Pei-Jane Huanga,1, You-Cheng Hseub,1, Meng-Shiou Leec, K.J. Senthil Kumarb, Chi-Rei Wuc, Li-Sung Hsud, Jiunn-Wang Liaoe, I-Shiung Chengf, Ya-Ting Kuoh, Shi-Ying Huangg,*, Hsin-Ling Yangh,*
aDepartment of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan
bDepartment of Cosmeceutics, College of Pharmacy, China Medical University, Taichung 40402, Taiwan
cSchool of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 40402, Taiwan
dInstitute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 40402, Taiwan
eGraduate Institute of Veterinary Pathology, National Chung Hsing University, Taichung 40402, Taiwan
fDepartment of Physical Education, National Taichung University of Education, Taichung 40402, Taiwan
gDepartment of pediatrics, Armed Force Taoyuan General Hospital, Taoyuan 32551, Taiwan
hInstitute of Nutrition, China Medical University, Taichung 40402, Taiwan
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*Corresponding authors. Tel.: +886 4 22053366 x 7503; fax: +886-4-22062891.
E-mail addresses: hlyang@mail.cmu.edu.tw (H-L, Yang); Huangsy56@yahoo.com.tw (S-Y, Huang)
1 Both authors contributed equally.
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Toona sinensis is one of the most popular vegetarian cuisines in Taiwan and it has been shown to induce apoptosis in cultured human premyelocytic leukemia (HL-60) cells. In the present study, we examined the effects of Toona sinensis leaf extracts (TS extracts) on tumor regression using in vitro cell culture and an in vivo athymic nude mice model. We found that TS extracts (10-75 g/mL) arrested HL-60 cells at the G1-S transition phase through the reductions of Cyclin D1, CDK4, Cyclin E, CDK2, and Cyclin A, and induction of CDK inhibitor p27KIP levels. Furthermore, VEGF expression and release was significantly inhibited by TS extracts. Notably, TS extracts treatment was effective in terms of delaying tumor incidence in the nude mice inoculated with HL-60 cells as well as reducing the tumor burden. Histological analysis confirmed that TS extracts significantly modulated tumor progression in xenograft tumor. Furthermore, a similar pattern of results were observed from gallic acid (5 and 10 g/mL), a major compound in TS, caused G1
arrest through regulations of cell-cycle regulatory proteins. Our data suggest that Toona sinensis exerts antiproliferative effects on HL-60 cells in vitro and in vivo due mainly to Xenografted nude mice
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Toona sinensis (A. Juss.) M. Roem popularly known as Chinese mahogany or Chinese toon, a member of Meliaceae family, and is a type of deciduous perennial tree widely distributed in Asia. In Chinese and Taiwanese culture it is one of the popular vegetarian cuisine. It has long been used as a traditional Chinese medicine for a wide variety of conditions. The edible leaves used as an oriental medicine for treating rheumatoid arthritis, cervicitis, urethritis, tympanitis, gastric ulcers, enteritis, dysentery, itchiness, and cancer (Edmonds and Staniforth, 1998; Hseu et al., 2011a). While the underlying pharmacological mechanisms of this new drug are still a matter of debate, previous scientific literatures have been reported that T. sinensis possessed variety of biological activities including anti-cancer (Chang et al., 2002; Chang et al., 2006; Yang et al., 2006a;
Chen et al., 2009; Wang et al., 2010; Yang et al., 2010a; Yang et al., 2010b; Chia et al., 2010), angiogenesis (Hseu et al., 2011a) inflammation (Bak et al., 2009), anti-diabetes (Hsu et al., 2003; Yang et al., 2003), and antioxidant (Cho et al., 2003; Hseu et al., 2008a; Jiang et al., 2009) effects, as well as inhibiting Leydig cell steroidogenesis and improving the dynamic activity of human sperm quality (Poon et al., 2005). Moreover, the safety levels and nontoxic characteristics of aqueous extracts of T. sinensis were evaluated using acute and sub-acute toxicity studies in mice (Liao et al., 2006).
Gallic acid (GA), a major phenolic compound that rich in TSL has a wide spectrum of biological and pharmacological effects. Various animal models or human studies proved that GA is extremely safe even at using high doses. Also, a few studies addressing the bioavailability of GA in human revealed that this compound is extremely well observed when compared to other polyphenols (Manach et al., 2005). When GA was given orally at a dose of 0.3 mmoL in Assam black tea (contained >93% of free form GA) to human, a maximum serum concentration of 2.08 µM was observed in plasma, whereas 39.6% of the
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GA dose were extracted in urine as a GA or GA metabolites (Shahrzad et al., 2001). The pharmacological safety and efficacy of GA makes it a potential compound for treatment or prevention of a wide variety of human diseases.
Chemoprevention, which refers to the administration of natural or synthetic agents to prevent initiation and promotion events associated with carcinogenesis, is being increasingly appreciated as an effective approach for the management of neoplasia (Ahmad et al., 2001). Many studies have shown a clear link between abnormal cell-cycle regulation and apoptosis with cancer, as much as the cell-cycle inhibitors and apoptosis-inducing agents are being appreciated as armaments for the management of cancer (Stewart et al., 2003; Schmitt, 2003; Hsu et al., 2003). Eukaryotic cell-cycle progression involves a sequential activation of cyclin-dependent kinases (CDKs) whose activation is dependent upon their association with cyclins (Youn et al., 2008). Progression through the mammalian mitotic cycle is controlled by multiple holoenzymes comprising a catalytic CDK and a cyclin regulatory subunit (Takahashi et al., 1999; Hseu et al., 2008b). These cyclin-CDK complexes are activated at specific intervals during the cell-cycle but can also be induced and regulated by exogenous factors. Cell-cycle progression is also regulated by the relative balance between the cellular concentrations of cyclins/CDKs and CDKs inhibitors, including p27KIP (Hseu et al., 2008b; Kim et al., 2006). The cyclin-CDK complexes are subjected to inhibition via binding with CDK inhibitors (Kim et al., 2006).
Recently, the relationship between cell-cycle arrest and cancer has been emphasized, with increasing evidence suggesting that the related processes of neoplastic transformation, progression and metastasis involve alteration of the normal cell-cycle regulation. Thus, anticancer (chemopreventive) agents may alter regulation of the cell-cycle machinery, resulting in an arrest of cells in different phases of the cell-cycle and, thereby, reducing growth and proliferation of cancerous cells, which may be useful in cancer therapy.
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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 (Yang et al., 2006a). In our previous study, we demonstrated that aqueous leaf extracts of T. sinensis (TS extracts, 10-75 g/mL) and gallic acid (3,4,5-trihydroxybenoic acid, 5-10 g/mL), a purified natural phenolic component, exhibited apoptosis against human premyelocytic leukemia (HL-60) cells (Yang et al., 2006a).
Notably, the significant inhibitory effects of tumor cell proliferation were observed only in leukemia HL-60 cells, whereas not in erythrocytes and human lymphocytes (Yang et al., 2006a). However, the effect of TS extracts against tumour cell-cycle regulation was poorly understood. Therefore, the present study aimed to investigate the anticancer effect of TS extracts and gallic acid in terms of tumor regression using in vitro cell culture model (HL-60 cells) or in vivo athymic nude mice model of leukemia cancer.
2. Materials and Methods 2.1. Chemicals
RPMI-1640 medium (Gibco BRL, Grand Island, NY), antibody against cyclin E, CDK2, cyclin B1, CDC2, caspase-8, Fas, FasL, VEGF, and β-actin (Santa Cruz Biotechnology Inc., Heidelberg, Germany) and antibody against cyclin D1, CDK4, cyclin A, p27KIP, p15, caspase-9, and Bid (Cell Signaling Technology Inc., Danvers, MA) were obtained from their respective suppliers. All other chemicals were of the reagent or HPLC grade supplied either by Merck (Darmstadt, Germany) or Sigma (St. Louis, MO).
2.2. Preparation of TS extracts
Leaves of T. sinensis were sourced from Fooyin University, Kaohsiung, Taiwan. A
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voucher specimen was characterized by Prof. Horng-Liang Lay, Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung County, Taiwan, and deposited at Fooyin University, Kaohsiung, Taiwan. The aqueous leaf extracts of T. sinensis (TS extracts) were prepared by adding 1000 mL of water to 1000 g of fresh T. sinensis leaves and boiled until 100 mL remained, as previously described (Chang et al., 2002; Hseu et al., 2008a). The crude extracts were centrifuged at 3000 × g for 12 min and the supernatant was used for this study. The crude extracts (50 g) were concentrated in a vacuum and freeze dried to form powder, with the stock subsequently stored at -20C for further analysis of its anticancer properties. The yield of TS extracts was 6%. The total phenolic content of the TS extracts was estimated to be 130
± 26 mg gallic acid (pyrocatechol) equivalents/g of plant extracts as described previously (Yang et al., 2006a).
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 high performance liquid chromatography (HPLC) 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 compounds (gallic acid, methyl gallate, ethyl gallate, kaempferol, kaempferol-3-O-β-D-glucoside, quercetin, quercitrin, quercetin-3-O-β-D-glucoside, and rutin) were isolated from the TS extracts. The identification of the compounds was fully characterized by comparison of their spectral data (IR, NMR, and mass) with the analogous information reported in the literature (Yang et al., 2006a; Hsu et al., 2003). Gallic acid, a natural phenolic component purified from TS extracts was subjected in this study at a yield of 6% (Yang et al., 2006a).
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2.4. Cell culture
Human acute promyeloblastic leukemia (HL-60) cell line was obtained from the American Type Culture Collection (ATCC, 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. Cultures were harvested and cell numbers were counted by hemocytometer.
2.5. Flow cytometry analysis
Cellular DNA content was determined by flow cytometric analysis of propidium iodide (PI)-labeled cells as described previously (Hseu et al., 2007). In brief, HL-60 cells (2 × 105 cells/mL) were cultured in 6 cm culture dishes. After treatment with TS extracts or gallic acid, cells were harvested, washed and suspended in PBS and fixed in ice-cold 70%
ethanol at -20 °C for overnight. After incubation, cells were re-suspended in PBS containing 1% Triton X-100, 0.5 mg/mL RNase, and 4 g/mL PI at 37 °C for 30 min. A FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) equipped with a single argon-ion laser (488 nm) was used for flow cytometric analysis. Forward and right-angle light scatter, which are correlated with the size of the cell and the cytoplasmic complexity, respectively, were used to establish size gates and exclude cellular debris from the analysis.
DNA content of 1 × 104 cells per analysis was monitored using the FACSCalibur system.
The cell-cycle was determined and analyzed using ModFit software (Verity Software House, Topsham, ME). Apoptotic nuclei were identified as a subploid DNA peak, and were distinguished from cell debris on the basis of forward light scatter and PI fluorescence.
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2.6. Protein isolation and immunoblot analysis
HL-60 cells (2 × 106 cells/ 10 cm dish) were washed once in cold PBS, and suspended in 100 L 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 was vortex and kept on ice for 20 min and then centrifuged at 15000 × g for 20 min at 4 °C. Total protein content was determined using Bio-Rad protein assay reagent, with bovine serum albumin (BSA) as the standard; protein extracts were reconstituted in sample buffer (0.062 M Tris-HCl, 2% SDS, 10% glycerol, and 5% β-mercaptoethanol), and the mixture was boiled at 94 °C for 5 min. Equal amounts (50 g) of the denatured proteins were loaded into each lane, separated by 10-15% SDS polyacrylamide gel, followed by transfer of the proteins to PVDF membranes overnight. Membranes were blocked with 0.1% Tween-20 in Tris-buffered saline containing 5% 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 anti-rabbit or anti-mouse antibody for 2 h before being developed by SuperSignal ULTRA chemiluminescence substrate (Pierce Biotechnology, Rockford, IL). For densitometry analysis band intensities were quantified by commercially available software AlpaEaseFc 4.0 (Genetic Technologies, Inc., Miami, FL).
2.7. Determination of VEGF release
To determine the effects of TS extracts on VEGF levels, HL-60 cells grown to 85%
confluence were treated with 0-75 g/mL of TS extracts for 6 h. Then, the medium was aspirated from the flasks and centrifuged at 500 × g for 10 min to remove cells from the medium. The level of VEGF released into the culture medium was estimated using commercially available VEGF ELISA kit (Chemicon International Inc., Temecula, CA).
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2.8. Animal experiments
Eight weeks old male or female athymic nude mice (BALB/c-nu were purchased from GlycoNex Inc., (Taipei, Taiwan) and were maintained in cage housing separately in a specifically designed pathogen-free isolation facility with a 12 h light and 12 h dark cycle;
the mice were provided rodent chow (Oriental Yeast Co, Tokyo, Japan) and water ad libitum. All experiments were conducted in accordance with the guidelines of the China Medical University Animal Ethics Research Board.
2.9. Tumor cell inoculation
HL-60 cells were grown in RPMI-1640 medium supplemented with 10% FBS, 2 mM glutamine, 1% penicillin-streptomycin-neomycin in a humidified incubator (5% CO2 in air at 37°C). Experiments were carried out using cells less than 15 passages. HL-60 cells (1 × 106 cells in 200 L matrix gel) were injected subcutaneously on the right hind flank of nude mice as described previously (Hseu et al., 2008b). Tumor volume, as determined by caliper measurements of tumor length, width and depth, were calculated using the formula:
length × width2 × 1/2 every 3 days (Collins et al., 2003). The two study groups received intraperitoneal injections of TS extracts (0.2 mL/mouse) dissolved in PBS buffer at 7.5 mg/kg and 10 mg/kg every 2 days, while the control group received vehicle only. After 21 days of treatment, the mice were sacrificed. The tumors were removed and weighed before fixing in 4% paraformaldehyde, sectioning and staining with hematoxylin-eosin for light microscopic analysis. Part of the tumor tissue was immediately frozen and the rest was fixed in 10% neutral-buffered formalin and embedded in paraffin. To monitor drug toxicity, the body weight of each animal was measured every 3 days. In addition, a pathologist examined the mouse organs, including liver, lungs and kidneys.
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2.10. Statistical analysis
The results of the in vitro and in vivo experiments are presented as mean and standard deviation (mean ± SD) or standard error (mean ± SE), respectively. All study data were analyzed using analysis of variance (ANOVA), followed by Dunnett’s test for pair-wise comparison. Statistical significance was defined as p <0.05 for all tests.
3. Results
This study has investigated the anticancer effect of aqueous leaf extracts of T. sinensis (0-75 g/mL) and gallic acid (0-10 g/mL) in vitro using HL-60 premyelocytic leukemia cell line or in vivo nude mice xenograft model. The crude TS extracts were prepared from fresh T. sinensis leaves, yielding 6% based on the initial weight of T. sinensis leaves and the total yield of gallic acid from the TS extracts was 6% (Yang et al., 2006a).
3.1. TS extracts induce G1 cell-cycle arrest in HL-60 cells
Flow cytometric analysis was used to obtain the profile of DNA content of the HL-60 cells treated with TS extracts to measure the fluorescence of PI-DNA complex. HL-60 cells with lower DNA staining relative to diploid analogs were considered apoptosis. A remarkable accumulation of subploid cells, the so-called sub-G1 peak, was noted in those treated with TS extracts (75 g/mL) for 0-18 h compared with the untreated group (Fig. 1).
Furthermore, the stage at which growth inhibition was induced by TS extracts in the HL-60 cell-cycle progression was determined, from cellular distribution in the different phases of post treatment. Fig. 1 showed that exposure of cells to the TS extracts resulted in a time-dependent progressive and sustained accumulation of cells in the G1 phase. Furthermore, in response to TS extracts the percentage of cells in the G1 phase was gradually increased
2 previously reported that TS extracts dose- and time-dependently inhibits the growth of HL-60 cells (Yang et al., 2006a). Consistent with our previous report, the current findings also suggest that TS extracts promote cell growth inhibition by inducing G1 transition phase arrest in HL-60 cells.
3.2. TS extracts down-regulate Cyclin D1, CDK4, Cyclin E, CDK2, and Cyclin A expression and up-regulates P27KIP expression
To examine the molecular mechanism(s) that may underlying changes in cell-cycle patterns, the effects of the TS extracts on various cyclins and cyclin-dependent kinases (CDKs) involved in cell-cycle control of the HL-60 cells were investigated. Our investigative approach was to treat the HL-60 cells with TS extracts (0-75 g/mL) for 0-6 h. Dose and time-dependent reduction in cyclin D1, CDK4, cyclin E, CDK2, and cyclin A expression were observed after treatment with TS extracts (Fig. 2). Moreover, the CDKs inhibitors.
3.3. Activation of Fas-associated apoptotic pathway by TS extracts
To assess whether TS extracts (0-75 g/mL for 0-6 h) promoted apoptosis via a death
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receptor-associated pathway, the Fas and Fas ligand (FasL) protein levels in HL-60 cells were determined by Western blotting. Results showed that TS extracts appreciably stimulate the expression of Fas and FasL in a dose- and time-independent manner (Fig. 3).
It is well understood that induction of Fas and FasL cleaves caspase-8 from procaspase-8, and the activated caspase-8 further stimulates caspase-3 via mitochondrial-dependent or – independent cascade (Nagata, 1997). Bear this in mind; furtherTherefore, we verified whether TS extracts augment caspase-8 cleavage in HL-60 cells. Western blot results showed that TS extracts dose-and time-dependently induced cleavage of caspase-8 from the procaspase-8 (Fig. 3). In mitochondrial pathway of apoptosis, caspase-8 proteolytically activates a pro-apoptotic protein Bid, which targets mitochondrial membrane permiabilization and represents the mail link between extrinsic and intrinsic apoptotic pathways (Eskes et al., 2000). Our results also showed that down-regulation of Bid induced by TS extracts occurred in a dose- and time-independent manner (Fig. 3). In addition, we observed TS extracts activates caspase-9, which was concomitant with our previous report that TS extracts induced apoptosis through the release of cytochrome c (Yang et al., 2006a). However, the signaling cascade still in debatemechanism is poorly understood. This data provided strong evidence that TS extracts-induced release of cyctochrome c further promotes apoptosome-mediated cleavage of caspace-9 from procaspase-9. With reference to our previous report, we assured that TS extracts-induced aberrant release of cytochrome c further amplified the cleavage of caspase-9 in HL-60 cells (Fig. 3).
3.4. Effect of catalase on TS extracts-induced cell-cycle arrest and apoptosis in HL-60 cells.
Our previous study demonstrated that catalase (H2O2 scavenger) significantly decreased
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T. sinensis-induced cytotoxicity, DNA fragmentation, and ROS generation in HL-60 cells (Yang et al., 2006a). Further to confirm this issue, in the present study we examined the effects of antioxidant catalase oncould effect TS extracts-induced cell-cycle arrest (cyclin D1, CDK4, cyclin E, CDK2, cyclin A, and p27KIP) and apoptosis (Fas/FasL, caspase-8, Bid, and caspase-9) in HL-60 cells. Cells were simultaneously treated with TS extracts (75
g/mL for 6 h) and catalase (10 U/mL) for indicated time period (Fig. 2 and 3). We found
that catalase treatment significantly reduced TS extracts-induced G1 arrest in HL-60 cells as evidenced by up-regulation of cell-cycle regulatory proteins including cyclin D1, CDK4, cyclin E, CDK2, cyclin A, and inhibits p27KIP. Furthermore, catalase treatment markedly down-regulates death signaling cascades and pro-apoptotic proteins Fas, FasL, caspase-8, Bid, and caspase-9 in HL-60 cells (Fig. 2 and 3). These results also provided a positive mechanism that TS extracts-induced HL-60 cell-cycle arrest (G1) and apoptosis was associated with the production of intracellular ROS, especially H2O2.
3.5. TS extracts induce down-regulation of VEGF in HL-60 cells
A number of studies have shown that VEGF is one the most important angiogenic factor closely associated with neovascularization in human tumors. Western blotting and ELISA assay were used to analyze the effects of TS extracts on the expression and release of angiogenic-related protein VEGF in HL-60 cells. As shown in Fig. 4A, treatment of HL-60 cells with TS extracts dose-dependently inhibits the expression of VEGF. In addition, control cells (without treatment) released detectable levels of VEGF into the serum-free culture media at approximately 27 pg/105 cells (Fig. 4B). A concomitant with protein level, TS extracts significantly inhibits VEGF release into culture media in a dose-dependent manner (Fig. 4B).
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3.6. Effect of TS extracts on tumor growth in HL-60 xenograft nude mice.
Nude mice were used to evaluate the in vivo effect of TS extracts on tumor growth. HL-60 cells were xenograft into nude mice as described in materials and methods. All the animals appeared healthy with no loss of body weight noted during treatment with TS
Nude mice were used to evaluate the in vivo effect of TS extracts on tumor growth. HL-60 cells were xenograft into nude mice as described in materials and methods. All the animals appeared healthy with no loss of body weight noted during treatment with TS