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Nutrition and Cancer, 61(5), 696–707Copyright © 2009, Taylor & Francis Group, LLCISSN: 0163-5581 print / 1532-7914 onlineDOI: 10.1080/01635580902898743

Anticancer Properties of Ganoderma Lucidum MethanolExtracts In Vitro and In Vivo

Ljubica M. Harhaji Trajkovic, Sanja A. Mijatovic, Danijela D.Maksimovic-Ivanic, Ivana D. Stojanovic, and Miljana B. MomcilovicInstitute for Biological Research “Sinisa Stankovic,” Belgrade University, Belgrade, Serbia

Srdjan J. TufegdzicInstitute of Chemistry, Technology and Metallurgy, Belgrade, Serbia

Vuk M. Maksimovic and Zaklina S. MarjanovicInstitute for Multidisciplinary Research, Belgrade, Serbia

Stanislava D. Stosic-GrujicicInstitute for Biological Research “Sinisa Stankovic,” Belgrade University, Belgrade, Serbia

Anticancer activities of various extracts of the medicinal mush-room, Ganoderma lucidum, have been widely demonstrated andare mainly associated with the presence of different bioactivepolysaccharides and triterpenoids. We have evaluated and com-pared in vitro and in vivo the antitumor effects of two prepa-rations from Ganoderma lucidum: a methanol extract containingtotal terpenoids (GLme) and a purified methanol extract contain-ing mainly acidic terpenoids (GLpme). Both extracts inhibitedtumor growth of B16 mouse melanoma cells inoculated subcu-taneously into syngeneic C57BL/6 mice and reduced viability ofB16 cells in vitro, whereby GLme exhibited stronger effect. Fur-thermore, anticancer activity of GLme was demonstrated for thefirst time against two other rodent tumor cell lines, L929-mousefibrosarcoma and C6-rat astrocytoma. The mechanism of antitu-mor activity of GLme comprised inhibition of cell proliferationand induction of caspase-dependent apoptotic cell death medi-ated by upregulated p53 and inhibited Bcl-2 expression. More-over, the antitumor effect of the GLme was associated with inten-sified production of reactive oxygen species, whereas their neutral-ization by the antioxidant, N-acetyl cysteine, resulted in partialrecovery of cell viability. Thus, our results suggest that GLmemight be a good candidate for treatment of diverse forms ofcancers.

Submitted 27 June 2008; accepted in final form 18 November 2008.Address correspondence to Stanislava D. Stosic-Grujicic, De-

partment of Immunology, Institute for Biological Research “SinisaStankovic,” Belgrade University, Bulevar Despota Stefana 142, 11060Belgrade, Serbia. Phone: +381 112657258. Fax: +381 112761433.E-mail: [email protected].

INTRODUCTIONMany modern drugs are derived from natural ancient folk

remedies, the use of which was based on longstanding obser-vation of their beneficial effects on human health. Mushroomsproduce a variety of bioactive ingredients (1). One of the mostimportant medicinal mushrooms, Ganoderma lucidum (G. lu-cidum), also known as Reishi in Japan or Lingzhi in China,has been used as a folk remedy for more than 4,000 yr, whichmakes it one of the oldest mushrooms known to have been usedin medicine. The experience of traditional medicine that thisfungus (usually in the form of tea or different extracts) helpsin the prevention and treatment of a variety of diseases hasrecently been confirmed scientifically. Thus, numerous studieshave shown that extracts of G. lucidum sporocarps, spores, andmycelia exhibited strong positive effects in the treatment of di-abetes, hepatitis, heart disease, hypertension, bacterial and viralinfections, neurasthenia, AIDS, and different forms of cancer(2).

Although the chemical composition of the mushroom is verycomplex (comprising nucleosides, steroids, fatty acids, alka-loids, proteins, peptides, amino acids, vitamins, and inorganicelements) (3), most studies have linked the antitumor effect ofG. lucidum extracts to the presence of different polysaccharidesand triterpenoids. During the past two decades more than 200polysaccharides and 130 triterpene structures have been iso-lated from its fruiting bodies, spores, and mycelia (4). It is be-lieved that these two classes of mushroom-derived compoundshave completely different modes of anti-tumor action. Whereastriterpenoids directly suppress growth and invasive behavior ofcancer cells, polysaccharides stimulate anticancer immune re-sponses (5). The immunostimulatory action of polysaccharides

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GANODERMA LUCIDUM METHANOL EXTRACTS IN VITRO AND IN VIVO 697

is exerted through increases in interleukin-2, interferon-γ , andantibody production as well as through stimulation of NK cellsand cytotoxic T lymphocytes (6,7). On the other hand, in vitrostudies have shown that the direct antitumor effects of various G.lucidum derived triterpenoids are mediated through inhibitionof tumor cell proliferation (8,9) and through induction of apop-totic cell death accompanied by disruption of the mitochondrialmembrane, cytosolic release of cytochrome c, and activationof caspase-3 (8). In addition to this, some triterpenoids derivedfrom this mushroom could inhibit metastasis and invasion bytumor cells (10,11).

In general, two solvent extraction approaches are employedfor the isolation of terpenoids from G. lucidum (12). In the first,total terpenoids are isolated using organic solvents and water(13). In the second approach, acidic terpenoids are selectivelyisolated from the total terpenoid fraction (14,15). In the presentstudy, both methods were used, giving G. lucidum extracts withdifferent chemical composition: a methanol extract containingtotal terpenoids (GLme) and a purified methanol extract con-taining mainly acidic terpenoids (GLpme). We further exploredthe possible antitumor effects of both preparations in vitro onmouse melanoma B16, mouse fibrosarcoma L929, and rat astro-cytoma C6 cell lines as well as in an in vivo model of melanomain C57BL/6 mice and showed for the first time that the morecomplex methanol extract from G. lucidum (GLme) possesses amuch stronger antitumor potential against all 3 tumor cell linestested.

MATERIALS AND METHODS

ReagentsFetal calf serum (FCS), RPMI 1640, phosphate-

buffered saline (PBS), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),trypan blue (TB), lactic acid, phenazine methosulfate, p-iodonitrotetrazolium, N-acetyl-cysteine (NAC), and dihy-drorhodamine 123 (DHR) were all obtained from Sigma (St.Louis, MO). Propidium iodide and Annexin V-FITC were ob-tained from BD Pharmingen (San Diego, CA). β-nicotinamideadenine dinucleotide (NAD) was purchased from ICN (CostaMesa, CA). Rabbit polyclonal anti (mouse, rat, and human) p53was purchased from Santa Cruz Biotechnology (Santa Cruz,CA), whereas horseradish peroxidase-conjugated (donkey) an-tirabbit IgG was from GE Healthcare (Buckinghamshire, UK).

Preparation and Characterization of the G. LucidumExtracts

Sporocarps of the strain of G. lucidum collected in North-Eastern Serbia were obtained from the cultivation base ofAGARICA d.o.o. (Bela Crkva, Serbia) and grown on substratesunder controlled conditions following the original protocol ofthe company. The experiments were focused on terpenoids, agroup of fungal compounds previously identified as possible

sources of bioactivity. To narrow the search for the possiblecarriers of pharmacological activity, a modified approach ofconsecutive extraction with solvents of rising polarity was used(16). Methanol was employed since it was reported to give fun-gal extracts with the highest bioactivity (16). The entire sporo-carp of G. lucidum was cut into 2 to 3 mm chips and macerated3 times with methanol at 50◦C for 6 h. Methanol was sub-sequently removed from the extract using a rotary evaporatorunder vacuum, and the residue refined using an adaptation ofthe extraction method of Folch et al. (17). The resulting extract,containing total lipids (polar and nonpolar) was labeled GLme.To extract free acids (targeting triterpenoids), GLme was furtherdissolved in chloroform and rinsed with concentrated aqueousNaHCO3 solution twice. The combined water phases were acid-ified, reextracted with a fresh portion of chloroform, and theorganic phases separated, combined, and dried over Na2SO4.After filtration, the solvent was evaporated off to obtain a puri-fied extract, expected to contain all lipid compounds possessingacidic functional groups, referred to as GLpme. The lyophilizedpowders were dissolved at 400 mg/ml in DMSO and stored at–20◦C until HPLC analysis. Alternatively, the stock solutionwas diluted in PBS or culture medium immediately before usefor in vivo and in vitro treatments, respectively, and the finalDMSO concentration never exceeded 0.2%. The extracts wereanalyzed on a Hewlett Packard 1100 HPLC (Palo Alto, CA)with a photodiode array detector adjusted at 252 nm for deter-mination of terpenoid compounds. Reversed phase separationswere made on a Waters (Milford, MA) Xterra column (250 ×4 mm and 5 µm particle size) with a corresponding precolumn.Mobile phases were 0.3% phosphoric acid (mobile phase A)and acetonitrile (mobile phase B) with the following gradientprofile: 30 min from 30–40% B, followed by a linear rise to60% of B in the next 30 min. Acetonitrile (J. T. Baker, Philips-burg, NJ, USA), pro analysis (p.a.) grade phosphoric acid and18 M� deionized water (Millipore, Bedford, MA) were used.Standard mixtures of ganoderic acids (forms A, C2, H, and F)were obtained from Planta Analytica (Danbury, CT).

Tumor Cells and MiceA transplantable B16 murine melanoma cell line of C57BL/6

origin was a kind gift from Dr Sinisa Radulovic (Institute for On-cology and Radiology, Belgrade, Serbia). Mouse fibrosarcomacell line L929 was purchased from the European Collection ofAnimal Cell Cultures (Salisbury, UK), whereas rat astrocytomacell line C6 was a kind gift from Dr Pedro Tranque (Univer-sidad de Castilla-La Mancha, Albacete, Spain). Rat primaryastrocytes were isolated from newborn Albino Oxford rats aspreviously described (18). All cell lines were maintained bytwice-a-wk passages in HEPES-buffered RPMI 1640 mediumsupplemented with 5% FCS, 2 mM l-glutamine, 5 × 10–5 M 2-mercaptoethanol, antibiotics, and 0.01% sodium pyruvate (cul-ture medium) at 37◦C in a humidified atmosphere with 5% CO2.Inbred C57BL/6 mice were originally purchased from Charles

698 L. M. HARHAJI TRAJKOVIC ET AL.

River Laboratories (L’Arbresle Cedex, France) and then bredand kept in our own colony at the facilities of the Institute forBiological Research “Sinisa Stankovic” (Belgrade, Serbia) un-der standard laboratory conditions (nonspecific pathogen free)with free access to food and water. All experiments involvedgroups of 8 to 10 mice, matched by age (8–10 wk) and weight(20–25 g). All procedures were performed under the control ofthe Institutional Animal Care and Use Committee approved bythe Ethical Animal Commission of the Instate for BiologicalResearch “Sinisa Stankovic,” Belgrade, Serbia.

Induction of Melanoma in C57BL/6 Mice and TreatmentRegimen

Primary tumors were induced by sc injection of 2.5 × 105

B16 melanoma cells in the dorsal lumbosacral region of syn-geneic C57BL/6 mice. Treatment of B16-bearing mice withmushroom extracts was initiated on the day of tumor implan-tation (Day 0). The GLme and GLpme regimens consisted ofa daily ip injection of 100 mg/kg body weight (b.w.) dilutedin DMSO/PBS, for 13 consecutive days. The group of tumor-bearing mice treated with vehicle (DMSO/PBS) served as thecontrol. Tumor growth was monitored every 2 to 3 days by two-dimensional measurement of individual tumors for each mouse.Tumor volume (cm3) was calculated according to the formula:(π /6) × tumor length × tumor width2 (19). The animals wereobserved until Day 24 after tumor implantation when the controlanimals had to be euthanized due to large tumors.

Determination of Cell Viability by MTT, Trypan Blue, andLactate Dehydrogenase Release Assay

For the assessment of the cytotoxic and cytostatic activitiesof G. lucidum extracts, tumor cells were seeded in 96-well, flat-bottom microplates (104 cells/well) in culture medium. After 18h to ensure cell attachment, different concentrations of GLmeor GLpme were added and incubated for 24 h. Cytotoxicity wasthen determined using the tetrazolium (MTT), trypan-blue (TB),and lactate dehydrogenase (LDH) release assays as previouslydescribed (20). In the MTT assay, cell respiration was assessedby the mitochondrial-dependent reduction of MTT to coloredformazan product (21), which reflected cell viability. Briefly,following incubation with the extracts, tumor cells were pulsedfor an additional hour with 0.5 mg/ml MTT, culture media wereaspirated, and the cells lysed in DMSO. The conversion of MTTto formazan was monitored on an automated microplate readerat 570 nm. The cell viability was expressed as a percentage ofthe control value (untreated cells), which was arbitrarily set to100%. In the TB assay, the ability of viable cells to excludethe stain was estimated. Following incubation with the extracts,tumor cells were collected by trypsinization and counted in0.2% TB solution using a hemocytometer. The relative numbersof dead cells were calculated as percentages of the numberof stained cells (22). As a marker of necrotic cell death, therelease of the intracellular enzyme, LDH, which mediates the

conversion 2,4-dinitrophenylhydrazine into a visible hydrazoneprecipitate in the presence of pyruvate, was measured (23). Celldeath was determined using the following formula: [(E – C)/(T– C)] × 100, where E is the experimental absorbance of treatedor untreated cultures measured at 492 nm, C is the absorbance ofthe medium without cells, and T is the absorbance correspondingto the maximal (100%) LDH release of Triton-lysed cells.

Detection of Cell Cycle, Apoptosis, and Necrosis by FlowCytometry

After 36 h or 24 h of incubation with or without GLme, flowcytometric analyses of the cell cycle, apoptosis, and necrosis oftumor cells were done exactly as previously described (20,24).Briefly, to assess cell distribution among the cell cycle phases,tumor cells were fixed in 70% ethanol and then incubated withRNase (50 µg/ml) and PI (40 µg/ml) for 30 min at 37◦C in thedark. Red fluorescence was analyzed on a FACSCalibur flowcytometer (BD) using a peak fluorescence gate to discriminateaggregates. Cell distribution among cell cycle phases was de-termined using Cell Quest Pro software (BD), and hypodiploidcells in the sub-G0/G1 compartment were considered apoptotic.Early apoptosis and necrosis were analyzed by double stainingwith Annexin V-FITC and PI in which Annexin V bound tothe apoptotic cells with exposed phosphatidylserine, whereasPI labeled the necrotic cells with membrane damage. The cells(1 × 104) were analyzed with a FACSCalibur flow cytometer,and the percentage of apoptotic (Annexin+ /PI−) and necrotic(Annexin+ /PI+) cells was determined with Cell Quest Pro soft-ware.

Measurement of ROS GenerationThe production of oxygen radicals was determined by mea-

suring the intensity of green fluorescence emitted by redox-sensitive dye, DHR, upon excitation at 488 nm. The cells werestained with 1 µM DHR for 10 min before the treatment withGLme. After 2 h incubation, cells were detached by trypsiniza-tion, washed in PBS, and the fluorescence intensity in the treatedcells was analyzed with a FACSCalibur flow cytometer usingCell Quest Pro software (25).

Detection of Caspase ActivationActivation of caspases was detected by flow cytometry after

labeling the cells with a cell-permeable, FITC-conjugated, pan-caspase inhibitor ApoStat (R&D Systems, Minneapolis, MN)according to the manufacturer’s instructions. Caspase activitycorresponded to green fluorescence (FL1). The results are ex-pressed as % of cells containing active caspases.

Detection of Caspase 3, Bax, Bcl-2, and p53 ProteinExpression by Cell-Based ELISA andImmunocytochemistry

The intracellular concentration of caspase 3, Bax, Bcl-2,and p53 was determined by the slightly modified method for


cell-based ELISA of Versteeg et al. (2000). B16, L929, and C6cells (1 × 104/well) were cultured for 10 h without or with 50or 100 µg/ml of GLme. Thereafter, the cells were fixed in 4%paraformaldehyde, endogenous peroxidase was quenched with1% H2O2 in PBS containing 0.1% Triton X-100 (PBS/T), andnonspecific binding of antibodies blocked with PBS/T solutioncontaining 10% FCS. Primary antibodies specific for caspase3 and Bcl-2 (mouse antimouse, eBioscience, San Diego, CA),Bax, and total p53 (rabbit antimouse, Santa Cruz Biotechnol-ogy, Santa Cruz, CA, all at a concentration 1:250) were ap-plied in PBS/T supplemented with 2% bovine serum albumin(PBS/TB). Secondary peroxidase-conjugated goat antimouseIgG (Healthcare, UK), or donkey antirabbit IgG (AmershamBiosciences, Buckinghamshire, UK) were added in 1:2500 di-lution in PBS/TB. Both incubations were at 37◦C for 1 h. Sub-sequently, the cells were washed and incubated with a solutioncontaining 0.4 mg/ml OPD, 11.8 mg/ml Na2HPO4× 2H2O, 7.3mg/ml citric acid, and 0.015% H2O2 for 30 min at room tem-perature in the dark. The reaction was stopped with 50 µl of3 M HCl, and the absorbance at 450 nm was determined ina microplate reader. The absorbance values were normalizedfor the cell number by crystal violet assay (26) as describedin the original protocol (27), and the results are presented asthe fold increase over the control values for untreated cellsarbitrarily set to 1. Alternatively, p53 protein expression wasdetected by immunocytochemistry. Briefly, at the end of the cul-tivation period, the untreated or treated cells were fixed in 4%paraformaldehyde, permeabilized with 0.5% Triton X-100 in

PBS and endogenous peroxidase was quenched with 3% H2O2-10% methanol in PBS. After blocking nonspecific binding of an-tibodies with PBS/T solution containing 5% FCS, the cells wereincubated with primary polyclonal anti-p53 (1:250) overnight at4◦C. A rabbit extravidin peroxidase staining kit was used for de-tection (Sigma, St. Louis, MO) according to the manufacturer’sinstructions with diaminobenzidine (R&D Systems, Minneapo-lis, MN) as the substrate. The cells were counterstained withMayer’s hematoxylin and slides were mounted with glycergelmounting medium (Dako, Glostrup, Denmark).

Statistical AnalysisThe results are presented as means ± SD of triplicate obser-

vations from one representative of at least 3 experiments withsimilar results, except if indicated otherwise. The significanceof the differences between various treatments was analyzed byanalysis of variance (ANOVA), followed by Student–Newman–Keuls test. The efficacy of in vivo treatments was evaluated byMann–Whitney U test. A P value less than 0.05 was consideredto be statistically significant.

RESULTS

HPLC Analysis of the Methanol Extracts of G. LucidumThe HPLC profiles of the two different methanol extracts,

GLme and GLpme, are presented in Fig. 1A and B. The chro-matograms revealed the complex composition of GLpme and

FIG. 1. HPLC profiling of two different extracts derived from G. lucidum. HPLC profiles of A: GLme and B: GLpme were obtained using a diode array detectoradjusted to 252 nm. Distinct classes of compounds were discerned by spectral evaluation of separated peaks.


especially of GLme. Standard mixtures of ganoderic acids(forms A, C2, H, and F) gave little or no match with the extractpeaks for spectral similarity and retention times. We registeredonly small amounts of ganoderic acids C2 and H in GLpme andGLme after detailed spectral evaluation of each peak but foundtwo more large peaks with UV spectra characteristic for gan-oderic acids (RT around 35 min). Comparison with publisheddata for similar chromatographic conditions suggested that thesepeaks may have originated from some other forms of ganodericacid. The highest peak in the chromatogram (RT 15 min) aswell as other abundantly retained compounds (RT > 40 min)showed spectra uncharacteristic for ganoderic acids but similarto UV spectra of estradiols or ganoderotriol, which are often re-ported as constituents of Ganoderma extracts. The most notice-able difference between GLpme and GLme was in the regioncharacteristic for less polar compounds where more complexterpenoid derivatives and polyphenols could be expected (RT >

40 min). Also GLpme lacked two strong ganoderic acid peaksat 35 min, withholding the strongest peak (possibly estradiols organoderotriol) at 15 min. Comparison of different sets of GLmeand GLpme extracts indicated that the extraction methods wereappropriate since they produced consistent chromatographic re-sults.

GLme Reduces B16 Melanoma Cell Growth in SyngeneicC57BL/6 Mice More Potently Than GLpme

The short-course treatment of tumor-induced mice withGLme resulted in a significant reduction of B16 tumor growth ascompared to the control tumor-bearing animals (Fig. 2A). The

FIG. 2. The effects of GLme and GLpme on melanoma growth in vivo. B16cells (2.5 × 105 cells/mouse) were injected subcutaneously into C57BL/6 mice.GLme and GLpme diluted in DMSO/PBS (100 mg/kg body weight/day), orDMSO/PBS alone (control) were administered intraperitoneally to mice suc-cessively for 13 days. Tumor volumes are presented as the mean size of tumorsper group at different time points after challenge. All data presented as mean± SD are representative of two independent experiments (n = 10 mice pergroup). A: *P < 0.05 refers to the control group. B: Representative tumorsfrom control, GLpme, and GLme treated mice were photographed at autopsy(Day 24).

effect was stable even after GLme withdrawal at 13 days aftertumor implantation. At the end of observation (Day 24 when thecontrol mice had to be euthanized), the average tumor volumein the group of mice treated with GLme was 14 times lowerthan that in the control group (Fig. 2A). In addition, treatmentof tumor-bearing animals with GLpme also reduced melanomagrowth almost twofold at autopsy; but this decrease was notstatistically significant (Fig. 2A). Representative tumors fromall 3 animal groups are shown in Fig. 2B. Together, the resultsimply that GLme exerted a much stronger antimelanoma effectin vivo than GLpme.

GLme Possesses Stronger Antitumor Activity Against B16Tumor Cells In Vitro Than GLpme

The antitumor effects of the two methanol extracts on B16tumor cells in vitro were compared. As evaluated by MTT assayafter 24 h of incubation (Fig. 3A), both extracts reduced mi-tochondrial respiration of B16 tumor cells in a dose-dependentmanner, but the antitumor effect of GLme was more pronounced,suggesting that additional purification of GLme resulted in lossof antitumor capacity. Since GLme demonstrated stronger anti-tumor activity than GLpme both in vitro and in vivo, this extractwas chosen for further experiments.

GLme Reduces Growth of Various Tumor Cells byInhibition of Proliferation and Induction of Cell Death

We next investigated whether the reduction of mitochondrialrespiration by GLme, seen in B16 cells, depended on the celltype tested. To this end, two other malignant rodent cell lines,mouse fibrosarcoma L929 and rat astrocytoma C6, togetherwith their nontransformed counterparts—rat astrocytes—weretreated with different concentrations of GLme. Treatment for 24h reduced the number of viable L929 (Fig. 3B), C6 cells, andastrocytes (Fig. 3C) in a dose-dependent manner as determinedby the MTT assay. Importantly, C6 cells were more sensitiveto the antiproliferative and/or cytotoxic activity of GLme thanprimary astrocytes (Fig. 3C), suggesting that, at least in somerespect, the growth inhibiting activity of this extract is tumor cellspecific. The surprisingly rapid decrease of cell mitochondrialrespiration induced by doubling the dose of 50 µg/ml of GLme(Fig. 3A–C) led us to investigate more precisely the mechanismsof antitumor actions of both doses (50 µg/ml – “lower” and 100µg/ml – “higher”). In order to assess if the decrease in numberof viable tumor cells was due to inhibition of cell proliferationor induction of tumor cell death by GLme, we performed TBand LDH assays, which are both based on features of disturbedmembrane permeability of dying cells. Fig. 3 D through F showsthat the higher dose of GLme was strongly cytotoxic to tumorcells in both tests, whereas cytotoxicity was not so pronouncedin the cultures treated with the lower dose. Thus, round anddetached dead tumor cells were observed under the microscopein cell cultures incubated with the higher GLme concentration,whereas treatment with lower GLme dose resulted mainly in


FIG. 3. The effect of GLme on viability of different tumor cells. B16 (A, D), L929 (B, E) and C6 (C, F) cells (1 × 104 cells/well) and C: astrocytes (3 × 104

cells/well) were cultured in the absence or presence of various concentrations of GLme (B–F), or GLme and GLpme (A). After 24 h, cell viability was determinedby MTT (A–C), trypan blue, and LDH assay (D–F). The results are presented as the mean ± SD of 3 independent experiments performed in triplicate. *P < 0.05refers to untreated cultures. Morphology of tumor cell cultures was assessed by light microscopy (D–F, lower panels).

tumor cell elongation and reduction of cell number (Fig. 3 D–F,lower panels). Altogether these results suggest that the antitu-mor effect of the lower GLme dose at 24 h ensued mostly fromantiproliferative action. However, tumor cell exposure to thisamount of GLme for 48 h also resulted in high cytotoxicity(data not shown), indicating that the inhibition of proliferationdetected at 24 h could be just the first step of the dying processinduced by the extract.

GLme Induces Both Apoptotic and Necrotic Death ofB16, L929, and C6 Tumor Cells

In order to explore the type of cell death induced by GLme,we performed flow cytometric analysis of Annexin V-FITC andPI stained cells after 24 h of cultivation with or without GLme.In agreement with our previous results, treatment with the higherdose strongly induced death of tumor cells by both apoptosis

(3.5, 1.9, and 0.8% in control cells vs. 29.1, 41.2, and 31.9%in treated cells for B16, L929, and C6 cells, respectively) andnecrosis (3.6, 5.6, and 2.0% in control cells vs. 12.2, 15.4, and17.5% in treated cells for B16, L929, and C6 cells, respectively),whereas the amount of neither apoptotic nor necrotic cells inB16, L929, and C6 cell cultures was elevated after treatmentwith the lower dose of GLme (Fig. 4A–C). To confirm furtherthe occurrence of apoptosis in cell cultures treated with GLme,we analyzed PI stained cells flow cytometrically after 36 h ofincubation. Analysis of cellular DNA content revealed a sig-nificant increase in the proportion of hypodiploid cells treatedwith higher dose (1.0, 0.5, and 0.9% in control cells vs. 61.2,36.9, and 40.4% in treated cells for B16, L929, and C6 cells,respectively), whereas the lower dose of GLme induced signif-icant accumulation in sub-G phase of C6 cells only (0.9% incontrol cells vs. 8.4% in treated cells; Fig. 4 D–F). In addi-tion, treatment with the lower GLme concentration modulated


FIG. 4. The effect of GLme on the cell cycle, apoptosis, and necrosis in different tumor cells. B16 (A, D), L929 (B, E), and C6 (C, F) cells (5 × 105 cells/well)were cultured in the absence or presence of two different concentrations of GLme as indicated. Cells were stained with Annexin V-FITC and PI after 24 h (A–C)or PI alone after 36 h (D–F) and analyzed by flow cytometry. Each histogram was conducted with data from at least 10,000 events. The results are presented as themean ± SD of 3 independent experiments. *P < 0.05 refers to untreated cultures.

(although not statistically significantly) the cell cycle of all 3tumor cell lines tested, and this cell cycle arrest was probablyresponsible for the previously shown inhibition of cell prolifer-ation. Interestingly, whereas 50 µg/ml of GLme induced B16cell cycle arrest mostly in the G0/G1 phase (54.7 vs. 65.8% forcontrol and treated cells, respectively), C6 and L929 tumor cellswere arrested in the G2/M phase (24.5 and 29.3% in controlcells vs. 34.5 and 36.3% in treated cells for L929 and C6 cells,respectively), suggesting that the cell cycle modulating activityof GLme could be cell-type specific and controlled by differentsignaling pathways. However, cell-based ELISA demonstratedthat GLme-induced apoptosis of B16, L929, and C6 tumor cellsis mediated by caspase activation, Bcl-2 downregulation, andp53 increase.

In order to determine mechanism of apoptotic cell death in-duced by GLme, we investigated expression and activation ofdifferent proapoptotic and antiapoptotic molecules in all 3 celllines. Flow cytometric analyses demonstrated that after 16 h,GLme stimulated activity of caspases, enzymes with essentialrole in apoptosis (Fig. 5A). Upregulation of activity of maineffector caspase–caspase 3 was also confirmed by cell basedELISA (Fig. 5B). However, as revealed by cell based ELISA,

after 12 h of incubation in the presence of GLme, expressionof proapoptotic molecule Bax was not affected. On the otherhand, the expression of antiapoptotic molecule Bcl-2 was down-regulated in all cell lines tested, thus resulting in an increasedBax/Bcl-2 ratio, and data obtained on L929 cells are presented asrepresentative (Fig. 5C–E). Since the tumor suppressor proteinp53 is involved in inhibition of proliferation and induction ofapoptosis (28), we next studied the effects of GLme on p53 ex-pression. In concordance with the results presented above, levelof total p53 protein expression detected by cell-based ELISAwas significantly higher in all 3 GLme-treated tumor cell lines(Fig. 5F). In addition, a strong increase in expression of p53protein was confirmed by immunocytochemical analysis andpresented on L929 cells as representative (Fig. 5G). Therefore,apoptotic cell death of tumor cells induced by GLme is probablymediated by increase of p53, Bax/Bcl-2 ratio, and activation ofcaspases.

Antitumor Effect of GLme Is Mediated by Oxidative StressIt has been known that oxidative stress activates cell death

pathways (29). We therefore investigated the involvement of


FIG. 5. The effect of GLme on caspase activation, Bax, Bcl-2, and p53 protein expression. Tumor cells (5 × 105 cells/well for A and 1 × 104 cells/well forB–G) were cultured in the absence or presence of two different concentrations of GLme as indicated. A: After 16 h of incubation, C6 cells were stained with FITC-conjugated pan-caspase inhibitor ApoStat and analyzed by flow cytometry. Cell-based ELISA of B16, L929, and C6 cells for caspase 3 detection was performedafter 16 h (B); of L929 cells for Bax and Bcl-2 detecton was performed after 12 h (C–E); and of B16, L929, and C6 cells for p53 Ab after 10 h of treatment withGLme (F). Immunocytochemical staining of L929 cells (G) with anti-p53 Ab was performed after 10 h of treatment with GLme. Immunocytochemical staining ofL929 cells for p53 was performed after 10 h of treatment with GLme (G). The results are presented as the mean ± SD of 3 independent experiments. *P < 0.05refers to untreated controls.

oxidative stress in the observed cytotoxic action of GLme. Flowcytometric analysis of DHR stained tumor cells after 2 h ofincubation with GLme showed that this treatment markedly el-evated the production of ROS (Fig. 6A–C). The induction ofROS by GLme was dose dependent and was partly blocked inthe presence of the antioxidant NAC (Fig. 6A–C). The rele-vance of oxidative stress for the antitumor activity of GLmewas confirmed by determination of tumor cell viability af-ter 24 h of treatment with GLme in the presence or absence

of the antioxidant. Fig. 6D through F shows that neutraliza-tion of ROS by the scavenger NAC resulted in partial recov-ery of cell viability, especially of cells treated with the higherdose of GLme, indicating that release of ROS was at leastpartly responsible for the tumoricidal effect of the extract. Therelevance of oxidative stress in the antitumor activity of thehigher rather than the lower GLme dose probably only relatesto the quantity of released ROS and the resulting intracellulardamage.


FIG. 6. The effect of GLme on oxidative stress in different tumor cells. B16 (A, D), L929 (B, E), and C6 (C, F) tumor cells [5 × 105 cells/well (A–C)or 1 × 104 cells/well (D–F)] were treated with two different concentrations of GLme, as indicated, in the absence or presence of 5 mM NAC. Production ofreactive oxidative species was examined by flow cytometry after 2 h (A–C), and cell viability was detected by the MTT test after 24 h (D–F). The results arepresented as the mean ± SD of 3 independent experiments. *P < 0.05 refers to corresponding controls without GLme (A–C), or without NAC (D–F). DHR,dihydrorhodamine 123.

DISCUSSIONIn this article, it is shown that the G. lucidum derived

methanol extract, GLme, which contained total terpenoids, ex-hibited strong antitumor activity against B16 mouse melanomacells both in vitro and in vivo, whereas the purified methanolextract, GLpme, which contained mainly acidic terpenoids, wasless effective. An anticancer effect of GLme in vitro againstmouse fibrosarcoma L929 and rat astrocytoma C6 cells wasalso demonstrated. This activity was mediated by both inhi-bition of cell proliferation and induction of programmed andaccidental cell death, depending on the extract concentration.Mechanisms of GLme compounds action were related to theirability to amplify ROS production, upregulate p53, and in-hibit Bcl-2 expression, promoting the death signal through cas-pase activation. The antitumor effect of G. lucidum derivedextracts has been widely demonstrated in both animals andhumans. In addition to polysaccharides, various triterpenoidswere found to be the main anticancer bioactive ingredients.For isolation of terpenoid enriched extracts, we used a mod-ified method combining extraction with organic solvents andsubsequent selective isolation of acidic terpenoids (30,31). The

first step yielded a fraction of total terpenoids (GLme), fol-lowed by refinement enriching the acidic terpenoids (GLpme).Since GLpme was derived from GLme, it was expected that itschemical composition would be less complex, and this wasconfirmed by HPLC analysis. Both extracts contained gan-oderotriols and small amounts of ganoderic acids C2 and H,which together constitute the terpenoid fraction (2). In addi-tion, as expected, GLpme did not contain less polar compoundssuch as more complex terpenoid derivatives and polyphenols(RT > 40 min). However, although acidic terpenoids were ex-pected in GLpme, this extract lacked two strong ganoderic acidpeaks close to 35 min. Moreover, both extracts obtained froma G. lucidum strain originating in Serbia contained lower quan-tities of ganoderic acids than similar extracts obtained fromstrains originating from other regions (30,11). However, a sim-ilar deficiency was reported in a strain originating from Japan(32), supporting the existence of strain specificity for terpenoidextract composition. Our results thus imply involvement ofsome other terpenoid molecules in the antitumor activity ofG. lucidum extracts besides the widely investigated ganodericacids.


In vitro experiments demonstrated that both extracts ex-erted powerful antimelanoma activity, but the antitumor ef-fect of GLme was stronger (IC50 values were 20.5 µg/ml and94.6 µg/ml for GLme and GLpme, respectively). Similarly,both extracts showed antitumor effects in the in vivo model ofmelanoma in C57BL/6 mice. However, although GLme treat-ment reduced average tumor volume 14-fold, the decrease intumor volume in the group of animals treated with GLpme didnot reach statistical significance. This suggests that further pu-rification of GLme reduced its biological activity both in vitroand in vivo. It is not clear whether the loss of two ganodericacids with peaks at 35 min, or of nonpolar terpenoids, was re-sponsible for this reduction of antitumor activity of GLpme. Itis possible that the loss of several different compounds togetherdiminished the biological effect. Namely, although numerousstudies on G. lucidum have shown its effectiveness in the treat-ment of a wide range of diseases and symptoms, none of theknown active components taken alone have produced results aspowerful as the original mixture of active compounds present inthe mushroom (33). Moreover, different compounds present inthe extracts can modulate unrelated signaling pathways and thusproduce a synergistic effect (34). Therefore, although the com-plexity of natural products is a major obstacle for their broadacceptance in Western medicine, our results support the ideathat this complexity is actually a great advantage and justifieswidespread use of the mushroom in the form of tea, powder, orextract instead of as single compounds.

In addition to the antimelanoma effects, we found that GLmealso inhibited growth of C6 and L929 cells in vitro. Althoughcytotoxicity of different G. lucidum components has been al-ready shown in a variety of tumor cells (35,36), this is the firststudy demonstrating its antimelanoma, antiglioma, and antifi-brosarcoma activity. The antitumor activity of GLme followeda similar mechanism in all 3 cell lines including inhibition oftumor cell proliferation and induction of programmed and ac-cidental cell death. Activation of cell death is beneficial in pre-neoplastic or tumor cells but may result in toxicity if it occursin normal cells too. However, we found that normal primaryastrocytes were less sensitive to the cytotoxic effect of GLmethan C6 astrocytoma cells. Moreover, in vivo application of theextract showed no signs of obvious toxicity, with regard to bodyweight and animal behavior, suggesting that GLme was verywell tolerated.

It is well known that reactive oxygen species can inhibit cellproliferation and induce apoptotic and necrotic cell death (37).We have demonstrated, for the first time, that the antitumor ef-fect of G. lucidum is mediated partly by oxidative stress, eventhough several studies have shown that G. lucidum possessesantioxidative properties. This difference could be explained bythe fact that our methanol extract was mainly composed of ter-penoids, whereas the antioxidative property of other extractsensued from compounds such as polysaccharides (38), peptides(39), amino-polysaccharides (40), and sterols (41). Neverthe-less, in one study, the antioxidative action of G. lucidum was

attributed to triterpenes (42). This discrepancy might be due touse of different cells and extracts that were similar but not iden-tical. Moreover, in our study, the terpenoid-containing extractaffected tumor cells directly, whereas Zhu et al. (42) demon-strated antioxidative properties of mushroom derived triterpeneson externally induced lipid peroxidation and oxidation of ery-throcyte membranes. One should also have in mind that therole of reactive oxygen species in tumor growth and progres-sion is context dependent and that although in our study ox-idative stress contributed to the eradication of tumor growthin vitro, oxidative stress can also drive cancer initiation andprogression through mutations caused by DNA damage and beinvolved in the promotion of angiogenesis and metastasis ofcancer (43).

Considering that terpenoids and terpenoid-containing ex-tracts derived from G. lucidum can arrest the tumor cell cyclein the G0/G1 (8,44) and G2/M (9) phases, it is not surprisingthat treatment with GLme partially blocked the cell cycle of C6and of L929 cells in the G2/M phase and that of B16 cells in theG0/G1 phase. It is well known that tumor suppressor protein p53is one of the main regulators of cell cycle progression and apop-tosis and that disruption of p53 expression in transformed cellsresults in apoptotic resistance and cancerogenesis, whereas over-expression leads to cell death (45). Accordingly, in this study,GLme probably directly and indirectly damaged DNA and cel-lular proteins, leading to upregulation of p53 expression andconsequent induction of cell cycle arrest and apoptosis. Havingin mind that the extract of GLme has numerous biologicallyactive compounds, we could suppose that some of them mayact on p53-dependent manner, but also the others are capableto promote different alternative intracellular signals with simi-lar outcome. Therefore, the role of p53 is important, but mostprobably not the exclusive, in GLme triggered death signals inmalignant cells. Since increased expression of p53 is connectedwith elevated sensitivity of tumor cells to various chemothera-peutics and the host immune response (46), the p53 upregulatingproperties of GLme could be also useful for chemosensitizationor immunosensitization of tumor cells.

Whereas the antitumor activity of GLme at 50 µg/ml wasexerted mainly through inhibition of tumor cell proliferation,doubling the concentration strongly induced tumor cell death,leading to a dramatic difference in the intensity of antitumor ac-tion of these two contiguous doses. This signifies the importanceof precise dose determination for eventual therapeutic use of theextract. However, the finding that the lower dose had mainly anantiproliferative effect was essentially correct only for the first24 h of treatment because after 48 h, a considerable quantity ofdead cells was also observed (data not shown). This suggeststhat there is no intrinsic difference between the antitumor mech-anisms of the two concentrations and that cell cycle arrest ob-served in the first 24 h in cell cultures treated with the lower doseprobably only represents an attempt by the tumor cells to repairtheir damaged DNA. When the injury develops so extensivelythat repair mechanisms become powerless, more cells begin to


die. GLme treatment (with the higher dose after 24 h and with thelower dose after 48 h) induced apoptotic cell death in all 3 tumorcell lines. Elevated caspase activity observed, besides exagger-ated p53 expression, was accompanied with abrogated Bcl-2protection and consequent domination of proapoptotic events.This proapoptotic-endorsed balance is particularly importantin melanoma and glioma cells relatively resistant to apoptoticstimuli (47,48), making the extract a promising candidate formelanoma and glioma treatment, either alone or in combina-tion with other antitumor therapies based on different modes ofaction. Besides apoptosis, necrosis was also detected in GLmetreated tumor cell cultures not only as appearance of Ann+ /PI+

cells in flow cytometric analyses but also as an increase in LDHrelease. However, only negligible LDH release was detected inthe first 18 h of cell incubation with GLme (not shown), im-plying that the observed necrosis might be secondary instead ofprimary. In the context of the antitumor response in vivo, celldeath by pure apoptosis or pure necrosis has some disadvan-tages. Namely, whereas apoptosis usually ends with ingestionof cell residues by nearby cells and is therefore relatively safefor local normal tissue, no inflammation is induced, accompa-nied with weak immunogenesis. On the other hand, necrotic celldeath ends with disruption of the cell membranes and release ofintracellular compounds into the microenvironment, promotingdestruction of normal local tissue but inducing a strong immuneresponse (49). Therefore, it seems that balanced occurrence ofboth types of cell death is preferable for an efficient antitumorresponse and that the capacity of GLme to trigger necrosis inaddition to apoptosis could actually represent an advantage foreventual anticancer therapy.

Cytotoxicity in cell culture systems is a property of mostclinically used antitumor agents. Accordingly, the melanomaregression demonstrated in vivo could be at least partly medi-ated by direct cytotoxic action of GLme. The effects of GLmetreatment on different components of the antitumor immuneresponse, metastasis and angiogenesis, were not investigatedhere. However, considering that the immunostimulating, an-tiangiogenic, and antimetastatic activity of G. lucidum derivedterpenoid-containing extracts have been demonstrated by oth-ers (10,11,15,50), it is possible that GLme possesses similarproperties.

The strong antimelanoma, antifibrosarcoma, and antigliomaproperties of GLme demonstrated in vitro, accompanied withregression of melanoma growth after well tolerated in vivo treat-ment, suggest that this extract is worth additional studies as apotential chemotherapeutic for treatment of various forms ofcancer. Having in mind that final outcome of tumor develop-ment depends not only on direct effect of this extract on tumorcells but also on various other antitumor mechanisms, our futurestudies would be comprised to investigate effects of GLme onthe immune system. In addition, in order to find most effectiveapproaches, which at the same time have less side effects, it is ofprimary interest to investigate the effects of combined treatmentof GLme with conventional chemotherapeutics.

ACKNOWLEDGMENTSThis work was supported by the Serbian Ministry of Science

(Grants No. 143029 and 143016). The authors thank Dr ZeljkoVucinic (Institute for Multidisciplinary Research) for criticallyperusing the manuscript and Marija Mojic for technical assis-tance.

REFERENCES1. Moradali MF, Mostafavi H, Ghods S, and Hedjaroude GA: Immunomod-

ulating and anticancer agents in the realm of macromycetes fungi (macro-fungi). Int Immunopharmacol 7, 701–724, 2007.

2. Paterson RR: Ganoderma—a therapeutic fungal biofactory. Phytochemistry67, 1985–2001, 2006.

3. Niu JF, Fang Z, Wang HJ, and Wang GC: The research advance on theeffective constituents of Ganoderma spp. J Agric Univ Hebei 25, 51–54,2002.

4. Zhou X, Lin J, Yin Y, Zhao J, Sun X, et al.: Ganodermataceae: naturalproducts and their related pharmacological functions. Am J Chin Med 35,559–574, 2007.

5. Sliva D: Ganoderma lucidum in cancer research. Leuk Res 30, 767–768,2006.

6. Lei LS and Lin ZB: Effect of Ganoderma polysaccharides on T cell sub-populations and production of interleukin 2 in mixed lymphocyte response.Yao Xue Xue Bao 27, 331–335, 1992.

7. Won SJ, Lin MT, and Wu WL: Ganoderma tsugae mycelium enhancessplenic natural killer cell activity and serum interferon production in mice.Jpn J Pharmacol 59, 171–176, 1992.

8. Tang W, Liu JW, Zhao WM, Wei DZ, and Zhong JJ: Ganoderic acid T fromGanoderma lucidum mycelia induces mitochondria mediated apoptosis inlung cancer cells. Life Sci 80, 205–211, 2006.

9. Lin SB, Li CH, Lee SS, and Kan LS: Triterpene-enriched extracts fromGanoderma lucidum inhibit growth of hepatoma cells via suppressing pro-tein kinase C, activating mitogen-activated protein kinases and G2-phasecell cycle arrest. Life Sci 72, 2381–2390, 2003.

10. Thyagarajan A, Jiang J, Hopf A, Adamec J, and Sliva D: Inhibition of ox-idative stress-induced invasiveness of cancer cells by Ganoderma lucidumis mediated through the suppression of interleukin-8 secretion. Int J MolMed 18, 657–664, 2006.

11. Kimura Y, Taniguchi M, and Baba K: Antitumor and antimetastatic effectson liver of triterpenoid fractions of Ganoderma lucidum: mechanism ofaction and isolation of an active substance. Anticancer Res 22, 3309–3318,2002.

12. Huie CW and Di X: Chromatographic and electrophoretic methods forLingzhi pharmacologically active components. J Chromatogr B AnalytTechnol Biomed Life Sci 812, 241–257, 2004.

13. Su HJ, Fann YF, Chung MI, Won SJ, and Lin CN: New lanostanoids ofGanoderma tsugae. J Nat Prod 63, 514–516, 2000.

14. Ma J, Ye Q, Hua Y, Zhang D, Cooper R, et al.: New lanostanoidsfrom the mushroom Ganoderma lucidum. J Nat Prod 65, 72–75,2002.

15. Luo J, Zhao YY, and Li ZB: A new lanostane-type triterpene from thefruiting bodies of Ganoderma lucidum. J Asian Nat Prod Res 4, 129–134,2002.

16. Al-Fatimi M, Wurster M, Kreisel H, and Lindenquist U: Antimicrobial,cytotoxic and antioxidant activity of selected basidiomycetes from Yemen.Pharmazie 60, 776–780, 2005.

17. Folch J, Lees M, and Sloane Stanley GH: A simple method for the isolationand purification of total lipids from animal tissues. J Biol Chem 226, 497–509, 1957.

18. McCarthy KD and de Vellis J: Preparation of separate astroglial and oligo-dendroglial cell cultures from rat cerebral tissue. J Cell Biol 85, 890–902,1980.


19. Buhtoiarov IN, Lum HE, Berke G, Sondel PM, and Rakhmilevich AL:Synergistic activation of macrophages via CD40 and TLR9 results in T cellindependent anti-tumor effects. J Immunol 176, 308–318, 2006.

20. Harhaji Lj, Mijatovic S, Maksimovic-Ivanic D, Stojanovic I, MomcilovicM, et al.: Anti-tumor effect of Coriolus versicolor methanol extract againstmouse B16 melanoma cells: in vitro and in vivo study. Food Chem Toxicol46, 1825–1833, 2008.

21. Mosmann T: Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. J Immunol Methods 65,55–63, 1983.

22. Cheon GJ, Chung HK, Choi JA, Lee SJ, Ahn SH, et al. : Cellular metabolicresponses of PET radiotracers to (188)Re radiation in an MCF7 cell linecontaining dominant-negative mutant p53. Nucl Med Biol 34, 425–432,2007.

23. Decker T and Lohmann-Matthes ML: A quick and simple method for thequantitation of lactate dehydrogenase release in measurements of cellularcytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods115, 61–69, 1988.

24. Mijatovic S, Maksimovic-Ivanic D, Radovic J, Miljkovic D, KaludjerovicGN, et al.: Aloe emodin decreases the ERK-dependent anticancer activityof cisplatin. Cell Mol Life Sci 62, 1275–1282, 2005.

25. Kaludjerovic GN, Miljkovic D, Momcilovic M, Djinovic VM, MostaricaStojkovic M, et al.: Novel platinum(IV) complexes induce rapid tumor celldeath in vitro. Int J Cancer 116, 479–486, 2005.

26. Flick DA and Gifford GE: Comparison of in vitro cell cytotoxic assays fortumor necrosis factor. J Immunol Methods 68, 167–175, 1984.

27. Versteeg HH, Nijhuis E, van den Brink GR, Evertzen M, Pynaert GN, et al.:A new phosphospecific cell-based ELISA for p42/p44 mitogen-activatedprotein kinase (MAPK), p38 MAPK, protein kinase B and cAMP-response-element-binding protein. Biochem J 3, 717–722, 2000.

28. Han X, Wang F, Yao W, Xing H, Weng D, et al.: Heat shock proteins andp53 play a critical role in K+ channel-mediated tumor cell proliferation andapoptosis. Apoptosis 12, 1837–1846, 2007.

29. Higuchi Y: Chromosomal DNA fragmentation in apoptosis and necro-sis induced by oxidative stress. Biochem Pharmacol 66, 1527–1535,2003.

30. Min BS, Gao JJ, Nakamura N, and Hattori M: Triterpenes from the spores ofGanoderma lucidum and their cytotoxicity against meth-A and LLC tumorcells. Chem Pharm Bull (Tokyo) 48, 1026–1033, 2000.

31. Linksel-Mekkawy S, Meselhy MR, Nakamura N, Tezuka Y, Hattori M,et al.: Anti-HIV-1 and anti-HIV-1-protease substances from Ganodermalucidum. Phytochemistry 49, 1651–1657, 1998.

32. Hirotani M, Ino C, and Furuya T: Comparative study on the strain-specifictriterpenoid components of Ganoderma lucidum. Phytochemistry 33, 379–382, 1993.

33. Huie CW: A review of modern sample preparation techniques for the ex-traction and analysis of medicinal plants. Anal Bioanal Chem 373, 23–30,2002.

34. Williamson EM: Synergy and other interactions in phytomedicines. Phy-tomedicine 8, 401–409, 2001.

35. Gonzalez AG, Leon F, Rivera A, Padron JI, Gonzalez-Plata J, et al.: Newlanostanoids from the fungus Ganoderma concinna. J Nat Prod 65, 417–421, 2002.

36. Jiang J, Slivova V, Valachovicova T, Harvey K, and Sliva D: Ganodermalucidum inhibits proliferation and induces apoptosis in human prostatecancer cells PC-3. Int J Oncol 24, 1093–1099, 2004.

37. Zhang SS, Zhang HQ, Li D, Sun LH, Ma CP, et al.: A novel benzotriazolederivative inhibits proliferation of human hepatocarcinoma cells by increas-ing oxidative stress concomitant mitochondrial damage. Eur J Pharmacol584, 144–152, 2008.

38. Lai KN, Chan LY, Tang SC, and Leung JC: Ganoderma extract preventsalbumin-induced oxidative damage and chemokines synthesis in culturedhuman proximal tubular epithelial cells. Nephrol Dial Transplant 21, 1188–1197, 2006.

39. Sun J, He H, and Xie BJ: Novel antioxidant peptides from fermentedmushroom Ganoderma lucidum. J Agric Food Chem 52, 6646–6652, 2004.

40. Lee JM, Kwon H, Jeong H, Lee JW, Lee SY, et al.: Inhibition of lipid per-oxidation and oxidative DNA damage by Ganoderma lucidum. PhytotherRes 15, 245–249, 2001.

41. Zhao HB, Wang SZ, He QH, Yuan L, Chen AF, et al.: Ganoderma to-tal sterol (GS) and GS1 protect rat cerebral cortical neurons from hy-poxia/reoxygenation injury. Life Sci 76, 1027–1037, 2005.

42. Zhu M, Chang Q, Wong LK, Chong FS, and Li RC: Triterpene antioxidantsfrom Ganoderma lucidum. Phytother Res 13, 529–531, 1999.

43. Brown NS and Bicknell R: Hypoxia and oxidative stress: its effects ongrowth, metastatic potential and response to therapy of breast cancer. BreastCancer Res 3, 323–327, 2001.

44. Yang HL: Ganoderic acid produced from submerged culture of Ganodermalucidum induces cell cycle arrest and cytotoxicity in human hepatoma cellline BEL7402. Biotechnol Lett 27, 835–838, 2005.

45. Kuerbitz SJ, Plunkett BS, Walsh WV, and Kastan MB: Wild-type p53 is acell cycle checkpoint determinant following irradiation. Proc Natl Acad SciUSA 89, 7491–7495, 1992.

46. Muret J, Yacoub M, Terrier P, Drusch F, Laplanche A, et al.: p53 status cor-relates with histopathological response in patients with soft tissue sarcomastreated using isolated limb perfusion with TNF-alpha and melphalan. AnnOncol 19, 793–800 2008.

47. Soengas MS and Lowe SW: Apoptosis and melanoma chemoresistance.Oncogene 22, 3138–3151, 2003.

48. Jendrossek V, Kugler W, Erdlenbruch B, Eibl H, Lang F, et al.:Erucylphosphocholine-induced apoptosis in chemoresistant glioblastomacell lines: involvement of caspase activation and mitochondrial alterations.Anticancer Res 21, 3389–3396, 2001.

49. Melcher A, Todryk S, Hardwick N, Ford M, Jacobson M, et al.: Tumorimmunogenicity is determined by the mechanism of cell death via inductionof heat shock protein expression. Nat Med 4, 581–587, 1998.

50. Wang G, Zhao J, Liu J, Huang Y, Zhong JJ, et al.: Enhancement of IL-2 andIFN-gamma expression and NK cells activity involved in the anti-tumoreffect of ganoderic acid Me in vivo. Int Immunopharmacol 7, 864–870,2007.