Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, Trypsin-EDTA, TRIzol reagent and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazol-carbocyanine iodide (JC-1), and caspase-3 and -9 colorimetric protease assays were purchased from Invitrogen (Carlsbad, CA, USA). SuperScript II reverse transcriptase was obtained from Promega (Madison, WI, USA). Antibodies for Bax and Bcl-2 were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), a fluorescein isothiocyanate (FITC)-conjugated Annexin V apoptosis detection kit was obtained from Becton-Dickinson (San Jose, CA, USA). All other chemicals, unless otherwise stated, were obtained from Sigma Chemicals (St. Louis, MO, USA).

The preparation of TARAP was performed as described (26). The roots of Rubus alceifolius Poir were collected from Anxi of Fujian Province, identified and authenticated by experts in our University, and the alkaloids were extracted. The herb powder (1 g) was extracted with 50 ml chloroform:methanol:ammonia solution (15:4:3) for 2 h in an ice bath, sonicated for 30 min, brought to room temperature and filtered. The filtered solution was collected and dessicated. The resultant residue was dissolved by 2 ml of 2% sulfuric acid solution and filtered. The filter paper and residue were re-washed with 2 ml of 2% sulfuric acid solution and with buffer solution (pH 3.6). Buffer was then added to make a final volume of 50 ml and the solution saved for future use. Acid dye colorimetry was used to measure total alkaloid content. Total alkaloid content was 0.81 mg of alkaloid per gram of initial herb powder.

Human hepatocellular carcinoma cell HepG2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were grown in DMEM containing 10% (v/v) FBS, and 100 U/ml penicillin and 100 μg/ml streptomycin in a 37°C humidified incubator with 5% CO₂. The cells were subcultured at 80–90% confluency.

HepG2 cells were grown in culture and then detached by trypsinization, washed and resuspended in serum-free DMEM. Six-week-old athymic BALB/c nu/nu male mice were given an s.c. injection of 4×10⁶ HepG2 cells mixed with Matrigel (1:1) in the right flank to initiate tumor growth. After 7 days of xenograft implantation when tumor size reached 3 mm in diameter, mice were randomly divided into two groups and gavaged with the following: i) control group (n=10), physiological saline (PS); and ii) TARAP group (n=10), 3 g/kg/d dose of TARAP in PS. All treatments were given 5 days a week for 21 days. Body weight and diet consumption were recorded twice weekly throughout the study. Tumor sizes were measured twice weekly and volume was calculated as reported recently (27). At the end of experiment, tumors were excised and weighed, and part of tumor was fixed in buffered formalin and the remaining was stored at −80°C for molecular analyses.

Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. HepG2 cells were seeded into 96-well plates at a density of 1×10⁵ cells/ml in 0.1 ml medium. The cells were treated with various concentrations of TARAP for 24, 48 and 72 h. Treatment with 0.5% DMSO was included as vehicle control. At the end of the treatment, 10 μl MTT [5 mg/ml in phosphate-buffered saline (PBS)] were added to each well, and the samples were incubated for an additional 4 h at 37°C. The purple-blue MTT formazan precipitate was dissolved in 100 μl DMSO. The absorbance was measured at 570 nm using an ELISA reader (BioTek, Model ELX800, Winooski, VT, USA).

HepG2 cells were seeded into 6-well plates at a density of 2×10⁵ cells/well in 2 ml medium. The cells were treated with various concentrations of TARAP for 48 h. Cell morphology was observed using a phase-contrast microscope (Olympus, Japan). The photographs were taken at a magnification of ×200.

The TUNEL reaction was carried out after treatment with TARAP as previously described (28). Briefly, sequential 4 μm tissue sections were adhered to silane-coated slides and allowed to dry at room temperature (RT). Subsequently, sections were deparaffinized and rehydrated. Protein digestion was done by incubating tissue sections in 20 mg/ml proteinase K (Worthington Co., Lakewood, CO, USA) for 15 min at RT. Endogenous peroxidase was inactivated with 2% H₂O₂ in distilled water (dH2O) for 5 min, RT. The labelling mixture containing biotinylated dUTP in TdT enzyme buffer was added to sections and incubated at 37°C in an humified chamber for 1 h. After stopping the enzymatic reaction, sections were rinsed with PBS, covered with anti-digoxigenin peroxidase conjugate and incubated for 30 min at RT in an humified chamber. Then, sections were incubated in TBS with 0.05% diaminobenzidine (DAB) plus 3% H₂O₂ until colour development was achieved. Finally, sections were washed, counterstained in haematoxylin, dehydrated and mounted with DPX (Panreac SA, Barcelona, Spain), and as a negative control active TdT buffer was replaced by the kit equilibration buffer.

After incubation with various concentrations of TARAP, apoptosis of HepG2 cells was determined by flow cytometry analysis using a fluorescence-activated cell sorting (FACS)Calibur (Becton-Dickinson) and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit. Staining was performed according to the manufacturer’s instructions. The percentage of cells in early apoptosis was calculated by Annexin V-positivity and PI-negativity, while the percentage of cells in late apoptosis was calculated by Annexin V-positivity and PI-positivity.

JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green to red, which thus can be used as an indicator of mitochondrial potential. In this experiment, 1×10⁶ treated HepG2 cells were resuspended after trypsinization in 1 ml of medium and incubated with 10 μg/ml of JC-1 at 37°C, 5% CO₂, for 30 min. Both red and green fluorescence emissions were analyzed by flow cytometry after JC-1 staining.

The activities of caspase-3 and -9 were determined by a colorimetric assay using the caspase-3 and -9 activation kits, following the manufacturer’s instructions. Briefly, after treatment with various concentrations of TARAP for 48 h, HepG2 cells were lysed with the lysis buffer provided by the manufacturer, for 30 min on ice. The lysed cells were centrifuged at 16,000 × g for 10 min. The protein concentration of the clarified supernatant was determined and 100 μg of the protein were incubated with 50 μl of the colorimetric tetrapeptides, Asp-Glu-Val-Asp (DEAD)-p-nitroaniline (pNA) (specific substrate of caspase-3) or Leu-Glu-His-Asp (LEHD)-pNA (specific substrate of caspase-9) at 37°C in the dark for 2 h. Samples were read at 405 nm in an ELISA plate reader (BioTek, Model ELX800). The data were normalized to the activity of the caspases in control cells (treated with 0.5% DMSO vehicle) and are presented as ‘fold of control’.

The expression of Bax and Bcl-2 genes were detected by RT-PCR. Total RNA was isolated with TRIzol Reagent. Oligo(dT)-primed RNA (1 μg) was reverse-transcribed with SuperScript II reverse transcriptase (Promega) according to the manufacturer’s instructions. The obtained cDNA was used to determine the mRNA amount of Bcl-2 or Bax by PCR. GAPDH was used as an internal control. The sequences of the primers used for amplification of Bcl-2, Bax and GAPDH transcripts are as follows: Bcl-2 forward: 5′-CAG CTG CAC CTG ACG CCC TT-3 and reverse: 5′-GCC TCC GTT ATC CTG GAT CC-3′; Bax forward: 5′-TGC TTC AGG GTT TCA TCC AGG-3′ and reverse: 5′-TGG CAA AGT AGA AAA GGG CGA-3′; GAPDH forward: 5′-GT CAT CCA TGA CAA CTT TGG-3′ and reverse: 5′-GA GCT TGA CAA AGT GGT CGT-3′.

Immunohistochemical staining for Bcl-2 and Bax was performed as previously (29). The sections were deparaffinised in xylene and hydrated through graded alcohols. Antigen unmasking was performed using heat treatment in a microwave oven at 750 W for 7 min in a container with 10 mM sodium citrate buffer, pH 6.0. Sections were allowed to cool in the buffer at room temperature for 30 min and were rinsed in deionised water three times for 2 min each. The endogenous peroxidase activity was blocked with 3% (v/v) hydrogen peroxide for 10 min. The sections were incubated with 1% bovine serum albumin in order to decrease non-specific staining and reduce endogenous peroxidase activity. The sections were then incubated with Bax or Bcl-2 antibody (all in 1:200 dilution, Santa Cruz Biotechnology Inc.) at 4°C overnight using a staining chamber. Primary antibodies were diluted (1:100) in PBS. After rinsing three times in PBS, sections were incubated in biotinylated goat anti-rabbit IgG (Boshide, Wuhan, China) followed by avidin-biotin-peroxidase complex (Vector). Immunostaining was visualized by incubation in 3,3-diaminobenzidine (DAB) as a chromogen. Sections were counterstained with haematoxylin. The Bax and Bcl-2 positive immunostainings were evaluated by the use of Nikon Eclipse 50i microscope (×40 objective). The evaluation of Bax and Bcl-2 expression was analysed in 6 different fields and the mean percentage of Bax or Bcl-2 positive staining was evaluated.

All data are the means of three determinations. The data were analyzed using the SPSS package for Windows (Version 11.5). Statistical analysis of the data was performed with Student’s t-test and ANOVA. Differences with P<0.05 were considered statistically significant.

The anticancer activity of TARAP in vivo was determined via examining tumor volume and weight in hepatocellular carcinoma (HCC) xenograft mice, whereas its side-effects were determined by measuring the body weight change. As shown in Fig. 1A, tumor volume per mouse was 1,900±167 mm³ or 1,370±98 mm³ in control or TARAP treated group, respectively, accounting for a 28% decrease in tumor volume (P<0.01). Consistently, TARAP treatment caused 39% decrease in tumor weight (P<0.01) compared with control (0.97±0.21 g or 0.59±0.16 g per mouse in control or TARAP-treated group). In contrast, TARAP treatment did not affect body weight gain. These findings together demonstrated the in vivo antitumor efficacy of TARAP against human HCC tumor xenograft in nude mice without any apparent sign of toxicity. To evaluate the in vitro antitumor activity of TARAP, we performed MTT assay to examine its effect on the viability of human hepatocellular carcinoma HepG2 cells. As shown in Fig. 2, treatment with 0.25–1.0 mg/ml of TARAP for 24, 48 or 72 h, respectively, reduced cell viability by 11–20, 26–53 or 39–65%, compared to untreated control cells (P<0.01), indicating that TARAP inhibits the growth of HepG2 cells in a dose- and time-dependent manner. To further verify these results, we evaluated the effect of TARAP on HepG2 cell morphology that represents the healthy status of cells in culture. As shown in Fig. 3, untreated HepG2 cells appeared as densely packed and disorganized multilayers, whereas, many of the TARAP-treated cells were rounded, shrunken and detached from adjacent cells adhering to the plate or floating in the medium. Taken together, these data demonstrate that TARAP inhibits the growth of HepG2 cells. Taken together, it is suggested that TARAP inhibits hepatocellular carcinoma growth both in vivo and in vitro, without apparent adverse effects.

Cell apoptosis in HCC tumor tissues was determined via immunohistochemical (IHC) staining for TUNEL. As shown in Fig. 4A, the percentage of TUNEL-positive cells in control or TARAP-treated mouse group was 22.23±6.13 or 83.83±12.24%, respectively, indicating that TARAP treatment significantly induced cell apoptosis in HCC tumors. The apoptosis of HepG2 cells was evaluated by FACS analysis with Annexin V/PI staining. As shown in Fig. 4B and C, the percentage of cells undergoing either early apoptosis or late apoptosis following treatment with 0, 0.25, 0.5, 0.75 and 1.0 mg/ml of TARAP was 9.18±1.32, 17.5±3.41, 21.92±3.49, 33.1±3.31 and 54.72±8.76%, respectively (P<0.05,). These data together demonstrate that TARAP promotes hepatocellular carcinoma cell apoptosis both in vivo and in vitro.

The effect of TARAP on the change of mitochondrial membrane potential in HepG2 cells was examined via JC-1 staining followed by FACS analysis. The membrane-permeant JC-1 dye displays potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (∼525 nm) to red (∼590 nm). Therefore, collapse of mitochondrial potentail during apoptosis is indicated by a decrease in the ratio of red/green fluorescence intensity. As shown in Fig. 5, after treatment with 0, 0.25, 0.5, 0.75 and 1.0 mg/ml of TARAP the JC-1 red/green fluorescent ratio in HepG2 cells was 9.08±1.896, 3.38±0.348, 2.69±0.275, 2.34±0.282 and 2.21±0.226, respectively, suggesting that TARAP dose-dependently induces the loss of mitochondrial membrane potential in hepatocellular carcinoma cells. To identify the downstream effectors in the apoptotic signaling pathway, the activation of caspase-9 and caspase-3 was examined by a colorimetric assay using specific chromophores, DEVD-pNA (specific substrate of caspase-3) and LEHD-pNA (specific substrate of caspase-9). As showed in Fig. 6, TARAP treatment significantly and dose-dependently induced activation of both caspase-9 and caspase-3 in HepG2 cells (P<0.05 vs. untreated control cells).

To further investigate the mechanism of TARAP’s pro-apoptotic activity, we performed RT-PCR and IHC analyses to, respectively, determined the mRNA or protein expression of Bcl-2 and Bax in HCC mice. As shown in Fig. 7A, TARAP treatment significantly reduced the mRNA expression of anti-apoptotic Bcl-2 in HCC tumors, whereas that of pro-apoptotic Bax was significantly increased after TARAP treatment. Consistently, results of IHC assay showed that the protein expression patterns of Bcl-2 and Bax were similar to their respective mRNA levels. The percentage of Bcl-2- or Bax-positive cells in control group was 45.6±2.67 or 51.67±12.37%, whereas that in TARAP-treated mice was 22.5±5.73 or 86±20.36% (Fig. 7B). Collectively, it is suggested that TARAP promotes hepatocellular carcinoma cell apoptosis via increasing the pro-apoptotic Bax/Bcl-2 ratio.