Ganoderma lucidum causes apoptosis in leukemia, lymphoma and multiple
myeloma cells

Claudia I. Müllera, b,
<img alt="Corresponding author contact information" src="http://origin-cdn.els-
cdn.com/sd/entities/REcor.gif">,
<img src="http://origin-cdn.els-cdn.com/sd/entities/REemail.gif" alt="E-
mail the corresponding author">,

Takashi Kumagaia,

James O’Kellya,

Navindra P. Seeramc,

David Heberc,

H. Phillip Koefflera

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 Stat
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 μg/ml), U937 (63 μg/ml), K562 (50 μg/ml), Blin-1 (38 μg/ml),
Nalm-6 (30 μg/ml) and RPMI8226 (40 μg/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.
Keywords

Ganoderma lucidum;

Leukemia;

Lymphoma;

Multiple myeloma;

Apoptosis;

Growth arrest
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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.
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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 μg/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.
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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 μg/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.
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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 μg/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.
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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.
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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 μg/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.
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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 μg/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×).
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1. Human cancer cell lines examined for anti-proliferative effects of
G. lucidum extract
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The six most sensitive cell lines are in bold.
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Table 2. Inhibition of the proliferation of hematopoietic cell lines by G. lucidum extract
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Cells were cultured for 96 h in the presence of 10, 20, 40, 60, 80 or 100 μg/ml 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 (ED 50),
was calculated for each cell line.
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<img border="0" alt="Corresponding author contact information" title="Corresponding author conact information" src="http://origin-cdn.els-cdn.com/sd/entities/REcor.gif">
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.