Animal experiments

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 P27^KIP 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). Therefore, 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 mechanism 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 T. sinensis-induced cytotoxicity, DNA fragmentation, and ROS generation in HL-60 cells

2 antioxidant catalase could effect TS extracts-induced cell-cycle arrest (cyclin D1, CDK4, cyclin E, CDK2, cyclin A, and p27^KIP) 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 p27^KIP. 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.