Triterpenes From Ganoderma Lucidum Induce Autophagy in Colon Cancer Through the Inhibition of p38 Mitogen-Activated Kinase (p38 MAPK)

Article (PDF Available)inNutrition and Cancer 62(5):630-40 · June 2010with77 Reads
DOI: 10.1080/01635580903532390 · Source: PubMed
Abstract
Medicinal mushroom Ganoderma lucidum is one of the most esteemed natural products that have been used in the traditional Chinese medicine. In this article, we demonstrate that G. lucidum triterpene extract (GLT) suppresses proliferation of human colon cancer cells HT-29 and inhibits tumor growth in a xenograft model of colon cancer. These effects of GLT are associated with the cell cycle arrest at G0/G1 and the induction of the programmed cell death Type II-autophagy in colon cancer cells. Here, we show that GLT induces formation of autophagic vacuoles and upregulates expression of Beclin-1 (1.3-fold increase) and LC-3 (7.3-fold increase) proteins in colon cancer cells and in tumors in a xenograft model (Beclin-1, 3.9-fold increase; LC-3, 1.9-fold increase). Autophagy is mediated through the inhibition of p38 mitogen-activated protein kinase (p38 MAPK) because p38 MAPK inhibitor, SB202190, induces autophagy and expression of Beclin-1 (1.2-fold increase) and LC-3 (7.4-fold increase), and GLT suppresses phosphorylation of p38 MAPK ( approximately 60% inhibition) in colon cancer cells. Taken together, our data demonstrate a novel mechanism responsible for the inhibition of colon cancer cells by G. lucidum and suggest GLT as natural product for the treatment of colon cancer.
Nutrition and Cancer, 62(5), 630–640
Copyright
C
2010, Taylor & Francis Group, LLC
ISSN: 0163-5581 print / 1532-7914 online
DOI: 10.1080/01635580903532390
Triterpenes From
Ganoderma Lucidum
Induce Autophagy
in Colon Cancer Through the Inhibition of p38
Mitogen-Activated Kinase (p38 MAPK)
Anita Thyagarajan, Andrej Jedinak, Hai Nguyen, and Colin Terry
Methodist Research Institute, Indianapolis, Indiana, USA
Lee Ann Baldridge
Indiana University School of Medicine, Indianapolis, USA
Jiahua Jiang
Methodist Research Institute, Indianapolis, Indiana, USA
Daniel Sliva
Methodist Research Institute, Indianapolis, Indiana, and Indiana University School of Medicine,
Indianapolis, Indiana, USA
Medicinal mushroom Ganoderma lucidum is one of the most
esteemed natural products that have been used in the traditional
Chinese medicine. In this article, we demonstrate that G. lucidum
triterpene extract (GLT) suppresses proliferation of human colon
cancer cells HT-29 and inhibits tumor growth in a xenograft model
of colon cancer. These effects of GLT are associated with the cell
cycle arrest at G0/G1 and the induction of the programmed cell
death Type II–autophagy in colon cancer cells. Here, we show that
GLT induces formation of autophagic vacuoles and upregulates ex-
pression of Beclin-1 (1.3-fold increase) and LC-3 (7.3-fold increase)
proteins in colon cancer cells and in tumors in a xenograft model
(Beclin-1, 3.9-fold increase; LC-3, 1.9-fold increase). Autophagy
is mediated through the inhibition of p38 mitogen-activated pro-
tein kinase (p38 MAPK) because p38 MAPK inhibitor, SB202190,
induces autophagy and expression of Beclin-1 (1.2-fold increase)
and LC-3 (7.4-fold increase), and GLT suppresses phosphoryla-
tion of p38 MAPK (60% inhibition) in colon cancer cells. Taken
together, our data demonstrate a novel mechanism responsible for
the inhibition of colon cancer cells by G. lucidum and suggest GLT
as natural product for the treatment of colon cancer.
INTRODUCTION
Colon cancer is a leading contributor to cancer prevalence
and mortality in the Western world, and in the United States,
it is the third most common diagnosed cancer with an esti-
Submitted 20 March 2009; accepted in final form 26 October 2009.
Address correspondence to Daniel Sliva, Cancer Research Labora-
tory, Methodist Research Institute, 1800 N. Capitol Ave., E504, Indi-
anapolis, IN 46202. Phone: 317-962-5731. Fax: 317-962-9369. E-mail:
dsliva@clarian.org
mated 50,000 deaths in 2009 (1). The association among diet,
colon cancer risk, and mortality is well established (2), and an
inverse correlation between mushroom intake and the risk of
gastric cancer has been demonstrated (3). Ganoderma lucidum
is one of the important Asian fungi that were recognized in
China and Korea as Ling Zhi (mushroom of immortality) and
in Japan as reishi mushroom or mannentake (10,000 yr mush-
room) more than 4,000 yr ago (4). Although G. lucidum was
originally used to improve health and promote longevity, its
potential therapeutic effects were recognized in traditional Chi-
nese medicine for the treatment of a variety of diseases. Two
major groups of biologically active compounds isolated from
G. lucidum are polysaccharides (mainly glucans and glycopro-
teins) and lanostane-type triterpenes (ganoderic acids, ganoderic
alcohols, and their derivatives) (5). Most of the anticancer ef-
fects of G. lucidum polysaccharides have been attributed to the
modulation of the immune system through the activation of
macrophages, neutrophils, dendritic cells, natural killer cells,
and T- and B-lymphocytes (6,7). Mechanistically, G. lucidum
polysaccharides (GLP) induced cytokine expression via Toll-
like receptors (TLR)-4 in macrophages and dendritic cells;
whereas immunoglobulin production was mediated through
TLR-4/TLR-2 in B-lymphocytes (8,9). The anticancer activi-
ties of G. lucidum triterpenes were originally demonstrated by
their cytotoxic/killing effects on hepatoma (10), naso-pharynx
carcinoma (11), lung carcinoma and sarcoma (12), breast cancer
(13), and leukemia cells (14). Mechanistically, these triterpenes:
1) inhibited activity of farnesyl protein transferase (FPT) (15);
2) demonstrated antioxidative activity (16); 3) inhibited the ac-
tivity of 5α-reductase (17); or 4) suppressed the activities of
630
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TRITERPENES FROM GANODERMA LUCIDUM AND COLON CANCER 631
HMG-CoA reductase and acyl-CoA acyltransferase in the
mevalonate pathway (18). G. lucidum extract (GLE), contain-
ing polysaccharides and triterpenes, was reported to suppress
growth and metastatic potential of the highly invasive human
breast cancer cells MDA-MB-231 by inhibiting the activity of
AKT kinase and transcription factors AP-1 and NF-kB resulting
in the downregulation of expression of cyclin D1 and urokinase
plasminogen activator (19,20). Moreover, GLE modulated es-
trogen receptor signaling (21) and inhibited the oxidative stress-
induced invasiveness by the suppression of interleukin-8 (IL-8)
secretion from breast cancer cells MCF-7 (22). GLE was found
to inhibit proliferation of prostate cancer cells PC-3 by down-
regulating cyclin B and Cdc2 and upregulating p21 expres-
sion, which was shown also by cell-cycle arrest at G2/M phase;
whereas the induction of apoptosis of PC-3 cells was associated
with the upregulation of expression of proapoptotic Bax (23).
GLE also inhibited angiogenesis of vascular endothelial cells
by suppressing secretion of the proangiogenic factors vascu-
lar endothelial growth factor (VEGF) and transforming growth
factor-
∗∗∗∗∗
b1 (TGF-b1) from PC-3 cells (24).
In this study, we evaluated G. lucidum triterpene extract
(GLT) on the in vitro growth and in the xenograft model of
human colon cancer cells HT-29 in vivo. Here, we show that
GLT suppresses growth of HT-29 cells through cell cycle arrest
at the G0/G1 phase and by the induction of the programmed cell
death Type II, autophagy. Moreover, GLT also inhibits growth
of tumors in a xenograft model of colon cancer. In summary, our
data suggest possible molecular mechanism by which triterpene
extract from medicinal mushroom Ganoderma lucidum inhibits
growth of colon cancer cells.
MATERIALS AND METHODS
Cell Culture and Reagents
Human colon cancer cells HT-29 were obtained from ATCC
(Manassas, VA) and were maintained in Dulbecco’s modified ea-
gle medium (DMEM) containing penicillin (50 U/ml), strepto-
mycin (50 U/ml), and 10% fetal bovine serum (FBS). Media and
supplements came from GIBCO BRL (Grand Island, NY). FBS
was obtained from Hyclone (Logan, UT). GLT was obtained
from Pharmanex (Provo, UT; batch number 050607; Shang-
hai R&D, Pharmanex). GLT contains a mixture of lanostanoid
triterpenes, which were previously isolated and identified by
nuclear magnetic resonance and mass spectrometry (18,25,26).
GLT was dissolved in DMSO (Sigma, St. Louis, MO) at the
concentration 40 mg/ml and stored at 4
C (the final concen-
tration of DMSO in controls and GLT was 0.3%). 3-methyl
adenine (3MA) was purchased from Sigma (St. Louis, MO) and
dissolved in Dulbecco’s phosphate-buffered saline (PBS; Cam-
brex Bio Science Walkersville, Inc., Walkersville, MD). Propid-
ium iodide, absolute ethanol, and cremophor were purchased
from Sigma. SB202190 was from Calbiochem (San Diego,
CA).
Cell Proliferation Assay
Cell proliferation was determined by the MTT assay, ac-
cording to the manufacturer’s instructions (Promega, Madison,
WI). Briefly, HT-29 cells were cultured in a 96-well plate and
treated at indicated times with GLT (0–1.0 mg/ml). At the end
of the incubation period, an absorption was determined with a
micro plate reader at 570 nm as described previously (19). Data
points represent mean ± SD in the representative experiment of
triplicate determinations. Similar results were obtained in two
independent experiments.
Flow Cytometry Analysis
HT-29 cells (1 × 10
6
) were seeded in 100 mm dishes and
after 24 h treated with GLT (0–0.25 mg/ml) for 0 to 48 h. The
cells were harvested, fixed with 3 ml ice-cold ethanol at 4
C
for 1 h and stained with 50 µg/ml of propidium iodide. Cell
cycle analysis was performed on a FACStar
PLUS
flow cytometer
(Becton-Dickinson, San Jose, CA) as previously described (27).
Data are the mean ± SD from 3 independent experiments.
Autophagy
HT-29 cells were plated in a 6-well plate and treated with
GLT (0–0.5 mg/ml) or SB202190 (20 µM) in the absence or
presence of 3MA (10 mM). The formation of autophagic vac-
uoles was observed under the phase contrast microscope at ×40
magnification over a period of 0 to 24 h.
Immunofluorescent Detection of Autophagy
HT-29 cells were plated into Lab-Tek II 8 chamber culture
slides (Nalge Nunc International, Naperville, IL) and allowed
to attach at 37
C for 48 h. Cells were then treated with GLT
(0.25 mg/ml) and incubated for 6 h at 37
C. Subsequently,
the cells were rinsed briefly with PBS and then fixed with 4%
formaldehyde-PBS for 15 min at room temperature. Later the
cells incubated with ice cold methanol for 10 minutes in the
freezer, followed by a brief rinse with PBS. Fixed cells were
then blocked using 5% normal goat serum in PBS/Triton for
1 h, followed by incubation in LC-3 primary antibody for 24 h
at 4
C, washed and incubated with AlexaFluor 488-conjugated
goat antirabbit secondary antibody for 2 h at room temperature
in the dark, rinsed with PBS, and counterstained with 4
,6-
diamidino-2-phenylindole (DAPI). Images were obtained using
an Olympus BX40 microscope at ×50 magnification with an
oil immersion lens. Picture frame software (Optronics, Goleta,
CA) was used to obtain all images at similar intensities.
Human Colon Tumor Xenograft Experiments
HT-29 cells (5 × 10
6
) in 0.2 ml DMEM were implanted sub-
cutaneously in the right flank on the ventral side of the 6-wk-old
male nude mice (Harlan, Indianapolis, IN). After a week of
implantation with tumor cells, when tumors reached approxi-
mately 20 to 30 mm
3
, the animals were randomized into control
and treatment groups (10 animals/group). The animals received
intraperitoneal injections (100 µl) of 10% DMSO, 70% mixture
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632 A. THYAGARAJAN ET AL.
of ethanol and cremophor (3:1), and 20% PBS (control) or 6 mg
GLT/kg of body weight dissolved in DMSO and adjusted to the
final concentration of 10% DMSO, 70% mixture of ethanol and
cremophor (3:1), and 20% PBS (treatment group) daily for 23
days. The tumor size was measured using calipers, and the tumor
volume was estimated by the formula tumor volume (mm
3
) =
(W × L) 2 × 1/2, where L is the length and W is the width of the
tumor. At the end of the experiment (Day 23), the tumors were
dissected, fixed in 10% neutral formalin or snap frozen, and
stored separately in liquid nitrogen. The tumor sections were
analyzed by immunohistochemistry. The protocol for animal
experiments was approved by the Animal Research Committee
at the Methodist Hospital according to the NIH guidelines for
the care and use of laboratory animals.
Tumor Extracts
Tumors from control and GLT-treated mice were washed with
ice-cold PBS, minced, and homogenized in the lysis buffer con-
taining 50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1%
Triton X-100, 1 mM PMSF, 0.1 mM sodium vanadate, 2.5 mM
sodium pyrophosphate, 1 mM b-glycerophosphate, and 50X
Complete solution (Roche, Indianapolis, IN). The homogenate
was incubated at 4
C for 30 min and the lysate cleared by cen-
trifugation at 14,000 rpm for 30 min.
Western Blot Analysis
HT-29 cells treated with GLT (0–1.0 mg/ml) as indicated
in the text and whole cell extracts prepared as previously de-
scribed (19). Equal amounts of cellular (20 µg/lane) and tissue
(30 µg/ml) proteins were separated on NuPAGE 4 to 12% Bis-
Tris gel or 12% Tris-Glycine gel (Invitrogen, Carlsbad, CA) and
transferred to a PVDF membrane (Millipore, Bedford, MA). The
protein expression was detected with the corresponding primary
antibodies: anti-Beclin-1, anti-LC-3, anti-p38, anti-phospho-
p38 (Cell Signaling, Beverly, MA), and anti-GAPDH antibody
(Santa Cruz Biotechnology, Santa Cruz, CA), respectively. Pro-
tein expression was visualized using the ECL Western Blotting
Detection System (Amersham Biosciences, Buckinghamshire,
UK).
Immunohistochemistry
Immunohistochemistry was performed on 5 µm sections
of formalin-fixed, paraffin-embedded samples. For antigen re-
trieval, the slides were boiled for 10 min in citrate buffer fol-
lowed by an overnight incubation at 4
Cwithaprimarymon-
oclonal antibody against Ki-67 (NeoMarker, Fremont, CA),
Beclin-1, and LC-3 (Abgent, San Diego, CA).
Densitometric Analysis
Autoradiograms of the Western blots were scanned with
HP Scanjet 5470c scanner (Hewlett-Packard, Palo Alto, CA).
The optical densities of Beclin-1, LC-3, phospho-p38, p38, and
GAPDH proteins on the films were quantified and analyzed
with the UN-SCAN-IT software (Silk Scientific, Orem, UT).
The ratios of specific proteins to GAPDH were calculated by
standardizing the ratios of each control to the unit value.
Statistical Analysis
Cell cycle distribution data and proliferation data were sum-
marized using descriptive statistics (mean, SD). For each cell
cycle phase, the effect of GLT over time on cell cycle distribution
was analyzed using a 2-way ANOVA model, and the interac-
tion between time and treatment was assessed. If a significant
interaction was found, Tukey’s post hoc test was then used to
compare mean values at different levels of the 2-way interac-
tion. Proliferation data was compared across treatment groups at
both time points (24 h and 48 h) using 1-way ANOVA models.
Dunnett’s post hoc test was used to compare mean values for
each treatment group to the control.
Tumor volume measurements were summarized by treatment
group using descriptive statistics (mean, SD). The comparison
of the change in tumor volume, immunohistochemical staining,
and vacuole formation between groups was performed using a
2-sample Student t-test. Results were considered significant at
P 0.05.
RESULTS
GLT Inhibits Proliferation of Colon Cancer Cells and
Growth of Xenograft Tumors In Vivo
As mentioned previously, G. lucidum demonstrated cyto-
static/cytotoxic effects against a variety of cancer cells. There-
fore, we evaluated whether GLT also affects growth of human
colon cancer cells HT-29. As seen in Fig. 1A, increased con-
centration of GLT (0–1.0 mg/ml) markedly suppressed prolifer-
ation of HT-29 cells. At the low/moderate dosage, GLT exhibits
cytostatic activity; whereas at higher concentrations, cytotoxic
activity is observed. Both the cytostatic and cytotoxic effects
of GLT are statistically significant (P < 0.001). To investigate
whether GLT suppresses growth of colon tumors in vivo, we
determined the effect of GLT on the growth of established colon
cancer HT-29 xenograft tumors. Therefore, HT-29 cells were
subcutaneously injected in the right flank of the nude mice as
described in Materials and Methods. After the tumors reached
size to 20 to 30 mm
3
, mice were randomly assigned to control
(vehicle) and GLT treatment groups. Mice bearing established
HT-29 tumors were given intraperitoneal injections of vehicle
only or 6 mg/kg of GLT daily for 23 days. As seen in Fig. 1B and
Table 1, GLT treatment significantly (P = 0.012) suppressed the
growth of HT-29 tumors (final volume = 650.2 ± 283.8 mm
3
;
change in volume = 631.7 ± 276.5 mm
3
) when compared with
the vehicle control group (final volume = 1,227.1 ± 584.3 mm
3
;
change in volume = 1,203.6 ± 589.9 mm
3
). In addition, GLT
markedly suppressed proliferation of colon cancer cells in vivo
as demonstrated by the decrease in the staining with Ki-67 in
the tumor tissue from the animals treated with GLT (Fig. 1C;
P < 0.005).
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TRITERPENES FROM GANODERMA LUCIDUM AND COLON CANCER 633
FIG. 1. Ganoderma lucidum triterpene extract (GLT) inhibits growth of colon cancer cells in vitro and in vivo. HT-29 cells were treated with GLT (0, 0.06,
0.125, 0.250, 0.5, and 1.0 mg/ml) for 24 and 48 h. Proliferation was assessed as described in Materials and Methods. Data are the mean ± SD of triplicate
determinations and compared across treatment groups at each time point using ANOVA. Dunnett’s post hoc test was used to compare each treatment group to
the control.
indicates a significant difference between treatment and control. Statistical significance,
, P < 0.001. Similar results were received in at least two
additional experiments. B: HT-29 cells were injected subcutaneously into nude mice and treated with vehicle (empty circles) or 6 mg GLT/kg of body weight daily
for 23 days as described in Materials and Methods. Data are the mean ± SD (n = 10 mice/group). Statistical analysis for the changes in the final tumor volumes
is described in the Table 1. C: Dissected tumors from animals treated with vehicle (control) or 6 mg GLT/kg of body weight (GLT) were sectioned and stained
with Ki-67 as described in Materials and Methods. Data are the mean ± SD (n = 4).
, P < 0.005. D: Body weight of nude mice with HT-29 xenografts, which
were treated with vehicle (empty circles) or 6 mg GLT/kg of body weight daily for 23 days as described in Materials and Methods. Data are the mean ± SD
(n = 10 mice/group).
Finally, the therapeutic dose (6 mg GLT/kg of body weight)
was not toxic to tested animals as assessed by body weight
determinations of the treated mice when compared with the
control group (Fig. 1D). In addition, GLT treatment did not
change the weight of spleen and liver as compared to the control
group (data not shown). Therefore, GLT inhibits growth of colon
cancer cells in vitro and suppresses growth of colon tumors in
animal experiments.
GLT Treatment Induces Cell Cycle Arrest at G0/G1 and
Autophagy in HT-29 Cells
Dietary/natural, agent-induced, cell cycle arrest is usually
prerequisite to the demise of cancer cells, which can be medi-
ated by the apoptotic or nonapoptotic, autophagy or necrosis,
pathways (28). To determine whether GLT induces cell cycle
arrest, HT-29 cells were treated with GLT at indicated times
and analyzed by flow cytometry as described in Materials and
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634 A. THYAGARAJAN ET AL.
TABLE 1
Effect of GLT on the tumor growth in vivo
a
Volume Control GLT (6 mg/kg)
Baseline (mm
3
)23.5 ± 7.818.5 ± 10.0
Final (mm
3
)1, 227.1 ± 584.3 650.2 ± 238.8
Change (mm
3
)1, 203.6 ± 589.9 631.7 ± 276.5
b
a
Abbreviation is as follows: GLT, Ganoderma lucidum triter-
pene extract. Tumor volume was summarized by treatment group
using descriptive statistics (mean, SD). The comparison of the
change in tumor volume between groups was performed using a
2-sample Student t-test.
b
Statistical significance P = 0.012 for the change in tumor vol-
ume in animals treated with GLT (6 mg/kg of body weight) vs.
control (0 mg/kg of body weight; n = 10 mice per group).
Methods. As seen in Table 2, the treatment of HT-29 cells with
0.25 mg/ml of GLT significantly increased the cell population at
G0/G1 after 24 and 48 h, respectively (68% compared to 48% in
control at 24 h and 73% compared to 62% in control at 48 h; P <
0.001). Interestingly, GLT treatment did not induce the amount
of cells at sub-G0/G1 phase, suggesting that the inhibition of
growth of HT-29 cells is independent of apoptosis (Table 2, Fig.
2). In addition, we were not able to detect any apoptotic DNA-
laddering in HT-29 cells treated with GLT (data not shown),
suggesting that nonapoptotic cell death can be induced by GLT
treatment. To examine this hypothesis, HT-29 cells were treated
with GLT (0–1.0 mg/ml) for 3, 6, and 24 h and the formation of
autophagic vacuoles evaluated by the phase microscopy. There-
fore, GLT induced the formation of vacuoles in HT-29 cells in
a time- and dose-response manner (Figs. 3a, 3b, 3c; only 6 h
treatment is shown; P < 0.001). As recently demonstrated, in-
hibition of p38α pathway can induce autophagic death in colon
cancer cells HT-29 (29). As expected, the treatment of HT-29
cells with p38 inhibitor SB202190 (20 µM, 6 h) also induced
autophagy (Fig. 3d; P < 0.001). The pretreatment of HT-29 cells
with 3MA, a well known inhibitor of autophagic process (30),
prevented the formation of vacuoles in cells treated with GLT
and SB202190 (Figs. 3e, 3f, 3g; P < 0.005).
Since the induction of autophagy in mammalian cells is as-
sociated with the upregulation of expression of autophagic pro-
teins Beclin-1 and LC-3 (31,32), we examined whether GLT
and SB2092190 induces their expression in colon cancer cells.
HT-29 cells were treated with GLT (0–1.0 mg/ml, 6 h), and the
expression of Beclin-1 and LC-3 was evaluated in the whole
cell lysates by Western blot analysis. As seen in Fig. 4A, al-
though the GLT treatment only slightly induced the expression
of Beclin-1, the expression of LC-3 was markedly induced in
HT-29 cells treated with GLT. To examine whether the inhibition
of p38 induces expression of Beclin-1 and LC-3 in colon cancer
cells, HT-29 cells were treated with p38 inhibitor SB202190
(20 µM) at indicated times and the expression of Beclin-1 and
LC-3 evaluated as described previously. As expected, the inhibi-
tion of p38 markedly induced expression of LC-3, whereas the
same treatment only slightly induced the expression of Beclin-
1 in HT-29 cells (Fig. 4B). To further evaluate whether GLT
itself modulates the activity of p38 in colon cancer cells, HT-
29 cells were treated with GLT (0–1.0 mg/ml) for 0, 3, and
6 h, and the expression of phospho-p38 was evaluated in the
whole cell lysates by Western blot analysis. As seen in Fig. 4C,
GLT markedly decreased phosphorylation of p38 in a dose- and
time-dependent manner.
The localization of LC-3 protein to the membranes of the
autophagic vacuoles is an additional confirmation of autophagy.
Therefore, we used an immunohistochemical approach to detect
the localization of LC-3 in HT-29 cells treated with GLT and
SB202190. As seen in Fig. 5, both GLT and SB202190 induced
the expression of LC-3 in HT-29 cells; and LC-3 was mainly
localized in the autophagic vacuoles. Therefore, our data suggest
TABLE 2
Effect of GLT on cell cycle distribution
a
Time (h) GLT (mg/ml) G0/G1 S G2/M sub-G0/G1
0040± 0.5
a
46 ± 0.8
a
14 ± 1.0
a
0.6 ± 0.28
24 0 48 ± 0.6
b
33 ± 0.9
b
19 ± 1.0
b
1.9 ± 0.30
48 0 62 ± 1.4
c
21 ± 1.3
c
16 ± 0.3
c
1.6 ± 0.17
00.2540± 0.5
a
47 ± 1.0
a
13 ± 1.2
a
1.6 ± 1.20
24 0.25 68 ± 1.1
d
19 ± 1.0
c
13 ± 0.6
a
2.1 ± 0.89
48 0.25 73 ± 1.2
e
10 ± 0.6
d
16 ± 0.6
c
2.4 ± 0.20
P value <0.001 <0.001 <0.001 0.520
a
Abbreviation is as follows: GLT, Ganoderma lucidum triterpene extract. Cell cycle distribution G0/G1, S, G2/M, and
sub-G0/G1 in percent. Data summarized using mean (±SD) and compared across all time points and treatment groups using a
2-way analysis of variance (ANOVA) for each cell cycle phase. The P value presented is for the interaction between time and
treatment from the ANOVA model. Post hoc tests between pairs of means were performed using Tukey’s multiple comparison
test. Within cell cycle phase, means sharing the same subscript letter are not significantly different. Those with differing
subscript letters were found to be significantly different.
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TRITERPENES FROM GANODERMA LUCIDUM AND COLON CANCER 635
FIG. 2. Ganoderma lucidum triterpene extract (GLT) induces cell cycle arrest at G0/G1. HT-29 cells were treated with A: 0 mg/ml of GLT or B: 0.25 mg/ml of
GLT for 48 h. Cell cycle analysis was performed as described in Materials and Methods. The results are representative of 3 separate experiments.
FIG. 3. Induction of autophagy in HT-29 cells treated with Ganoderma lucidum triterpene extract (GLT). HT-29 cells were treated with a: vehicle, b: 0.25 mg/ml
GLT, c: 0.5 mg/ml GLT, d: 20 µM SB202190 in the absence (b, c, d) or presence of 10 mM 3-methyl adenine (3MA) for 6 h. The autophagic vacuoles were
detected under the phase microscope as described in Materials and Methods. Data are representative from 3 independent experiments. The amount of vacuoles in
b, c, and d were compared with the control;
, P < 0.001. The amount of vacuoles in the absence or presence of 3MA (b vs. e; c vs. f; d vs. g) was also compared;
∗∗
, P < 0.005.
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636 A. THYAGARAJAN ET AL.
FIG. 4. Ganoderma lucidum triterpene extract (GLT) induces expression of Beclin-1 and LC-3 and inhibits p38 mitogen-activated protein kinase in HT-29 cells.
A: HT-29 cells were treated with GLT (0–1.0 mg/ml) for 6 h, and the expression of Beclin-1 and LC-3 was evaluated by Western blot analysis as described in
Materials and Methods. The same blots were stripped and probed for glyderaldehyde-3-phosphate dehydrogenase (GAPDH) for the loading of equal proteins.
The results are representative of 3 separate experiments. B: HT-29 cells were treated with p38 inhibitor SB202190 (20 µM) for 0 to 6 h, and the expression of
Beclin-1 and LC-3 was evaluated by Western blot analysis as described in Materials and Methods. The same blots were stripped and probed for GAPDH for the
loading of equal proteins. The results are representative of 3 separate experiments. C: HT-29 cells were treated with GLT (0–1.0 mg/ml) for 0, 3, and 6 h;andthe
expression of phospho-p38 and p38 was evaluated by Western blot analysis as described in Materials and Methods. The results are representative of 3 separate
experiments.
that GLT induces autophagy in HT-29 cells, and this process is
associated with increased expression of LC-3 protein and its
localization in vacuoles.
GLT Induces Autophagy in Tumors
As we demonstrated previously (Fig. 1C, Table 1), GLT sup-
presses the growth of colon cancer HT-29 xenograft tumors. To
examine whether GLT also induces autophagy in vivo, we ex-
amined the expression of LC-3 and Beclin-1 in colon tumors. To
evaluate whether Beclin-1 and LC-3 expression is induced in the
tumor tissue from the animals treated with GLT, the tumor tissue
lysates were prepared and analyzed by Western blotting. GLT
treatment induced expression of Beclin-1 and LC-3 in colon
tumors in a xenograft model of HT-29 cells in mice (Fig. 6A;
only representative samples are shown). Moreover, immunohis-
tochemical analysis of the tissue sections stained with Beclin-1
and LC-3 antibodies confirmed increased levels of Beclin-1 (P
< 0.001) and LC-3 (P < 0.005) in tumors in animals treated with
GLT (Fig. 6B). Therefore, GLT treatment induces autophagy of
colon cancer cells in vivo, and this effect is associated with
increased expression of Beclin-1 and LC-3, respectively.
DISCUSSION
In spite of the early detection by colonoscopy, surgical in-
tervention, chemotherapy, and radiation therapy, colorectal can-
cer remains the third leading cause of cancer mortality in the
United States (1). Colorectal cancer is mainly a disease of high
income countries (North America, parts of Europe, Australia,
New Zealand, and Japan), where overall rates are nearly 4 times
higher than in middle- to low-income countries (Africa, Cen-
tral America, and parts of Asia) (2). In addition to the “non-
Westernized” dietary patterns some of the natural products are
commonly used in the forms of extracts or teas in Asian coun-
tries. One of these natural products is a medicinal mushroom
Ganoderma lucidum, a well known natural agent recognized in
traditional Chinese medicine (4). Although recent studies have
demonstrated chemopreventative effects of G. lucidum in chem-
ically induced colon carcinogenesis in animals (33,34), and the
inhibition of growth and induction of apoptosis in human colon
cancer cells (35), the molecular mechanism(s) responsible for
the demise of colon cancer cells by G. lucidum have not been
fully addressed.
In this study, we showed that GLT suppresses proliferation
of HT-29 colon cancer cells in vitro and inhibits growth of
colon cancer tumors in an animal xenograft model through the
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TRITERPENES FROM GANODERMA LUCIDUM AND COLON CANCER 637
FIG. 5. Ganoderma lucidum triterpene extract (GLT) induces expression of LC-3 to autophagic vacuoles. HT-29 cells were treated with a: vehicle, b: 0.25 mg/ml
GLT, and c: 20 µM SB202190 for 6 h; and the expression of LC-3 was evaluated by the immunofluorescent staining as described in Materials and Methods.
Data are representative from 3 independent experiments. DAPI, 4
,6-diamidino-2-phenylindole.
induction of programmed cell death (PCD): autophagy. Apop-
tosis is a well-known form of PCD and is widely accepted as
the main mechanism of cancer cell death. Apoptosis is classi-
fied as a Type I PCD, whereas autophagy is classified as Type
II PCD (28). Apoptosis as a demise of cancer cells induced by
G. lucidum extracts or isolated triterpenes has been previously
described (23,35–44). However, we were unable to detect apop-
tosis in human colon cancer cells treated with GLT. Instead, we
have found that GLT induces autophagy in HT-29 cells and in
colon tumors as we confirmed by the presence of autophagic
vacuoles and by the increased expression of proautophagic pro-
teins Beclin-1 and LC-3. Comes et al. (29) demonstrated in-
duction of autophagy of colon cancer cells by the inhibition
of p38 MAPK. As we mentioned previously, our data clearly
demonstrate that GLT inhibits activity of p38 in HT-29 cells.
Therefore, inhibition of p38 by GLT may result in the induction
of expression of Beclin-1 and LC-3 proteins and formation of
autophagic vacuoles, finally leading to the autophagy of colon
cancer cells. The induction of autophagy of colon cancer cells by
the soybean B-group triterpenoid saponins though the induction
of LC-3 expression has been previously reported (45). However,
the autophagy has been linked to the inhibition of AKT signaling
and enhanced extracellular-regulated kinase 1/2 activity (46). In
addition, a plant triterpenoid, avicin D, induced autophagy of
osteosarcoma cells by activation of adenosine monophosphate-
activated protein kinase (47). Alternatively, activation of p38
induced autophagy in astrocytes (48) and melanoma cells (49).
Therefore, autophagy can be induced by a variety of stimuli
through different cellular targets and signaling pathways, and
the activation or inhibition of specific signaling pathway de-
pends on the molecular characteristic of the particular cancer
cell (50).
Here, we demonstrated the anticancer effect of G. lucidum
triterpenes after the intraperitoneal injection of GLT. Although
this route of administration is not optimal for dietary interven-
tion, we are able to demonstrate that GLT can suppress growth
of human colon cancer cells in vivo and that the effect of GLT
is systemic. Moreover, a study evaluating an oral administra-
tion of GLT in an animal model of the food-borne, carcinogen-
and inflammation-induced colon carcinogenesis is in progress.
G. lucidum is generally used in the forms of tea, powder, and
dietary supplements to promote health (51). Although there is
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638 A. THYAGARAJAN ET AL.
FIG. 6. Ganoderma lucidum triterpene extract (GLT) induces autophagy in colon tumors. A: Protein extracts were prepared from tumors from animals treated
with vehicle (-) or 6 mg GLT/kg of body weight (GLT). The expression of Beclin-1 and LC-3 was evaluated by Western blot analysis as described in Materials
and Methods. Representative sample is shown. B: Dissected tumors from animals treated with vehicle (control) or 6 mg GLT/kg of body weight (GLT) were
sectioned and stained with Beclin-1 and LC-3 as described in Materials and Methods. Each section from 3 different tumors for each group was analyzed at
×200 magnification, and the stain positive cells were scored from 4 independent places on the slide depending upon the intensity of the brown coloration (0 = no
change, 1 = slight brown, 2 = light brown, 3 = brown, and 4 = dark brown). Statistical significance was Beclin-1:
, P < 0.001; LC-3:
, P < 0.005. GAPDH,
glyderaldehyde-3-phosphate dehydrogenase.
anecdotal evidence of the use of G. lucidum in humans, and the
recommended dosage in the Pharmacopeia of the Republic of
China is 6 to 12 g/day, only a recent controlled human supple-
mentation study demonstrated that an oral application of 1.44
gofG. lucidum extract/day for 4 wk did not show any sign of
toxicity (52). Moreover, daily oral use of an herbal extract con-
taining4gofG. lucidum for 24 wk by patients with rheumatoid
arthritis and an oral application of 5.4 g of G. lucidum polysac-
charide extract for 12 wk by patients with advanced lung cancer
was well tolerated, respectively (53,54). Based on our current
finding, further studies that have evaluated the bioavailability of
GLT are justified and needed prior to initiating clinical studies
with GLT.
In summary, our report suggests GLT as a natural agent that
possesses the activity to inhibit growth of colon cancer cells in
vitro and in vivo by the induction of autophagy in colon cancer
cells.
ACKNOWLEDGMENTS
This work was supported by the Methodist Research Institute,
Clarian Health, Inc. We thank Shailesh Dudhgaonkar for his help
with animal experiments.
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    • "Ligand profile module was utilized to reversely identify targets for six isolated and two known positive GTs in Discovery Studio 4.0 (DS, Accelrys Inc., San Diego, CA, USA). Previous researches indicated GA-A and GA-D have the clear anti-cancer activity [18,41]. Therefore, in this paper, GA-A and GA-D were selected to be the typical positive compounds for guiding the anti-cancer bioactive prediction of six isolated GTs. "
    [Show abstract] [Hide abstract] ABSTRACT: Ganoderma triterpenes (GTs) are the major secondary metabolites of Ganoderma lucidum, which is a popularly used traditional Chinese medicine for complementary cancer therapy. In the present study, systematic isolation, and in silico pharmacological prediction are implemented to discover potential anti-cancer active GTs from G. lucidum. Nineteen GTs, three steroids, one cerebroside, and one thymidine were isolated from G. lucidum. Six GTs were first isolated from the fruiting bodies of G. lucidum, including 3β,7β,15β-trihydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid methyl ester (1), 3β,7β,15β-trihydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid (2), 3β,7β,15α,28-tetrahydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid (3), ganotropic acid (4), 26-nor-11,23-dioxo-5α-lanost-8-en-3β,7β,15α,25-tetrol (5) and (3β,7α)-dihydroxy-lanosta-8,24-dien- 11-one (6). (4E,8E)-N-d-2'-hydroxypalmitoyl-l-O-β-d-glucopyranosyl-9-methyl-4,8-spingodienine (7), and stigmasta-7,22-dien-3β,5α,6α-triol (8) were first reported from the genus Ganodema. By using reverse pharmacophoric profiling of the six GTs, thirty potential anti-cancer therapeutic targets were identified and utilized to construct their ingredient-target interaction network. Then nineteen high frequency targets of GTs were selected from thirty potential targets to construct a protein interaction network (PIN). In order to cluster the pharmacological activity of GTs, twelve function modules were identified by molecular complex detection (MCODE) and gene ontology (GO) enrichment analysis. The results indicated that anti-cancer effect of GTs might be related to histone acetylation and interphase of mitotic cell cycle by regulating general control non-derepressible 5 (GCN5) and cyclin-dependent kinase-2 (CDK2), respectively. This research mode of extraction, isolation, pharmacological prediction, and PIN analysis might be beneficial to rapidly predict and discover pharmacological activities of novel compounds.
    Full-text · Article · May 2016
    • "Additionally, it was also reported that Ganoderma lucidum triterpene extract (GLT) suppressed the proliferation of human colon cancer cells and inhibited tumor growth in a xenograft model, which were associated with the induction of autophagic cell death [397]. Furthermore, ganoderic acid activated autophagy, which facilitates immune recognition of CD4 + T cells; induced autophagic cell death and apoptosis of melanoma [398] Ganoderma lucidum triterpene extract induced autophagy which inhibited the development of colon cancer via p38 MAPK signaling [397]; and suppressed gastric cancer cells through the repression of p62 [399]. All these findings suggest that the traditional therapeutic role of Ganoderma lucidum is in part related to autophagy regulation, which may also be responsible for the novel use of the herb in cancer treatment. "
    [Show abstract] [Hide abstract] ABSTRACT: Autophagy is a universal catabolic cellular process for quality control of cytoplasm and maintenance of cellular homeostasis upon nutrient deprivation and environmental stimulus. It involves the lysosomal degradation of cellular components such as misfolded proteins or damaged organelles. Defects in autophagy are implicated in the pathogenesis of diseases including cancers, myopathy, neurodegenerations, infections and cardiovascular diseases. In the recent decade, traditional drugs with new clinical applications are not only commonly found in Western medicines, but also highlighted in Chinese herbal medicines (CHM). For instance, pharmacological studies have revealed that active components or fractions from Chaihu (Radix bupleuri), Hu Zhang (Rhizoma polygoni cuspidati), Donglingcao (Rabdosia rubesens), Hou po (Cortex magnoliae officinalis) and Chuan xiong (Rhizoma chuanxiong) modulate cancers, neurodegeneration and cardiovascular disease via autophagy. These findings shed light on the potential new applications and formulation of CHM decoctions via regulation of autophagy. This article reviews the roles of autophagy in the pharmacological actions of CHM and discusses their new potential clinical applications in various human diseases.
    Full-text · Article · Mar 2016
    • "However, inhibition of p38 MAPK increased the level of LC3-II compared to cells treated with 50 μM CPF. Previous other reports demonstrated that a deficiency of p38 activity in HT29 human colon cancer cells [55] and a myelogenous leukemia K562 cell line [56] are associated with an increase in pro-autophagic gene expression and autophagy induction. A selective p38 MAPK inhibitor, such as SB203580 or SB202190, induces vacuole formation and LC3-II accumulation [57]. "
    [Show abstract] [Hide abstract] ABSTRACT: Mitochondrial quality control and clearance of damaged mitochondria through mitophagy are important cellular activities. Studies have shown that PTEN-induced putative protein kinase 1 (PINK1) and Parkin play central roles in triggering mitophagy; however, little is known regarding the mechanism by which PINK1 modulates mitophagy in response to reactive oxygen species (ROS)-induced stress. In this study, chlorpyrifos (CPF)-induced ROS caused mitochondrial damage and subsequent engulfing of mitochondria in double-membrane autophagic vesicles, indicating that clearance of damaged mitochondria is due to mitophagy. CPF treatment resulted in PINK1 stabilization on the outer mitochondrial membrane and subsequently increased Parkin recruitment from the cytosol to the abnormal mitochondria. We found that PINK1 physically interacts with Parkin in the mitochondria of CPF-treated cells. Furthermore, a knockdown of PINK1 strongly inhibited the LC3-II protein level by blocking Parkin recruitment. This indicates that CPF-induced mitophagy is due to PINK1 stabilization in mitochondria. We observed that PINK1 stabilization was selectively regulated by ROS-mediated c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling activation but not p38 signaling. In the mitochondria of CPF-exposed cells, pretreatment with specific inhibitors of JNK and ERK1/2 significantly decreased PINK1 stabilization and Parkin recruitment and blocked the LC3-II protein level. Specifically, JNK and ERK1/2 inhibition also dramatically blocked the interaction between PINK1 and Parkin. Our results demonstrated that PINK1 regulation plays a critical role in CPF-induced mitophagy. The simple interpretation of these results is that JNK and ERK1/2 signaling regulates PINK1/Parkin-dependent mitophagy in the mitochondria of CPF-treated cells. Overall, this study proposes a novel molecular regulatory mechanism of PINK1 stabilization under CPF exposure.
    Full-text · Article · Feb 2016
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