Puerarin was obtained from Sigma-Aldrich (P5555; Sigma-Aldrich Co. LLC, Shanghai, China). The puerarin stock solution was prepared at 100 mM in serum-free culture medium and stored at −20°C. 5-FU (F6627; Sigma-Aldrich), was dissolved in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Gaithersburg, MD, USA) to yield a 20 mM stock solution, which was stored at −20°C. The Eca-109 esophageal cancer cell line was obtained from the China Center for Type Culture Collection (Wuhan, China). The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (100 μg/ml streptomycin and 100 IU penicillin; Genom, Hangzhou, China) in a humidified incubator at 37°C under 5% CO₂.

The inhibitive effects of puerarin and 5-FU on the in vitro growth of Eca-109 cells were determined using the cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kunamoto, Japan). The cells (5×10³ cells per well) were seeded in 96-well microtitre plates. Following exposure to puerarin (400, 800, 1,600, 3,200, 6,400 μM), 5-FU (40, 80, 160, 320, 640 μM) or puerarin + 5-FU for 48 h, 10 μl CCK-8 solution [5 mg/ml in phosphate-buffered saline (PBS)] was added to each well and the plates were incubated for an additional 2 h at 37°C. The CCK-8 solution in the medium was removed and the optical density at 450 nm was determined by an iMark Microplate Absorbance Reader (Bio-Rad, Hercules, CA, USA). Each assay was performed in triplicate. The results are expressed as the inhibition rate (IR): IR = (A×B)/A×100%, where A and B are the absorbance of the control and sample groups, respectively, after 48 h of incubation.

Apoptotic cells were detected by Hoechst 33258 staining following the manufacturer’s instructions (C0003; Beyotime Institute of Biotechnology, Shanghai, China). The cells were cultured in DMEM for 12 h following seeding in a sterile six-well plate, and then fixed in 0.5 ml methanol for 30 min and washed with PBS twice. 0.5 ml Hoechst 33258 reagent was used to stain the apoptotic cells in the dark at room temperature for 5 min following exposure to puerarin (160 μM), 5-FU (160 μM) alone or puerarin and 5-FU combined for 48 h. The cells were then rinsed with PBS twice. Apoptotic cells were identified on the basis of morphological changes in the nuclear assembly by observing chromatin condensation and fragment staining using Hoechst 33258. The stained cells were examined and photographed under a fluorescence microscope (Olympus 1X71; Olympus Corporation, Tokyo, Japan) at an excitation wavelength of 330–380 nm. In each group, ten microscopic fields were selected randomly and the cells were counted.

To quantify the percentage of cells undergoing apoptosis, the Annexin V-fluorescein isothiocyanate (FITC) kit (Multi-Sciences Biotechnology Co., Ltd., Hangzhou, China) was used according to the manufacturer’s instructions. Briefly, cells were incubated for either 24 h or 48 h with puerarin and 5-FU alone or in combination. The cells were then washed twice with cold PBS and resuspended in fluorescein isothiocyanate conjugated annexin V binding buffer at a concentration of 1×10⁶ cells/ml. Following incubation, 100 μl solution was transferred to a 5-ml culture tube, and 5 μl Annexin V-FITC and 10 μl PI were added. The tube was gently centrifuged at 1,000 × g and incubated for 15 min at room temperature in the dark. At the end of the incubation, 400 μl binding buffer was added and the cells were analyzed immediately by flow cytometry (FACSAria™; Beckman Coulter, Inc., Fullerton, CA, USA). Flow cytometric analysis was performed using CellQuest^™ software (BD Biosciences, Franklin Lakes, NJ, USA).

All procedures were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the study was approved by the Ethics Committee for Animal Research of Wuhan University, China. Male BALB/c nude mice, aged 5–6 weeks, were purchased from the Center for Animal Experiments of Wuhan University (Wuhan, China). The mice weighed 16–18 g at the beginning of the experiment. The mice were maintained in autoclaved filter-top micro-isolator cages with autoclaved water and sterile food provided ad libitum. The cages were maintained in an isolator unit provided with filtered air. Tumor cells used for inoculation were grown in culture and harvested as described above. A total of 24 mice were inoculated subcutaneously with injections of 1×10⁷ cells/mouse; a further six mice inoculated with saline acted as a control group. Tumor sizes were determined using micrometer calipers and mice with similarly sized tumors were randomly divided into four groups (with six mice/group): Saline control group; puerarin 25 mg/kg/day group; 5-FU 12 mg/kg/day group; and puerarin + 5-FU combination group. Following 3 weeks, all mice were sacrificed by spinal dislocation and the xenograft tumors were removed and measured. Tumor volume (TV) was calculated using the following formula: TV (mm³) = d²xD/2, where d and D signify the shortest and the longest diameters, respectively.

For histological examination, the tumor tissues were fixed in 10% buffered formalin and embedded in paraffin, and 4 μm tissue sections were prepared. The TUNEL assay was performed with an in situ apoptosis detection kit (Roche, Branchburg, NJ, USA) according to the manufacturer’s instructions. Positive cells were identified, counted (six random fields per slide) and analyzed by an Olympus-BX53 upright fluorescence microscope (Olympus).

The livers and kidneys of the mice from the different groups were fixed in 10% buffered formalin, and the preserved tissues were cleaned in running water, processed for histological examination according to the conventional methods and stained with haematoxylin and eosin. The morphology of any lesions observed was classified and registered by a skilled histologist who was blinded to the treatment groups. Blood was collected by cardiac puncture using heparin-rinsed 1-ml syringes (20 gauge needles). The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and serum creatinine (Cr), biomarkers of liver and renal injury were detected by a Beckman 700 spectrophotometer (Beckman-Coulter, Chicago, IL, USA).

Data were subjected to non-parametric analysis using the Mann-Whitney rank sum test. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).

The inhibition of proliferation by puerarin and 5-FU in the Eca-109 cells was assessed after 48 h of drug exposure, following 24 h culture in drug-free medium. As shown in Fig. 1, after 48 h of treatment, growth of the Eca-109 cells was significantly inhibited in a dose-dependent manner (P<0.01). The mean [± standard deviation (SD)] inhibition rate was 6.56±0.04% at 400 μM puerarin and 90.76±0.83% at 6,400 μM. 5-FU at 40 μM exhibited an inhibition rate of 7.10±0.06%, while the rate at 640 μM was 76.56±4.71%. In addition, the effect of puerarin and 5-FU combined was higher than that of puerarin or 5-FU alone and the difference was identified to be statistically significant (P<0.05). This indicated that puerarin and 5-FU exhibited a synergistic effect on inhibiting the proliferation of Eca-109 cells.

Apoptosis induced by puerarin and 5-FU was confirmed using Annexin V/PI staining to detect externalization of phosphatidylserine on the cell membrane. As shown in Fig. 2, the proportion of Annexin V-positive/PI-negative cells increased progressively in Eca-109 cells incubated at low concentrations of puerarin (400 μM) and/or 5-FU (40 μM) for 48 h. Puerarin and 5-FU alone significantly promoted apoptosis compared with the control group (P<0.05), although the combined effects of the two drugs were greater than the effects of puerarin or 5-FU alone (P<0.05).

Hoechst 33258 staining was used to detect the morphological features of apoptotic cells induced by puerarin and 5-FU in vitro, which revealed that apoptotic bodies containing nuclear fragments were generated in apoptotic cells. The mean (±SD) of apoptotic cells in the control, puerarin, 5-FU and combined group were 3.67±2.16, 51.67±8.45, 55.17±10.17 and 83.0±9.21, respectively. Therefore, the percentages of apoptotic cells induced by either puerarin and 5-FU alone or the two drugs combined were significantly elevated compared with that in the control group (P<0.05, Fig. 3). Furthermore, the apoptotic rate in the combined treatment group was significantly greater than that of either respective treatment alone (P<0.05, Fig. 3).

Following the investigation of apoptosis in Eca-109 cells in vitro, the antitumor effect of puerarin and 5-FU was evaluated in xenograft tumor mouse models. None of the mice died over the course of treatment and all 24 mice successfully developed tumor xenografts. On day 14, the tumor xenografts reached a mean size of 216.53±32.29 mm³. The 24 mice were randomly divided into four groups as described above. No statistically significant differences were detected among the sizes of the tumors in the different groups. Subsequently, the mice were administered the different treatments. The results revealed that puerarin and 5-FU administered either in combination or individually exhibited significant inhibitory effects in vivo, with tumor volumes and weights in the mice in these groups all significantly reduced as compared with the saline control group (P<0.05, Table I), and the average tumor volume in the combination group was significantly lower than that in either the puerarin or the 5-FU group (P<0.05, Table I). The mean (±SD) tumor volume in the control group was 1,015.26±108.88 mm³ at the end of the experiment, and the tumor inhibition rate of puerarin combined with 5-FU was 89.06%, whereas the inhibition rates of puerarin and 5-FU alone were 76.93 and 72.21%, respectively (Table I). The mean tumor weights in the different groups are shown in Table I; the inhibition rates were 87.48, 65.93 and 61.81%, for the combined treatment, puerarin only and 5-FU only groups, respectively. These results demonstrated that the antitumor effect of puerarin combined with 5-FU was superior to the effects of the drugs when used individually.

Tumor tissues isolated from the xenograft mice of the four groups were assessed using the TUNEL assay; representative micrographs are shown in Fig. 4. The tumors derived from combination-treated mice exhibited a markedly higher count of apoptotic bodies compared with the control tumors, suggesting that puerarin potentiates the activity of 5-FU, which includes the suppression of cellular viability and increased apoptosis of tumor cells in vivo.

At the end of the experiment, the nude mice were necropsied. No clear metastasis, peptic ulcer and haemorrhage, or injury to the liver and kidney was visible to the naked eye.

Hepatic toxicity was monitored by quantitative analysis of the ALT and AST expression levels that served as biochemical markers of liver injury (20). The hepatic toxicity induced by different treatments is shown in Table II. ALT and AST activities in the serum were not significantly elevated compared with the control group (P>0.05), and no differences between the combination, puerarin and 5-FU groups compared with the control groups were identified (P>0.05). No observable gross or histological changes were observed in the livers of either the treated groups or the control groups; similar results were obtained for renal injury.