups were assessed by one-way analysis Journal of Environmental Pathology, Toxicology and Oncology

5 Pu-erh Tea Increases Anticancer and Antiangiogenesis Effects 279 of variance with Duncan s multiple range tests. Differences were considered significant when P < SAS version 9.1 statistical software (SAS Institute Inc., Cary, NC) was used to perform these analyses. III. RESULTS A. In Vitro Anticancer Effect of Fermented Pu-erh Tea in HT-29 Cells An MTT assay was used to analyze the dose-dependent effects on HT-29 cells. As the tea concentrations increased, HT-29 cell growth was increasingly inhibited. At concentrations of µg/ml, the growth inhibitory effects of the F-Pu-erh tea X sample increased. According to our preliminary results, the growth inhibitory rate of Pu-erh tea was 100% for concentrations greater than 400 µg/ml. Therefore, concentrations of 100 and 200 µg/ml were chosen to evaluate the in vitro anticancer effect of Pu-erh tea. The anticancer effects of Pu-erh tea and green tea extracts on HT-29 cells were examined. The growth inhibitory rates of HT-29 cells treated with 100 µg/ml of green tea, U-Pu-erh tea S, F-Pu-erh tea S, U-Pu-erh tea X, and F-Pu-erh tea X were 12%, 13%, 37%, 25%, and 52%, respectively. At 200 µg/ml, the growth inhibitory rates of the cells treated with these samples were 53%, 55%, 70%, 67%, and 85%, respectively (P < 0.05). The anticancer effect of F-Pu-erh tea on the HT-29 cells was clearly stronger than that of U-Pu-erh tea; the effects of both types of F-Pu-erh tea also were greater than that of green tea (Table 2). B. Induction of Apoptosis by F-Pu-erh Tea To determine whether the growth inhibitory activity of Pu-erh tea in HT-29 human colon carcinoma cells was related to the induction of apoptosis, chromatin condensation first was analyzed by fluorescent microscopy using the DNA-binding fluorescent dye DAPI and flow cytometric analysis. HT-29 cells normally contain nuclei with a homogeneous chromatin distribution. Treatment with Pu-erh tea extracts (200 µg/ml) induced chromatin condensation and nuclear fragmentation, suggesting the presence of apoptotic cells (Fig. 1A). Chromatin condensation and the formation of apoptotic bodies, characteristics of apoptosis, were observed in the cells cultured with F-Pu-erh tea X and to a lesser extent in those cultured with U-Pu-erh tea X. These results suggest that F-Pu-erh tea X was more effective than U-Pu-erh tea X in inducing the condensation and formation of apoptotic bodies. Green tea also was shown to induce the formation of apoptotic bodies, but the extent of formation was weaker than that of U-Pu-erh tea X. The sub-g1 DNA content of the cancer cells was evaluated by flow cytometric analysis, which revealed enhanced apoptosis (increased apoptotic cells) of HT-29 cancer cells when treated with F-Pu-erh tea X compared with those treated with U-Pu-erh tea X and green tea (Fig. 1B and C). C. Expressions of the Apoptosis-Associated Bax, Bcl-2, and Caspase Genes To determine which type of apoptotic pathways was induced by treatment with tea, the expression of the Bax, Bcl-2, and caspase genes in HT-29 cells were examined by reverse transcriptase PCR (RT- PCR). As shown in Fig. 2A, treatment with 200 µg/ml of F-Pu-erh tea significantly changed the expression of proapoptotic Bax and antiapoptotic Bcl-2. These results suggest that the tea induced apoptosis in HT-29 cells via Bax-dependent and Bcl-2-dependent pathways. Moreover, the apoptosis induced by F-Pu-erh tea X was associated with increased Bax and decreased Bcl-2 messenger RNA (mrna) expression when compared to the other types of tea. The levels of expression of caspase-9 and -3 in mrna were low in untreated control HT-29 cells but were detected after cells were treated with 200 µg/ml of F-Pu-erh tea X. With the F-Pu-erh tea treatment, the expression of caspase-9 and -3 in mrna were significantly upregulated (Fig. 2B). In particular, a 2- to 3-fold difference in the expression of these genes was observed when cells treated with F-Pu-erh tea X were compared with Volume 32, Number 4, 2013

6 280 Zhao et al. TABLE 2: Inhibition of the Growth of HT-29 Human Colon Carcinoma Cells by Methanol Extracts of Tea Samples, as Evaluated by an MTT Assay Treatment OD 540 (µg/ml) Control (untreated) ± a,a ± a,a Green tea ± (12) b ± (53) B U-Pu-erh tea S ± (13) b ± (55) B F-Pu-erh tea S ± (37) c ± (70) C U-Pu-erh tea X ± (25) d ± (67) D F-Pu-erh tea X ± (52) e ± (85) E The values in parentheses are inhibition rates (%). Mean values with different letters (a e, A E) in the same column are significantly different (P < 0.05) according to Duncan s multiple range test. F, fermented; MTT, 3-(4,5-dimetyl-thiazol)-2,5-diphenyl tetrazolium; OD 540, optical density at 540 nm (concentration of sample); S, Seven-son tea cake Pu-erh tea; U, unfermented; X, Xiaguan bowl tea. those treated with green tea. The results suggested that F-Pu-erh tea X had the strongest anticancer effect, that U-Pu-erh tea X exerted a stronger effect than the green tea control, and that green tea had a slight anticancer effect on HT-29 cells. D. Expressions of Inflammation-Related NF-κB-p65, IκB-α, inos, and COX-2 Genes The inflammation induced by F-Pu-erh tea X was associated with decreased NF-κB-p65 and increased IκB-α expressions in mrna when compared to the other types of teas (Fig. 3A). Inhibition of inos and COX-2 activity predisposes a number of human cell lines to inflammation stimuli. Therefore, the anticancer actions of F-Pu-erh tea associated with the inhibition of the activation of inos and COX-2 gene expression were examined. As shown in Fig. 3B, the expression of COX-2 and inos in mrna was detected in untreated control HT-29 cells but was not detected after cells were treated with 200 µg/ml of F-Puerh teas. With the tea treatment, the expression of both COX-2 and inos in mrna was reduced gradually. These findings indicated that F-Pu-erh tea X may contribute to the prevention of cancer by reducing susceptibility to inflammation. The results also suggested that F-Pu-erh tea X has stronger anti-inflammatory properties than U-Pu-erh tea X and green tea. E. Antiangiogenesis Effect of Pu-erh Tea Tube formation by endothelial cells in Matrigel was analyzed quantitatively by examining 5 photomicrographs obtained from random fields in cell cultures in each well. Incubation of HUVECs with Pu-erh tea inhibited the formation of microtubular structures. Fig. 4 shows the antiangiogenetic effect of the tea at a concentration of 50 µg/ml. Incubation with the 2 kinds of U-Pu-erh tea (U- Pu-erh tea S and U-Pu-erh tea X) and green tea inhibited the formation of microtubular structures to similar degrees (53.0%, 63.5%, and 67.2%, respectively). The 2 types of F-Pu-erh tea (F-Pu-erh tea S and F-Pu-erh tea X) significantly inhibited tube formation (86.3% and 89.3%, respectively; P < 0.05). In this study, Pu-erh tea was found to have stronger antiangiogenetic effects than green tea; thus, Pu-erh tea is able to inhibit the growth of Journal of Environmental Pathology, Toxicology and Oncology

7 Pu-erh Tea Increases Anticancer and Antiangiogenesis Effects 281 FIG 1: Induction of apoptosis in HT-29 human colon carcinoma cells by treatment with green tea and Pu-erh tea. A: Cells were incubated with tea extracts (200 μg/ml) for 48 hours and then stained with DAPI. After 10 minutes of incubation at room temperature, the cells were washed with phosphate-buffered saline and photographed with a fluorescence microscope using a blue filter (400 original magnification). The arrows in Fig. 1 mean the apoptotic cells. B: To quantify the degree of apoptosis induced by tea samples (200 μg/ml), the cells were evaluated for the sub-g1 DNA (apoptotic cells) content using a flow cytometer. C: Amount of the apoptotic cells in each group. The letters (a c) mean the significant differences (P < 0.05) of apoptotic cells in each group according to Duncan s multiple range test. GT, green tea; FPX, fermented Pu-erh tea X; UPX, unfermented Pu-erh tea X. solid tumors. This finding implies that drinking Pu-erh tea, especially F-Pu-erh tea, may be quite beneficial for preventing cancer that can be considered an angiogenesis-dependent disease. F. Analysis of Pu-erh Tea Composition An initial LC-MS/MS analysis of the methanol extracts of U-Pu-erh tea X and F-Pu-erh tea X showed that there were 12 important peaks for U-Pu-erh tea X. These peaks (Fig. 5) were for gallic acid (1.70 minutes, m/z 169), epigallocatechin (9.10 minutes, m/z 305), catechin (9.30 minutes, m/z 289), caffeine (9.62 minutes, m/z 195), epicatechin (10.73 minutes, m/z 289), epigallocatechin gallate (10.87 minutes, m/z 457), epicatechin gallate (12.51 minutes, m/z 441), quercetin-3-galactoside (12.74 minutes, m/z 463), kaempferol-3-rutinoside (13.24 minutes, m/z 593), kaempferol-3-glucoside (13.51 min- Volume 32, Number 4, 2013

8 282 Zhao et al. FIG. 2: Effects of tea (200 μg/ml) on the expression of Bax and Bcl-2 (A), and caspase-9 and caspase-3 (B) in HT- 29 human colon carcinoma cells. Cells were incubated with tea samples for 48 hours. Reverse transcriptase polymerase chain reaction (PCR) was performed on isolated total RNA to measure Bax and Bcl-2 expression. The PCR products were separated on a 1% agarose gel and visualized by ethidium bromide staining. GAPDH was used as a housekeeping control gene. Band intensity was measured with a densitometer and expressed as the fold change over the control. Fold ratio was determined using the following formula: gene expression/gapdh control numerical value (control fold ratio of 1) Mean values with different letters (a d) are significantly different (P < 0.05) according to Duncan s multiple range test. GT, green tea; FPX, fermented Pu-erh tea X; UPX, unfermented Pu-erh tea X. utes, m/z 447), quercetin (15.39 minutes, m/z 301), and lutein (25.09 minutes, m/z 568). The spectra for F-Pu-erh tea X also contained the 12 peaks observed for the U-Pu-erh tea X extract. In addition, 2 peaks occurred at 3.43 and minutes on the LC chromatogram (Fig. 6). The 2 new compounds had molecular weights of 154 (m/z 153, [M-H] - ) and 286 (m/z 285, [M-H] - ) and were identified as resorcylic acid (3.43 minutes) and kaempferol (17.37 minutes), respectively (Fig. 5). This analysis demonstrated that gallic acid, resorcylic acid, quercetin, and kaempferol concentrations were increased in F-Pu-erh tea X but that epigallocatechin gallate levels were decreased. Furthermore, glycosides such as quercetin-3-galactoside, kaempferol-3-rutinoside, and kaempferol-3-glucoside were converted into aglycones during fermentation (Fig. 5B). In this Journal of Environmental Pathology, Toxicology and Oncology

9 Pu-erh Tea Increases Anticancer and Antiangiogenesis Effects 283 study we performed an LC-MS analysis to confirm that the fermentation process increased gallic acid, resorcylic acid, quercetin, and kaempferol concentrations in Pu-erh tea. IV. DISCUSSION Apoptosis is a fundamental cell event, and understanding its mechanisms of action will have a significant effect on antitumor therapy. The Bcl-2 family, which includes promoters (Bax and Bid) and inhibitors (Bcl-2 and Bcl-xL), is a key regulator in mitochondria-mediated apoptosis. 23 Bcl-2 is especially important to preserve the integrity of the outer mitochondrial membrane, thereby preventing the release of proapoptotic factors from mitochondria and inhibiting apoptosis. 24,25 In this study, apoptosis induced by F-Pu-erh tea is relat- FIG. 3: Effects of tea (200 µg/ml) on the expression of nuclear factor (NF)-κB-p65, IκB-α, inducible nitric oxide synthase (inos), and COX-2 in HT-29 human colon carcinoma cells. Cells were incubated with tea samples for 48 hours. Reverse transcriptase polymerase chain reaction (PCR) was performed on isolated total RNA to measure Bax and Bcl-2 expression. The PCR products were separated on a 1% agarose gel and visualized by ethidium bromide staining. GAPDH was used as a housekeeping control gene. Band intensity was measured with a densitometer and expressed as the fold change over the control. Fold ratio was determined using the following formula: gene expression/gapdh control numerical value (control fold ratio of 1). Mean values with different letters (a d) are significantly different (P < 0.05) according to Duncan s multiple range test. GT, green tea; FPX, fermented Pu-erh tea X; UPX, unfermented Pu-erh tea X. Volume 32, Number 4, 2013

10 284 Zhao et al. FIG. 4: Antiangiogenesis effects of the different tea extracts (50 µg/ml). Mean values with different letters (a f) are significantly different (P < 0.05) according to Duncan s multiple range test. GT, green tea; FPS, fermented Pu-erh tea S; FPX, fermented Pu-erh tea X; UPS: unfermented Pu-erh tea S; UPX, unfermented Pu-erh tea X. Journal of Environmental Pathology, Toxicology and Oncology

11 Pu-erh Tea Increases Anticancer and Antiangiogenesis Effects 285 FIG. 5: High-performance liquid chromatography chromatogram using a photodiode array detector ( nm) for unfermented Pu-erh tea X (A) and fermented Pu-erh tea X (B) extracts. FIG. 6: Identification and structures of the new compounds discovered in fermented Pu-erh tea X extract. Mass spectra (upper ones of each peak), mass spectrometry (MS)/MS spectra (lower ones of each peak), and molecular structures (bottom) of resorcylic acid (A) and kaempferol (B). Volume 32, Number 4, 2013

12 286 Zhao et al. ed to the downregulation of Bcl-2 and increased levels of Bax, thus promoting apoptosis in HT-29 cells. The caspase family of aspartate-specific cystine protease also plays a critical role in regulating apoptosis. 24 Capase-3 is an especially critical executioner of apoptosis. Activated caspase-3 can cleave poly (ADP-ribose) polymerase, a cell survival protein that is often deactivated by cleavage during apoptosis. 25 F-Pu-erh tea increases the mrna expression of caspase-3 and -9 compared with normal HT-29 cells. These data suggest that apoptosis induced by F-Pu-erh tea is caused by caspase-3-dependent cell death. Nuclear factor (NF)-κB-p65 is a ubiquitous transcription factor that presents in cytosol and normally binds to its natural inhibitory protein, inhibitor of κb (IκB). Following its induction by a variety of agents, NF-κB is released from IκB and translocated to the nucleus and regulates the gene expression required for cell proliferation, inflammation, and cell adhesion In this study, F-Puerh tea significantly downregulated the expression of NF-κB in mrna and upregulated the expression of IκB in mrna, showing anti-inflammatory activity in HT-29 human colon cancer cells (Fig. 3A). COX-2 and inducible nitric oxide synthase are important enzymes that mediate inflammatory processes. Improper upregulation of COX-2 and/ or inos has been associated with the pathophysiology of certain types of human cancers as well as inflammatory disorders. Since inflammation is closely linked to tumor promotion, inhibiting the activities of COX-2 and inos may be a promising approach for colon cancer chemoprevention. 29,30 Treatment with tea samples, especially F-Pu-erh tea, significantly decreased the expression of COX- 2 and inos in mrna (Fig. 3B). Thus F-Pu-erh tea may contribute to the prevention of cancer by increasing anti-inflammatory effects as well. A correlation has been noted between the level of angiogenesis and the process of metastasis in many varieties of malignant tumor 31,32 Pathological angiogenesis is a hallmark of cancer, as well as various ischemic and inflammatory diseases. 33 Antiangiogenic therapy is one of the most promising approaches for arresting tumor growth and cancer metastasis in cancer treatment. 34 F-Pu-erh tea significantly inhibited angiogenesis in HUVECs compared with U-Pu-erh tea and green tea. Although the anticancer effect of 2 different F-Pu-erh tea products were slightly different, F-Pu-erh tea showed the highest anticancer activity, followed by U-Pu-erh tea and green tea (Fig. 4). Tea has been reported to exert anticancer effects, which are associated with its many functional compounds, such as polypenols, catechines, amino acids, and vitamins The total catechin content in F-Pu-erh tea was less than that of U-Puerh tea, but the levels of gallic acid, resorcylic acid, quercetin, and kaempferol were higher than those in U-Pu-erh tea (Fig. 5). The amount of gallic acid increased during fermentation because of its liberation from catechin gallates. Gallic acid is cytotoxic against cancer cells and induces apoptosis without affecting normal cells. 39 Resorcylic acid lactones have been identified as a new class of kinase inhibitor and have been approved for the clinical treatment of different types of cancers. 40 Quercetin, a natural plant product, may be an inhibitor of cancer in tissues such as colon, lung, and intestine. 41 Evidence also suggests that kaempferol, which may be converted to an aglycone during the fermentation process, may have chemopreventative properties. 42 After fermentation, kimchi (fermented cabbage) and doenjang (fermented soybeans) had greater functional composition than unfermented cabbage and soybeans. 6,7 New bioactive compounds and aglycone flavonoids could form during fermentation. These compounds can increase the anticancer effects of the fermented foods. 43 V. CONCLUSION We used various in vitro experimental methods, such as MTT assay, DAPI staining, and RT-PCR, to evaluate the anticancer effect of teas, especially F-Pu-erh tea. Our results showed that Pu-erh tea had stronger anticancer activities after fermentation than did U-Pu-erh tea and green tea. Additional functional compounds were generated by fermentation and may have contributed to these findings. The results of this study suggest that fu- Journal of Environmental Pathology, Toxicology and Oncology

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