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  • NUTRITION AND CANCER, 49(2). 209-216 Copyright © 2(H)4, Lawrence Edbaum Associates, Inc.

    Ganoderma lucidum Suppresses Growth of Breast Cancer Cells Through the Inhibition of Akt/NF-icB Signaling

    Jiahua Jiang, Veronika Slivova, Kevin Harvey, Tatiana Valachovicova, and Daniel Sliva

    Abstract: Ganoderma lucidum (Reishi. Lingzhi) is a popular Asian mushroom thai has been used for more than 2 millen- nia for ihe general promotion of health and was therefore called the "Mushroom of Immortality." Ganoderma lucidum was also used in traditional Chinese medicine to prevent or treat a variety of diseases, including cancer. We previously demonstrated that Ganoderma lucidum suppresses the inva- sive behavior of breast cancer cells by inhibiting the tran- scription factor NF-KB. However, the molecular mechanisms responsible for the inhibitory effects o/Ganoderma lucidum on the growth of highly invasive and metastatic breast cancer cells has not been fully elucidated. Here, we show that Ganoderma lucidum inhibits proliferation of breast cancer MDA-MB-23I cells by downregulating Akt/NF-icB signaling. Ganoderma lucidum suppresses phosphorylation of Akt on Ser^^-^ and downregulates the expression of Akt. which results in the inhibition of NF-KB activity in MDA-MB-23I cells. The biological effect of Ganoderma lucidum was demon- strated by cell cycle arrest at GO/Gl. which was the result of the downregulation of expression of NF-KB-regulated cyclin Dl. followed by the Inhibition of cdk4. Our results suggest that Ganoderma lucidum inhibits the growth of MDA-MB'23l breast cancer cells by modulating Akt/NF-KB signaling and could have potential therapeutic use for the treatment of breast cancer.


    One third of all newly diagnosed cancers among women in the United States are breast cancers (1). Because breast cancer often progresses from the therapy-responsive pheno- type to the highly invasive and metastatic phenotype, breast cancer is the second leading cause of cancer death in the U.S. female population (2). A comprehensive review by the World Cancer Research Fund and the American Institute of Cancer Research clearly demonstrates the importance of nutrition in the prevention of cancer, which could also contribute to the low incidence of breast cancers among Asian women (3). Therefore, it was suggested that some nutritional products

    have chemopreventive and therapeutic effects against cancer. These anticancer effects also significantly increased the pop- ularity of dietary supplements in cancer patients (4).

    The popular edible mushroom Ganoderma lucidum has been widely used in eastern Asia to promote health and lon- gevity. The regular consumption oi Ganoderma lucidum in the form of teas was believed to fortify the mind and the body, and therefore Ganoderma hicidum was called the "Mushroom of Immortality" (5). The dried powder of Ganoderma lucidum has been used in traditional Chinese medicine for more than 2.000 years to prevent or treat different diseases, including cancer (6). The anticancer properties of Ganoderma lucidum have been attributed to either the isolated poly sac charides, which are responsible for the stimulation of the immune sys- tem, or triterpenes, which demonstrate cytotoxic activity against cancer cells (for review, see ref. 7). However, Ganoderma lucidum is currently available as a dietary supple- ment in the form of extracts or capsules containing fruiting bodies and/or spores of this mushroom. We recently reported that Ganoderma lucidum suppresses constitutively active transcription factors AP-I and NF-KB, which resulted in the downregulation of expression of urokinase-type plasminogen activator (uPA) and its receptor uPAR in human breast and prostate cancer cells (8). The anticancer effect of Ganoderma lucidum was also demonstrated hy the inhibition of adhesion, migration, and invasion of the highly metastatic breast cancer cellsMDA-MB-231 (9).

    The highly invasive potential of breast cancers was linked to the overexpression of epidermal growth factor receptors (EGFRs) (10), which control signaling through the activation of the phosphatidylinosito! 3-kinase (PI3K)/NF-KB pathway responsible for the aberrant cell cycle progression of breast cancer cells (11). In addition, PI3K also activates serine/threonine protein kinase Akt (12), which can be specifi- cally phosphorylated on Thr'"*̂ or Ser^^' (13). Furthermore, activated Akt can subsequently regulate the NF-KB pathway (14,15), and NF-KB controls the expression of the cell cycle regulator cyclin Dl, which is responsible for the transition from the GI to the S phase during cell cycle progression (16). Finally, PI3K. Akt, and NF-KB are constitutively active in

    All authors are affiliated with the Cancer Research Laboratory, Methodist Research Institute. Indianapolis, IN 46202. D. Sliva is also affiliated with the De- partment of Medicine, School of Medicine, Indiana University. Indianapolis. IN 46202.

  • highly invasive human MDA-MB-231 breast cancer cells (17,18) and therefore they are suitable targets for cancer treat- ment (19). In the present study, we show that Ganoderma hicidum inhibits the growth of breast cancer cells by inducing cell cycle arrest at GO/Gl by suppressing NF-KB activity through the inhibition of Akt phosphorylation in MDA-MB-231 cells. We also report that Ganoderma lucidum suppresses the expression of cyclin DI followed by the inhibi- tion of cyclin-dependent protein kinase cdk4.

    Materials and Methods


    Ganoderma lucidum (Reishimax) was purchased from Pharmanex (Provo, UT). According to the manufacturer, this sample contains powdered extract (20:1) with spores and is standardized to 13.5% polysaccharides and 6% triterpenes. Stock solution was prepared by dissolving Ganoderma lucidum in sterile water at a concentration of 50 mg/ml and stored at 4°C.

    Cell Culture

    The human breast cells MCF- lOA and breast cancer cells MDA-MB-231 were obtained from ATCC (Manassas, VA). MCF-lOA cells were maintained in DMEM/FI2 medium containing 5% horse serum (HS), insulin (10 |ig/ml), epider- mal growtb factor (EGF; 20 ng/ml), cholera toxin (100 |Xg/ml). hydrocortisone (0.5 |ig/ml), penicillin (50 U/ml). and streptomycin (50 U/ml). MDA-MB-231 cells were main- tained in DMEM medium containing penicillin (50 U/ml), streptomycin (50 U/ml), and 10% fetal bovine serum (FBS). Media and supplements came from GIBCO BRL (Grand Is- land, NY). HS and FBS were obtained from Hyclone (Logan, UT).

    Cell Proliferation Assay

    Cell proliferation was determined by the tetrazolium salt method, according to the manufacturer's instructions (Promega, Madison, WI). Briefly, MCF-lOA and MDA- MB-231 cells (5 X lOVwell) were cultured in a 96-well plate and treated at indicated times with Ganoderma lucidum (0-1.0 mg/ml). At the end of the incubation period, the cells were harvested and absorption was determined with an ELISA plate reader at 570 nm. Data points represent mean ± standard deviation in one experiment repeated at least twice.

    Cell Cycle Analysis

    MDA-MB-231 cells (1 x 10'') were seeded and after 24 h treated with Ganoderma lucidum (0.5 mg/ml) for the indi- cated period of time (0-48 h). After incubation, the cells were harvested by trypsinization, washed with Dulbecco's phosphate buffered saline (DPBS) containing 2% FBS, and

    resuspended in propidium iodine (50 |Jg/ml). Cell cycle anal- ysis was performed on a FACStarf- '̂̂ flow cytometer (Becton-Dickinson, San Jose, CA), as previously described (20). Data are the mean ± standard deviation from 3 inde- pendent experiments.

    DNA T^ansfection and Chloramphenicol Acetyltransferase (CAT) Assay

    MDA-MB'23I cells were transfected with NF-icB-CAT reporter constructs and p-galactosidase expression vector pCHl 10, as previously described (8). Twenty-four hours af- ter transfection, ceils were treated with Ganoderma lucidum for an additional 24 h at 37°C, as indicated in the text. Cell lysates were prepared and CAT assays performed, as de- scribed (8). Data points represent the mean ± standard devia- tion of three independent transfection experiments.

    Western Blot Analysis

    MDA-MB-231 cells (1 x 10 )̂ were treated with Gano- derma lucidum (1.0 mg/ml) for 24,48, 72, and 96 h. After in- cubation, cells were washed twice with ice-cold DPBS. lysed with I ml of ice-cold lysis buffer (50 mM Tris-HCI pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EGTA. 1 mM EDTA, and protease inhibitor cocktail Complete'^ [Boehringer Mann- heim, Indianapolis. IN]) at 4°C for 30 min. The lysates were collected and cleared of nuclei by centrifugation for 10 min at 14,000 g. The equal amounts of proteins (20 |ag/lane) were separated on 15% SDS-PAGE and transferred to a PVDE membrane (Millipore, Bedford, MA). The protein expres- sion was detected with the corresponding primary antibod- ies: anti-Akt, anti-phospho-Akt (Thr'̂ ***), anti-phospho-Akt (Ser^"; Cell Signaling, Beverly, MA), anti-cyclin Dl, anti-Cdk4, and anti-^-actin antibody (Santa Cruz Biotech- nology, Santa Cruz, CA). Protein expression was visualized using the ECL Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, UK).


    Ganoderma lucidum Inhibits Proliferation of Highly Invasive Human Breast Cancer Cells by Cell Cycle Arrest at GO/Gl

    We recently reported that Ganoderma lucidum inhibits the invasiveness of breast cancer cells by suppressing ceil ad- hesion, cell migration, cell invasion, and colony formation (9). To demonstrate the effect of Ganoderma lucidum on cell growth, we treated MDA-MB-231 breast cancer cells with increasing concentrations of Ganoderma lucidum (0-1.0 mg/ml) for 24, 48, and 72 h, and cell proliferation was deter- mined. As expected, Ganoderma lucidum suppressed the proliferation of MDA MB-231


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