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
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