Antitumor effects and mechanisms of Ganoderma extracts and spores oil (2024)

Preparations of Ganoderma extracts and spores oil

Ganoderma extracts (GanoHerb™) and Ganoderma spores oil were provided by Fujian Xianzhilou Biological Science and Technology Co., Ltd. (Fuzhou, China). Ganoderma extract, a brown powder, was dissolved in double distilled water to prepare solutions of various concentrations, which were brown suspensions. Ganoderma spores oil was a soft capsule with 0.5 g/0.5 ml golden oil in each capsule. The stock solution of Ganoderma spores oil were prepared using double distilled water that contained 6 µl/ml (v/v) Tween 80.

Cell lines and culture

The human acute myeloid leukemia HL-60, human chronic myeloid leukemia K562, human gastric carcinoma SGC7901, murine sarcoma S180 and murine hepatoma H22 cell lines were purchased from the Shanghai Cell Institute of the Chinese Academy of Sciences (Shanghai, China). All tumor cells were maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal calf serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) and antibiotics (100 kU/l penicillin and 100 mg/l streptomycin) at 37°C in a humidified atmosphere of 5% CO₂.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

The cell cytotoxicity of Ganoderma spores oil in vitro was detected by MTT assay. The MTT assay was based on the ability of live cells to utilize thiazolyl blue and convert it into dark blue formazan. Exponentially growing cells (2×10⁴/well) were seeded into 96-well plates and treated with a series of concentrations of Ganoderma spores oil (0.31, 0.63, 1.25, 2.5, 5 and 10 mg/ml) for 24 h. Control cells were exposed to double distilled water containing the same concentration of Tween 80. Subsequently, 20 µl of 5 mg/ml MTT (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated for 4 h. The dark blue formazan crystals were solubilized in lysis buffer containing 10% sodium dodecyl sulfate (SDS), 5% isobutyl alcohol and 12 mmol/l HCl, and the optical density (OD) was detected at 492 nm with a spectrophotometer. The rate of growth inhibition was calculated according to the formula: Inhibition rate (%) = (1 - mean OD value of treated group/mean OD value of control group) × 100. The half maximal inhibitory concentration (IC₅₀) value was calculated by linear regression method.

Trypan blue exclusion method

The cell cytotoxicity of Ganoderma extract in vitro was assessed using the trypan blue exclusion method. Briefly, exponentially growing cells were treated with a series of concentrations of Ganoderma extract (0.22, 0.44, 0.67, 0.89 and 1.10 mg/ml) for 24 h. Control cells were exposed to double distilled water. Then cells were dyed with 0.4% trypan blue. Viable (trypan blue dye-excluding) cells were counted using a light microscope. Inhibition rate was calculated according to the formula: Inhibition rate (%) = (1 - viable cells in treated group / viable cells in control group) × 100.

Antitumor effects in vivo

A total of 60 ICR mice (6–8 weeks old; male and female; body weight, 18–20 g) were obtained from the Animal Centre of Fujian Medical University (Fuzhou, China) and maintained in specific pathogen-free conditions. Animals were exposed to a 12 h light/dark cycle and fed a standard diet. All animal studies were approved by the ethics committee of Fujian Medical University. S180 or H22 tumor cells in suspension (1×10⁶ cells in 200 µl solution) were carefully inoculated intradermally into the left axilla of mice. The tumor-bearing mice were then randomly assigned to treatment experimental groups (n=10 per group). At 24 h following the tumor inoculation, mice were intragastrically administrated with i) extract of Ganoderma, 4 g/kg; ii) extract of Ganoderma, 2 g/kg; iii) extract of Ganoderma, 1 g/kg; iv) Ganoderma spores oil, 1.2 g/kg; v) fluorouracil (5-FU), 25 mg/kg (positive control); or vi) 0.9% NaCl solution 0.2 ml/10 g (negative control) once per day for 10 consecutive days. At the end of the experiments (day 12), mice were weighed and then sacrificed by cervical dislocation. The tumors, thymus glands and spleens of the mice were excised carefully and weighed. The following formulae were used: i) Tumor suppression rate (%) = (1 - mean tumor weight in treated group / mean tumor weight in control group) × 100; ii) Thymus index = 100 × thymus weight / body weight; and iii) Spleen index = 100 × spleen weight / body weight.

Catalytic activity of topoisomerases

The catalytic activity of topoisomerases was measured as previously described (10). K562 cells (10⁷) were washed with phosphate-buffered saline (PBS) and treated with lysis buffer containing 20 mM Tris, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 25 mM KCl, 5 mM MgCl₂, 250 mM sucrose and 0.5% NP40 (pH 7.2) for 10 min at 4°C, then centrifuged at 4,000 × g for 2 min. The pellet was resuspended in 50 µl of extraction buffer containing 20 mM Tris, 1 mM EGTA, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM dithiothreitol (DTT) and 400 mM NaCl (pH 7.2) and incubated for 30 min at 4°C. After centrifugation at 22,000 × g for 15 min, the supernatant containing extracted topoisomerases was obtained and its protein concentration was determined using the Bradford Protein Assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's instructions, prior to storage at −20°C.

The catalytic activity of topoisomerase II in Ganoderma extracts was evaluated as follows: The total reaction volume was 10 µl, containing 2.5 µl 4X reaction buffer [50 mM Tris (pH 8.0), 120 mM KCl, 0.5 mM DTT, 0.5 mM adenosine triphosphate (ATP), 10 mM MgCl₂ and 30 µg/ml bovine serum albumin (BSA; Beyotime Institute of Biotechnology)], 0.1 mM ATP (Promega Corporation, Madison, WI, USA), 0.2 µg pBR322 DNA (Promega Corporation), various amounts of topoisomerase II-containing crude extract and double distilled water to make the total volume up to 10 µl. Following incubation at 37°C for 30 min, the reaction was stopped by the addition of 5 µl of a solution (50 mM EDTA, 50% glycerol and 0.25 mg/ml bromphenol blue) maintained at 4°C. Samples were separated through a 1% agarose gel. After staining with ethidium bromide, gels were photographed under ultraviolet (UV) illumination and analyzed using Syngene Genesnap Tools software (version 3.00.22; Syngene; Synoptics Ltd., Cambridge, UK).

The catalytic activity of topoisomerase I in Ganoderma extracts was evaluated as follows: The total reaction volume was 10 µl, containing 1 µl 10X reaction buffer [10 mM Tris-HCl (pH 7.9), 0.15 M NaCl, 1 mM EDTA, 0.1 mM spermidine, 0.1% BSA and 5% glycerol], 0.2 µg pBR322 DNA, various amounts of topoisomerase I-containing crude extract and double distilled water to make the total volume up to 10 µl. The rest of the procedures were the same as the aforementioned topoisomerase II method.

The inhibitory effects of Ganoderma extracts and Ganoderma spores oil on topoisomerase I and II were then detected. After selecting the amounts of crude extract that unwound 0.2 µg pBR322 DNA to establish topoisomerase I and topoisomerase II reaction systems, 1, 2 and 4 mg/ml extracts and 4.5, 9 and 18 mg/ml spores oil was added to each reaction system and double distilled water was used to make the total volume up to 10 µl. Following incubation at 37°C for 30 min, the reaction was stopped and separated through 1% agarose gel. The staining gel was photographed under ultraviolet (UV) illumination and analyzed using Syngene Genesnap Tools software (version 3.00.22; Syngene).

Cell cycle analysis

The cell cycle was analyzed by flow cytometry. K562 cells were seeded in 6-well plates at a density of 1×10⁵/ml, and treated with 1.25 and 2.5 mg/ml Ganoderma spores oil for 12 h. Then cells were washed twice with PBS and fixed in 70% ethanol at 4°C overnight. Prior to analysis, cells were washed and resuspended in PBS, then incubated with 10 mg/ml RNase A for 3–5 min and 50 µg/ml propidium iodide (Sigma-Aldrich) at 4°C for 30 min in a dark chamber. The cell cycle distribution (percentage of cells in various phases) was analyzed by flow cytometry (Epics XL; Beckman Coulter, Inc., Brea, CA, USA) using Multicycle software (version 3.11; Phoenix Flow Systems; San Diego, CA, USA).

Western blot analysis

K562 cells (1×10⁷) were washed with PBS and then treated with lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM phenylmethane sulfonyl fluoride, 1 mM aprotitin and 1% NP40 for 30 min at 4°C. Following centrifugation at 11,000 × g for 20 min, the protein concentration of the supernatant was determined; equal amounts (30 µg) of protein were separated by 10% SDS-polyacrylamide gel electrophoresis and then electro- transferred onto nitrocellulose membranes in transfer buffer. The membrane was blocked with 5% BSA for 2 h and then incubated with monoclonal mouse anti-human cyclin D1 (cat. no. sc-20044; 1:500), monoclonal mouse anti-human cyclin E (cat. no. sc-247; 1:500) and polyclonal rabbit anti-human actin (cat. no. sc-7210; 1:2,000) primary antibodies (Santa Cruz Biotechnology Inc., Dallas, TX, USA) in blocking solution at 4°C for 2 h, followed by extensive washing with PBS twice. The membranes were then incubated with alkaline-phosphatase conjugated goat anti-mouse (cat. no. sc-2047; 1:2,500) and goat anti-rabbit (cat. no. sc-2034; 1:2,000) IgG secondary antibodies (Santa Cruz Biotechnology Inc.) in blocking solution at room temperature, followed by washing with Tris-buffered saline 3 times. The target proteins became visible following the addition of Alkaline Phosphatase Substrate Solution BCIP/NBT (Promega Corporation).

Statistical analysis

Statistical analysis of the data was performed with the Student's t-test. Data were expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Inhibitory effects on tumor cells in vitro

The anti-proliferative activities of Ganoderma extracts and spores oil on the K562, HL60 and SGC-7901 cell lines were determined. Using the trypan blue exclusion method, dose-dependent inhibitory effects were observed when the Ganoderma extract concentrations were in the range of 0.22–1.10 mg/ml. The IC₅₀ value of Ganoderma extracts for the K562, HL60 and SGC-7901 cells were 0.44, 0.39 and 0.90 mg/ml, respectively (Table I).

The MTT assays revealed that treatment with Ganoderma spores oil concentrations of 0.31–10.0 mg/ml caused dose-dependent cytotoxicity in K562, HL60 and SGC-7901 cells, with IC₅₀ values of 1.13, 2.27 and 6.29 mg/ml, respectively (Table II).

Suppression of tumor growth in vivo

The effects of intragastric administration of Ganoderma extracts and spores oil on S180 cells in mice were examined (Fig. 1; Table III). Table III shows that the 4 g/kg extracts and 1.2 g/kg oil groups resulted in significant inhibitions of tumor growth by comparing the average tumor weight with the control group at the end of the experiment (P=0.013 and P=0.032, respectively); however, the inhibitory rates of the 1 and 2 g/kg extract groups were 15.6 and 19.0%, respectively (P=0.253 and P=0.170, respectively). The average body weights were not significantly different among treatment groups, with the exception of the 5-FU group (positive control).

The suppressive effects of Ganoderma extracts and spores oil on H22 cells in mice are shown in Fig. 2 and Table IV. Data in Table IV showed that Ganoderma extracts and spores oil exhibited relatively strong inhibitory effects on H22 cells in mice. The inhibitory rates of the 1, 2 and 4 g/kg extract groups on H22 were 28.8, 38.5 and 44.6%, respectively (P=0.008, P=0.008 and P=0.002, respectively, compared with control group), and of the 1.2 g/kg oil group was 44.9% (P=0.004). The average body weights were significantly decreased in the 4 g/kg extracts (P=0.038) and 5-FU (P=0.002) groups compared with the control group.

Effects on immunity indexes of mice bearing tumors

Table V shows that Ganoderma extracts increased the immunity indexes of mice bearing S180: The spleen index of the 1 g/kg extracts group was significantly increased compared with the control group (P=0.041); the thymus indexes in the 1, 2 and 4 g/kg extracts groups were all improved significantly (P=0.029, P=0.021 and P=0.038, respectively); while in the 5-FU group, the thymus index was decreased significantly (P=0.002). Ganoderma spores oil exerted no significant effects on spleen index and thymus index.

After treatment with Ganoderma extract and spores oil, there were no significant changes in the immunity indexes of mice bearing H22 cells (Table VI).

Inhibitory effects on topoisomerases activities

The activities of topoisomerase I and II are determined by the conversion of a fixed amount of supercoiled DNA into relaxed and broken DNA. Topoisomerase inhibitors could decrease the amount of relaxed and broken DNA and increased supercoiled DNA by inhibiting the catalytic activities of topoisomerases. The minimal concentration of crude topoisomerases from K562 cells required to fully relax 0.2 µg pBR322 DNA was used to test Ganoderma extracts and spores oil.

The results showed that the Ganoderma extracts and spores oil inhibited the activities of topoisomerase I and II. Treated with Ganoderma extracts, 2 mg/ml, topoisomerase I and II were inhibited, as reflected by the increased amount of supercoiled DNA and decreased amount of relaxed and broken DNA (Fig. 3). When Ganoderma spores oil, 4.5 mg/ml, was added into the reaction system of topoisomerase I, supercoiled DNA was observed. As the concentration of Ganoderma spores oil was increased, the inhibition of topoisomerase in dose-dependent manner was observed. Using Ganoderma spores oil, 18 mg/ml, the activity of topoisomerase II was then suppressed (Fig. 4).

Effect of Ganoderma spores oil on cell cycle

The cell cycle was analyzed by flow cytometry. As shown in Table VII, Ganoderma spores oil induced a G1 arrest in K562 cells. With the treatment of Ganoderma spores oil, 1.25 mg/ml, for 12 h, the percentage of S cells in K562 was decreased from 55.7±0.6 to 42.3±0.2 (P=0.038). When the concentration of Ganoderma spores oil was raised to 2.5 mg/ml, the percentage of S cells was further decreased to 38.7±3.2 (P=0.017), and the percentage of G1 cells rose from 32.7±0.5 to 43.7±2.2 (P=0.022).

Results of Ganoderma spores oil on cyclin D1 and cyclin E

Cyclin D1 and E are important regulators of G1-S phase cell cycle transition. The expression levels of cyclin D1 and E were determined by Western blotting. The results showed that after the treatment of Ganoderma spores oil 1.25–5.0 mg/ml for 12 h, cyclin D1 expression in K562 decreased, but cyclin E expression exhibited no obvious change (Fig. 5).