Telomerase associated apoptotic events by mushroom - Ganoderma lucidum on pre-malignant human urothelial cells.

Telomerase associated apoptotic events by mushroom - Ganoderma lucidum on
pre-malignant human urothelial cells.
John W.M. Yuen1, Mayur Danny I. Gohel1, and Doris Wai-Ting Au2
Number of manuscript pages: 18
Number of figures: 5
Number of tables: 2
Keywords:
Ganoderma lucidum, Triterpenes, Bladder cancer, Apoptosis, Telomerase, Prevention
1J.W.M. Yuen and M.D.I. Gohel are affiliated with the Dept. of Health Technology &
Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR,
China.
2D.W.T. Au is affiliated with the Dept. of Biology and Chemistry, City University of
Hong Kong, Kowloon, Hong Kong Special Administrative Region, China.
Page 1 of 30 PDF proof only
This is the Pre-Published Version.
Yuen 2
ABSTRACT
Relapse of transitional bladder tumors remains a challenge despite advances in
immunological therapies. The chemopreventive effects of Ganoderma. lucidum on the
tumorigenic phase of bladder cancer were tested, using a 4-aminobiphenyl (ABP)
tumorigenic transformable human urothelial cell (HUC-PC) model. Our in vitro data
clearly show that G. lucidum inhibited the viability and growth of HUC-PC. This
could be explained by a concomitant induction of apoptosis and inhibition of
telomerase activity in G. lucidum treated HUC-PC culture. Significant exteriorization
of phosphatidylserine was detected by Annexin-V on cell surface at 3h upon G.
lucidum incubation, and the HUC-PC cells subsequently lost the membrane integrity
for uptake of DNA-specific 7-AAD dye, reaching 100% apoptotic in 48 hours.
Moreover, the presence of G. lucidum in HUC-PC culture significantly elevated the
levels of hydrogen peroxide and 8-hydroxy-2’-deoxyguanosine (8-OHdG) production.
Taken together, physiological doses of G. lucidum promote apoptosis and oxidative
DNA damage as well as suppress telomerase activity in HUC-PC cultures, which are
essential to explain its anti-HUC-PC growth properties. The findings of this study
strongly supports that G. lucidum is a potential source of chemopreventive agents for
bladder cancer, based on its effectiveness on controlling the pre-malignant urothelial
cell growth and carcinogen-induced transformation. (199)
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INTRODUCTION
The carcinoma of the urinary bladder is the most common urologic malignancy
encountered (1,2). The most frequent form (up to 90%) of bladder cancer on clinical
presentation is urothelial carcinoma, i.e. transitional cell carcinoma (TCC). Risk
factors for TCC include tobacco exposure, chemicals such as N-nitrosamines and
aromatic amines (3) and genetic susceptibility (4). Majority of TCC are superficial
disease (90%) and standard clinical management calls for transurethral resection with
or without intravesical immunotherapy or chemotherapy (5). The most commonly
used immunotherapeutic agent - Bacillus Calmette-Guérin (BCG), is based on the
urothelial internalization to trigger inflammatory response, which ultimately results in
TNF--induced apoptosis (6). Despite the positive impact (reduction by 20-30% of
recurrence) and historical applications (30 years) of BCG, recurrence rate of bladder
cancer remains high with lethal side-effects, as well as with approximately 10-30% of
recurrent cancers progressing to invasive muscle disease to threaten survival rate (2).
Therefore, powerful chemopreventive agents are demanded for prevention bladder
cancer recurrence and progression.
Ganoderma lucidum, called “Red Lingzhi” in China has been used for longevity and
tonicity in the East for over two thousand years, and because of its perceived health
benefits, the mushroom are nowadays widely consumed as a supplement for different
kinds of diseases and health maintenance (7). G. lucidum is commonly used for
immune boosting and cancer prevention, whereas the evidence of anticancer
properties comes from experimental studies in vitro and animals to humans’ in vivo
(8). Triterpenes contain skeletons of lanostane and are responsible for the bitter taste
of G. lucidum, exerting cytotoxic-based carcinostatic activities (9,10). Cytoxicity and
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Yuen 4
growth inhibition on various cancer cells have been well documented, and most of
them are based on controlling the cell cycle and signaling pathways (11-13).
Apoptosis has also been demonstrated by G. lucidum in malignant cell lines, such as
leukemia (14-17), and skin (18). Recently, it was reported that G. lucidum inhibits the
growth of a “chemically tumorigenic transformable” human urothelial cell line (HUCPC)
(19). However, the toxic mechanisms of G. lucidum on urothelial cells remain
virtually unknown. The HUC-PC cell line is a clonal line derived from the simian
virus 40 (SV40) - immortalized urothelial cell line “SV-HUC” cells (20), thus
providing a relevant model, akin to the intravesical contact exposure of carcinogens
and candidate therapeutic agents, for understanding the cause-and-effects between
carcinogenesis and chemoprevention.
Unlimited replicative potential is an essential element for carcinogenesis. The enzyme
telomerase plays a vital role controlling cell proliferation (21,22), and telomerase
expression is found in over 85% human cancers, including 95% of all advanced
malignancies (23). Up-regulation of telomerase can be detected in transformed
bladder cancer cells (24-26), voided urine (27) and bladder-wash specimens (28) of
most patients with urothelial cancer. Telomerase has been proposed as a urine-based


marker for bladder cancer with 70-85% sensitivity and 80-95% specificity (29).
Emerging evidences show the protective effects of telomerase on cell growth and
survival (30). In this report concentrated extract of G. lucidum (GLE) at physiological
doses were found to reduce viability and growth of HUC-PC cells, which could be
explained by a concomitant suppression of telomerase activity and increase of
apoptosis under oxidative stress (elevated hydrogen peroxide (H2O2) and oxidative
DNA damage).
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Yuen 5
Telomerase associated apoptotic events by mushroom - Ganoderma lucidum on
pre-malignant human urothelial cells.
John W.M. Yuen1, Mayur Danny I. Gohel1, and Doris Wai-Ting Au2
Number of manuscript pages: 18
Number of figures: 5
Number of tables: 2
Keywords:
Ganoderma lucidum, Triterpenes, Bladder cancer, Apoptosis, Telomerase, Prevention
1J.W.M. Yuen and M.D.I. Gohel are affiliated with the Dept. of Health Technology &
Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR,
China.
2D.W.T. Au is affiliated with the Dept. of Biology and Chemistry, City University of
Hong Kong, Kowloon, Hong Kong Special Administrative Region, China.
Page 1 of 30 PDF proof only
This is the Pre-Published Version.
Yuen 2
ABSTRACT
Relapse of transitional bladder tumors remains a challenge despite advances in
immunological therapies. The chemopreventive effects of Ganoderma. lucidum on the
tumorigenic phase of bladder cancer were tested, using a 4-aminobiphenyl (ABP)
tumorigenic transformable human urothelial cell (HUC-PC) model. Our in vitro data
clearly show that G. lucidum inhibited the viability and growth of HUC-PC. This
could be explained by a concomitant induction of apoptosis and inhibition of
telomerase activity in G. lucidum treated HUC-PC culture. Significant exteriorization
of phosphatidylserine was detected by Annexin-V on cell surface at 3h upon G.
lucidum incubation, and the HUC-PC cells subsequently lost the membrane integrity
for uptake of DNA-specific 7-AAD dye, reaching 100% apoptotic in 48 hours.
Moreover, the presence of G. lucidum in HUC-PC culture significantly elevated the
levels of hydrogen peroxide and 8-hydroxy-2’-deoxyguanosine (8-OHdG) production.
Taken together, physiological doses of G. lucidum promote apoptosis and oxidative
DNA damage as well as suppress telomerase activity in HUC-PC cultures, which are
essential to explain its anti-HUC-PC growth properties. The findings of this study
strongly supports that G. lucidum is a potential source of chemopreventive agents for
bladder cancer, based on its effectiveness on controlling the pre-malignant urothelial
cell growth and carcinogen-induced transformation. (199)
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INTRODUCTION
The carcinoma of the urinary bladder is the most common urologic malignancy
encountered (1,2). The most frequent form (up to 90%) of bladder cancer on clinical
presentation is urothelial carcinoma, i.e. transitional cell carcinoma (TCC). Risk
factors for TCC include tobacco exposure, chemicals such as N-nitrosamines and
aromatic amines (3) and genetic susceptibility (4). Majority of TCC are superficial
disease (90%) and standard clinical management calls for transurethral resection with
or without intravesical immunotherapy or chemotherapy (5). The most commonly
used immunotherapeutic agent - Bacillus Calmette-Guérin (BCG), is based on the
urothelial internalization to trigger inflammatory response, which ultimately results in
TNF--induced apoptosis (6). Despite the positive impact (reduction by 20-30% of
recurrence) and historical applications (30 years) of BCG, recurrence rate of bladder
cancer remains high with lethal side-effects, as well as with approximately 10-30% of
recurrent cancers progressing to invasive muscle disease to threaten survival rate (2).
Therefore, powerful chemopreventive agents are demanded for prevention bladder
cancer recurrence and progression.
Ganoderma lucidum, called “Red Lingzhi” in China has been used for longevity and
tonicity in the East for over two thousand years, and because of its perceived health
benefits, the mushroom are nowadays widely consumed as a supplement for different
kinds of diseases and health maintenance (7). G. lucidum is commonly used for
immune boosting and cancer prevention, whereas the evidence of anticancer
properties comes from experimental studies in vitro and animals to humans’ in vivo
(8). Triterpenes contain skeletons of lanostane and are responsible for the bitter taste
of G. lucidum, exerting cytotoxic-based carcinostatic activities (9,10). Cytoxicity and
Page 3 of 30 PDF proof only
Yuen 4
growth inhibition on various cancer cells have been well documented, and most of
them are based on controlling the cell cycle and signaling pathways (11-13).
Apoptosis has also been demonstrated by G. lucidum in malignant cell lines, such as
leukemia (14-17), and skin (18). Recently, it was reported that G. lucidum inhibits the
growth of a “chemically tumorigenic transformable” human urothelial cell line (HUCPC)
(19). However, the toxic mechanisms of G. lucidum on urothelial cells remain
virtually unknown. The HUC-PC cell line is a clonal line derived from the simian
virus 40 (SV40) - immortalized urothelial cell line “SV-HUC” cells (20), thus
providing a relevant model, akin to the intravesical contact exposure of carcinogens
and candidate therapeutic agents, for understanding the cause-and-effects between
carcinogenesis and chemoprevention.
Unlimited replicative potential is an essential element for carcinogenesis. The enzyme
telomerase plays a vital role controlling cell proliferation (21,22), and telomerase
expression is found in over 85% human cancers, including 95% of all advanced
malignancies (23). Up-regulation of telomerase can be detected in transformed
bladder cancer cells (24-26), voided urine (27) and bladder-wash specimens (28) of
most patients with urothelial cancer. Telomerase has been proposed as a urine-based
marker for bladder cancer with 70-85% sensitivity and 80-95% specificity (29).
Emerging evidences show the protective effects of telomerase on cell growth and
survival (30). In this report concentrated extract of G. lucidum (GLE) at physiological
doses were found to reduce viability and growth of HUC-PC cells, which could be
explained by a concomitant suppression of telomerase activity and increase of
apoptosis under oxidative stress (elevated hydrogen peroxide (H2O2) and oxidative
DNA damage).
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MATERIALS AND METHODS
Preparation of concentrated extract from G. lucidum
A proprietary extract composed of G. lucidum fruiting bodies and cracked spores,
branded ReishiMax GLpTM, was purchased from Pharmanex Inc. (Hong Kong, China).
The active ingredients of the product was standardized as 13.5% polysaccharides (-
1,3-glucans) and 6% triterpenes (ganoderic acids and other), which is the highest level
of extractable activities, whereas the remaining 80% composed of nucleosides, fatty
acids, and amino acids, according to the manufacturer’s technical bulletin. The
powdered extract was sonicated with 95% ethanol for 30 minutes, and the supernatant
was further extracted by successive sonication using absolute ethanol (19). The waterinsoluble
brown powder, i.e. GLE was retrieved from the filtrate (through 0.45 μm
polypropylene filter) under reduced pressure.
Cell culture
SV-HUC-PC cell line originates from Department of Human Oncology, University of
Wisconsin Medical School, gifted by Dr. Rao from the University of California, Los
Angeles. The cell line was cultured in F12 Ham enriched Dulbecco’s Modified
Eagle’s Medium (Sigma, St. Louis, MO) with 1% penicillin and streptomycin (10,000
μg/ml penicillin, 10 mg/ml streptomycin) and 10% Fetal Bovine Serum (GIBCO BRL
Island, New York, U.S.A.). Logarithmically growing cells were harvested and seeded
at concentration of 1 x 106 cells per 100-mm culture dish for assays. Cultures were
maintained at 37oC in a water-saturated atmosphere containing 5% CO2. For assays,
various concentrations (0, 40, 80 and 100 μM) of GLE (in assay media containing at
most 0.01% absolute ethanol) were inhibited with HUC-PC cells, in the presence or
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absence of 100 μM ABP (Sigma, St. Louis, MO), which dissolved in DMSO (Sigma,
St. Louis, MO) with final concentration at not more than 0.02% in assay media.
LDH cytotoxicity assay and Cell viability
The cytotoxicity of test substances, i.e. 4-aminobiphenyl (ABP, CAS. No. 92-67-1)
and GLE, were tested on the cell lines by using LDH Cytotoxicity Detection kit
(TaKaRa Bio Inc., Shiga, Japan), preformed on a 96-well microplate. In addition, no
interference and cytotoxicity was observed from any test substances and solvent
concentrations used for assays. Cell viability was assessed by using an automated
Beckman Coulter Vi-CELLTM XR Cell Viability Analyzer with its reagent pack
(Miami, FL). Percentage cell growth inhibition (%GI) was calculated using the
following formulae:
( Mean control
viable cell number
- Experimental viable
cell number )
%GI
=
( Mean control
Viable cell number
- Initial seeded cell
number )
X 100
All cell counting results were verified between the manual and automated methods.
Apoptosis assay
The Beckman Coulter annexin V-FITC /7-AAD kit (Immunotech, France) was used
to measure apoptosis. Briefly, cells were treated with various assay media and assayed
periodically (at 1, 2, 3, 4, 5, 6, 8, 12 and 48 hours) during the 48-hour incubation. Cell
analysis was performed on Beckman Coulter COULTER® EPICS® XLTH (Miami, FL)
equipped with the Elite software version 5. A minimum of 10,000 events were
collected and measured at FL1 (525nm) and FL4 (675nm). Cells incubated with 3%
formaldehyde- containing PBS for 30 minutes on ice was used as positive control.
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Untreated cells without staining were used as negative control, and those with staining
were used as background control.
Real Time Quantitative –Telomeric Repeat Amplification Protocol (RTQ-TRAP)
Cells (1-5 x 106) were lysed in 1x CHAPS buffer containing RNase inhibitor, and
incubated on ice for 30 minutes. The lysate was then centrifuged at 12,000g for 40
minutes at 4oC, and the supernatant was collected (31). Total protein concentration of
the cell extract was determined using a Bio-Rad Bradford protein assay kit (Hercules,
CA). The protein level of each extract was adjusted to 10 ng/μl for telomerase activity
analysis. The telomeric repeat amplification protocol (TRAP) is a landmark method
for measuring telomerase activity, and a real-time PCR technique has been
incorporated to allow rapid and precise quantitation (31). TS and ACX Primers were
purchased from Molecular Information Laboratory (South Korea). 10X TRAP buffer
containing 200 mM Tris, 630 mM KCl, 35 mM MgCl2, 10 mM EGTA, 1 mg/ml BSA,
and 0.05% Tween 20 was prepared and stored at -20oC until use. The total volume of
the reaction mixture was 25 μl, containing 40X SYBR Green (Invitrogen), 1X
Fluorescein (Bio-Rad), 10mM dNTP (Promega), 0.25 μl Hot Star Taq polymerase
(Qiagen), 14.75 μl RNase/DNase free distilled water, 0.1 μg each of TS (5'-
AATCCGTCGAGCAGAGTTAG-3') and ACX (5'-GCGCGG(CTTACC)4-3') primers,
2 μl 10X TRAP buffer, and 5 μl protein extract. The PCR was performed in a 96-well
microtiter plate on a MyiQ Single-Color Real-Time PCR Detection System. The
reaction mixture was first incubated at 25oC for 30 minutes to elongate the TS primer.
The PCR was started at 95oC for 15 minutes to activate the Hot Star Taq polymerase,
followed by 30-cycle amplification (95oC for 30 seconds, 60oC for 30 seconds, and
72oC for 60 seconds). Fluorescence signal generated from SYBR green was collected
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and analyzed with iCycler iQ Detector software (Ver. 3.0a; Bio-Rad), where
measurable threshold intensity (threshold cycle; Ct) was achieved or at maximum 30
cycles. Relative telomerase activity was calculated using the derived equation 2-Ct (1).
Extracellular H2O2 assay in culture media
Culture media from each study group was collected after 48-hour incubation, and
filtered using Centricon YM-10 microconcentrator (Millipore Co., Bedford, MA) with
10nm pore size for the H2O2 assay. The method was based on the FOX assay as
published elsewhere (32), with minor modifications. Briefly, 100 μl of sample was
added to 170 μl of working reagent in a 96 microplate well, mixed and left for 20
minutes at room temperature, and the absorbance at 590 nm was read against a
complete media blank, using microplate reader (TECAN, Austria). The H2O2
concentration of each sample was read from a standard curve, with 0, 0.5, 1.0, 2.5 and
5.0 μM working standards freshly prepared from a 30% H2O2 stock solution (RDH).
8-OHdG ELISA assay
Oxidative DNA damage was analyzed by measuring free 8-OHdG present in the
culture media. Very low quantity of 8-OHdG is expected in the culture media.
Filtered culture media were assayed immediately using the Highly Sensitive 8-OHdG
Check Kit (Japan Institute for Control of Aging, Shizuoka, Japan). The
manufacturer’s instructions are strictly followed. Duplicated samples of each study
group were run in triplicate. The outer most wells of the ELISA plate were not used to
avoid edge effect, and phosphate buffer was added to the unused wells to maintain the
uniform temperature within the wells.
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Statistical Analysis
All experiments were performed in duplicate for reproducibility. Descriptive statistics,
such as mean and standard error, were used to summarize the results. GraphPad Prism
(GraphPad software, version 3.0 for windows, U.S.A.) was used to perform Student’s
t test for statistical comparisons. Pearson’s correlation test was carried out to measure
the relationship between measured parameters. Statistical significance was sought at
two-tailed P-value 0.05.
RESULTS
Effects on HUC-PC cell growth
Cell growth was enhanced by 16%(±4%) with ABP incubation for 48 hours (Table 1).
22%(±6%) more of the cell growth was shown on ABP-pretreated cells (24 hours)
followed by 48 a hour-culture in complete media, as compared with control (Table 2).
The GLE re-extracted from the capsule powder is water-insoluble. As determined by
LDH release from cells, no direct cytotoxicity was observed from GLE at 80 μg/ml or
below after 24 hour-incubation, but 14%(±2%) and 100%(±6%) of the cells were
killed with 100 μg/ml and 200 μg/ml GLE, respectively. Growth inhibition was
demonstrated at 100% by 80 μg/ml GLE, as initial cell seeding number (i.e. 1 x 106
cell / 100-mm dish) was maintained after 48 hour-incubation, regardless of the
presence or absence of ABP (Table 1). Table 1 and 2 also summarize the growth
inhibitory effects of GLE at different concentrations, with ABP treatments, in a dosedependent
manner.
Apoptotic effects of GLE on HUC-PC cells
As shown in Figure 1a, cell shrinkage, elongation and blebs formation appeared in 48
hours for GLE-treated cells. A time-dependent progressive apoptotic effect of GLE on
the cells is shown in Figure 2a & b. When incubated with 80 μg/ml of GLE, about
30% of the cells were apoptotic at 3 hours, and gradually progressed to reach 70% at
8 hours, predominantly Annexin-V positive but 7-AAD negative phenotype. At 12
hours, the apoptotic cell population was maintained at 70%, however, the phenotype
became both Annexin-V and 7-AAD positive. The cell culture was continued and
reached 100% apoptotic with almost 60% Annexin-V + 7-AAD positive phenotype.
10-20% of control cells were shown apoptotic (data not shown). Similar trend of the
apoptotic process was also shown on the cells treated simultaneously with GLE and
ABP, which was in a dose-dependent response (Figure 3).
Inhibitory effects of GLE on HUC-PC telomerase activity
Relative telomerase activity was enhanced significantly (P<0.001) by 33%(±4%) for
ABP-pretreated cells (Figure 4a). When incubated with GLE, 30% of relative
telomerase activity of HUC-PC was inhibited significantly (P < 0.001), and 40% was
inhibited for the ABP-pretreated cells (P<0.001), and this inhibitory effect was in a
dose-dependent manner (Figure 4a & b). However, the relative telomerase activity of
the cells was inversely proportional to the growth inhibition exerted by GLE (Fig. 4c).
Oxidative stress in apoptotic HUC-PC cells
In the presence of GLE, there was significant increase of H2O2 (P<0.05) and 8-OHdG
(P<0.01), by 20% and 22% respectively, in the ABP-cultured media (Fig. 5a). The 8-
OHdG formation induced by GLE was in a clear dose-dependent manner (Fig. 5b). In
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the absence of ABP, 43% of H2O2 in culture media was significantly increased
(P<0.05) by TRE, but no increment of 8-OHdG was found (data not shown).
DISCUSSION
Findings of the present study support G. lucidum as a source of chemopreventive
candidate for urothelial cancer. Apoptosis, as measured by morphological and
cytometric changes, is evident to be one of the main cause contributing for HUC-PC
growth inhibition. In the early phase of apoptosis, the cells bind positively with
annexin V, because of the lost of the asymmetry of cell membrane that causes the
exteriorization of negatively charged phospholipids - phosphatidylserine (PS) from
the inner leaflet. As shown in the results, the process induced by GLE progressed to
late apoptosis, irrespective of the presence or absence of ABP, in dose- and timedependent
manner such that the cells were continuously losing the cell membrane
integrity and stained positive with DNA specific viability dye –7-amino-actinomycin
D (7-AAD). This is the first report that G. lucidum suppress pre-malignant urothelial
cells by promoting apoptosis. These results are consistent with other reports, that
complex extract or pure Ganoderma triterpenes induce apoptosis on cancer cell lines,
including the hematopoietic cell lines (14), the highly metastic 95-D lung cancer cells
(17), the human hepatoma HuH-7 cells (33), and pure triterpene compounds, such as
ganoderic acid T and X (17,33), reported to be active in causing apoptosis. Over
hundreds of triterpene molecules have been identified in G. lucidum (8). In fact, many
commercially available G. lucidum products contain complex mixtures of triterpenes
and polysaccharides as their active ingredients. We, therefore, accentuate the
importance of studying a re-extracted fraction of active ingredient rich rather than a
selected pure compound for apoptotic activity.
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The inhibitory effect of G. lucidum on telomerase activity is also demonstrated in the
present study. Both the basal and ABP-induced telomerase activities were
significantly reduced to similar levels, in dose-dependent manner. The GLE-induced
telomerase inhibition is shown to be directly proportional to the growth inhibitory
effect after 48 hour-incubation. Inhibition of telomerase has been shown to limit
human cancer cell growth by disrupting telomere maintenance (34). Human tumor
cells with shortened telomeres are effectively and rapidly killed after inhibiting the
telomerase enzyme (35). Interestingly, according to the Fenton’s reaction, H2O2 is
being dissociated to generate hydroxyl radicals that are able to oxidize C-8 position of
G bases to form 8-OHdG (36). In the present study, the increment of H2O2
concentration could be a possible mechanism that attacks the telomere repeats
(TTAAGGG) of the telomeres, to generate 8-OHdG (dose-dependent) and accelerate
telomere shortening, which is unlikely to be compensated when telomerase activity is
being suppressed.
Although oxidative DNA damage, in particular 8-OHdG is widely accepted to be
mutagenic that promotes carcinogenesis, it has been also proposed as a therapeutic
strategy through induction of cell cycle arrests (37-39). It is well-known that G2-M
transition of the cell cycle is a critical checkpoint to verify genomic instability, by
blocking DNA-damaged cells to enter mitosis (40). Some chemotherapeutic agents
are genotoxic to the DNA during the synthesis (S) phase of cell cycle to cause the G2-
M arrest (41,42). For G. lucidum, the regulation of G2-M related proteins have been
uncovered recently to suppress cell growth of prostate cancer (13) and hepatoma (43).
In the study of Lu et al, only 6-10% of the HUC-PC cells undergoing G2-M phase cell
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cycle arrest were induced by G. lucidum, which was insufficient to explain the
exhibited growth inhibition (19). A combination of apoptosis and oxidative DNA
damage associated with telomerase inhibition as demonstrated in this study are useful
to explain significant growth inhibition by GLE on pre-tumorigenic HUC-PC cells
and other cancer cells.
The worthiness of using cells in pre-cancerous stage is particularly important for
chemoprevention. Nothing is better than eliminating the adverse cells before it turns
malignant, especially looking at bladder carcinoma where rate of recurrences remain
exceptionally high even after complete transurethral resection. The non-tumorigenic
but ABP-transformable characteristic of HUC-PC cell line provides an excellent
model for studying the chemoprevention of bladder cancer, since majority of bladder
cancer are diagnosed as TCC at presentation and residual cells after resection are
regarded as the key stimulus factor for recurrence. The superficial mucosal urothelial
lining is targeted for attack by carcinogens. The HUC-PC cell line has been proven to
be sensitive to ABP for tumorigenic transformation (20). It was demonstrated by
several investigators that the exposure of ABP and its metabolites to HUC-PC cells
caused changes in proteomic profile (44), alteration of F/G-actin ratio (45), formation
of DNA adducts (46), and instability of the genome (47). In addition, Hahn and
associates (26) have shown that direct tumorigenic conversion of normal human
epithelial cells can be achieved by combining ectopic expression of human telomerase
catalytic subunit with SV40 large-T oncoprotein. Therefore, the enhancement of
telomerase activity and cell proliferation demonstrated in the present study, provides
evidence that the carcinogenic transformation of HUC-PC cells requires telomerase
activation.
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Conclusively, data presented in the current study provide mechanistic insight on the
chemopreventive effects of G. lucidum on pre-cancerous human urothelial cells.
Growth inhibition induced by GLE is mediated via apoptosis associated with
suppression of telomerase activity and oxidative DNA damage. Our findings support
G. lucidum is a source of chemopreventive candidate for bladder cancer. Detailed
evaluations are essential before positioning it into the category of chemoprevention.
Last but not the least, studies of immunological events including chemotaxis and
phagocytes are ongoing to define the efficacy and effectiveness of G. lucidum on premalignant
cell clearance.
ACKNOWLEDGEMENTS
The authors would like to acknowledge the Research Committee of the Hong Kong
Polytechnic University for the postgraduate scholarship (RGH8) and Sir Edward
Youde Memorial Fellowship awarded to Mr. John Yuen to conduct this study. The
authors are grateful to Dr. J.Y. Rao (UCLA Medical Center, USA) for providing the
SV40-HUC-PC cell line and professional advices. The authors would also like to
special thank Mr. Mike Chiu (Hong Kong Polytechnic University) and research staff
of City University of Hong Kong, Ms. Helen Mok and Ms. Wai Yu for technical
assistances.
MAILING ADDRESS FOR REPRINTS
Address correspondence to Dr. Danny Gohel, Department of Health Technology &
Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR,
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China. Phone: +852-34008584. Fax: +852-23624365. E-mail:
danny.gohel@polyu.edu.hk
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