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ReishiMax clinical study

ReishiMax clinical study

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Published by nsagelocspa
The study has shown Pharmanex's Reishimax as a potential agent for the prevention and/or treatment of prostate cancer.
The study has shown Pharmanex's Reishimax as a potential agent for the prevention and/or treatment of prostate cancer.

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Published by: nsagelocspa on Nov 04, 2009
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01/19/2014

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Abstract.
 
Ganoderma lucidum
 
(Reishi), an oriental medicalmushroom, has been widely used in Asian countries forcenturies to prevent or treat different diseases, including cancer.However, the mechanism(s) responsible for the effects of 
Ganoderma lucidum
on cancer cells remain to be elucidated.We have previously demonstrated that
Ganoderma lucidum
down-regulated the expression of NF-
 
κ
B-regulated urokinaseplasminogen activator (uPA) and uPA receptor (uPAR),which resulted in suppression of cell migration of highlyinvasive human breast and prostate cancer cells. In this study,we investigated the effects of 
Ganoderma lucidum
on cellproliferation, cell cycle, and apoptosis in human prostate cancercells PC-3. Our data demonstrate that
Ganoderma lucidum
inhibits cell proliferation in a dose- and time-dependent mannerby the down-regulation of expression of cyclin B and Cdc2and by the up-regulation of p21 expression. The inhibition of cell growth was also demonstrated by cell cycle arrest at G2/Mphase. Furthermore,
Ganoderma lucidum
induced apoptosisof PC-3 cells with a slight decrease in the expression of NF-
κ
B-regulated Bcl-2 and Bcl-xl. However, the expression of pro-apoptotic Bax protein was markedly up-regulated, resultingin the enhancement of the ratio of Bax/Bcl-2 and Bax/Bcl-xl.Thus,
Ganoderma lucidum
exerts its effect on cancer cells bymultiple mechanisms and may have potential therapeutic usefor the prevention and treatment of cancer.
Introduction
Prostate cancer is the most common malignancy in men inthe United States, accounting for about 33% of all cancersdiagnosed in males (1). Prostate cancer cells initially respondto androgen ablation therapy, but long-term antiandrogentreatment of prostate cancer patients results in loss of responsiveness. Prostate cancers finally progress fromandrogen-dependent to androgen-independent with highlymetastatic behavior. The transcription factor, nuclear factor-
κ
B (NF-
κ
B), is overexpressed in highly invasive prostatecancers (2-4), and its activity has been linked to cancer chemo-resistance (5). NF-
κ
B is also associated with tumor cellproliferation, invasion, and angiogenesis (6,7), and furtheractivation of NF-
κ
B has been demonstrated by various carcino-gens and tumor promoters, such as benzopyrene, UV radiation,and phorbol esters (8-10). Therefore, activation of NF-
κ
Bpromotes cell proliferation and survival, while suppressionof NF-
κ
B decreases cell proliferation and sensitizes the cellsto apoptosis induced by cytokines and chemotherapeuticagents (11-13). Furthermore, constitutively active NF-
κ
Bcontributes to the resistance of tumors to conventional therapyby mechanisms employing the up-regulation of antiapoptoticgenes such as Bcl-2, Bcl-xl, and the cell cycle regulatorcyclin D1, all of which are overexpressed in highly invasiveprostate cancer cells (14-17). We have recently demonstratedthat constitutively active NF-
κ
B controls cell adhesion andmigration in highly invasive prostate cancer cells (18),suggesting that the inhibition of NF-
κ
B may be especiallyimportant for the treatment of highly invasive and androgen-independent prostate cancer.
Ganoderma lucidum
(Reishi), a basidiomycetous fungus, iswidely used in China and other Asian countries to treat varioushuman diseases, such as hepatitis, hepatopathy, hypertension,nephritis, and cancers (19-21). In addition, many bioactivecomponents isolated from
Ganoderma lucidum
have beendemonstrated to possess antioxidative, antihypertensive, andanticancer effects (22-25). For example, polysaccharides from
Ganoderma lucidum
exert anticancer effects against HL-60and U937 leukemic cell lines (26). Some of the triterpenesisolated from
Ganoderma lucidum
also exhibit cytotoxicactivity against mouse sarcoma and mouse lung carcinomacells
in vitro
(27). Furthermore, we have recently reportedthat
Ganoderma lucidum
suppresses cell migration of highlyinvasive human breast and prostate cancer cells by inhibitingconstitutively active NF-
κ
B, which resulted in the down-regulation of expression of urokinase-type plasminogenactivator (uPA) and its receptor uPAR (28). Therefore, theNF-
κ
B-regulated genes may be suitable targets of 
Ganodermalucidum
for treatment of prostate cancer.
INTERNATIONAL JOURNAL OF ONCOLOGY 24: 1093-1099, 2004
 
Ganoderma lucidum
inhibits proliferation and inducesapoptosis in human prostate cancer cells PC-3
JIAHUA JIANG
1
, VERONIKA SLIVOVA
1
, TATIANA VALACHOVICOVA
1
,KEVIN HARVEY
1
andDANIEL SLIVA
1,21
Cancer Research Laboratory, Methodist Research Institute, 1800 N Capitol Avenue, E504, Indianapolis, IN 46202;
2
Department of Medicine, School of Medicine, Indiana University, Indianapolis, IN, USAReceived December 5, 2003; Accepted January 16, 2004
_________________________________________
Correspondence to
:
Dr D. Sliva, Cancer Research Laboratory,Methodist Research Institute, 1800 N Capitol Avenue, E504,Indianapolis, IN 46202, USAE-mail: dsliva@clarian.org
Key words:
Ganoderma lucidum
, prostate cancer, NF-
κ
B, cell cyclearrest, apoptosis
 
The present study was undertaken to further characterizethe effect of 
Ganoderma lucidum
on prostate cancer cellsand to determine its effect on NF-
κ
B-regulated genes,which are involved in cell proliferation and apoptosis. Herewe demonstrate that
Ganoderma lucidum
inhibits cellproliferation through cell cycle arrest at G2/M phase andinduces apoptosis in highly invasive prostate cancer cellsPC-3. Growth inhibition is linked to the down-regulation of expression of cyclin B and Cdc2 and to the up-regulation of p21 expression.
Ganoderma lucidum
also induced apoptosiswith moderate down-regulation of expression of antiapoptoticBcl-2 and Bcl-xl. Furthermore, the expression of proapoptoticBax protein was increased. Thus,
Ganoderma lucidum
exertsits effect on cancer cells by multiple mechanisms and mayhave potential therapeutic use for the prevention and treatmentof cancer.
Materials and methods
 Materials.Ganoderma lucidum
(Reishimax) was purchasedfrom Pharmanex (Provo, UT). According to the manufacturer,this sample contains 13.5% polysaccharidesand 6%triterpenes.
Ganoderma lucidum
was dissolved in boiledwater, stored at 4˚C, and reheated to 70˚C for 10 min beforeevery experiment.
Cell culture.
The human prostate cancer cell line PC-3 wasobtained from ATCC (Manassas, VA). PC-3 cells weremaintained in F-12 medium containing penicillin (50 U/ml),streptomycin (50 U/ml), and 10% fetal bovine serum (FBS).Media and supplements came from Gibco BRL (GrandIsland, NY). FBS was obtained from Hyclone (Logan, UT).
Cell proliferation assay.
Cell proliferation was determined bythe tetrazolium salt method, according to the manufacturer'sinstructions (Promega, Madison, WI). Briefly, PC-3 cells(5x10
3
/well) were cultured in a 96-well plate and treated atindicated times with
Ganoderma lucidum
(0-0.5 mg/ml). Atthe end of the incubation period, the cells were harvested andabsorption was determined with an ELISA plate reader at570 nm. Data points represent mean
±
SD in one experimentrepeated at least twice.
Cell cycle analysis.
PC-3 cells (1x10
6
) were seeded and after24 h treated with
Ganoderma lucidum
(0.5 mg/ml) for theindicated period of time (0-48 h). After incubation, the cellswere harvested by trypsinization, washed with Dulbecco'sphosphate-buffered saline (DPBS) containing 2% FBS, andresuspended in propidium iodine (50
µ
g/ml). Cell cycle analysiswas performed on a FACStar
PLUS
flow cytometer (Becton-Dickinson, San Jose, CA), as previously described (29). Dataare the mean
±
SD from six independent experiments.
 Nuclear fragmentation assay
. PC-3 cells (1x10
4
/well) weregrown in multichamber slides (Nalgene Nunc Inc., Naperville,IL) and treated with
Ganoderma lucidum
(0-1.0 mg/ml) for48 h. After incubation, the cells were quickly washed in ice-cold DPBS, fixed in methanol at -20˚C for 15 min, and driedand stained with DNA-specific fluorochrome DAPI (2
µ
g/ml)for 5 min. Stained cells were washed twice with DPBS, andthe changes in nuclei were observed with a Leica fluorescencemicroscope through UV filter.
 DNA laddering.
PC-3 cells (1x10
6
/well) were cultured in100-mm dish and treated with
Ganoderma lucidum
(1.0 mg/ml)for different times (0-72 h). After treatment, adherent andnon-adherent cells were collected, centrifuged at 5,000 g for5 min, and lysed with cold lysis buffer [5 mM Tris (pH 8.0),20 mM EDTA, 0.5% Triton X-100] on ice for 45 min. DNAwas extracted with phenol:chloroform:isoamyl alcohol(25:24:1), again extracted with chloroform, and precipitatedwith ethanol at -20˚C. The DNA pellet was resuspended in TEbuffer (pH 8.0) with 100
µ
g/ml RNase A and incubated at 37˚Cfor 1 h. DNA laddering was detected by electrophoresis on1.5% agarose gels containing ethidium bromide and visualizedby ultraviolet light.
 Annexin V staining.
PC-3 cells (2.5x10
5
) were treated with
Ganoderma lucidum
(0-1.0 mg/ml) for 48 h. After incubation,the cells were harvested and labeled with annexin V conjugatedto fluoroscein (Roche Diagnostics, Indianapolis, IN). Thelabeled apoptotic cells were analyzed by flow cytometry, aspreviously described, on a FACStar
PLUS
flow cytometer (29).
 DNA transfection and chloramphenicol acetyltransferase(CAT) assay
. PC-3 cells were transfected with NF-
κ
B-CATreporter constructs and ß-galactosidase expression vectorpCH110, as previously described (28). Twenty-four hoursafter transfection, cells were treated with
Ganoderma lucidum
for an additional 24 h at 37˚C, as indicated in the text. Celllysates were prepared and CAT assays performed, as described(28). Data points represent the mean
±
SD of three independenttransfection experiments.
Western blot analysis
. PC-3 cells (1x10
7
) were treated with
Ganoderma lucidum
(1.0 mg/ml) for 24, 48, 72, and 96 h.After incubation, cells were washed twice with ice-coldDPBS, lysed with 1 ml of ice-cold lysis buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EGTA, 1 mMEDTA, and protease inhibitor cocktail Complete
(Boehringer Mannheim, Indianapolis, IN)]. Cells were lysedat 4˚C for 30 min with occasional vortexing. The lysates werecollected and cleared of nuclei by centrifugation for 10 minat 14,000 rpm. The protein concentration was determinedaccording to the manufacturer's protocol (Bio-Rad Laboratories,Hercules, CA). For Western blot analysis, equal amounts of proteins (20
µ
g/lane) were separated on 15% SDS-PAGE andtransferred to a PVDF membrane (Millipore, Bedford, MA).The membrane was incubated with the corresponding primaryantibodies diluted 1:1,000 in blocking solution, as follows:amouse anti-Bax monoclonal antibody (Biomol ResearchLaboratories Inc., Plymouth Meeting, PA); a mouse anti-Bcl-2monoclonal antibody (Zymed Laboratories Inc., South SanFrancisco, CA); a rabbit anti-Bcl-xl polyclonal antibody(Transduction Laboratories, Lexington, KY); and a mouseanti-cyclin D1 monoclonal antibody, a rabbit anti-Cdk4 poly-clonal antibody, a mouse anti-cyclin B monoclonal antibody,a mouse anti-Cdc2 monoclonal antibody, a rabbit anti-p21polyclonal antibody, and a mouse anti-actin monoclonalantibody (Santa Cruz Biotechnology, Santa Cruz, CA).
JIANG
et al
:
Ganoderma lucidum
AND PROSTATE CANCER CELLS
 
1094
 
Anti-mouse or anti-rabbit secondary antibody horseradishperoxidase conjugate (1:5,000) (Amersham Biosciences,Buckinghamshire, UK) was used to detect and visualize bythe ECL Western Blotting Detection system (AmershamBiosciences, Buckinghamshire, UK).
Results
Ganoderma lucidum inhibits proliferation of human prostatecancer cells
. We have recently shown that
Ganoderma lucidum
inhibits cell migration of highly invasive human prostate cancercells PC-3 (28). To examine the effect of 
Ganoderma lucidum
on cell proliferation, PC-3 cells were treated with increasingconcentrations of 
Ganoderma lucidum
(0-0.5 mg/ml), andcell growth was determined. As seen in Fig. 1,
Ganodermalucidum
markedly inhibited proliferation of PC-3 cells in atime-dependent manner. Growth inhibition was also dose-dependent because 96 h of treatment with 0.125, 0.25, and0.5 mg/ml of 
Ganoderma lucidum
inhibited proliferation of PC-3 cells by 7.6%, 53.8%, and 79.6%, respectively (Fig. 1).Therefore, our current data clearly demonstrate the anti-proliferative effect of 
Ganoderma lucidum
on highly invasivehuman prostate cancer cells.
Ganoderma lucidum arrests PC-3 cells at G2/M phase of thecell cycle
. Since a recent report demonstrated that alcoholextract of 
Ganoderma lucidum
inhibited cell proliferation of breast cancer cells through cell cycle arrest at G1 phase (30),we examined whether the same mechanism is also employedin prostate cancer cells. PC-3 cells were treated with 0.5 mg/mlof 
Ganoderma lucidum
and cell cycle distribution wasdetermined by flow cytometry after 24 and 48 h. As shown inTable I,
Ganoderma lucidum
induced cell cycle arrest atG2/M phase in PC-3 cells. Thus, the treatment of PC-3 cellsdid not significantly change the amount of cells at G0/G1phase of the cell cycle, but resulted in an increase of 57.1%and 86.3% of the cell population in G2/M phase after 24 and48 h, respectively. These data suggest that
Ganoderma lucidum
inhibits the growth of prostate cancer cells by cell cycle arrestat G2/M phase.
 Effects of Ganoderma lucidum on cell cycle regulatory proteins.
Since the dysregulation of cell cycle control is ahallmark of cancer (31), and since, as shown above,
Gano-derma lucidum
induced cell cycle arrest of PC-3 cells at G2/Mphase, we investigated how
Ganoderma lucidum
modulatesexpression of cell cycle regulatory proteins. Therefore, weexamined the expression of cyclin D1 and Cdk4, which controlG1/S transition, and the expression of cyclin B and Cdc2,which are involved in G2/M transition (31). PC-3 cells weretreated with
Ganoderma lucidum
(0-96 h) and whole cellextracts were prepared and subjected to Western blot analysiswith specific antibodies against cell cycle regulatory proteins.As seen in Fig. 2,
Ganoderma lucidum
moderately down-regulated the expression of the G0/G1 regulatory proteins,cyclin D1 and Cdk4. However, the same treatment markedlydecreased expression of cyclin B and Cdc2, which control
INTERNATIONAL JOURNAL OF ONCOLOGY 24: 1093-1099, 2004
1095
Figure 1.
Ganoderma lucidum
inhibits proliferation of PC-3 cells. PC-3cells were treated with 0, 0.125, 0.25, and 0.5 mg/ml of 
Ganodermalucidum
. Proliferation was assessed after 5, 12, 24, 48, 72 and 96 h, asdescribed in Materials and methods. Each bar represents the mean
±
SD of three experiments.
Table I. Effect of 
Ganoderma lucidum
on cell cycledistribution. –––––––––––––––––––––––––––––––––––––––––––––––––Time (h)G0/G1SG2/M –––––––––––––––––––––––––––––––––––––––––––––––––021.3
±
6.855.4
±
4.423.3
±
5.52420.3
±
11.641.7
±
6.0
a
36.0
±
6.0
a
4819.4
±
5.137.2
±
8.7
a
43.4
±
5.2
a
 –––––––––––––––––––––––––––––––––––––––––––––––––
a
p<0.01
 –––––––––––––––––––––––––––––––––––––––––––––––––
Figure 2. Effect of 
Ganoderma lucidum
on cell cycle regulatory proteins.PC-3 cells were treated with
Ganoderma lucidum
(1.0 mg/ml) for indicatedtimes. Whole cell extracts were subjected to Western blot analysis with anti-cyclin D1, anti-Cdk4, anti-cyclin B, anti-Cdc2, and anti-p21 antibodies. Theequivalent amount of protein was verified by reprobing the blot with anti-ß-actin antibody. The results are representative of three separate experiments.

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