Leukemia Research 30 (2006) 841–848

Ganoderma lucidum causes apoptosis in leukemia,

lymphoma and multiple myeloma cells
Claudia I. Müller a,b,∗ , Takashi Kumagai a , James O’Kelly a ,
Navindra P. Seeram c , David Heber c , H. Phillip Koeffler a
a Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States
b Division of Haematology/Oncology, University Hospital, Freiburg, Germany
c Center for Human Nutrition, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States

Received 4 May 2005; received in revised form 5 December 2005; accepted 6 December 2005
Available online 19 January 2006

Abstract

Over many centuries, herbal remedies have treated a variety of ailments. This empiric observational approach has produced a number
of leads for formulated medicines. Ganoderma lucidum extract was screened for its anti-proliferative activity using a panel of 26 human
cancer cell lines. The six most sensitive hematologic cell lines were: HL-60 (ED50 26 ␮g/ml), U937 (63 ␮g/ml), K562 (50 ␮g/ml), Blin-1
(38 ␮g/ml), Nalm-6 (30 ␮g/ml) and RPMI8226 (40 ␮g/ml). Cell cycle analyses revealed a G2/M arrest, most prominently in HL-60 cells. Four
hematopoietic cell lines (HL-60, Blin-1, U937, RPMI8226) were examined for apoptosis, which ranged between 21 and 92%. After exposure
to G. lucidum extract, HL-60 cells became multinucleated with an increased DNA content. These results indicate that G. lucidum extract
has a profound activity against leukemia, lymphoma and multiple myeloma cells and may be a novel adjunctive therapy for the treatment of
hematologic malignancies.
© 2005 Elsevier Ltd. All rights reserved.

Keywords: Ganoderma lucidum; Leukemia; Lymphoma; Multiple myeloma; Apoptosis; Growth arrest

1. Introduction
Ganoderma lucidum (Fr.) Karst (common names: Reishei,
Lingzhi) is a herbal mushroom that has been used for cen-
turies in Traditional Chinese Medicine (TCM) for its health
promoting properties [1]. The fruiting bodies, spores and cul-
tivated mycelia of G. lucidum as well as its extracts are used
world-wide as ingredients in health foods, herbal medicines
and dietary supplements (extracts and powdered forms) and
have been used as anti-cancer agents and for prevention and
treatment of various other diseases in ancient China (100 bc)
[2–6]. Previous reports revealed that G. lucidum extract inhib-
∗ Corresponding author at: Division of Hematology/Oncology, Davis
Building 5065, Cedars-Sinai Medical Center, David Geffen School of
Medicine at UCLA, 8700 Beverly Boulevard, Los Angeles, CA 90048,
United States. Tel.: +1 310 423 7759; fax: +1 310 423 0225.
E-mail address: MullerCI@cshs.org (C.I. Müller).
ited growth of several cancer cell lines including the human
prostate PC-3 cancer cells, and several human bladder can-
cer cell lines. These cells were arrested in the G2/M phase
of their cell cycle [7,8]. Also, Ganoderma in a dose- and
time-dependent manner inhibited the cell proliferation and
induced apoptosis of HT-29, a human colon carcinoma cell
line and MDA-MB-231 and MCF-7 breast cancer cell lines
[5,9,10].
The phytochemical constituents of Ganoderma include
polysaccharides, proteins, nucleosides, fatty acids, sterols,
cerebrosides and triterpenes [11]. The triterpenoids com-
mon to Ganoderma include the ganoderic acids that may be
considered as a chemical marker compound to authenticate
and/or standardize Ganoderma extracts and products [12,13].
The polysaccharide fraction of Ganoderma was shown
to slow growth of sarcoma cells growing in mice [14]. The
polysaccharides were able to induce the expression of IL-
1, IL-6, IL-12, IFN-␥, TNF-␣, GM-CSF, G-CSF, M-CSF in

0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.leukres.2005.12.004
842 C.I. Müller et al. / Leukemia Research 30 (2006) 841–848

monocytes–macrophages and T lymphocytes which might, 2. Materials and methods

in part, mediate some of their anti-tumor activity [1,15,16].
Furthermore, these polysaccharides may also have immune 2.1. Extraction of G. lucidum and standardization by
enhancing properties perhaps as a consequence of their ability HPLC analysis
to stimulate cytokine production [15].
Triterpenes have been shown to inhibit growth of human G. lucidum fruiting bodies were collected and authen-
hepatoma Huh-7 cells associated with G2 cell cycle arrest, but ticated in China by Phytomedical Research Inc. (Beijing,
the compounds had no effect on growth of “normal” liver cell China), and Botanica Biosciences (Ojai, CA). A commercial
lines in vitro [2]. Moreover, the triterpene-fraction of Gano- standard of ganoderic acid C2 was purchased from Chro-
derma inhibited the primary and metastatic tumor growth of madex (Santa Ana, CA). G. lucidum fruiting bodies were
Lewis lung carcinoma (LLC) cells implanted in mice [17]. extracted in water and lyophilized to yield a dark brown
This study investigated the anti-cancer effects of G. powder.
lucidum using a panel of 26 human cancer cell lines (16 hema- All solvents were HPLC (high performance liquid chro-
tologic malignancies; see Section 2). A detailed description matography) grade and were purchased from Fisher (Tustin,
of the characteristics of each cell line is also given in Table 1. CA). Ganoderma extract (22.28 mg) was sonicated for
30 min with 4 ml (MeOH:water, 1:1 v/v). Ganoderic acid C2
Table 1 was dissolved in MeOH to make a stock solution of 1 mg/ml
Human cancer cell lines examined for anti-proliferative effects of G. lucidum that was further serially diluted with MeOH:water (1:1 v/v).
extract Quantitation was done by peak area measurements in compar-
Cell line Characteristics ison with a standard curve generated for ganoderic acid C2.
HL-60 Acute myeloblastic leukemia 16 hematological All samples (25 ␮l injection volume) were filtered (0.22 ␮m)
(FAB M2) cell lines before analysis on a Waters 2690 HPLC system equipped
U937 Diffuse histiocytic lymphoma with a 996 PDA detector (Waters, Milford, MA). Compounds
(monocytic leukemia cells)
NB4 Promyelocytic leukemia (FAB
were eluted at 1.00 ml/min on a Novapak (Waters) RP-18 col-
M3) umn (150 mm × 3.9 mm i.d., 5 ␮m) with a Symmetry C18
THP-1 Acute monocytic leukemia (FAB guard column (20 mm × 3.9 mm i.d., 5 ␮m). The eluent con-
M5) sisted of 2% aqueous acetic acid (A) and acetonitrile (B). A
K562 Erythroid chronic myeloid gradient solvent system was used as follows: 0–5 min 95% A
leukemia (blast crisis)
Blin-1 Pre-B acute lymphoblastic
in B; 6–60 min 5% A in B. The wavelength of detection was
leukemia 254 nm.
Nalm-6 Non-T, non-B acute
lymphoblastic leukemia 2.2. Cell lines
Jurkat T-cell acute lymphoblastic
leukemia
RPMI8226 Multiple myeloma (IgG␭)
Cell lines that were studied included: myeloid leukemia
ARH77 Multiple myeloma (IgG␬) (HL-60, U937, K562, THP-1, NB4), acute lymphoblastic
U266 Multiple myeloma (IgE␭) leukemia (B- and T-ALL) (Blin-1, Nalm-6, Jurkat), mul-
NCI-H929 Multiple myeloma (IgA␬) tiple myeloma (RPMI8226, ARH77, U266, NCI-H929),
Daudi Burkitt’s lymphoma Burkitt’s lymphoma (Daudi, Ramos), non-Hodgkin’s lym-
Ramos Burkitt’s lymphoma
NCEB-1 Centroblastic–centrocytic, diffuse
phoma (NCEB-1, SUDHL6), prostate cancer (LNCaP, PC-3,
lymphoma DU145), breast cancer (MCF-7, MDA-MB-231), colorectal
SUDHL6 Diffuse large-B-cell lymphoma cancer (HT-29), pancreatic cancer (PANCI, ASPC1, BxPC-
LNCaP Prostate cancer (androgen 10 solid tumor 3) and non-small cell lung cancer (NSCLC) (NCI-H520) (see
receptor positive) cell lines Table 1). Most cell lines were obtained from American Type
PC-3 Prostate cancer (androgen Culture Collection (ATCC, Rockville, MD), Blin-1, NCEB-
receptor negative) 1 and SUDHL6 cells were generously provided by Sven de
DU145 Prostate cancer (androgen
Vos (University of California, Los Angeles). NB4 cells were a
receptor negative)
MCF-7 Breast cancer (estrogen receptor kind gift from M. Lanotte (St. Louis Hospital, Paris, France).
positive) Cells were maintained in culture according to recommenda-
MDA-MB231 Breast cancer (estrogen receptor tions.
negative)
HT29 Colorectal cancer
2.3. MTT assays
PANCI Pancreatic cancer
ASPC1 Pancreatic cancer
BxPC-3 Pancreatic cancer An initial screening was done to explore the anti-
NCI-H520 Non-small cell lung cancer proliferative effects of the herbal extract on the 26 human
(squamous cell carcinoma) cancer cell lines. Cells (3 × 104 ml−1 ) were cultured in
The six most sensitive cell lines are in bold. the presence of 50 and 100 ␮g/ml G. lucidum extract for
 C.I. Müller et al. / Leukemia Research 30 (2006) 841–848
96 h in 96-well plates (Flow Laboratories, Irvine, CA).
Thereafter, 10 ␮l of MTT solution (in 5 mg/ml PBS,
Roche) was added to each well and incubated for 4 h
in a humidified atmosphere at 37 ◦ C according to the
manufacturer’s protocol. Consecutively, 50 ␮l solubilization
solution (20% SDS) was added into the wells and incubated
overnight (16 h). After culture, cell number and viability
were evaluated by measuring the mitochondrial-dependent
conversion of the yellow tetrazolium salt MTT to purple
formazan crystals by metabolic active cells. The resulting
colored solution was quantified at 540 nm using an enzyme-
linked immunoabsorbent assay reader (ELISA reader,
Bio-Rad).
2.4. Cell cycle analysis
Cells (3 × 104 ml−1 ) were incubated for 72 h either with
or without G. lucidum (100 ␮g/ml). They were collected,
washed, suspended in cold 1× PBS, fixed in 75% methanol
and stained with propidium iodide (PI). Cell cycle analysis
was performed using the Becton Dickinson Flow Cytometer.
2.5. Assessment of apoptosis by Annexin V assay
Apoptotic cell death was examined by Annexin V-
apoptosis detection kit (Pharmingen Inc., San Diego, CA).
During early stages of apoptosis, phosphatidylserine (PS)
becomes externalized on the outer plasma membrane. In
order to be able to distinguish between apoptosis and
necrosis, cells were stained with FITC-labeled Annexin
V and propidium iodide (PI). Annexin V binds to the
externalized PS, whereas PI is able to penetrate the
increasingly permeable plasma membrane during necrosis
or later stages of apoptosis and binds to cellular DNA.
Cells were analyzed by fluorescence activated cell sorting
(FACS).
Four hematological cell lines (HL-60, U937, Blin-1 and
RPMI8226) were treated with G. lucidum at four different
concentrations (50, 100, 150 and 200 ␮g/ml) for 72 h. Control
cells and treated cells were analyzed for staining by Annexin
V and PI.
2.6. Assessment of apoptosis by determination of
mitochondrial membrane potential
Apoptosis was further investigated by analysis of
the mitochondrial membrane potential (
Ψ ) by JC-1
assay according to the manufacturer’s protocol (Cell
Technology, MN). JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 -
tetraethylbenzimidazolcarbocyanine iodide) is a lipophilic
cationic dye which selectively incorporates into intact
mitochondria and undergoes a reversible change in flu-
orescence emission from green to red as mitochondrial
membrane potential increases. During apoptosis, loss of the
mitochondrial membrane potential occurs. In cells with high
membrane potential, the formation of dimers of the dye is
843
promoted, resulting in red fluorescence. In contrast, in cells
with low membrane potential, JC-1 is in its monomeric form,
which then fluoresces green. The red/green fluorescence
intensity ratio is used to analyze whether a cell is apoptotic
or not. A decrease in red/green ratio indicates an increase
in apoptotic cells. Fluorescence of the cells is measured
by flow cytometry. Formation of JC-1 aggregates within
the mitochondria results in emission of red fluorescence
in the 590 nm spectrum. If the mitochondriae collapse
during apoptosis, the fluorescence shifts to the green 510 nm
spectrum.
2.7. Western blot analysis
Total cell lysate (25 ␮g) was electrophoresed on 10–20%
SDS-polyacrylamide gel (Bio-Rad, Hercules, CA) and
transferred by electroblotting to a polyvinylidene fluoride
membrane (Pall Corporation, Pensacola, FL). The mem-
brane was incubated overnight with the following anti-
bodies: anti-p21WAF1 (SC-397, 1:500 dilution) and anti-
p27KIP1 (SC-528, 1:500 dilution), both rabbit polyclonal
antibodies from Santa Cruz Biotechnology (Santa Cruz,
CA) followed by a secondary horseradish peroxidase-
conjugated donkey anti-rabbit antibody (Amersham Bio-
sciences). Detection was performed using the SuperSignal®
chemiluminescence substrate (Pierce, Rockford, IL). Blots
were also subjected to overnight incubation with a
murine monoclonal GAPDH antibody (RDI-TRK5G4-
6C5, 1:10,000 dilution; Research Diagnostics, Flanders,
NJ) followed by a secondary horseradish peroxidase-
conjugated sheep anti-mouse antibody (Amersham Bio-
sciences). Western blots were stripped between hybridiza-
tions with stripping buffer (10 mM Tris–HCl pH 2.3, 150 mM
NaCl).
2.8. Cytospin preparations and staining
Cytospin preparations were performed for seven hemato-
logic (NCI-H929, RPMI8226, Blin-1, Nalm-6, Daudi, U937,
HL-60) and six solid tumor (ARO, BHP2-7, PC3, DU145,
NCEB-1, MCF-7) cell lines after 96 h treatment with G.
lucidum (100 ␮g/ml). Slides were consecutively stained with
Diff Quik® (Dade Behring, Switzerland) staining solution
according to the manufacturer’s protocol.
3. Results
3.1. HPLC analysis of G. lucidum extract
The G. lucidum extract was standardized to 0.15% gan-
oderic acid C2 content (Fig. 1a). Fig. 1b shows the HPLC
chromatogram of ganoderic acid C2 standard eluting at a
retention time of 32.9 min. Fig. 1c shows the G. lucidum
extract spiked with ganoderic acid C2 standard confirming
its presence at the retention time of 32.9 min.
844 C.I. Müller et al. / Leukemia Research 30 (2006) 841–848

Fig. 1. (a) HPLC chromatogram of G. lucidum extract showing ganoderic acid C2 standard eluting at 32.9 min. (b) HPLC chromatogram of ganoderic acid C2
standard eluting at 32.9 min. (c) HPLC chromatogram of G. lucidum extract spiked with ganoderic acid C2 standard confirming its presence at 32.9 min.

Fig. 2. Growth arrest of hematologic cell lines induced by G. lucidum extract. Cells included: panel a, acute myeloid leukemia (HL-60, U937); panel b, erythroid
chronic myeloid leukemia (K562); panel c, acute lymphoblastic leukemia (Blin-1, Nalm-6); panel d, multiple myeloma (RPMI8226). Cells of each line were
treated with G. lucidum (10, 20, 40, 60, 80 and 100 ␮g/mg) for 96 h. MTT assay was performed. Viable cells were expressed as a percentage of untreated
control cultures for each line. Results represent the mean ± S.D. of three different experiments performed in triplicates.
 C.I. Müller et al. / Leukemia Research 30 (2006) 841–848 845

Table 2 3.3. G2/M arrest induced by G. lucidum extract

Inhibition of the proliferation of hematopoietic cell lines by G. lucidum
extract
Cell cycle analysis was performed for HL-60, U937,
Cell line ED50 (␮g/ml) Hematologic malignancy RPMI8226, Nalm-6 and Blin-1 cells (G. lucidum, 100 ␮g/ml,
HL-60 26 Acute myeloblastic leukemia (FAB M2) 72 h). Results revealed a G2/M arrest, most prominently in
U937 63 Diffuse histiocytic lymphoma HL-60 cells (29% in G2/M compared to 12% in control cells).
(monocytic leukemia cells)
Moreover, an increase of DNA content occurred in the G.
K562 50 Erythroid chronic myeloid leukemia
(blast crisis) lucidum treated HL-60 cells (Fig. 3). Only a slight increase
Blin-1 38 Pre-B acute lymphoblastic leukemia of cells in the G2/M phase occurred in the RPMI8226 cells
Nalm-6 30 Non-T, non-B acute lymphoblastic (16%, untreated control compared to 20% treated cells) and
leukemia the Nalm-6 cells (12%, untreated control versus 15% treated
RPMI8226 40 Multiple myeloma (IgG␭)
cells), whereas Blin-1 and U937 cells showed no increase of
Cells were cultured for 96 h in the presence of 10, 20, 40, 60, 80 or 100 ␮g/ml cells in G2/M phase (data not shown).
of G. lucidum, and cell growth was analyzed by MTT assay. Percent growth
in experimental wells compared to untreated control wells was graphed, and
the effective dose which inhibited 50% growth (ED50 ), was calculated for 3.4. Pro-apoptotic effects of G. lucidum extract
each cell line.
We analyzed whether the anti-proliferative effects of G.
lucidum could be explained in part by apoptosis. HL-60,
3.2. Anti-proliferative effects of G. lucidum extract in a U937, Blin-1 and RPMI8226 were cultured with G. lucidum
variety of cancer cell lines (50, 100, 150 and 200 ␮g/mg, 72 h) and analyzed for apopto-
sis by Annexin V staining. G. lucidum, in a dose-dependent
The initial screening of 26 cancer cell lines (Table 1) was manner induced apoptosis for each of the cell lines, espe-
performed culturing these cells with 50 and 100 ␮g/ml of cially HL-60 (Fig. 4) and Blin-1 cells. In the case of HL-60
G. lucidum for 96 h and measuring their growth by MTT cells, approximately 4% of the diluent control cells were
(data not shown). Only those lines which achieved 50% Annexin V positive, which increased in a dose–response fash-
inhibition of growth (hematopoietic cells) were studied fur- ion with 92% positivity in the presence of 200 ␮g/ml of G.
ther. The effect of G. lucidum in a dose-dependent fashion lucidum (Fig. 4). Blin-1 cells were less sensitive with approx-
on the growth of the six most sensitive hematological cell imately 42% of the cells being apoptotic after treatment with
lines (HL-60, U937, K562, Blin-1, Nalm-6, RPMI8226) was Ganoderma (200 ␮g/ml, 72 h) (data not shown). Consistent
examined at 96 h of culture. Dose–response curves were gen- with their lower sensitivity to G. lucidum as measured by
erated (Fig. 2). The effective dose, which inhibited 50% cell MTT assay, U937 and RPMI8226 cells had less apoptosis
growth (ED50 ), was calculated and ranged between 26 and (32 and 21%, respectively) in response to the herbal extract
63 ␮g/ml (Table 2). (200 ␮g/ml, 72 h) (Fig. 4 and data not shown, respectively).

Fig. 3. Induction of G2/M arrest in HL-60 cells by G. lucidum extract. Panel a shows cell cycle analysis by flow cytometry after PI staining for untreated
control cells. Panel b depicts cell cycle analysis for cells treated with G. lucidum, 100 ␮g/ml for 72 h. Increasing number of HL-60 cells in the G2/M phase of
the cell cycle were detected. Horizontal and vertical axis represent DNA content and cell number, respectively. Percentages of the different cell cycle phases
are calculated for the population of cells within the dashed lines. Representative of one of two experiments with similar results.
846 C.I. Müller et al. / Leukemia Research 30 (2006) 841–848

Fig. 4. Induction of apoptosis in HL-60 and U937 cells by G. lucidum extract. HL-60 (panel a) and U937 (panel b) cells were cultured with G. lucidum (50,
100, 150 and 200 ␮g/mg) for 72 h, stained with FITC-conjugated Annexin V and propidium iodide (PI). Percentage of apoptotic cells was measured by flow
cytometry in comparison to diluent-treated control cells. Bar graphs reflect the cells exclusively stained with FITC. Results represent the mean ± S.D. of three
different experiments.

3.5. Apoptosis coincident with a decrease of
mitochondrial membrane potential
Apoptosis begins when a cell activates its own destruc-
tion by initiating a series of complex cascading events that
include the depolarization of the mitochondrial membrane
potential. The ability of G. lucidum to alter the mitochon-
drial transmembrane electrical potential was investigated in
HL-60 and U937 myeloid cells. As the cells lose electrical
potential, the fluorescence of the JC-1 dye changes from red
to green. An increase of monomeric JC-1 molecules (green
fluorescence) due to a decrease of mitochondrial membrane
potential occurred in a dose-dependent manner. After 48 h of
culture with G. lucidum, a dose-dependent decrease of the
ratio of red to green fluorescence occurred as the mitochon-
dria became progressively depolarized (Fig. 5).
3.6. Upregulation of p21WAF1 and p27KIP1 by G.
lucidum extract
We examined the effect of G. lucidum on the expression
of cell cycle- and apoptosis-related proteins by Western blot
analysis. U937 cells were treated with G. lucidum at four dif-
ferent concentrations (50, 100, 150 and 200 ␮g/ml) for 48 and
72 h. Western blotting showed that an increase in p21WAF1
protein expression occurred in a dose- and time-dependent
fashion compared to diluent only treated cells (Fig. 6a). G.
lucidum upregulated the expression of p27KIP1 proteins at
48 h of exposure; effects did not further increase at 72 h
(Fig. 6b and data not shown).
3.7. Multinucleation of HL-60 cells after treatment with
G. lucidum extract
Cytospin preparations and staining were performed for
morphologic analysis after culture of 13 cell lines with G.
lucidum (100 ␮g/ml, 96 h). This treatment caused prominent
multinucleation of only HL-60 cells (Fig. 7). Further dose-
dependent studies revealed that approximately 1% of HL-
60 cells became multinucleated in the presence of 1 ␮g/ml
G. lucidum (96 h), 5% cells in the presence of 10 ␮g/ml,
10% cells with 50 ␮g/ml, 40% cells with 75 ␮g/ml and 80%
cells with 100 ␮g/ml (data not shown). Untreated HL-60 had
<0.5% multinucleated cells. Multinucleation did not occur in
the six other hematologic and six solid tumor cell lines ana-
lyzed (100 ␮g/ml, 96 h, data not shown). FACS analysis of
PI stained HL-60 cells confirmed the increased DNA content
of these cells (see Fig. 3).
4. Discussion
Because G. lucidum is traditionally consumed in cooked
aqueous forms such as soups and tea, we conducted our

Fig. 5. Induction of mitochondrial membrane collapse in U937 cells cultured with G. lucidum extract. HL-60 (panel a) and U937 (panel b) cells were incubated
with G. lucidum for 48 h. Mitochondrial membrane potential of cells treated with G. lucidum and diluent treated control cells was assessed by flow cytometry
after staining with JC-1. JC-1 dimers fluoresce red in stable mitochondria and form green fluorescent monomers when the mitochondrial membrane is decreasing
in potential. The decrease of the red/green fluorescence reflects increasing apoptotic cells. Results represent ratios of the mean of three different experiments.
 C.I. Müller et al. / Leukemia Research 30 (2006) 841–848 847

Fig. 6. Upregulation of p21WAF1 and p27KIP1 by G. lucidum extract in U937 cells. U937 cells were treated with increasing doses of G. lucidum extract (50,
100, 150 and 200 ␮g/ml) for 48 and/or 72 h. Protein lysates were analyzed by Western blot with p21WAF1 (panel a) or p27KIP1 (panel b) specific antibodies. The
blots were stripped and rehybridized with a GAPDH antibody as control for equal loading.

evaluation on the aqueous extract of the fruiting bodies of
the mushroom. We demonstrate the anti-tumor properties
of an extract of G. lucidum against a variety of human
leukemic, lymphoma and myeloma cell lines. After the initial
screening of five herbs with known anti-cancer activities (G.
lucidum, Isatis indigotica, Dendranthema morifolium, Rab-
dosia rubescens and Panax pseudoginseng), G. lucidum was
found to be the overall most active extract (data not shown).
The 16 hematologic cell lines were more sensitive to the
growth inhibiting properties of Ganoderma than were the 10
solid tumor lines (data not shown). Further studies focused
on the anti-cancer activities of Ganoderma in the six most
sensitive hematopoietic cell lines.
These hematopoietic cell lines underwent growth arrest
in a dose-dependent manner in response to treatment with
Ganoderma with ED50 values for HL-60, Nalm-6, and
Blin-1 ranging between 26 and 38 ␮g/ml. The RPMI8226
(ED50 , 40 ␮g/ml), K562 (ED50 , 50 ␮g/ml) and U937 (ED50 ,
63 ␮g/ml) were slightly less sensitive to the drug. Growth
inhibition was associated with cell cycle retardation in the
G2/M phase especially in the HL-60 cells.
Apoptosis as shown by Annexin V staining was particu-
larly prominent in HL-60 acute myeloid leukemia cells. Mea-
surement of mitochondrial membrane potential (JC-1 assay)
paralleled the apoptosis data consistent with G. lucidum caus-
ing mitochondrial induced cell death.
Recent reports showed that G. lucidum increased the
expression of p21WAF1 and p27KIP1 in the PC-3 prostate
cancer cell line and MCF-7 breast cancer cell line [7,10].
We detected increased protein expression of p21WAF1 and
p27KIP1 in U937 cells after their treatment with G. lucidum
for 48 and 72 h, respectively (Fig. 6a and b). Even though no
G2/M arrest was observed in U937 cells after treatment with
G. lucidum, p21WAF1 and p27KIP1 might play a role in these
cells having decreased proliferation and increased apoptosis.
No change in expression levels of p21WAF1 and p27KIP1 was
detected in HL-60 cells.
Multinucleation in HL-60 cells was reported to occur after
exposure to several different substances [18–20]. No uniform
explanation has been provided to explain the mechanism.
Cholesterol starvation has been noted to cause formation of
polyploid HL-60 cells; this was reversed by the readdition

Fig. 7. Multinucleation of HL-60 cells caused by G. lucidum extract. Untreated HL-60 cells (panel a) and HL-60 cells treated with G. lucidum (100 ␮g/mg,
96 h) (panel b). Arrowheads point to several of the multinucleated cells present in the photograph. Cells were cytocentrifuged, fixed, stained as described in
Section 2 (magnification 400×).