Toona sinensis extracts induces apoptosis via reactive oxygen species in human premyelocytic leukemia cells

Food and Chemical Toxicology 44 (2006) 1978–1988 www.elsevier.com/locate/foodchemtox

Toona sinensis extracts induces apoptosis via reactive oxygen species in human premyelocytic leukemia cells Hsin-Ling Yang a, Wen-Huei Chang b, Yi-Chen Chia c, Chin-Jung Huang a, Fung-Jou Lu b, Hseng-Kuang Hsu d, You-Cheng Hseu e,* a Institute of Nutrition, China Medical University, Taichung, Taiwan School of Applied Chemistry, Chung Shan University, Taichung, Taiwan c Department of Food Technology, Tajen University, Pingtung, Taiwan d Department of Physiology, Kaohsiung Medical College, Kaohsiung, Taiwan Department of Cosmeceutics, China Medical University, 91 Hsueh Shih Road, Taichung 40421, Taiwan b

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Received 2 December 2005; accepted 22 June 2006

Abstract Toona sinensis (T. sinensis), well known in Taiwan as a traditional Chinese medicine, has been shown to exhibit antioxidant effects. In this study, therefore, the ability of T. sinensis to induce apoptosis was studied in cultured human premyelocytic leukemia HL-60 cells. Treatment of the HL-60 cells with a variety of concentrations of the aqueous extracts of T. sinensis (TS extracts) (10–75 lg/ml) and gallic acid (5–10 lg/ml), the natural phenolic components purified from TS extracts, resulted in dose- and time-dependent sequences of events marked by apoptosis, as shown by loss of cell viability and internucleosomal DNA fragmentation. Furthermore, apoptosis in the HL-60 cells was accompanied by the release of cytochrome c, caspase 3 activation and specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP). This increase in TS extracts- and gallic acid-induced apoptosis was also associated with a reduction in the levels of Bcl-2, a potent cell-death inhibitor, and an increase in those of the Bax protein, which heterodimerizes with and thereby inhibits Bcl-2. Interestingly, TS extracts- and gallic acid-induced dose-dependent reactive oxygen species (ROS) generation in HL-60 cells. We found that catalase significantly decreased TS extracts- or gallic acid-induced cytotoxicity, DNA fragmentation, and ROS production, however, slight reduction was observed with vitamins C and E. Our results indicate that TS extracts- or gallic acid-induced HL-60 apoptotic cell death could be due to the generation of ROS, especially H2O2. The data suggest that T. sinensis exerts antiproliferative action and growth inhibition on HL-60 cells through apoptosis induction, and, therefore, that it may have anticancer properties valuable for application in food and drug products.  2006 Elsevier Ltd. All rights reserved. Keywords: Toona sinensis; Gallic acid; HL-60 cells; Reactive oxygen species; Apoptosis

1. Introduction Toona sinensis Roem. (Meliaceae; T. sinensis), a kind of arbor, is widely distributed in Asia. Long used as a nutri-

Abbreviations: T. sinensis, Toona sinensis; TS extracts, Aqueous extracts of T. sinensis; PARP, poly (ADP-ribose) polymerase; ROS, reactive oxygen species. * Corresponding author. Tel.: +886 4 22070414; fax: +886 4 22078083. E-mail address: [email protected] (Y.-C. Hseu). 0278-6915/$ - see front matter  2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2006.06.027

tious food in Chinese society. T. sinensis is very popular for vegetarian cuisine in Taiwan. The edible leaves have also been used as an oriental medicine for treating enteritis, dysentery and itchiness (Edmonds and Staniforth, 1998) with no irreversible side effects observed after treatment. It has also been reported that the crude extracts of T. sinensis induce apoptosis of human lung cancer cells (Chang et al., 1998), reduce plasma glucose in diabetic rats (Yu, 2002), and improve lipolysis and glucose in 3T3-L1 adipocytes (Hsu et al., 2003; Yang et al., 2003). Further, T. sinensis

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can improve the dynamic activity of human sperm (Yang et al., 2003) and inhibit steroidogenesis by suppressing the activities of steroidogenic enzymes in normal mouse Leydig cells (Poon et al., 2005). Strong DPPH radical-scavenging activities and inhibitory effects on lipid peroxidation have been demonstrated for MeOH extracts of T. sinensis (Cho et al., 2003). Methyl gallate from T. sinensis roots acts against hydrogen peroxide-induced oxidative stress and DNA damage in MDCK cells (Hsieh et al., 2004). Previous phytochemical work on Toona species has led to the isolation of triterpenes and phenolic compounds (Edmonds and Staniforth, 1998). To further the search for novel bioactive agents from Meliaceae plants, T. sinensis was chosen for phytochemical investigation (Park et al., 1996; Tsai et al., 2001; Hsieh et al., 1999). Known compounds, including gallic acid, methyl gallate, kaempferol, quercitin, quercitrin, rutin, catechin, epicatechin, oleic acid, palmitic acid, linoleic acid, linolenic acid, a mixture of b-sitosterol and stigmasterol, and b-sitosteryl-glucoside, were isolated and identified from this plant (Park et al., 1996; Tsai et al., 2001; Hsieh et al., 1999). Although it remains unclear which of the components of T. sinensis are active compounds, polyphenols have received increasing attention recently because of some interesting new findings regarding their biological activities. Of these compounds, it has been demonstrated that gallic acid (3,4,5-trihydroxybenoic acid) possesses antioxidant and anticancer activities (Wu et al., 1998; Lo´pez-Ve´lez et al., 2003; Ow and Stupans, 2003; Inoue et al., 1994, 1995). Gallic acid and its structurally related compounds are widely distributed in fruits and plants. Gallic acid, gallic acid ester, and its catechin derivatives are also some of the main phenolic components of both black and green tea, and red wine (Lo´pez-Ve´lez et al., 2003). Esters of gallic acid have a diverse range of industrial uses, as antioxidants in food, in cosmetics, and in the pharmaceutical industry (Ow and Stupans, 2003). Studies utilizing these compounds have found that they possess many potential therapeutic properties including anticancer properties (Inoue et al., 1994, 1995). Leukemia is one of the most threatening diseases today. Given that most adult leukemia patients are not candidates for transplantation, and that a more rational therapy is not adequately defined, they are typically treated with regimens that are based on (or at least include) chemotherapy. Recently, considerable attention has been devoted to the sequence of events referred to as apoptotic cell death, and the role of this process in mediation of the lethal effects of the diverse antineoplastic agents present in leukemia cells (Kaufman, 1989). Apoptosis is a highly regulated process that involves activation of a cascade of molecular events leading to cell death, characterized by cell shrinkage, membrane blebbing, chromatin condensation, and formation of a DNA ladder with multiple fragments (180– 200 bp) caused by internucleosomal DNA cleavage (Steller, 1995). Experiments in animals and studies of circulating blasts from leukemia patients have yielded evidence that

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apoptosis also occurs in response to chemotherapy in vivo. Human acute-leukemia cell lines (HL-60 cells) have proven particularly informative in study of chemotherapyassociated apoptotic proteolytic events. Plants possess many phytochemicals with various bioactivities, which include antioxidant, anti-inflammatory and anticancer activities. Therefore, many plants have been investigated to identify new anticancer compounds, as well as to elucidate the mechanisms of cancer prevention and apoptosis. In this study, the effects of the aqueous extracts of T. sinensis (TS extracts) and gallic acid, the natural phenolic components purified from TS extracts, on cultured human premyelocytic leukemia HL-60 cells was investigated due to their interesting biological activities and potential clinical application. The data reported herein show that TS extracts and gallic acid induce massive death in HL-60 cells, the dying cells exhibiting the ultrastructural and biochemical features that characterize apoptosis. Additionally, the biochemical steps linking TS extracts and gallic acid to the apoptotic process in these cells have been investigated. 2. Materials and methods 2.1. Chemicals Fetal bovine serum (FBS), RPMI 1640, penicillin–streptomycin (PS) and glutamine were obtained from GIBCO Laboratories (GIBCO BRL, Grand Island, NY). Rabbit polyclonal antibody against Bcl-2, Bax, cytochrome c and caspase 3 was purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany). PARP rabbit polyclonal antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal antibody against actin was obtained from Sigma Chemical Co. (St. Louis, MO). The 2 0 , 7 0 -dihydrofluorescein-diacetate was acquired from Molecular Probes Inc. (Eugene, OR, USA). All other chemicals were of the highest grade commercially available and were supplied either by Merck (Darmstadt, Germany) or Sigma chemicals (St. Louis, MO).

2.2. T. sinensis preparation and extraction The leaves of T. sinensis were sourced from Fooyin University, Kaohsiung Hsien, Taiwan. A voucher specimen (FY-001) was characterized by Dr. Horng-Liang Lay, Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung County, Taiwan, and deposited at Fooyin University, Kaohsiung Hsien, Taiwan (Hsieh et al., 2004). The aqueous extracts of T. sinensis (TS extracts) was prepared by adding 1000 ml water to the leaves of fresh T. sinensis (1000 g) then boiling them until 100 ml remained, as previously described (Chang et al., 2002). The crude extracts were centrifuged at 3000 rpm for 12 min and the supernatant used for this study. The crude extracts (50 g) were concentrated in a vacuum and freeze dried to form a powder, and the stock was then stored at 20 C for subsequent analysis of its anticancer properties. The yield of TS extracts was 5%.

2.3. Isolation of gallic acid from TS extracts TS extracts were dissolved in a mobile phase consisting of methanol– water (50:50, v/v) before HPLC (high performance liquid chromatography) analysis and separation. Chromatographic separation was achieved with a mobile phase consisting of methanol–water (50:50, v/v) in the first 15 min, gradually increasing the methanol to 100% in the next 10 min. A flow-rate of 4.0 ml/min at room temperature was used. Eight

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compounds (gallic acid, methyl gallate, ethyl gallate, kaempferol, kaempferol-3-O-b-D-glucoside, quercetin, quercitrin, quercetin-3-O-b-Dglucoside and rutin) were isolated from TS extracts. The identification of the compounds was fully characterized by comparison of their spectral data (IR, NMR, and mass) with analogous information reported in the literature (Hsieh et al., 2004; Park et al., 1996). Gallic acid, the natural phenolic component purified from TS extracts, was collected for use in this study. The yield of gallic acid from TS extracts was 6%.

2.4. Cell culture and assessment of cell growth and cell viability HL-60 cells, a human acute promyeloblastic leukemic cell line, were obtained from the American Type Culture Collection (Rockville, MD). These cells were grown in RPMI-1640 supplemented with 10% heat-inactivated FBS, 2 mM glutamine, and 1% penicillin–streptomycin–neomycin in a 5% CO2 humidified incubator at 37 C. The HL-60 cells were incubated with TS extracts (0, 10, 25, 50, 75 lg/ml) and gallic acid (0, 5, 10 lg/ml) for 2, 4 and 6 h. Cultures were harvested and cell numbers enumerated by hemocytometer analysis of cell suspensions. Cell growth (2.0 · 105 cells/ml) and viability (1.0 · 106 cells/ml) were checked after treatment using trypan blue exclusion and examined using phase contrast microscopy. Taxol is a diterpenoid plant product that exhibits antitumor activity against various malignant cells such as leukemias (Rowinsky et al., 1989). In this study, the effects of taxol on the growth of HL-60 cells were also examined.

2.5. DNA gel electrophoresis (DNA laddering) The presence of internucleosomal DNA cleavage in HL-60 cells (5.0 · 105 cells/ml) was investigated using DNA gel electrophoresis. HL-60 cells were harvested, washed twice with cold phosphate-buffered saline (PBS), and then resuspended in DNA lysis buffer (50 mM Tris–HCl [pH 8.0], 10 mM EDTA, and 0.5% sarkosyl) supplemented with proteinase K at a final concentration of 100 lg/ml. The mixture was incubated at 50 C for 3 h and then DNase-free RNase added for a further 3 h. The DNA was extracted with equal amounts of phenol, chloroform and isoamyl alcohol (25:24:1, v:v:v). DNA purity and concentration were determined by electrophoresis through a 1.5% agarose gel containing ethidium bromide, followed by observation under ultraviolet illumination.

2.6. Preparation of total cell extract and immunoblot analysis HL-60 cells (2.0 · 105 cells/ml) were washed once with PBS and suspended in 100 ll lysis buffer (10 mM Tris–HCl [pH 8], 0.32 M sucrose, 1% Triton X-100, 5 mM EDTA, 2 mM DTT, and 1 mM phenylmethyl sulfony flouride). The suspension were put on ice for 20 min and then centrifuged at 17,400g for 20 min at 4 C. Total protein content was determined with Bio-Rad protein assay reagent using bovine serum albumin as a standard, and protein extracts were reconstituted in sample buffer (0.062 M Tris– HCl, 2% SDS, 10% glycerol, and 5% b-mercaptoethanol) and the mixture boiled for 5 min. Equal amounts (50 lg) of the denatured proteins were loaded into each lane, separated on 10% SDS polyacrylamide gel, followed by overnight protein transfer to the PVDF membranes. The membranes were blocked with 0.1% Tween-20 in Tris-buffered saline containing 2% non-fat dry milk for 20 min at room temperature, and the membranes were reacted with primary antibodies for 2 h. They were then incubated with a horseradish peroxidase-conjugated goat antirabbit or antimouse Ab for 2 h before development with ECL Western blotting reagents (Amersham, NJ, USA).

2.7. Measurement of ROS generation by flow cytometry Intracellular ROS production was detected by flow cytometry using 2 0 ,7 0 -dihydrofluorescein-diacetate (DCFH-DA) (Rothe and Valet, 1990). The HL-60 cells were cultured in 60-mm tissue-culture dishes (1.0 · 106 cells/ml), with renewal of the culture medium when the cells were 80% confluent. HL-60 cells were incubated with 10 lM DCFH-DA

in culture medium at 37 C for 15 min before the end of time point of each experiment. These cells were collected by centrifugation, washed and resuspended as 1 · 106 cells/ml in PBS containing 5 lg/ml of propidium iodide (PI) and submitted for flow cytometic analysis. PI treatment differentiates between integrated and non-integrated cell membranes because the latter permit intracellular dye penetration and the former do not. The intracellular ROS, as indicated by dichlorofluorescein (DCF) fluorescence, was measured with a Becton–Dickinson FACS-Calibur flow cytometer (Becton Dickinson, NJ).

2.8. Determination of total phenolic compounds Total phenolic content of aqueous extracts of T. sinensis extracts were determined approximately by using the Folin–Ciocalteu reagent method. 0.1 ml of TS extracts at different doses (0, 25, 50, 75 and 100 lg/ml), 0.1 ml of Folin–Ciocalteu reagent (50%), and 2 ml of Na2CO3 (2%) was added and mixed, and then was allowed to stand for 30 min. The absorbance was measured at 750 nm in a spectrophotometer. The total phenolic content was expressed as gallic acid equivalents (GAE) in milligrams per gram of sample, using a standard curve generated with gallic acid.

2.9. Human mononuclear cell preparation Cells were isolated from freshly donated human peripheral blood of healthy volunteers as described previously (Aliverti et al., 1995). Cell viability (1.0 · 106 cells/ml) were checked after treatment using trypan blue exclusion and examined using phase contrast microscopy.

2.10. Statistical analysis Mean data values are presented with their deviation (mean ± SD). Analysis of variance (ANOVA) was followed by Dunnett’s test for pairwise comparison. Statistical significance was defined as p < 0.05 for all tests.

3. Results In this study, the human promyelocytic leukemia cell line, HL-60, was used to investigate the capability of the aqueous extracts of T. sinensis (TS extracts) (0–75 lg/ml) and gallic acid (0–10 lg/ml), the natural phenolic components purified from TS extracts, to induce apoptosis and elaborate the molecular mechanism. The crude TS extracts separated from Fresh T. sinensis leaves, yielding 5%, based on the initial weight of the crude extracts. The total phenolic content of TS extracts was estimated to be 130 ± 26 mg gallic acid (pyrocatechol) equivalents/g of plant extracts from triplicate measurements. The yield of gallic acid from TS extracts was 6%. 3.1. Effect of TS extracts and gallic acid on HL-60 cell growth To investigate the potential effects of TS extracts on proliferation and survival of HL-60 cells, the cells were exposed to 0–75 lg/ml of TS extracts and 0–10 lg/ml gallic acid for 2, 4 and 6 h. TS extracts and gallic acid-induced cell death in a dose- and time-dependent manner, as determined using trypan blue exclusion (Fig. 1). Further, phasecontract micrographs revealed that TS extracts and gallic acid causes cell shrinkage (Fig. 2).

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Time (h) Fig. 1. Effects of TS extracts and gallic acid from TS extracts on HL-60 cell growth. HL-60 cells were treated with 0, 10, 25, 50 and 75 lg/ml of TS extracts (A) and 0, 5, and 10 lg/ml of gallic acid (B) for 2, 4 and 6 h. Control cells were maintained in the vehicle for the indicated time periods. Taxol was dissolved in 0.1% DMSO. The presence of 0.1% DMSO had no effect on any of the parameters monitored. Results are presented as the mean ± SD of three assays. * Indicates significant difference in comparison to control group (p < 0.05).

3.2. Induction of apoptotic DNA fragmentation by TS extracts and gallic acid Agarose-gel electrophoresis of chromosomal DNA-treated TS extracts (at 0, 10, 25, 50, 75 lg/ml) or gallic acid (at 0, 5, 10 lg/ml) showed a ladder-like pattern of DNA fragments consisting of multiples of approximately 180–200 base pairs. The apoptosis-inducing activity of TS extracts (Fig. 3A) and gallic acid (Fig. 3B) was dose-dependent and time-dependent. 3.3. Effect of TS extracts and gallic acid on cytochrome c release, caspase 3 activity, and PARP cleavage Treatment of HL-60 cells with a variety of chemotherapeutic agents is accompanied by increased cytosolic translocation of cytochrome c, activation of caspase 3, and PARP

Fig. 2. Phase-contrast micrographs of TS extracts and gallic acid-treated HL-60 cells. HL-60 cells were treated with (A) control, (B) 75 lg/ml of TS extracts, and (C) 10 lg/ml of gallic acid for 6 h. Typical result from three independent experiments is shown. Bar represents 50 lm.

degradation (Lazebnik et al., 1994;Tewari et al., 1995). In the present study, the cytosol levels of cytochrome c were examined using western blot analysis. Our results reveal that TS extracts and gallic acid induce cytosolic cytochrome c release (Fig. 4). Since cytochrome c is reportedly involved in the activation of the caspases that trigger apoptosis, we investigated the role of caspase 3 in the cell response to TS extracts and gallic acid. Caspase 3 (CPP32) is a cytosolic protein that normally exists as a 32-KDa inactive precursor. It is cleaved proteolytically into a heterodimer when the cell undergoes apoptosis (Nicholson et al., 1995). As shown in Fig. 4, the involvement of caspase 3 activation is further supported by immunoblotting analysis in which TS extracts and

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Fig. 3. DNA fragmentation of HL-60 cells exposed to TS extracts and gallic acid. (A) HL-60 cells were incubated with 0, 10, 25, 50 and 75 lg/ml of TS extracts for 6 h and with 75 lg/ml of TS extracts for 2, 4 and 6 h. (B) HL-60 cells were incubated with 0, 5 or 10 lg/ml of gallic acid for the indicated time periods (2, 4, or 6 h). DNA ladders reflecting the presence of DNA fragments were viewed on ethidium-bromide-stained gel. Typical result from three independent experiments is shown. M: molecular-weight markers.

Fig. 4. Western blot analysis of cytochrome c, caspase 3, and PARP protein levels exposed to TS extracts and gallic acid. (A) HL-60 cells were incubated with 0, 10, 25, 50 and 75 lg/ml of TS extracts for 6 h and with 75 lg/ml of TS extracts for 2, 4 and 6 h. (B) HL-60 cells were treated with 0, 5, and 10 lg/ml of gallic acid for 6 h. Protein (50 lg) from each sample was resolved on 10% SDS-PAGE, and western blot performed. Typical result from three independent experiments is shown.

gallic acid evidently induces proteolytic cleavage of procaspase 3 into its active form, a 17-KDa fragment. Since PARP-specific proteolytic cleavage by caspase 3 is consid-

ered a biochemical characteristic of apoptosis (Nicholson et al., 1995), a western blotting experiment was conducted using the antibody against PARP. PARP is a nuclear

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enzyme which is involved in DNA repair, and it has been demonstrated that the 116-kDa PARP protein is cleaved into a 85-kDa fragment in cytosol (Nicholson et al., 1995). PARP is cleaved into a 85-kDa fragment after the addition of TS extracts and gallic acid (Fig. 4). 3.4. Effect of TS extracts and gallic acid on Bcl-2 and Bax protein Recently, it has been shown that the Bcl-2 family plays an important regulatory role in apoptosis, either as activator (Bax) or as inhibitor (Bcl-2) (Green and Reed, 1998; Adams and Cory, 1998). Bcl-2 and Bax protein levels were studied in cultured HL-60 cells to examine the involvement of Bcl-2 and Bax in TS extracts- and gallic acid-mediated apoptosis. Western blot analysis of Bcl-2 and Bax exposed to TS extracts and gallic acid was resolved on 10% SDSPAGE. Incubation of HL-60 cells with TS extracts and gallic acid reduced Bcl-2, a potent cell-death inhibitor, and increased Bax protein levels (Fig. 5). The increase in TS extracts- and gallic acid-induced apoptosis was associated with the reduction in Bcl-2 and an increase in Bax. These results indicate that TS extracts and gallic acid induces dysregulation of Bcl-2 and Bax in HL-60 cells. 3.5. Effect of TS extracts and gallic acid on ROS production in HL-60 cells ROS production can be detected by flow cytometric analysis using DCFH-DA as a fluorescence probe for estimating ROS generation (Rothe and Valet, 1990). DCFH-

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DA, a non-fluorescent cell-membrane permeable probe, was used to penetrate the cells, react with cellular esterases and ROS, and then metabolize into fluorescent DCF. A basal level of DCFH-DA fluorescence was demonstrated for the untreated HL-60 cells (control). Incubation of HL-60 cells for 0, 0.5, 1 and 2 h with 75 lg/ml of TS extracts or 10 lg/ml of gallic acid caused a significant increase in fluorescence response (Fig. 6). The maximum ROS increase was observed at 2 h after treatment. However, the DCF fluorescence declined subsequently because of significant cell death (data not shown). The results indicate that TS extracts or gallic acid induces ROS generation in HL-60 cells. 3.6. Effect of vitamin C, vitamin E, and catalase on TS extracts- and gallic acid-induced cell viability, DNA fragmentation, and ROS production in HL-60 cells The effects of several antioxidants, vitamin C (10 lg/ml), vitamin E (10 lg/ml), and catalase (28 U/ml) on TS extracts- and gallic acid-induced cell viability, DNA fragmentation, and ROS production were examined. HL-60 cells were treated simultaneously with TS extracts (75 lg/ ml) or gallic acid (10 lg/ml) plus various antioxidants (independently) (Fig. 7). We found that catalase significantly decreased TS extracts- and gallic acid-cytotoxicity, DNA fragmentation, and ROS production, however, only slight reductions were observed with vitamins C and E. Our results indicate that the TS extracts- or gallic acid-induced HL-60 apoptotic cell death may be due to the generation of ROS, especially H2O2.

Fig. 5. Western blot analysis of Bcl-2 and Bax protein levels exposed to TS extracts and gallic acid. (A) HL-60 cells were incubated with 0, 10, 25, 50 and 75 lg/ml of TS extracts for 6 h and with 75 lg/ml of TS extracts for 2, 4 and 6 h. (B) HL-60 cells were treated with 0, 5, and 10 lg/ml of gallic acid for 6 h. Protein (50 lg) from each sample was resolved on 10% SDS-PAGE, and western blot performed. Typical result from three independent experiments is shown. Relative changes in Bcl-2 and Bax protein bands were measured using densitometric analysis. Results are presented as mean ± SD of three assays. * Indicates significant difference in comparison to control group (p < 0.05).

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Fig. 6. Effects of TS extracts and gallic acid on intracellular ROS Level in HL-60 cells. HL-60 cells were incubated with 75 lg/ml of TS extracts (A) or 10 lg/ml of gallic acid (B) for 0, 0.5, 1 and 2 h. DCFH-DA at a final concentration of 10 lM was added to the culture medium 15 min before the end of each experiment. DCF fluorescence was measured by flow cytometer analysis. Typical result from three independent experiments is shown.

3.7. Effects of TS extracts and gallic acid on the growth of lymphocytes To test whether TS extracts or gallic acid affects normal cells, its effects on the growth of human lymphocytes were examined. The number of lymphocytes was not affected by TS extracts at 0, 10, 25, 50 or 75 lg/ml and gallic acid at 0, 5 or 10 lg/ml after 12 h of incubation (Fig. 8). Furthermore, the addition of TS extracts and gallic acid did not cause significant hemolysis in RBCs up to 15 h of incubation (data not shown), and RBCs will begin to autolyse after 15 h. 4. Discussion Recently, the relationship between apoptosis and cancer has been emphasized, with increasing evidence suggesting that the related processes of neoplastic transformation, progression and metastasis involve alteration of normal apoptotic pathways (Bold et al., 1997). Apoptosis provides a number of clues with respect to effective anticancer therapy, and many chemotherapeutic agents reportedly exert their antitumor effects by inducing apoptosis in cancer cells. The results reported herein reveal that TS extracts (0–75 lg/ml) and gallic acid (0–10 lg/ml), the natural

phenolic components purified from TS extracts exert antiproliferative action and growth inhibition in cultured human premyelocytic leukemia HL-60 cells. The dying cells exhibit the ultrastructural and biochemical features that characterize apoptosis, as shown by loss of cell viability and internucleosomal DNA fragmentation. This study also defines those events, most of which are used as biomarkers of apoptosis, that are associated with TS extracts- and gallic acid-induced HL-60 apoptotic cell death. Cells undergoing apoptosis were found to have a cytosolic elevation of cytochrome c, with a corresponding decrease in the mitochondria (Yang et al., 1997). After the release of mitochondrial cytochrome c, the cysteine protease 32-kDa proenzyme CPP32, a caspase 3, is activated by proteolytic cleavage into an active heterodimer (Nicholson et al., 1995). Activated caspase 3 is responsible for the proteolytic degradation of poly ADP-ribose polymerase, which occurs at apoptosis onset (Nicholson et al., 1995; Lazebnik et al., 1994). In this study, we produce evidence demonstrating that T. sinensisinduced apoptosis of HL-60 cells is mediated by increased cytosolic translocation of cytochrome c, activation of caspase 3, and degradation of PARP. It has been demonstrated that the gene products of Bcl-2 and Bax play important roles in apoptotic cell death (Jacobson and Raff, 1995; Oltvai et al., 1993). In Bcl-2 family members, the Bcl-2 and Bax

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Fig. 7. Effects of vitamin C (10 lg/ml), vitamin E (10 lg/ml), and catalase (28 U/ml) on cell growth (A), DNA fragmentation (B), and intracellular ROS Level (C) in TS extracts- or gallic acid-treated HL-60 cells. (A) Cells were incubated with 75 lg/ml of TS extracts or 10 lg/ml of gallic acid for 6 h. Results are presented as mean ± SD of three assays. * Indicates significant difference in comparison to TS extracts- or gallic acid-treated group (p < 0.05). (B) Cells were incubated with 75 lg/ml of TS extracts or 10 lg/ml of gallic acid for 6 h. N: untreated cell; C: cells treated with vitamin C, vitamin E, or catalase alone; T: TS extracts; G: gallic acid. Typical result from three independent experiments is shown. (C) Cells were incubated with 75 lg/ml of TS extracts or 10 lg/ml of gallic acid for 2 h. Typical result from three independent experiments is shown.

protein ratio has been recognized as a key factor in regulation of the apoptotic process (Adams and Cory, 1998; Green, 1998). Evidence indicates that dysregulation of Bcl-2 and Bax may contribute to cytochrome c release, cas-

pase 3 activation, and resultant apoptosis (Jacobson and Raff, 1995; Buttke and Sandstrom, 1994). In the present study, the increase in TS extracts- and gallic acid-induced apoptosis was associated with Bcl-2 reduction and Bax

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Fig. 8. Effects of TS extracts on the growth of human lymphocytes. Lymphocytes were incubated with 0, 10, 25, 50, 75 and 100 lg/ml of TS extracts and 0, 5, and 10 lg/ml gallic acid for 12 h. Cell numbers were obtained by counting cell suspensions with a hemocytometer. Results are presented as mean ± SD of three assays. * Indicates a significant difference in comparison to the control group (p < 0.05).

increase. Analysis of our data indicates that T. sinensis may disturb the Bcl-2/Bax ratio and, therefore, lead to apoptosis of HL-60 cells. Recently, many researchers have reported that internucleosomal DNA fragmentation is not essential to apoptosis, and that it is typically accompanied by some necrotic cell death, suggesting the possibility that this fragmentation may not be an adequate indicator of this natural process (Cohen et al., 1992; Schulze-Osthoff et al., 1994). It is clear, however, that the central mechanism of apoptosis is evolutionarily conserved, and that caspase activation is an essential step in this complex apoptotic pathway (Thornberry and Lazebnik, 1998). The presented data, therefore, provide more important evidence that T. sinensis-induced cell death is apoptosis. It has been reported that extracts from natural products, such as fruits, vegetables and medicinal herbs, have positive effects against cancer, compared with chemotherapy or recent hormonal treatments (Pezzuto, 1997). There is increasing evidence that many natural isolated compounds and Chinese medicinal herbs are promising biological modifiers for cancer treatment. In this study, a number of compounds, including gallic acid, methyl gallate, ethyl gallate, kaempferol, kaempferol-3-O-b-D-glucoside, quercetin, quercitrin, quercetin-3-O-b-D-glucoside, and rutin, have been isolated from the leaves of T. sinensis, as determined by HPLC (data not shown). The yield of gallic acid, the natural phenolic component purified from TS extracts, was about 6%. Other studies have found that gallic acid, a naturally occurring plant phenol and oxidant, induces apoptosis in cancer cells (such as HL-60RG, Hela, dRLh-84, PLC/ PRF/5 and KB cells) with higher sensitivity than normal analogs (such as rat primary cultured hepatocytes, macrophages, endothelial cells and fibroblasts) (Inoue et al., 1994, 1995). Gallic acid exhibits an antiproliferative effect in cells through induction of apoptosis associated with cytochrome c translocation, caspase 3 activation, PARP degradation, and dysregulation of Bcl-2 and Bax (Isuzugawa

et al. 2001a,b; Sohi et al., 2003). Other workers have shown that intracellular gallic acid-induced ROS, especially H2O2, play an important role in eliciting an early signal in apoptosis (Inoue et al., 2000; Sakaguchi et al., 1998), and that catalase significantly reduces gallic acid-induced apoptotic cell death (Isuzugawa et al. 2001a,b; Nogaki et al., 1998). Further, it was determined that the cytotoxic activity demonstrated for gallic acid was not a feature common to phenolic compounds, but rather a fairly specific characteristic. These results imply that natural gallic acid in T. sinensis possibly acts as a chemopreventive agent with respect to inhibition of cancer cell growth through apoptosis induction, functionally rendering this the most-effective fraction of T. sinensis. However, full characterization of possible compounds which may account for the anticancer activity of T. sinensis requires further study. Many of the agents that induce apoptosis are oxidants or stimulators of cellular oxidative metabolism, while antioxidant activity has been revealed for many inhibitors of apoptosis (Buttke and Sandstrom, 1994). Indeed, factors for oxidative stress, such as ROS production (Garcia-Ruiz et al., 1997; Coyle and Puttfarcken, 1993), lipid peroxidation (Hockenbery et al., 1993), downregulation of the antioxidant defenses characterized by reduced glutathione levels (Marchetti et al., 1996), and reduced transcription of superoxide dismutase, catalase and thioredoxin, have been observed in some apoptotic processes (Briehl and Baker, 1996). Moreover, ROS can also play an important role in apoptosis by regulating the activity of certain enzymes involved in the cell-death pathway (Garcia-Ruiz et al., 1997; Coyle and Puttfarcken, 1993). All these factors point to a significant role for intracellular oxidative metabolites in the regulation of apoptosis. Other recent studies appear to support the notion that T. sinensis may possess protective antioxidant properties (Cho et al., 2003; Hsieh et al., 2004). As described above, however, TS extracts and gallic acid induces ROS generation in the HL-60 cellular environment. Thus, T. sinensis might serve as a mediator for the reactive oxygen-scavenging system and potentially act as both a pro-oxidant and an antioxidant, depending on the redox state of the biological environment. This dual-property role for antioxidants has also been reported by other workers (Turley et al., 2000; Sahu and Gray, 1997). In addition, several researchers have shown that antioxidants, such as retinoids and vitamin E, produce genetic changes that cause apoptosis in cancer cells by mechanisms other than antioxidant effect (Zou et al., 2001; Turley et al., 1997). In this study, we found that catalase significantly decreased TS extracts- and gallic acid-induced cytotoxicity, DNA fragmentation and ROS production, however, vitamins C and E only produced a slight reduction. Our results indicate that the T. sinensis-induced HL-60 apoptotic cell death may be due to the generation of ROS, especially H2O2. It is suggested that T. sinensis-induced intracellular ROS plays an important role in eliciting an early signal for triggering apoptosis. In particular, H2O2, which is derived from superoxide anions generated extracellularly,

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