G. lucidum fruiting bodies were extracted with 95% (v/v) aqueous ethanol at room temperature. Combined ethanolic extracts were evaporated to dryness, redissolved in water and extracted with chloroform. After addition of a saturated NaHCO₃ solution, the chloroform layer containing non-acidic triterpenoids was collected. The crude extracts were purified using silica gel (200–300 mesh) column chromatography. The column was eluted with petrol ether/acetone (v/v=1:0, 50:1, 30:1, 10:1, 3:1 sequentially) and eight fractions were collected. The second fraction was separated further by MCI chromatography and the elution gradient was 40–100% methanol in water. The fifth fraction, obtained with 80% methanol elution, was termed GLAI. Triterpenes were visualized as fluorescent spots under long wavelength UV light.

Human tumor cell lines, SW620 cells (colon), MCF-7 (breast), K562 (bone marrow), and mouse lymphocytic leukemia cell line L1210 were obtained from the American Type Culture Collection (ATCC) and maintained at 37°C in RPMI-1640 containing 10% fetal calf serum (FCS) (Kraeber, Wedel, Germany), 100 U/ml penicillin and 100 μg/ml streptomycin.

Cells were adjusted to a concentration of 1×10⁴ cells/ml. Cell suspension (180 μl) and different test agents (20 μl) were added to each well of a 96-well microplate reader. After incubation at 37°C in a 5% CO₂ atmosphere for a defined time, 20 μl Alamar Blue reagent (Biosource, Nivelles, Belgium) were added to each well and incubation continued for another 6 h. The extinction was measured using a micro ELISA autoreader at 570 and 600 nm. The proliferation rate was calculated according to the Biosource protocol.

SW620 cells (1×10⁴ cells/ml) were treated for 24 h with a control and 10 μM GLAI. Morphological observations of cultured cells were made by inverted, phase-contrast microscopy. Samples treated with DMSO only served as controls. For nuclear staining, SW620 cells (1×10⁵ cells/ml) were treated for 24 h with DMSO and 10, 50 and 100 μM GLAI. After treatment, cells were harvested and washed with ice-cold phosphate-buffered saline (PBS). The cells were then incubated in nuclear fluorochrome Hoechst 33258 at a final concentration of 10 μg/ml at room temperature for 10 min in the dark. Nuclear morphology was then examined with an Olympus fluorescent microscope.

To confirm the nature of the effects of GLAI on SW620 cells, dual-staining [propidium iodide (PI) and annexin V (AV)] flow cytometry was used to measure the externalization of phosphatidylserine (PS). Aliquots (5×10⁶) of SW620 cells cultured as described above were treated with 20, 50 or 100 μmol/l GLAI for 24 h. Controls were treated with DMSO only. After washing and trypsinization, cell samples were collected by centrifugation (400 g, 3 min, 4°C) and double-stained using the apoptosis detection kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Cells were incubated for 30 min at 25°C in 100 μl 1X buffer solution, 5 μl AV-FITC and 5 μl PI, and then a further 400 μl of 1X solution was added. The green fluorescence of AV-FITC-bound PS and the red fluorescence of DNA-bound PI in individual cells were measured at 525 and 575 nm, respectively, using a BD FACSCalibur. Cell populations were classified as: AV⁻/PI⁻, viable cells; AV⁺/PI⁻, early apoptotic cells; AV⁺/PI⁺, apoptotic cells; and AV⁻/PI⁺, residual damaged cells.

Caspase-3 activity in the lysates of SW620 cells was measured using the Caspase-3 cellular activity assay (Calbiochem, Darmstadt, Germany). SW620 cells were cultured as described above and suspensions (2×10⁷ cells) were treated with different concentrations of GLAI (0, 10, 25 and 50 μmol/l) for 24 h. Controls were treated with DMSO only. After washing and trypsinization, cell suspensions were centrifuged (400 g, 3 min, 4°C) and cell pellets were re-suspended in 1 ml ice-cold cell lysis buffer for 5 min. Following centrifugation (400 g, 3 min, 4°C), cytosol supernatants were collected and enzyme activity was measured according to the manufacturer’s instructions. Reaction mixtures (total volume 100 μl) were incubated at 37°C for 10 min and the optical density value was measured for 15 h at 405 nm using an ELISA reader (Bio-Tek, Atlanta, GA, USA).

For RNA extraction, two experiments were performed. Firstly, SW620 cells (3.6×10⁶ cells/ml) were incubated in RPMI-1640 containing 10% FCS and different concentrations of GLAI (10, 50 and 100 μg/ml). The entry with 0 μg/ml GLAI, treated with DMSO, was used as a negative control. The cells were cultured for 24 h at 37°C, 5% CO₂, in a humidified incubator. Secondly, SW620 cells (2×10⁶/ml) were incubated in RPMI-1640 containing 10% FCS and GLAI (50 μg/ml). The cells were cultured for 0, 6, 12, 18 and 24 h at 37°C, 5% CO₂.

The cells were lysed in TRIzol reagent (Invitrogen Life Technologies). Total RNA was extracted according to the manufacturer’s instructions. The RNA pellet was dissolved in diethyl pyrocarbonate (DEPC)-treated water prior to use for reverse transcription, electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide (EB) staining under an ultraviolet light, and two bands (28S and 18S) are evident.

RNA was primed with oligo(dT)15 and converted into complementary DNA (cDNA) by Moloney murine leukemia virus (MMLV) reverse transcriptase. The reaction mixture for reverse transcription contained 5.0X buffer, 2.5 mM of each deoxynucleotriphosphate (dNTP, i.e., dATP, dCTP, dGTP and dTTP), 40 U/μl RNase inhibitor, 200 U/μl reverse transcriptase, 10 μM oligo(dT)15 and 2 μg total RNA (equal amounts of starting RNA were used for each condition in the different experiments). The final volume of the reaction was 30 μl. The program parameters were 70°C for 5 min to heat, 37°C for 1.5 h for reverse transcription reaction and 95°C for 5 min to inactivate the reverse transcriptase. cDNA generated by reverse transcription was either used immediately for PCR experiments for the cytokines of interest or stored at −20°C.

Oligonucleotide primers for GAPDH, Bcl-2 and Bax were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Table I shows the sequence primers and the sizes of the fragments generated by the PCR reactions.

The components added to the sample to make up 20 μl reaction mixture were: 1 μl cDNA, 2 μl 10X Taq DNA polymerase buffer, 2 μl 25 mM Mg²⁺ (Promega, Madison, WI, USA), 0.5 μl 10 mM mixture of all four deoxynucleotide triphosphates (Promega), 0.5 μl each of 5′ and 3′ primer (10 μM) and 1 unit Taq DNA polymerase (Promega). PCR was performed for 35 cycles. Temperature cycling was initiated with each cycle as follows: for GAPDH, 95°C for 45 sec (denaturation), 58°C for 45 sec (annealing), 72°C for 30 sec (extension); for Bcl-2, 95°C for 45 sec, 56°C for 45 sec, 72°C for 30 sec; for Bax, 95°C for 45 sec, 58°C for 45 sec and 72°C for 30 sec. The amplified products were detected in 1.5% agarose gels stained with EB and visualized under UV light.

For the analysis of p53 and XIAP, SW620 cells (3.6×10⁶/ml) were incubated in RPMI-1640 containing 10% FCS and different concentrations of GLAI (10, 20, 50 and 100 μg/ml). The entry with 0 μg/ml GLAI, treated with DMSO, was used as a negative control. The cells were cultured for 24 h at 37°C, 5% CO₂, in a humidified incubator. Cells were washed twice with PBS and lysed in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100 and 100 μg/ml PMSF at 4°C overnight. The suspensions were then centrifuged at 12,000 rpm for 5 min; All lysates were subjected to BCA protein assay reagent (Pierce, Rockford, IL, USA) for the quantification of protein concentration, and then Western blot analysis was performed. Total proteins (20–50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis using a 10% polyacrylamide gel. The proteins in the gel were transferred to a PVDF membrane. The membrane was blocked with 0.1% BSA in TBST for 1 h. Membranes were incubated with primary antibody (1:2,000) at 4°C overnight and then with secondary antibody (1:2,000) for 1 h. The membranes were washed three times in TBST for 10 min between each step. The signal was detected using the Amersham ECL system (Amersham-Pharmacia Biotech, Arlington Heights, IL, USA).

The data are shown as the means ± SD. The Student’s t-test was used to determine the significance of differences between population means with results considered as: significant, p<0.05; very significant, p<0.01; extremely significant, p<0.001.

To determine the effect of GLAI on different tumor cells, the proliferation assay was performed using the Alamar blue. As shown in Fig. 1, GLAI at a concentration of 2 μmol/l inhibited the growth of SW620, K562 and L1210 cells to ~19, 12 and 60%, respectively. Exposure of the SW620, K562 and L1210 cells to 10 μmol/l GLAI inhibited cell growth by 35, 52 and 86%. respectively. However, no additional effect was observed with MCF7 cells at these concentrations (Fig. 1). However, increased growth inhibition of SW620, K562, MCF7 and L1210 cells (to 55, 90 and 98%, respectively) was observed following exposure to 50 μmol/l GLAI. Treatment with 100 μmol/l GLAI resulted in almost total inhibition of cell growth and few viable cells (see below) in all cases. Positive controls treated with 5-fluorouracil were inhibited 88–90% under these conditions.

Normally adhesive SW620 cells were readily suspended following treatment with 10 μmol/l GLAI for 24 h, and few viable cells were observed following exposure to 50 μmol/l GLAI (data not shown). Light microscopy showed that cells exposed to DMSO (Fig. 2A) or 10 μmol/l GLAI exhibited distinct morphological features, such as apoptotic bodies, associated with programmed cell death (Fig. 2B).

To further determine the nuclear morphology of SW620 cells treated with GLAI, Hoechst 33258 staining was performed. Following treatment of SW620 cells with different concentrations of GLAI for 24 h, the chromatin stained with Hoechst 33258 had a characteristic condensed and fragmented appearance (Fig. 2C-F).

The staining patterns of SW620 cells exposed to GLAI for 24 h and treated with AV-FITC and PI are shown in Fig. 3. More than 94% of cells remained viable following treatment for 24 h with DMSO alone (negative control) and almost no apoptotic events were detected (Fig. 3, lower left quadrant). However, the proportion of cells with externalized PS increased in cells following treatment for 24 h with different concentrations of GLAI (Fig. 3).

Caspases are significant mediators of apoptosis in mammalian cells (21), therefore, we measured caspase-3 activities. When SW620 cells were treated with different concentrations of GLAI, intracellular caspase-3 activity was analyzed (Fig. 4). Caspase-3 activity was found to be up-regulated in SW620 cells in a dose-dependent manner.

In order to understand the induction mechanism of apoptosis by GLAI, we examined the expression levels of Bcl-2 and Bax using RT-PCR, as well as p53 and XIAP using Western blotting. The treatment of the SW620 cells with GLAI resulted in a marked decrease of Bcl-2 at mRNA levels in a dose-dependent manner (Fig. 5A). By contrast, the Bax mRNA expression level was increased in a dose-dependent manner (Fig. 5B). The results of Western blotting showed that the p53 protein expression level was up-regulated after SW620 cells were treated with different concentrations of GLAI. By contrast, the XIAP protein expression level was down-regulated (Fig. 5C).