The human SMMC-7721 HCC cell line was obtained from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (Gibco Life Technologies, Carlsbad, CA, USA) in a humidified atmosphere of 5% CO₂ at 37°C.

NC (98% purity) was purchased from the National Institute of Food and Drug Control (Beijing, China). NC was prepared as a 10 mM stock solution in dimethyl sulfoxide (DMSO) and stored in aliquots at 4°C prior to use. Cytoxan [cyclophosphamide, (CTX)] was obtained from the Jiangsu Hengrui Medicine Co., Ltd. (China). CTX was prepared in 0.9% sterile sodium chloride solution at a concentration of 20 mg/ml.

The antiproliferative effect of NC on SMMC-7721 cells was evaluated by determining the IC₅₀ value, which is defined as the concentration required for 50% inhibition of cell growth. Logarithmically growing cells were seeded at a density of 1–5×10⁴ cells/well in 96-well plates (Corning Inc., Corning, NY, USA) and allowed to adhere for 12 h at 37°C. The medium was then replaced with fresh medium containing various concentrations of NC. The cells were maintained for 2 days, and the antiproliferative activity of NC was determined using MTT (Amresco, Solon, OH, USA). MTT (10 μl) (5 mg/ml stock) was added to each well for 4 h, the seeding medium was discarded and 0.15 ml DMSO was added to each well. Absorbance was read at 570 nm. The cell inhibition ratio was determined as: (1 − A_{treated group}/A_{control group}) × 100.

For the morphological examination, the cells were cultured in 24-well plates. After 24-h treatment with NC, the cells were observed and photographed using a Leica DMI3000B inverted microscope (Leica, Wetzlar, Germany).

Cells were counted, plated in triplicate at 1×10³ cells/well in 6-well plates and allowed to adhere for 12 h. The cells were then cultured in culture medium containing various concentrations of NC for 10 days. After most of the colonies expanded to ~50 cells, they were washed twice with phosphate-buffered saline (PBS) and fixed in methanol for 15 min. The plates were photographed, and colonies >50 cells were counted using Quantity One 4.6.2 (Bio-Rad, Hercules, CA, USA). At least three independent experiments were carried out for each assay.

After exposure to NC, the cells were fixed in ice-cold 2.5% glutaraldahyde and preserved at 4°C for further processing. The cells were then post fixed in 1% osmium tetroxide in 2.5% glutaraldahyde, dehydrated in graded alcohol, embedded in Epon 812, sectioned with an ultramicrotome, and stained with uranyl acetate and lead citrate. The sections were examined with a transmission electron microscope (JEM-1200EX, JEOL Ltd., Tokyo, Japan).

Apoptotic nuclear morphology was assessed by acridine orange (AO) staining (Sigma, St. Louis, MO, USA). Cells treated with 0, 0.5 or 1.0 μg/ml NC for 48 h were collected, washed with PBS and then fixed with 95% ethanol for 15 min. After being acidified with 1% acetic acid for 30 sec, the coverslips were dyed with 0.1 g/l AO for 10 min. The slips were differentiated with 0.1 mol/l CaCl₂ for 2 min, washed with PBS three times, and the nuclear morphology was examined under a fluorescent microscope (Leica).

The percentage of cells undergoing apoptotic cell death induced by NC was measured by flow cytometry using the Annexin V-FITC/PI kit (Nanjing KeyGen Biotech Co., Ltd., China) according to the manufacturer’s instructions. Cells treated with NC for 24 h were collected and washed twice with PBS. The cells were resuspended in 500 μl binding buffer, and then 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI) were added into the cell suspension, followed by gentle vortexing. The stained samples were incubated for 15 min at room temperature in the dark and then analyzed using a Beckman Coulter FACSCalibur-500 flow cytometer within 1 h. Events (100,000) were obtained using the green channel FL1 for Annexin V-FITC and the red channel FL3 for PI.

The Caspase-3 Activity Assay kit (Beyotime Institute of Biotechnology, Jiangsu, China) was used for the detection of caspase-3 activity according to the manufacturer’s instructions. Briefly, control and treated cells were collected, washed with ice-cold PBS, and resuspended in 50 μl of chilled cell lysis buffer for 15 min on ice. Lysates were clarified by centrifugation (20,000 × g, 10 min, 4°C) and assayed immediately.

Cells were cultured to the logarithmic growth phase, and the cell numbers were adjusted to 1×10⁴/ml after trypsinization. Six-well plates were seeded with 1×10⁴ cells/well and cultured for 12 h. After treatment with NC (2.0 μg/ml) for 24 h, immunohistochemical staining was performed to detect Bax and Bcl-2 protein. Briefly, the activity of endogenous peroxidase in the samples was quenched by incubation in 3% hydrogen peroxide solution for 10 min. The coverslips were then incubated with antibody against Bax and Bcl-2 (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) at 4°C overnight. The staining was completed by incubation with substrate-chromogen 3,3′-diaminobenzidine (DAB; Zhongshan Golden Bridge Biotechnology Co., Ltd). The coverslips were then stained, dehydrated and mounted. Negative control slides were prepared by replacing the primary antibody with PBS. Five different zones of each slide were selected at random for microscopic visualization, and the positive cell numbers and average optical density (A_OD), defined as the ratio of the positive target optical density and the positive area percentage (I_OD/Area), were analyzed using a pathological image analysis system (Leica DMR + Q550).

After exposure of SMMC-7721 cells to NC, the concentrations of p53 and p21 in cell supernatants were determined using the human p53 and human p21 ELISA kits (Shanghai XINUO Biotechnology Co., Ltd., Shanghai, China), according to the manufacturer’s instructions. Optical density was read using a microplate reader (Bio-Rad) at 450 nm. A standard curve was generated by correlating the original concentrations of the targeted factor and the corresponding optical densities. The concentrations of p53 and p21 in samples were calculated according to the standard curve.

Primers for quantitative PCR (qPCR) were designed and synthesized by Sangon (Shanghai, China) (Table I). Total RNA was isolated from cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). cDNA was synthesized from the isolated RNA using the RevertAid™ First Strand cDNA Synthesis kit (Tiangen Biotech Co., Ltd., Beijing, China). Reverse transcription was performed using oligo(dT) primers at 25°C for 10 min, 42°C for 60 min, and then 70°C for 10 min. qPCR was performed using a SYBR-Green qPCR kit [Tiangen Biotech (Beijing) Co., Ltd.] according to the manufacturer’s instructions in an iCycler iQ 7500 real-time PCR system (Bio-Rad) for 35 cycles. A gene expression normalization factor was calculated for each sample based on the geometric mean of β-actin as a user-defined housekeeping gene.

Four- to 6-week female BALB/c nude mice (weighing 18–20 g) were provided by the Experimental Animal Center of Guangxi Medical University (Nanning, China). The mice were bred and maintained at our institute under specific pathogen-free conditions, with continuous free access to sterilized food and autoclaved water under a controlled 12 h light/dark cycle at 22–25°C with 50% relative humidity. All the animal procedures were approved by the Animal Ethics Committee of Guangxi Medical University.

Immunodeficient BALB/c nude mice were randomized and then subcutaneously implanted with 0.2 ml SMMC-7721 cells at a density of 1×10⁷ cells/ml into the right axilla. Nude mice bearing tumors of 150–300 mm³ were selected for experimentation. Mice bearing human SMMC-7721 HCCs were randomized into the NC group (10 mg/kg NC/day), the CTX group (20 mg/kg cytoxan/day) and the control normal saline group (0.2 ml saline/day). Drug administration (i.p. injection once per day for 14 days) was commenced on the day after tumor implantation. The tumor dimensions were measured at 7-day intervals using a caliper for length and width. Tumor size was then calculated as described previously (21) using the equation:

where W is the width and L is the length. After 14 days of treatment, the animals were euthanized and the tumors were collected.

Apoptosis in tumor tissue sections was determined using the In Situ Cell Death Detection kit (Roche, Bromma, Sweden). Briefly, tumor tissue sections of formalin-fixed, paraffin-embedded specimens were dewaxed in xylene and rehydrated in a graded series of ethanol. The tumor samples were incubated with proteinase K (2 mg/ml), and TUNEL staining was performed according to the manufacturer’s instructions.

Endogenous levels of p53 were depleted by RNA interference-mediated suppression using the following double-stranded RNA oligonucleotides: forward, 5′-CUA CUUCCUGAAAACAACGTT-3′ and reverse, 5′-CGUUGU UUUCAGGAAGUAGTT-3 for human p53 mRNA (GenBank accession no. AB082923.1). Short hairpin interfering RNA (shRNA) molecules that target the same sequences of human p53 were cloned into the eukaryotic expression vector pGPU6/GFP/neo, which was purchased from GeneChem (Shanghai, China). Additionally, a pGPU6/GFP/neo plasmid carrying an shRNA targeting luciferase (target sequence: forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′) was used as a negative control.

SMMC-7721 cells (1×10⁵/well) were plated in 6-well plates in RPMI-1640 medium supplemented with 10% FBS. After 24 h, 1.5 μg of DNA was transfected using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. After 24-h transfection, the cells were treated with 1.0 μg/ml NC for an additional 24 or 48 h for MTT assay, AO staining and ELISA analysis. Transfected cells were as designated 7721-pGPU6/GFP-C control shRNA (con shRNA, control), 7721-pGPU6/GFP-p53shRNA (p53^−/−).

Data were presented as means ± standard deviation (SD) in triplicate and analyzed using SPSS software (version 15.0; SPSS, Inc., Chicago, IL, USA). The difference among various groups was analyzed by ANOVA, and P<0.05 was considered to indicate a statistically significant result.

To verify the effects of NC on inhibiting HCC cell proliferation and to determine an effective concentration of NC, we performed MTT assays following treatment of SMMC-7721 cells at a range of doses. NC caused a dose-dependent decrease in SMMC-7221 cell growth, with an IC₅₀ values of 1.08 μg/ml (Fig. 1B). For practical purposes, NC concentrations that cause a 25% reduction in cell viability with no apparent pro-apoptotic effect after 24-h treatment (0.5 μg/ml for SMMC-7221 cells) are considered to be non-cytotoxic and have been used to study the effects on cell cycle progression (22), while the IC₅₀ values at 24 h are regarded as cytotoxic. To examine the effects of NC at a range of doses, we included doses of 0.5, 1.0 and 2.0 μg/ml in subsequent in vitro studies. The NC inhibition of SMMC-7221 cells at these three concentrations was time-dependent, as shown by treatment for 1, 2, 3, 4 and 5 days (Fig. 1C).

To further examine the morphological changes following NC treatment at doses that span the IC₅₀ values, we treated SMMC-7221 cells with 0.5, 1.0 and 2.0 μg/ml of NC for 48 h, and assessed the morphology using light microscopy. SMMC-7721 cells in the control group exhibited a normal and healthy shape, demonstrated by clear cytoskeletons. By contrast, NC-treated cells exhibited cell shrinkage, which was obvious for the 2.0 μg/ml NC group (Fig. 1D). The number of SMMC-7721 colones was also strongly reduced following NC treatment (Fig. 1E). These findings verify that NC affects the morphology and growth of SMMC-7221 cells, and that its cytotoxic effect is dose- and time-dependent.

To determine whether NC effectively inhibits HCC tumor growth in vivo, SMMC-7221 cells were transplanted into nude mice and 10 mg/kg/day NC was injected. As a control chemotherapeutic, the nitrogen mustard alkylating agent, CTX was injected into a separate group of mice. After 7 or 14 days treatment, transplanted SMMC-7721 tumors were significantly smaller than the untreated or CTX-treated tumors (Fig. 2A). The inhibition ratio was 30.90%, suggesting a significant difference from the control (P<0.05; Table II). Furthermore, the volume of tumor tissue in the NC group was less than that of the control (P<0.05; Fig. 2B). These results suggested that NC is efficacious in the treatment of HCC in an in vivo model.

A variety of agents trigger growth inhibition as well as cell death (23). Cell cycle arrest commonly leads to growth inhibition and is therefore a good marker for chemopreventive or antitumor activity (24). However, no reports have determined a checkpoint for the cell cycle arrest of SMMC-7721 cells by NC. To determine the effects of NC on SMMC-7221 cell cycle distribution, we performed flow cytometry. Untreated cells (controls) showed the expected pattern of continuously growing cells, in which a high peak was observed in the G₁ phase region and a lower peak in the G₂/M phase. However, following treatment with 0.5, 1.0 or 2.0 μg/ml NC, the G₁ percentage progressively decreased while the G₂/M percentage increased, with a statistical increase in the G₂/M phase distribution after 1.0 and 2.0 μg/ml of NC (Table III). This finding suggests that NC inhibits cell proliferation in a dose-dependent manner in SMMC-7721 cells by inducing G₂/M arrest.

The mode of tumor cell killing by most anticancer agents is apoptotic cell death, and DNA fragmentation is a hallmark of this process (25). To further examine whether apoptosis occurs in cultured SMMC-7721 cells in response to NC treatment, control and cells treated with 0.5 or 1.0 μg/ml NC were stained with AO and visualized under a fluorescent microscope. Cells incubated with AO had green nuclei and yellow chromatin, whereas the cells treated with NC showed typical apoptotic changes, such as chromatin condensation and deformed and fragmented nuclei, particularly in the 1.0 μg/ml NC treatment group (Fig. 3A). These results suggested that the cytotoxicity of NC involves the induction of apoptotic cell death.

To confirm the cell apoptosis induced by NC, we used TEM to examine ultrastructural changes in SMMC-7721 cells after NC treatment. The nuclei and chromatin of control cells appeared normal (Fig. 3B). However, after 24-h treatment with NC (2.0 μg/ml), chromatin margination, chromatin condensation, vacuolation in the cytoplasm and apoptotic body formation were observed (Fig. 3B). These results provided further evidence for the morphological induction of apoptosis as a consequence of NC treatment.

To confirm that NC induces apoptosis, SMMC-7721 cells were stained with Annexin V-FITC and PI, and subsequently analyzed by flow cytometry. In untreated cells, only (2.00±0.06%) of SMMC-7721 cells were Annexin V⁺/PI⁻ at 24 h of incubation. However, following exposure to 2.0 μg/ml NC, the level of apoptotic cells was increased to (18.9±0.23%) (Fig. 4A and B).

Caspase-3 activation is a critical event in apoptosis induction. To verify that cell death in SMMC-7221 cells is activated by NC through a caspase-3-dependent pathway, we measured caspase-3 activity in SMMC-7721 cells following NC treatment. Increased caspase-3 activation was observed at the NC doses tested, with the greatest increase at the highest NC doses (Fig. 4C).

To test whether apoptosis also occurs in vivo following the treatment of mice with NC, we prepared tissue sections from the tumors of nude mice transplanted with SMMC-7721 cells. TUNEL assays revealed increased levels of apoptosis in the tumor tissue of mice that were treated with NC (Fig. 4D). Collectively, these results verify that NC-induced cell death occurs in vitro and in vivo by the activation of apoptosis.

To investigate the molecular mechanism of NC-induced apoptosis in SMMC-7721 cells, the expression levels of several apoptosis-related proteins were examined. As assessed by ELISA, NC-induced a dose-dependent increase in the expression of the tumor suppressor p53 and the cyclin-dependent kinase inhibitor p21 after exposure to NC for 24 h (Fig. 5A). The increase in the two proteins was verified by qPCR (P<0.01; Fig. 5B).

The role of the Bcl-2 family proteins, Bcl-2 and Bax was also assessed. Immunohistochemical staining results showed that NC induced an increase in the proapoptotic Bax protein and a decrease in the apoptosis regulatory protein, Bcl-2 (Fig. 5C, Table IV). These results defined a pathway of apoptosis that involves the modulated expression of p53, p21, Bax and Bcl-2.

To investigate the role of p53 on the effect of NC on SMMC-7721 cells, we transfected SMMC-7721 cells with shRNA against p53 and assessed growth inhibition by NC. The results showed that the effects of NC in inhibiting cell growth in p53 knockdown cells were reduced at 24 and 48 h following treatment (Fig. 6A). Furthermore, p53 knockdown cells had less bright AO staining, further suggesting protection from NC-induced cell death (Fig. 6B). To verify the effects of p53 knockdown on p53 expression levels, we assessed these levels using ELISA. The results confirmed that p53 was increased significantly after NC treatment, but that this increase was ablated by knockdown (Fig. 6C). Notably, p21 expression also showed a similar pattern of increase by NC and reduction following p53 knockdown (Fig. 6D). These results suggested that p53 is important in NC-induced apoptosis of SMMC-7721 and that p21 expression is concomitant, and may be regulated by p53 expression.