Puerarin (P5555) was obtained from Sigma-Aldrich (St. Louis, MO, USA) with a purity of 98%, as assessed by reverse-phase high-performance liquid chromatography. The puerarin was stored as a 100-mM stock solution in dimethyl sulfoxide at −20°C and diluted with serum-free culture medium for use in experiments. The compound 5-FU was obtained from Sigma-Aldrich (F6627) and diluted to various concentrations in serum-free culture medium. The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies, Inc. (Kumamato, Japan).

The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit was obtained from Beijing MultiSciences Biotech Co., Ltd (Beijing, China) and the Hoechst staining kit was obtained from the Beyotime Institute of Biotechnology (Shanghai, China).

The HCC SMMC7721 cell line was obtained from the China Center for Type Culture Collection (Wuhan, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL), 50 mg/ml streptomycin (Beyotime Institute of Biotechnology), 50 IU/ml penicillin (Beyotime Institute of Biotechnology), and 2 mM glutamine (Beyotime Institute of Biotechnology) in a humidified atmosphere of 5% CO₂ at 37°C.

The inhibitory effect of puerarin on SMMC7721 cell growth in vitro was determined using CCK-8 dye, which is only absorbed by living cells. Briefly, 5,000 cells/well were seeded into 96-well microtiter plates. Following exposure to puerarin (400, 800, 1,600, 3,200 or 6,400 μM), 5-FU (40, 80, 160, 320 or 640 μM) or puerarin and 5-FU (400:40; 800:80; 1600:60; 3200:320 μM, respectively) for 48 h, 10 μl CCK-8 solution was added to each well, and the plates were incubated for an additional 2 h (at 37°C with 5% CO₂). The optical density was then determined at a wavelength of 450 nm using a microplate reader (iMark; Bio-Rad, Hercules, CA, USA). Each experiment was performed in triplicate and the results were presented as the inhibition rate (IR), which was calculated using the following formula: IR (%) = [(A-B)/A] × 100, where A and B are the absorbance of the control and sample groups following 48 h of incubation, respectively.

The following equation (18,19) was used to evaluate the nature of the interaction between puerarin and 5-FU: D = D_m[fa/(1 - fa)]^1/m, where D is the dose, D_m is the dose required to produce the median effect (analogous to the IC₅₀), fa is the fraction of the system affected by D, and m is a Hill-type coefficient signifying the sigmoidicity of the dose-effect curve. The combination index (CI) values were obtained using Biosoft CalcuSyn software (Biosoft, Cambridge, UK) written in BASIC for automatic graphing of the CI with respect to fa, to determine whether the two drugs were non-exclusive. Synergism was indicated by a CI value of <1, summation by a CI value equal to 1 and antagonism by a CI value of >1.

Apoptotic cells were detected by Hoechst 33258 staining as follows. Following exposure to puerarin (1,600 μM), 5-FU (160 μM) or puerarin and 5-FU for 48 h, the SMMC7721 cells were incubated with 20 μM Hoechst 33258 (Beyotime Institute of Biotechnology) for 10 min at room temperature. The cells were then washed twice with phosphate-buffered saline (PBS) and examined under a fluorescence microscope (BX53F; Olympus, Tokoyo, Japan). The apoptotic cells were identified by Hoechst 33258 staining of the condensed chromatin and nuclear fragments. A total of 10 random fields were counted for each sample.

An Annexin V/PI apoptosis kit was used according to the manufacturer’s instructions to quantify the percentage of cells undergoing apoptosis. Firstly, SMMC7721 cells were incubated for 48 h with puerarin (1,600 μM) or 5-FU (160 μM) alone or in combination. Next, the cells were washed twice with cold PBS and resuspended in binding buffer at a concentration of 1×10⁶ cells/μl. Then, 5 μl of Annexin V-FITC and 10 μl PI were added, and the cells were incubated for 5 min at room temperature in the dark. Following incubation, 200 μl of binding buffer was added and the cells were analyzed immediately by flow cytometry (FACSAriaIII; BD Biosciences, Franklin Lakes, NJ, USA). The flow cytometry analysis was performed using the Cell Quest software (BD Biosciences). The Annexin V⁺/PI⁻ cells were identified as apoptotic cells, and the Annexin V⁻/PI⁺ cells were identified as necrotic cells (20). The entire procedure was repeated three times for each sample.

All animal procedures, which complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (21), were approved by the Committee on Animal Experimentation of Wuhan University (Wuhan, China). Four- to six-week-old male BALB/c-nu/nu nude mice were purchased from the Center for Experimental Animals of Wuhan University (Wuhan, China). All mice weighed 16–18 g and were bred in autoclaved, filter-top, microisolator cages, which were kept in an isolator unit with filtered air. The mice had access to water and food ad libitum. The human HCC SMMC7721 cell line used for inoculation was cultured as previously described. The mice were inoculated subcutaneously with 1×10⁷ SMMC7721 cells per mouse and the tumor sizes were measured using micrometer calipers. The mice were randomly divided into the following four groups, with six mice per group: Saline tumor control, puerarin (50 mg/kg/day), 5-FU (12 mg/kg/day), and puerarin (50 mg/kg/day) in combination with 5-FU (12 mg/kg/day).

The tumor volume (TV) was calculated using the following formula: TV (mm³) = d²x(D/2), where d and D are the shortest and longest diameters, respectively. The tumor size was measured with calipers every three days. The mice were sacrificed by extracting the eyeball at the end of four weeks and the tumor xenografts were removed, weighed and measured for additional analyses.

Sections of each tumor xenograft were fixed in 4% formaldehyde, dehydrated with an ethanol gradient, embedded in paraffin, dewaxed and rehydrated with a decreasing ethanol gradient (100, 95, 90, 80 and 70%), according to standard instructions. An in situ apoptosis detection kit (Roche Molecular Systems Inc., Branchburg, NJ, USA) was used to detect apoptosis. All procedures were performed according to the manufacturer’s instructions. The specimens were incubated with proteinase K [15 μg/ml in 10 mM Tris/HCl (pH 7.5)] for 20 min at room temperature after being dewaxed and rehydrated. Next, the specimens were rinsed with 3% H₂O₂, and incubated with equilibration buffer and terminal deoxynucleotidyl transferase (Beyotime Institute of Biotechnology). The specimens were then incubated with an anti-digoxigenin-peroxidase conjugate. Finally, the 3,3′-diaminobenzidine substrate was added to react with the peroxidase and the specimens were counterstained with hematoxylin, mounted and analyzed using a light microscope (BX53F).

Following the completion of the study, blood was collected from the nude mice by cardiac puncture using heparin-rinsed 5-ml syringes and 22-gauge needles. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and serum creatinine (Cr) levels were measured to evaluate liver and renal injury using an Olympus AU5400 Immuno Analyzer (Olympus). All mice were sacrificed and dissected following the completion of the study. In addition, metastasis, hemorrhage and liver and kidney morphology were assessed by an observer blinded to the treatment groups. This study was approved by the Institutional Review Board of Renmin Hospital at Wuhan University.

Statistical analyses were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). All data are presented as the mean ± standard deviation (SD). The means of the different groups were compared using non-parametric analysis (Mann-Whitney rank-sum test) and P<0.05 was considered to indicate a statistically significant difference.

The inhibition of SMMC7721 proliferation by puerarin and 5-FU was assessed following 48 h of drug exposure and 24 h of culture in drug-free medium. After 48 h of treatment, in vitro SMMC7721 cell growth was significantly inhibited in a dose-dependent manner (P<0.01; Fig. 1). The mean ± SD rate of inhibition by puerarin was 19.51±0.72% at a concentration of 400 μM, and 95.56±0.81% at a concentration of 6,400 μM. The rate of growth inhibition by 5-FU at 40 μM was 38.83±0.63%, while the rate was 75.91±3.54% at 640 μM.

The SMMC7721 cells were exposed to the two drugs in combination at a fixed molar ratio (puerarin to 5-FU, 10:1) for 48 h. The effect on cell proliferation was then assessed by CCK-8 assay and the rate of growth inhibition was calculated according to the method used by Chou and Talalay (18,19). All experiments were repeated in triplicate. The CI values were <1 when the fraction of affected cells was between 0.2555 and 0.7420 (Fig. 2).

Morphological changes in the SMMC7721 cells indicating apoptosis were detected by Hoechst 33258 staining. The classical characteristics of apoptotic cells are nuclear fragmentation and chromatin condensation. Distinct chromatin condensation and nuclear fragmentation were identified in the treatment groups, while the nuclei of the control cells stained a weak homogeneous blue (Fig. 3).

An Annexin V/PI apoptosis kit was used to quantify the percentage of cells undergoing apoptosis. The proportion of Annexin V-positive/PI-negative cells increased progressively over 48 h in the SMMC7721 cells incubated with low concentrations of puerarin (1,600 μM) and 5-FU (160 μM) (Fig. 4). The cells treated with puerarin or 5-FU demonstrated a significantly higher rate of apoptosis when compared with that of the control group (P<0.01; Fig. 4), and the rate of apoptosis was significantly higher in the combined treatment group when compared with that of the individual treatment groups (P<0.01; Fig. 4).

The effect of puerarin and 5-FU on the growth of primary tumor xenografts was investigated in nude mice. None of the mice succumbed to the disease during the study, and 18 mice successfully grew tumor xenografts. The mice were randomly divided into three groups as previously described, and a saline-only control group was included. No significant differences were identified in the tumor sizes among the four groups at the start of treatment. The mice were treated with puerarin, 5-FU, puerarin and 5-FU in combination, or saline only. Puerarin or 5-FU alone, as well as the combined treatment with the two drugs, significantly inhibited the SMMC7721 tumor growth rate (as determined by tumor volume and weight) when compared with that of the control group (P<0.05; Tables I and II).

The TUNEL assay demonstrated that significant cell death had occurred in the tumor masses from the treatment groups when compared with that of the saline-only control group (P<0.01; Fig. 5); however, the degree of apoptosis differed in each group. The tumors from the combined treatment group demonstrated a significantly increased level of apoptosis when compared with that of the puerarin and 5-FU groups (P<0.01; Fig. 5).

At the end of the study, blood was collected from the mice by cardiac puncture using heparin-rinsed 5-ml syringes and 22-gauge needles. The ALT, AST, BUN and Cr levels were measured to evaluate liver and renal injury (22). Serum ALT, AST, BUN and Cr levels were not identified to be significantly elevated in the treated mice when compared with those of the control group (P>0.05, Table III), and no significant difference in the levels of these biomarkers was identified between the puerarin, 5-FU and combined treatment groups (P>0.05, Table III). All mice were sacrificed and dissected following completion of the study. In addition, no liver or kidney injury, obvious metastasis or hemorrhage was observed. Furthermore, no significant difference was identified in liver or kidney weight between the treated groups and the control group, and no evident lesions were observed by histopathological examination.