Kudingcha was purchased in Chongqing, China. The Kudingcha was stored at −80°C and freeze-dried to produce a powder. A 20-fold volume of boiling water was added to the powdered sample and extracted twice. The water extract was evaporated using a rotary evaporator (N-1100; Eywla, Tokyo, Japan), concentrated and then dissolved in dimethylsulfoxide (DMSO; Amresco, Solon, OH, USA) to adjust to the stock concentration (20%, w/v).

MCF-7 human breast adenocarcinoma cells obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) were used for the experiments. The cells were cultured in RPMI-1640 medium (Gibco Co., Birmingham, MI, USA) supplemented with 10% fetal bovine serum (FBS; Gibco Co.) and 1% penicillin-streptomycin (Gibco Co.) at 37°C in a humidified atmosphere containing 5% CO₂ (model, 311 S/N29035; Forma, Waltham, MA, USA). The medium was changed two or three times each week.

The anticancer effects of Kudingcha were assessed by MTT assay. The MCF-7 human breast adenocarcinoma cells were seeded in a 96-well plate at a density of 2×10⁴cells/ml in a volume of 180 μl per well. Kudingcha solutions (20 μl) with concentrations of 50, 100 and 200 μg/ml were added and then the cells were incubated at 37°C in 5% CO₂ for 48 h. An MTT solution (200 μl, 5 mg/ml; Amresco) was added and the cells were cultured for a further 4 h under the same conditions. Subsequent to removing the supernatant, 150 μl of DMSO was added per well and mixed for 30 min. Finally, the absorbance of each well was measured with an ELISA reader (model 680; Bio-Rad, Hercules, CA, USA) at 540 nm (12).

Total RNA was isolated from the MCF-7 human breast adenocarcinoma cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The RNA was digested with RNase-free DNase (Roche, Basel, Switzerland) for 15 min at 37°C and purified using an RNeasy kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. cDNA was synthesized from 2 μg total RNA by incubation at 37°C for l h with avian myeloblastosis virus (AMV) reverse transcriptase (GE Healthcare, Little Chalfont, Buckinghamshire, UK) with random hexanucleotides, according to the manufacturer’s instructions. The sequences of the primers that were used to specifically amplify the genes of interest are shown in Table I. Amplification was performed in a thermal cycler (Eppendorf, Hamburg, Germany). The PCR products were separated in 1.0% agarose gels and visualized using ethidium bromide (EtBr) staining (13).

A quantity of 26-M3.1 colon carcinoma cells were obtained from Professor Yoon at the Department of Food and Nutrition, Yuhan University, Bucheon, South Korea. These highly metastatic cells were maintained as monolayers in Eagle’s minimal essential medium (EMEM; Gibco Co.) supplemented with 7.5% FBS, a vitamin solution, sodium pyruvate, non-essential amino acids and L-glutamine (Gibco Co.). The cultures were maintained in a humidified atmosphere of 5% CO₂ at 37°C. Experimental lung metastasis was induced by injecting the colon 26-M3.1 cells into the lateral tail vein of 6-week-old female Balb/c mice (Experimental Animal Center of Chongqing Medical University, Chongqing, China) (14). Kudingcha solutions (400, 800 and 1,600 mg/kg) were subcutaneously injected into the mice and after 2 days the animals were intravenously inoculated with the 26-M-3.1 cells (2.5×10⁴/mouse). After 2 weeks the mice were sacrificed and their lungs were fixed in Bouin’s solution (saturated picric acid:formalin:acetic acid, 15:5:1; v/v/v). The rate of metastasis was assessed by counting the lung tumor colonies (tumors on the lung surface) as observed under the naked eye using a digital camera (Canon D550, Tokyo, Japan). The inhibitory rate of metastasis was assessed using the formula: Inhibitory rate = (Number of control mouse metastatic tumors − number of kudingcha mouse metastatic tumors) / number of control mouse metastatic tumors × 100. The protocol for these experiments was approved by the Animal Ethics Committee of Chongqing Medical University.

Data are presented as the mean ± SD. Differences between the mean values for individual groups were assessed using a one-way ANOVA with Duncan’s multiple range test. P<0.05 was considered to indicate a statistically significant difference. SAS version 9.1 (SAS Institute Inc., Cary, NC, USA) was used for statistical analysis.

The anti-cancer effects of Kudingcha on the MCF-7 cells were evaluated using an MTT assay. The growth inhibitory rates of the MCF-7 cells treated with the varying concentrations of Kudingcha are shown in Table II. When solutions of the Kudingcha were administered to the MCF-7 cells, the growth inhibitory rates observed at concentrations of 50, 100 and 200 μg/ml were 19, 58 and 81%, respectively (P<0.05). These results demonstrated that Kudingcha had marked anti-proliferative effects on the MCF-7 cells. In addition, it was observed that the higher the concentration of Kudingcha, the stronger the anticancer effects.

In order to determine a possible mechanism underlying the growth inhibitory activity of Kudingcha in the MCF-7 cancer cells, the induction of apoptosis was monitored. The extent of chromatin condensation was analyzed by fluorescence microscopy of cells stained with the DNA-binding fluorescent dye, 4,6-diamidino-2-phenylindole (DAPI). While the untreated MCF-7 cells presented nuclei with homogeneous chromatin distribution, treatment with Kudingcha induced chromatin condensation and nuclear fragmentation, suggesting the presence of apoptotic cells (Fig. 1).

To elucidate the mechanisms underlying the inhibition of cancer cell growth by Kudingcha, the expression of Bax, Bcl-2, and caspase-3 and -9 was measured in the MCF-7 cells by RT-PCR analysis following a 48-h incubation with the various concentrations of Kudingcha solution. As shown in Fig. 2, the expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 showed significant changes in the presence of 200 μg/ml Kudingcha. These results suggest that Kudingcha induced apoptosis in the MCF-7 cells via a Bax- and Bcl-2-dependent pathway. The mRNA expressions levels of caspase-3 and -9 were extremely low in the untreated control MCF-7 cells, but significantly increased once the cells were treated with 200 μg/ml Kudingcha. With increased concentrations of the Kudingcha treatment, the mRNA expression of caspase-9 and -3 was gradually elevated. More specifically, the apoptotic induction caused by Kudingcha was correlated with the upregulation of Bax, caspase-3 and caspase-9, and the down-regulation of Bcl-2 in terms of mRNA expression. The effects of 200 μg/ml Kudingcha were greater than those of the 100 and 50 μg/ml Kudingcha solutions.

The present study also determined whether the anticancer actions of Kudingcha were associated with the inhibition of NF-κB, IκB-α, iNOS and COX-2 gene expression. As shown in Fig. 3, the mRNA expression of NF-κB and IκB-α was reduced in the MCF-7 cells treated with 200 μg/ml Kudingcha solution. Kudingcha significantly modulated the expression of the genes associated with inflammation. The mRNA expression of NF-κB was decreased, while IκB-α mRNA expression levels were increased. Additionally, the mRNA expression of COX-2 and iNOS was gradually decreased in the presence of Kudingcha depending on the concentrations. These observations indicate that Kudingcha may help prevent cancer in the early stages by increasing anti-inflammatory activities. Overall, the results of this experiment demonstrated that a higher concentration of Kudingcha had a stronger anti-inflammatory effect on the human breast adenocarcinoma cells than lower concentration solutions.

The prophylactic inhibition of tumor metastasis by Kudingcha was evaluated using an experimental mouse metastasis model (Table III). All Kudingcha-treated mice had significantly fewer lung metastatic colonies than those of the control mice (number of metastatic tumors, 57±6, n=10; P<0.05). Kudingcha was most effective at inhibiting lung metastasis at a concentration of 1600 mg/kg. This concentration (inhibitory rate, 33.3%; number of metastatic tumors, 38±6) inhibited tumor formation and lung metastasis to a greater degree than the 800 mg/kg (inhibitory rate, 22.8%; number of metastatic tumors, 44±6) and 4000 mg/kg solutions (inhibitory rate, 8.8%; number of metastatic tumors, 52±6).