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Full-length article Acta Pharmacologica Sinica 2008 May; 29 (5): 620-627 Reversal effect of Ganoderma lucidum polysaccharide on multidrug resistance in K562/ADM cell line1 Wei-dong LI2,4, Bo-di
ZHANG2, Ran WEI2, Ji-hong
LIU3, Zhi-bin LIN2
2Department of Pharmacology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100083, China;
3Confocal Microscope Laboratory, Peking University Health Science Center, Beijing 100083, China
1 This research was supported by a research fund from Shanghai Green Valley Holding.
Abstract
Aim: To investigate the reversal effect
of Ganoderma lucidum polysaccharides
(Gl-PS) on multidrug resistance (MDR) in the
adriamycin (ADM)-resistant leukemic cell line K562/ADM.
Methods: Cytotoxicity was assayed by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method; the ADM concentration in cells was determined by flow cytometry and confocal
laser scanning microscopy techniques; the expression of P-glycoprotein was assayed by flow cytometry; and the mRNA
expression levels of MDR-1 and MDR-associated protein (MRP)1 were determined by RT-PCR.
Results: Gl-PS reversed MDR in K562/ADM cells.
Gl-PS obviously reversed the resistance of K562/ADM to doxorubicin. The
reversing factors of Gl-PS at 10 and 20 mg/L were 6.46 and 6.80, respectively.
MDR-1 and MRP1 transcription were downregulated by 10 and 50 mg/L
Gl-PS. Conclusion: Gl-PS can reverse the MDR by downregulating the expression of MDR-1 and MRP1 in K562/ADM cells.
Key words multidrug resistance; Ganoderma lucidum polysaccharides; K562/adriamycin;
P-glycoprotein; multidrug resistance-1; multidrug
resistance-associated protein-1
Note: Please read the complete
full text with Figures and Tables at
Introduction
Multidrug resistance (MDR), a major cause of cancer
treatment failure, is a phenomenon whereby cancer cells
develop resistance to a wide variety of chemotherapeutic drugs.
MDR has been associated with the overexpression of
P-glycoprotein (P-gp) or MDR-associated protein (MRP), 2
transmembrane transporters that act as pumps to remove toxic
drugs from tumor cells. The MDR-1 overexpression has been
reported in many tumors and in vitro-selected,
drug-resistant cell lines[1_4]. Meanwhile, MRP1 has also been
identified as having the ability to induce MDR. It has been found
that the accumulation of chemotherapeutic agents in tumor
cells with overexpressed P-gp170 or MRP1 was significantly
reduced as compared to the MDR antagonist-treated tumor
cells[5_7]. The other approach for MDR regulation is the
modulation of the MDR-1 gene. Studies on the MDR-1 gene
promoter suggest that the inhibition of the P-gp expression may
occur at the gene level[8]. The MDR modulators may either
block the induction of MDR-1 or MRP1 gene expression or
downregulate P-gp expression. Up to now, there have been
many drugs used to decrease the incidence of MDR clinically.
Unfortunately, the drugs, such as verapamil
(VER)[9], have distinct toxicity at MDR treatment concentrations, so their
clinical treatments are restricted. It is necessary to find more
effective and less toxic drugs that can be used for cancer
therapy.
Ganoderma lucidum (Gl; Lingzhi) is a traditional
Chinese medicine and has been widely used in China and other
Oriental countries to prevent and treat various diseases for a
long time. Gl polysaccharides (Gl-PS) are the main
pharmacological ingredient extracted from the fruit body and
mycelium of mushroom Gl (Fr) Karst. A lot of experimental
evidence has been accumulated in the past several decades,
and the data suggest that Gl-PS have wide activities, such
as immune modulation, and
antitumor[10,11], anti-atherosclerosis, antidiabetic, and anti-aging
activities[12].
Gl is possibly effective in cancer patients receiving
conventional chemotherapy. Recently, investigators have
focused attention on the effect of Gl-PS on antitumor and
immunologic regulation. The water extract and the
polysaccharide fraction of Gl showed significant antitumor effects
in several tumor-bearing animals, mainly through its
immunoenhancing activity. Recent studies also showed that
the alcohol extract or the triterpene fraction of
Gl also had antitumor effects, which may be directly related to the
cytotoxic activity against tumor
cells[13_20]. So far, it is not clear whether
Gl-PS have effects on conversing MDR. The
purpose of this study is to investigate the effects of
Gl-PS on MDR and the influence on the gene expression of MDR-1
and MRP1 in human K562/adriamycin (ADM) cells.
Materials and methods
Reagents and drugs Gl-PS were extracted from the fruit
body of Gl (Leyss ex Fr) Karst by boiling water, followed
by ethanol precipitation, dialysis, and protein depletion.
Gl contains polysaccharide peptides with a molecular
weight of 584 900; the ratio of polysaccharides to peptides
is 93.51%:6.49%. The polysaccharides consisted of
D-hamnose, D-xylose, D-fructose,
D-galactose, D-mannose, and D-glucose with a molar ratio of
0.793:0.964:2.944:0.167:0.389:7.94 and linked together by b-glycosidic linkages. The
peptides contained 16 kinds of amino acids. RPMI-1640
medium was purchased from Gibco (Grand Island, NY, USA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) and DMSO were purchased from Sigma (St Louis,
MO, USA). ADM and VER were purchased from PRC Navy
General Hospital (Beijing, China), and the P-gp monoclonal
antibody was purchased from Santa Cruz (Santa Cruz, CA,
USA). Total RNA isolation kits and one-step RT-PCR kits
were purchased from SBS (Beijing, China).
Cell lines The K562 cell line (drug sensitive) was a
generous gift from Prof Wen-jie WANG (Institute of Materia
Medica, Chinese Academy of Medical Science, Beijing,
China), and the multidrug cell line K562/ADM was purchased
from the Chinese Medicine Academy (Beijing, China). These
2 cell lines were seeded in RPMI-1640 with 15% newborn
bovine serum. K562/ADM was treated with ADM 2 weeks
before the experiment. The cells in all of the experiments
were gathered when they were in the exponential growth
phrase.
Measurement of cytotoxicity by MTT
method The cells were incubated in 96-well plates
(1.0×105 /well), in 100 μL medium. ADM and
Gl-PS were added at different concentrations
(Gl-PS: 5, 10, 20, and 40 mg/L). VER was added as a
positive control. After 44 h incubation at 37 ºC, 20
μL MTT (5 g/L in phosphate-buffered saline [PBS]) was added. The
plates were incubated for 4 h, and the blue dye was
dissolved in 100 μL DMSO. The absorbance at 570 nm was
determined using an ELISA multiscan
reader[21].
The survival rate of the cells was calculated as follows:
Survival rate (%)=(T_B)/(U_B)×100%,
where T represents treated: the absorbance of tumor cells
exposed to drugs; U is untreated: the absorbance of
untreated cells; B is blank: the absorbance of cells without
drugs and MTT.
The IC50 for ADM in the Gl-PS-treated or untreated
tumor cells was determined, and the reversing factor (RF) was
calculated as follows:
RF=IC50 (control
group)/IC50 (drug-treated group).
Analysis of intracellular accumulation of ADM by
confocal laser scanning microscopy The reversing effect of
Gl-PS was analyzed based on the inherent fluorescence of ADM,
which accumulates in sensitive cells and is actively exported
from cells expressing a functional P-gp and MRP1. The cells
were maintained in medium for 48 h (K562/A cells were treated
with 10 and 50 mg/L Gl-PS) prior to the ADM accumulation
studies. The cells (100 000 cells/well) were seeded in
chambered borosilicate cover glass slides (Nunc, Naperville, Ill)
and incubated for 2 h (37 °C, humidified 5%
CO2) to allow the cells to adhere to the chamber slides. Then ADM (10 mg/L)
was added to the chamber slides. The chamber slides
containing cells without drugs were used as negative controls.
The cells were incubated with drugs for approximately 2 h,
washed once with Dulbecco's PBS, and examined
immediately by confocal to quantitative intracellular fluorescence
intensity. To determine the fluorescence intensities of ADM
obtained by confocal microscopy from different treatments,
excitation and detection parameters were kept constant.
Correction for differences in optical thickness between the
suspension analysis and intracellular ADM concentration was
accomplished according to previously-described
methods[22]. The laser cytometer was set at an excitation wavelength of
512 nm, and the emitted fluorescence was detected with a
barrier filter (band pass 530/30). Ten microscopic fields, each
containing aggregates of 10_15 cells, were analyzed for each
treatment. At least 2 experiments were performed on
different days.
Flow cytometric analysis of intracellular accumulation
of ADM The K562 and K562/ADM cells were separately
seeded in 5 mL of medium at a density of
1×106 cells/mL. In total, 100 μL drug solutions were added to obtain a final
ADM concentration of 10 mg/L in the absence or presence
of 10 mg/L Gl-PS. The cells were incubated at 37 ºC for 3 h,
by which time ADM accumulation had reached a steady state.
Aliquots of cellular suspension were collected. The cells
were immediately centrifuged for 10 min at 1000 r/min
(153×g), and washed 3 times with cold PBS, and then the cell pellets were
resuspended in PBS. The intensity of ADM was determined by
the auto-fluorescence of ADM using FACS
analysis[23].
Flow cytometric analysis of P-gp The K562/ADM cells
and K562 cells were plated at a density of
1×105 cell/mL in RPMI-1640. At 24 h after plating, the medium was replaced
with fresh medium containing 10 and 50 mg/L
Gl-PS alone. After 24 h, the cells were collected by trypsinization and
adjusted to a density of 1×106 cells/mL and centrifuged at
1000 r/min (153×g) for 8 min at 20 ºC. The cell pellet was
suspended in 20 μL anti-P-gp antibody (5 μL every
1×105 cells) and then incubated at 4 ºC for 30 min. After 2 washes
with cold PBS containing l% bovine serum albumin, the cells
were incubated under protection from light at 4 ºC for 30 min
with fluorescein-isothiocyanate-conjugated goat antimouse
immunoglobulin G (1:100). Then the cells were centrifuged
at 1000 r/min (153×g) for 10 min at 20 ºC, and suspended in
PBS. The fluorescence intensity in the cells was analyzed
by the flow cytometer.
RT-PCR assay for checking the mRNA level of MDR
and MRP
Isolation of total RNA The K562/ADM cells were seeded
with 10 mg/L Gl-PS for 48 h. Then
5×106 cells were collected. The total RNA isolation kits from SBS were used. All
operations followed the protocol in the kit.
Determination of RNA quality and
concentration The concentration and purity of total RNA was determined by
UV light absorption using a GeneQuant pro RNA/DNA
calculator (Biochrom, Cambridge, England). Preparations were
discarded if they had a ratio of optical densities at 260
nm/280 nm that was lower than 1.6[24]. To assess RNA quality, 5
μg of total RNA from each sample was loaded onto 1%
agarose-formaldehyde gels and subjected to electrophoresis.
Following ethidium bromide staining, RNA isolates were
considered good quality if the UV fluorescence of the 28S rRNA
band intensity was 2-fold of the 18S rRNA band, and no UV
fluorescence was detected below the 18S rRNA band.
Oligonucleotide PCR primers Sense and
antisense-specific primers were synthesized at SBS. All primers were
designed to span intron-exon boundaries to distinguish
between the amplification of mRNA and genomic DNA, and
were based on published human cDNA sequences.
cDNA synthesis and PCR All RT-PCR processes were
performed by the one-step RT-PCR kits purchased from SBS.
All operations followed the RT-PCR kit protocol. In total, 5
µg total RNA from the K562/ADM cells treated with
Gl-PS at 10 and 50 mg/mL and the control were treated by using the
following parameters: a reverse transcription step of 1 h at
42 °C, an initial denaturing step of 10 min at 94 °C,
denaturing at 94 °C for 1 min, and annealing at 58 °C for 50 s,
extending at 72 °C for 1 min. The final polymerization step was
extended for an additional 10 min. In total, 32 cycles of PCR
were performed. PCR amplification reactions were evaluated
through electrophoresis on 1% agarose gels containing
ethidium bromide and visualized by UV transillumination on
a GeneGenius imager (Frederick, MD, USA). The initial
product identification was made by comparison to the
myometrial control and the molecular weight ladder, photographed
by GeneGenius, and quantified by BandScan software
(Beijing, China). All RT_PCR experiments were carried out at
least 3 times.
Statistical analysis
Data are showed as mean±SEM from triplicate samples
of 3 independent experiments. Differences between the means
were analyzed by ANOVA. Results were considered to be
statistically significant when P<0.05.
Results
Reversal effect of Gl-PS on the multidrug K562/ADM
cells To determine if Gl-PS are toxic to normal cells, the
effects of Gl-PS on the K562 cells were assayed as described
in Materials and methods. Figure 1 shows that
Gl-PS did not have significant cytotoxity on normal cells, but VER and
Gl-PS obviously reversed the resistance of K562/ADM to ADM.
The RF were 2.96, 6.46, 6.80, and 3.35 in the 5, 10, 20, and 40
mg/L Gl-PS-treatment groups, respectively (Figure 2). In the
same condition, the RF of VER was 10.76. Both VER and
Gl-PS did not change the IC50 of sensitive K562 cells. These
results indicated that Gl-PS can reverse the drug resistance of
K562/ADM cells and enhance the cytotoxity of other
antitumor drugs, such as ADM.
Effect of Gl-PS on ADM accumulation in K562/ADM
cells assayed by confocal laser scanning microscopy
In order to examine whether Gl-PS have a reversing effect on
MDR, intracellular ADM accumulation was assessed by
confocal laser scanning microscopy. Figure 3 shows the images
of the fluorescence intensity representing intracellular ADM
accumulation. Intracellular ADM accumulation in the
K562/ADM cells was significantly less than that that in the K562
cells. The distribution of ADM accumulation was also
different between the K562 cells and K562/ADM cells. After
treatment with Gl-PS, ADM was distributed in the K562/ADM cells
at higher concentrations than that in the K562 cells. We also
found that in the K562 cells and K562/ADM plus
Gl-PS, the ADM concentration was higher in nucleolus zones than in
plasma, but not for K562/ADM cells. These results indicate
that Gl-PS can also downregulate the expression of MRP1.
Influence of Gl-PS on ADM accumulation in K562/ADM
cells determined by flow cytometric analysis The effect of
Gl-PS modulation on ADM efflux was also studied in
resistant cells overexpressing P-gp and MRP1.
Gl-PS were initially tested at 10 and 50 mg/L. The
Gl-PS did not have inherent fluorescence and did not modulate ADM in the
non-MDR-overexpressing K562 cells. Following the uptake of
ADM, the K562/ADM cells removed most of their
intracellular ADM content during 90 min incubation in the medium
alone; the intracellular ADM concentration was down
to 2.39-folds that of the K562 cells. With the existence of
Gl-PS, the efflux of ADM in the K562/ADM cells can be modulated.
A total of 10 mg/L Gl-PS caused a 2.4-fold increase in ADM
accumulation in the K562/ADM, cells and 50 mg/L
Gl-PS caused a 2.2-fold increase (Figure 4).
Effect of Gl-PS on expression of P-gp in K562 and
K562/ADM cell lines To evaluate the P-gp expression level, the
K562/ADM cells were analyzed with the anti-P-gp
monoclonal antibody. VER was used as the positive control. As
shown in Figure 5, the expression rates of P-gp were 78.54%
in untreated K562/ADM cells, and 36.73% and 51.73% in 10
and 50 mg/L Gl-PS-treated K562/ADM cells, respectively.
The VER group showed a level at 60.09%. So we can
conclude that after treatment with Gl-PS, the expression rate of
P-gp was reduced distinctly (Figure 5).
Gl-PS downregulating MDR-1 and MRP1 gene
expression in MDR K562/ADM cells The K562/ADM cells were
shown by RT-PCR to express MDR-1 and MRP genes at a
rate of 56.58% and 37.59%, respectively. Neither of the genes
was detectable in the K562 cells (data not shown).
Treatment with Gl-PS (10 mg/L) for 24 h led to the downregulation
of MDR-1 and MRP mRNA expression in MDR cells. The
levels of MDR-1 and MRP1 mRNA nearly disappeared to a
level of 8.89% and 7.95%, respectively; Gl-PS (50 mg/L) was
also detected. The expression levels of MDR-1 and MRP-1
were also greatly reduced to approximately 11.12% and
10.05%, respectively (Figure 6).
Discussion
Nearly 50% of human cancers are either completely
resistant to chemotherapy or respond to chemotherapy only
transiently, after which they are no longer affected by
common anticancer drugs. This phenomenon is referred to as
MDR and is inherently expressed by some tumor types, while
others acquire MDR after exposure to chemotherapy treatment. We are actively investigating the mechanisms
involved in MDR, as well as developing therapeutic
strategies to circumvent these resistance mechanisms. We
evaluated tumor cells for MDR related to several resistance
processes and have made significant breakthroughs in
reversing MDR mediated by the drug efflux pump P-glycoprotein.
Phenotypic and functional analyses of MDR mechanisms
presented in human tumor specimens are also being
conducted in order to relate specific MDR mechanisms with
resistance to chemotherapy in specific tumor types.
MDR has been explained by the overexpression of a
family of ATP-binding cassette (ABC) transporters called MDR
proteins. ABC transporters are an ever-increasing family of
proteins[25]. The human MDR-1 gene encoding the MDR
transporter (P-gp-170) belongs to the ABC transporter
superfamily, of which there are 9 members. Uptake and/or
efflux of isotope-labeled drugs or rhodamine123 are
frequently used for the functional assay of P-gp-170 in tumor
cells. Several classes of MDR modulators that inhibit
P-gp-mediated efflux have been identified. P-gp and MRP1 are
constitutively expressed at many tumor cell lines and pump
drugs out of cells. Such a spatial distribution of this efflux
transporter has defined it as a functionally-important
element in reducing the effects of antitumor drugs.
The Gl-PS, a traditional Chinese medicine, has been
proven to be effective in tumor therapy. Our previous
research proved that Gl-PS have antitumor activity both
in vivo and in vitro[24]. There have been many studies on the
antitumor activity of Gl-PS and their possible mechanisms,
and many exciting results have been obtained. However,
due to its complexity in structure, the mechanisms by which
the Gl-PS act in the cells are unclear.
Gl-PS have low toxicity in humans, which is in contrast to other drugs, such as VRPL,
that leads to 40% lethality in mice at 150 mg/kg per day for 3 d.
The remarkably low toxicity of Gl-PS coupled with their
potent MDR-reversing activity as demonstrated earlier,
renders them appropriate for further study and development.
In the present study, we sought to use several different,
but complementary experimental approaches to examine
whether Gl-PS can reverse MDR. K562 cells repelling ADM
are the best models for MDR research and have been
selected in many previous studies[26] We have validated that
this cell line really has high-level expression of P-gp and
MRP1 (unpublished data). Depending on P-gp and MRP1
activity, ADM was pumped out of the K562/ADM cells and
formed a ring adhering to the cells. The condition in the
K562 cells was different: ADM was distributed into the cells
at a high concentration. After incubating the K562/ADM
cells with both ADM and Gl-PS, the ADM-accumulating rings
disappeared and the distribution conditions were greatly
similar to that of the K562 cells. So we can conclude that
Gl-PS has the ability to downregulate the effect of P-gp by an
unknown mechanism. However, we chose ADM as the
antitumor drug because of its auto-fluorescence that can be easily
assayed in the experiment. It is interesting
that Gl-PS has a prominent effect on reversing MDR. The cytotoxity assay
results indicated that proper concentrations of
Gl-PS can reduce the viability of K562/ADM cells in ADM. The results
of the confocal assay supported this conclusion and showed
that the rate of pumping drugs out of K562/ADM cells was
also reduced. To determine whether Gl-PS could
downregulate the P-gp and MRP1 functional expression in the K562/ADM
cells, we tested the P-gp expression level on the cell
membrane using the flow cytometric assay. Our results confirmed
the hypothesis that the P-gp expression was downregulated
under Gl-PS treatment. Furthermore, the results of the
RT-PCR experiments confirmed that MRP1 and P-gp were
significantly inhibited at the mRNA level. Our studies suggest
that Gl-PS can reverse MDR by
downregulating the expressions of P-gp and MRP1.
Acknowledgements
Gl-PS was kindly provided by Prof Shu-qian LIN and
Sai-zhen WANG of the Fuzhou Institute of Green Valley
Bio-Pharm Technology, Fuzhou, China.
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