Trypsin-EDTA (1X), Dulbecco’s minimal essential medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium, penicillin-streptomycin (PS) antibiotic mixture (100X) and qualified fetal bovine serum (FBS) were purchased from Invitrogen. Chemicals and reagents were of analytical grade purchased from Sigma Chemicals, St. Louis, MO, USA. Dimethylsulfoxide (DMSO) was purchased from Fisher Scientific, USA. Bromophenol Blue, Agarose, Tris-Base, Boric Acid, NaCl and SDS powder were ordered from USB, Cleveland, OH, USA. Ethanol was purchased from BDH.

Hep3B, Huh7 and WRL68 were purchased from ATCC and cultured according to their protocols. GA was a generous gift from the Chinese Medicine Laboratory, Hong Kong Jockey Club Institute of Chinese Medicine, and was isolated by the established method (2). GA was prepared by dissolving 4 mg of dry GA into 1 ml of DMSO. Cancer cells were seeded in a 96-well plate at the density of 5.0×10⁵ cells/well and treated with various concentrations of GA for 24 h. Methylthiazoletetrazolium (MTT) solution (5 mg/ml) was added to the assay mixture and incubated for 4 h. The culture media was removed prior to addition of DMSO. Each experiment with GA treatment was repeated three times. By comparing the absorbance of the wells of cells treated with different concentrations of GA with the control, the viability of cells after GA treatment was calculated. The concentration of GA that reduced the cell viability of 50% (IC₅₀) was recorded.

The 100 bp DNA ladder was from Fermentas and the Cell Death Detection ELISA kit was purchased from Roche Applied Science. The cell samples were processed according to the manufacturer’s protocols. The supernatant was subsequently removed and the DNA pellet was allowed to air dry for 15 to 20 min. TE buffer (20–50 μl) containing 0.2 mg/ml of RNase A was added to the DNA pellet. The samples were incubated at 37°C for 90 min to completely dissolve the sample. The DNA solution (2 μl) was added to 998 μl TE buffer and the concentration was measured using UV spectrophotometry (DU 650; Beckman-Coulter) with OD₂₆₀.

Ten microliters of the dissolved DNA samples were mixed with 2 μl 6X DNA loading dye. The mixtures were loaded on the 2% agarose gel and run at 75V for 1 h. The DNA bands were examined under UV illuminator and the gel was photographed for documentation.

Changes in cell morphology during apoptosis were examined by fluorescence microscopy of DAPI-stained cells. The monolayer of cells was washed in PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. The fixed cells were permeabilized with 0.2% Triton X-100 in PBS three times and incubated with 1 μg/ml of DAPI for 30 min. The cells were washed with PBS three times. The apoptotic nuclei (intensely stained, fragmented nuclei and condensed chromatin) were examined under ×400 magnification using a fluorescent microscope with a 340/380 nm excitation filter for at least two investigations.

ELISA was carried out according to the manufacturer’s protocol (Invitrogen). The cells were incubated with different concentrations of GA in a 96-well plate for 24 h prior to centrifugation at 200 × g for 10 min. The lysis buffer (200 μl) was added to lyse the cell pellet. The plate was kept at room temperature for 30 min prior to centrifugation at 200 × g for 10 min. The supernatant (20 μl) was placed in the streptavidin-coated plate for analysis. The immunoreagent (80 μl) was added to the well and covered by aluminum foil with shaking at 300 rpm for 2 h. The solution in each well was removed and the well was rinsed with 250 μl incubation buffer three times. ABTS solution of 100 μl was added into the wells prior to shaking in the plate shaker at 250 rpm for color development. After 10–20 min, 100 μl ABTS Stop Solution was added to stop the reaction and the well was measured at 405 nm. Absorbance of wells was recorded and the enrichment factor, which shows the extent of apoptosis in the GA-treated cells, was calculated by the following expression: (absorbance of the GA-treated sample-blank)/(absorbance of the vehicle-treated sample-blank).

The antibodies, anti-p53, anti-Bcl-2, anti-Bax, anti-caspase-8 and anti-PARP, were purchased from Santa Cruz Biotechnology, Inc. Anti-cytochrome c, anti-Bid and anti-GAPDH were purchased from BD Pharmingen. For HRP-secondary antibodies, anti-mouse and anti-rabbit antibodies were obtained from Invitrogen. Cells (1×10⁶) were seeded into a 100 mm dish. Different concentrations of GA were added prior to incubation for 4, 8, 12 and 24 h. The cell samples were spun at 14,000 × g for 15 min. The supernatant of the samples was collected for protein concentration determination according to the Bio-Rad Protein Assay protocol.

Cells were collected and counted prior to the addition of the cytosolic protein lysis buffer (50 μl). The sample was vortexed for 30 sec and centrifuged at 14,000 × g for 1 min at 4°C. A volume of 2X SDS sample loading buffer was added to the supernatant. The whole cell lysis buffer and 2X SDS sample loading buffer were added to completely lyse the sample.

The electrophoresis system, Mini-PROTEAN^® II cell from Bio-Rad, was used to perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resolving gel solution (10%) was set and poured into the gel casting form. The top of the gel was layered with 50 μl of isopropanol and the gel was left for 30 min. Stacking gel solution (4%) was added onto the top of the resolving gel. A 15-tooth comb was inserted and the gel was allowed to polymerize for 40 min. The samples mixed with 2X sample loading dye were boiled at 100°C for 10 min. After samples were loaded onto the wells, the gel was run at constant voltage at 150 V for 1 h in 1X running buffer.

The SDS-PAGE was completed when the dye front (blue in color) reached the bottom of the gel. The gel was removed and immersed into transfer buffer. The Whatman 3 MM paper (6 pieces/gel) and PVDF membrane were cut into the same dimension as the resolving gel. The PVDF membrane was washed with 100% methanol for 1 min and was immersed into the transfer buffer. The Whatman papers were soaked into the transfer buffer. A gel sandwich was assembled with the PVDF membrane and the resolving gel layered between two stacks of Whatman papers in the semi-dry Trans-Blot electroblotter (Bio-Rad). Proteins were transferred to the membrane at constant voltage at 10 V for 1.5 h. The membrane was collected and rinsed briefly with TBST buffer. Then, the membrane was immersed into blocking solution with primary antibody for 16 h at 4°C with continuous agitation. The primary antibody used was mouse monoclonal anti-p53 (1:1,000), anti-BID (1:1,000), anti-Bcl-2 (1:1,000), anti-Bax (1:500), anti-cytochrome c (1:2,000), anti-caspase-8 (1:1,000), anti-PARP (1:1,000), anti-transferrin receptor (1:1,000) and anti-GAPDH (1:5,000). The unbound primary antibody was removed by washing with TBST for 20 min, thrice. The membrane was immersed into secondary antibody (goat anti-mouse HRP/mouse anti-rabbit HRP) for 1 h at room temperature with slow agitation. The excess antibody was removed by washing with TBST three times (20 min). Millipore Immobilon Western HRP kit (0.5 ml of each reagent) was applied to the membrane for 3 to 5 min. After removing excess reagent, protein bands on the membrane were visualized and recorded on Fuji Super RX film (Fujifilm). Intensity of the bands was measured using ImageJ program.

Cells were seeded into 100 mm dishes. After 24 h, different concentrations of GA (2, 1, 0.5 μM) were added before incubation for another 24 h. The cells were collected and lysed in CHAPS buffer. The concentration of the lysate was measured and diluted 500 μg protein/500 μl buffer. The anti-Bax 6A7 monoclonal antibody (2 μg) was added to the diluted lysate and the mixture was incubated overnight at 4°C. After the incubation, 25 μl of protein A-agarose gel bead was added and the lysate was incubated for 3 h. The gel beads were washed three times using the CHAPS buffer and collected by centrifugation at 14,000 × g for 10 min. The sample loading dye (50 μl) was added to the beads prior to boiling for 10 min, followed by a centrifugation of 14,000 × g at 4°C. The supernatant was subjected to western blot analysis using mouse anti-Bax antibody.

Apo-One™ caspase-3/7 assay kit was purchased from Promega. Caspase inhibitor z-IETD-fmk and z-LEDH-fmk were from Calbiochem. Trypan blue was purchased from Invitrogen. Hemocytometer was obtained from Sigma Chemicals. Caspase-3/7 activity was examined using Apo-One™ Caspase-3/7 Assay kit (Promega). Cells (Hep3B or Huh7) were seeded in a 96-well plate in the medium for 24 h followed by GA treatment of the cell lines. Subsequently, z-DEVD-rhodamine 110 from the assay kit was added into the well. After 2 h of incubation at room temperature, the fluorescence intensity of the wells was measured by a fluorescence plate reader at excitation 492 nm and emission 535 nm.

The effects of GA on the viability of Hep3B and Huh7 were investigated using MTT assay. GA effectively reduced the viability of cancer cells after incubation for 24 h. The IC₅₀ of GA for Hep3B was 1.8 and 2.2 μM for Huh7 (Figs. 2 and 3). After treatment of cells with GA, cell shrinkage and budding were recorded. DNA fragmentation and condensation were visualized by the DAPI staining investigation using fluorescence microscopy (Figs. 4 and 5). DNA fragmentation was detected after GA treatment in HCC cell lines (Fig. 5). The amount of cytosolic DNA fragment was determined with cell ELISA kit (Fig. 6). The results showed that the amount of DNA in the cytosol significantly increased after addition of GA. Apoptosis is characterized by the occurrence of cell shrinkage and membrane blebbing (7). In addition, chromatin condensation and DNA cleavage shown by DAPI staining (Fig. 4) also indicated apoptosis (8). To avoid ambiguity, DNA fragmentation, another hallmark of apoptosis, was examined after the treatment of GA on the cell lines for 24 h. The result of agarose gel electrophoresis showed the presence of DNA ladders (Fig. 5). During apoptosis, mono- and oligo-nucleosomes are released into the cytoplasm before the plasma membrane breaks down (9,10). The cytoplasmic content of the tested cells was collected and the amount of released DNA was detected by the ELISA kit. The results showed that following GA treatment, the levels of cytoplasmic DNA in HCC cells was markedly elevated (Fig. 7). These results provide evidence that GA mediated cell death through apoptosis.

To study caspase activity in the GA-induced apoptosis, Apo-One™ Caspase-3/7 Assay was used. An increase of caspase-3/7 activity was found in the two HCC cells after GA treatment (Fig. 7). The activation of caspase-8 in the death receptor pathway and caspase-9 in the mitochondrial pathway were determined using western blot analysis (Figs. 8–10). The results indicate that the cleavage of the active form of caspases-8 and -9 after GA-treatment occurred in a time- and dose-dependent manner (Fig. 11).

The involvement of Bid and its relationship with caspase-8 activation in GA-induced apoptosis were investigated. Full length Bid (24 kDa) was found to be cleaved in a time- and dose-dependent manner upon GA treatment (Fig. 12). A cell fractionation experiment was performed to investigate the level of tBid in the mitochondria. Fig. 13 indicates an accumulation of tBid in the mitochondria in the HCC cell line.

It has been reported that Bax plays an important role in Bid-mediated apoptosis through conformational change and mediation of signaling process (11,12). Fig. 14 shows that the expression level of Bax was constant in GA-induced apoptosis. In order to further examine the role of Bax in GA-induced apoptosis, immunoprecipitation (IP) of Bax using Bax (6A7) monoclonal antibody was performed. This antibody recognizes the Bax protein with conformational change but not the native form (11). It was reported that the conformational change of Bax was triggered by the truncated form of Bid (tBid) (13). This tBid is subsequently translocated to the mitochondria with Bax. The translocation of tBid to mitochondria was shown in western blot analysis (Fig. 15). Cytochrome c, which is originally present in the mitochondria, was released.