ganoderma lucidum suppresses growth of breast cancer cells ... · we recently reported that...

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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 ofAkt. 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. Introduction 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.

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Page 1: Ganoderma lucidum Suppresses Growth of Breast Cancer Cells ... · We recently reported that Ganoderma lucidum inhibits the invasiveness of breast cancer cells by suppressing ceil

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

Ganoderma lucidum Suppresses Growth of Breast Cancer CellsThrough 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 popularAsian mushroom thai has been used for more than 2 millen-nia for ihe general promotion of health and was thereforecalled the "Mushroom of Immortality." Ganoderma lucidumwas also used in traditional Chinese medicine to prevent ortreat a variety of diseases, including cancer. We previouslydemonstrated that Ganoderma lucidum suppresses the inva-sive behavior of breast cancer cells by inhibiting the tran-scription factor NF-KB. However, the molecular mechanismsresponsible for the inhibitory effects o/Ganoderma lucidumon the growth of highly invasive and metastatic breast cancercells has not been fully elucidated. Here, we show thatGanoderma lucidum inhibits proliferation of breast cancerMDA-MB-23I cells by downregulating Akt/NF-icB signaling.Ganoderma lucidum suppresses phosphorylation of Akt onSer^^-^ and downregulates the expression of Akt. which resultsin 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 ofthe downregulation of expression of NF-KB-regulated cyclinDl. followed by the Inhibition of cdk4. Our results suggestthat Ganoderma lucidum inhibits the growth ofMDA-MB'23l breast cancer cells by modulating Akt/NF-KBsignaling and could have potential therapeutic use for thetreatment of breast cancer.

Introduction

One third of all newly diagnosed cancers among womenin the United States are breast cancers (1). Because breastcancer often progresses from the therapy-responsive pheno-type to the highly invasive and metastatic phenotype, breastcancer is the second leading cause of cancer death in the U.S.female population (2). A comprehensive review by the WorldCancer Research Fund and the American Institute of CancerResearch clearly demonstrates the importance of nutrition inthe prevention of cancer, which could also contribute to thelow 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 hasbeen widely used in eastern Asia to promote health and lon-gevity. The regular consumption oi Ganoderma lucidum in theform of teas was believed to fortify the mind and the body, andtherefore Ganoderma hicidum was called the "Mushroom ofImmortality" (5). The dried powder of Ganoderma lucidumhas been used in traditional Chinese medicine for more than2.000 years to prevent or treat different diseases, includingcancer (6). The anticancer properties of Ganoderma lucidumhave 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 activityagainst 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 fruitingbodies and/or spores of this mushroom. We recently reportedthat Ganoderma lucidum suppresses constitutively activetranscription factors AP-I and NF-KB, which resulted in thedownregulation of expression of urokinase-type plasminogenactivator (uPA) and its receptor uPAR in human breast andprostate cancer cells (8). The anticancer effect of Ganodermalucidum was also demonstrated hy the inhibition of adhesion,migration, and invasion of the highly metastatic breast cancercellsMDA-MB-231 (9).

The highly invasive potential of breast cancers was linkedto the overexpression of epidermal growth factor receptors(EGFRs) (10), which control signaling through the activationof the phosphatidylinosito! 3-kinase (PI3K)/NF-KB pathwayresponsible for the aberrant cell cycle progression of breastcancer cells (11). In addition, PI3K also activatesserine/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 cycleregulator cyclin Dl, which is responsible for the transitionfrom 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.

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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 Ganodermahicidum inhibits the growth of breast cancer cells by inducingcell cycle arrest at GO/Gl by suppressing NF-KB activitythrough the inhibition of Akt phosphorylation inMDA-MB-231 cells. We also report that Ganoderma lucidumsuppresses the expression of cyclin DI followed by the inhibi-tion of cyclin-dependent protein kinase cdk4.

Materials and Methods

Materials

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

Cell Culture

The human breast cells MCF- lOA and breast cancer cellsMDA-MB-231 were obtained from ATCC (Manassas, VA).MCF-lOA cells were maintained in DMEM/FI2 mediumcontaining 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). andstreptomycin (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 saltmethod, 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 plateand treated at indicated times with Ganoderma lucidum(0-1.0 mg/ml). At the end of the incubation period, the cellswere harvested and absorption was determined with anELISA 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 htreated with Ganoderma lucidum (0.5 mg/ml) for the indi-cated period of time (0-48 h). After incubation, the cellswere harvested by trypsinization, washed with Dulbecco'sphosphate 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 ChloramphenicolAcetyltransferase (CAT) Assay

MDA-MB'23I cells were transfected with NF-icB-CATreporter constructs and p-galactosidase expression vectorpCHl 10, as previously described (8). Twenty-four hours af-ter transfection, ceils were treated with Ganoderma lucidumfor an additional 24 h at 37°C, as indicated in the text. Celllysates 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. lysedwith 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, andprotease inhibitor cocktail Complete'^ [Boehringer Mann-heim, Indianapolis. IN]) at 4°C for 30 min. The lysates werecollected and cleared of nuclei by centrifugation for 10 minat 14,000 g. The equal amounts of proteins (20 |ag/lane) wereseparated on 15% SDS-PAGE and transferred to a PVDEmembrane (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 visualizedusing the ECL Western Blotting Detection System(Amersham Biosciences, Buckinghamshire, UK).

Results

Ganoderma lucidum Inhibits Proliferation ofHighly Invasive Human Breast Cancer Cellsby Cell Cycle Arrest at GO/Gl

We recently reported that Ganoderma lucidum inhibitsthe 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 cellgrowth, we treated MDA-MB-231 breast cancer cells withincreasing concentrations of Ganoderma lucidum (0-1.0mg/ml) for 24, 48, and 72 h, and cell proliferation was deter-mined. As expected, Ganoderma lucidum suppressed theproliferation of MDA MB-231 cells in a dose- and time-de-pendent manner (Fig. I). To investigate the mechanism bywhich Ganoderma lucidum inhibits cell growth, we analyzed

210 Nutrition and Cancer 2004

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1.0

- • - Control- O - 0.2S malm\-Y~ '••8 mg/ml

Time [hours]

Figure 1. Ganodenna lucidum inhibits proliferation of MDA-MB-231cells. MDA-MB-3 cells were treated with 0. 0.25. 0.5. and !.O mg/ml ofGanodemui lucidum. Proliferation was assessed after 24.48. and 72 h. as de-scribed in Materials and Methods. Each bar represents the mean ± standarddeviation of 3 experimenis.

GO/CI S G2/M GO/GI S G2/M

24

Time [hours]

GO/GI S G2/M

48

Figure 2. Effect of Ganodenna tucidum on cell cycle distribution.MDA-MB-231 cells were treated with Ganoderma lucidum {0.5 mg/ml) for0, 24. and4S h. and the amoimi of cellsai GO/Gl. S. and G2/M phases weredetermined hy flow cytomeiry, as described in Materials and Methods.Each bar represents the mean + standard deviation of 3 experiments.

cell cycle distribution by flow cytometry. As shown in Fig. 2,treatment with Ganoderma lucidum (0.5 mg/ml) caused cellcycle arrest at the GO/GI phase, where the amount ofMDA-MB-231 cells at GO/GI increased from38.7% (Oh) to66.5% {24 h) and 63.9% (48 h).

Ganoderma lucidum Suppresses NF-KBActivity by Inhibiting Akt Kinase

We have previously demonstrated that purified spores orfruitin" bodies of Ganoderma lucidum decrease NF-KB ac-

tivity at the DNA-binding and at the transactivation levels inbreast cancer cells (8). However, the amounts of biologicallyactive components in our original samples of Ganodermalucidum were not determined, which could result in a widerangeof their potency to inhibit cancer cells (8.21). Thus, inthe present study, we used Ganoderma lucidum in the form ofpowdered extract (20:1) with spores, which contains 13.5%polysaccharides and 6% triterpenes. In accordance with ourprevious data, Ganoderma lucidum inhibited constitutivelyactive NF-icB in the reporter gene assay in MDA-MB-231cells in a dose-response manner (Fig. 3).

Because Akt serine threonine kinase controls the activityof NF-KB (14,15), we investigated whether the inhibitory ef-fect oi Ganoderma lucidum on NF-KB is mediated through thesuppression of Akt in breast cancer cells. MDA-MB-231 ceilswere treated with Ganoderma lucidum (1.0 mg/ml) for 0, 24,48. 72. and 96 h. and the expression of Akt was determined inwhole cell extracts by Western blot analysis. As seen in Fig.4A, Ganoderma lucidum inhibits the expression of Akt kinasein a time-response manner. However, the activity of Akt re-quires phosphorylation at Thr̂ "** and Ser*'̂ ^ (22). Therefore,we investigated whether Ganoderma lucidum affects thephosphorylation of Akt. Although we did not observe signifi-cant inhibition of p-Akt- Thr̂ *̂ ^ (not shown), we found thatGanodenna lucidum markedly decreased phosphorylation ofAkt at Ser̂ ''̂ (Fig. 4B). To determine whether the effect ofGanodetTna lucidum on the activity of Akt is reversible, wetreated MDA-MB-231 cells with Ganoderma lucidum (1.0mg/ml). and after 24 and 48 h of incubation, culture mediawere replaced with media without Ganoderma lucidum, andthe incubation continued for an additional 24 and 48 h. Asshown in Fig. 4C, removal of Ganoderma lucidum restored theactivity of Akt in MDA-MB-231 cells, as demonstrated by the

120 n

0.25 0.5 1.0

Ganoderma lucidum [mg/ml]

Figure 3. Ganodermu lucidum inhibits NF-KB in MDA-MB-231 cells,MDA-MB-23i cells were transfected with NF-KB-CAT reporter constructand p-galactosidase expression vector Twenty-lour hours after transfection,the cells were treated with Ganoderma lucidum and NF-KB was measured,as described in Materials and Methods. The results are expressed as thepercentage of relative NF-icB activity. Each bar represents the mean ± stan-dard deviation of 3 experiments.

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a0 24 48 72 96 [h]

Akt

P-actin

0 24 48 72 96 [h]

p-actin

P-actin

4 5

Figure 4. Ganodermu hiiiditm inhibils Aki in MDA-MB-231 cells. MDA-MB-231 cells were treated with Ganndermu lucidiim (Gl. 1.0 mg/ml) for ihe indi-cated limeii. Whole cell extracts were prepared and subjected to Western blul analysis with A; anti-Akt and B: antl-p-Akt-Sef*'' antibodies, as described in Ma-terials and Methods. C; MDA-MB-231 cells were untreated for 24 h (I), and 48 h (2), or treated with Gl for 24 h (.3), and 48 h (4), or treated with Gl tor 24 h foi-lowed by the incubation with fresh media for an additional 24 h (5) and 48 h (6). or treated with G/for 48 h followed by the incubation with fresh media for anadditional 24 h (7) and 48 h (8). Western blot analysis with anti-p-Akt-Ser^^-' antibodies was performed as described previously. The equivalent amount of pro-tein was verified by reprobing the blots wilh anti-P-actin antibody. The results are representative of 3 separate experiments.

phosphorylation of Akt at Ser '̂'̂ . Thus. Ganoderma luciduminhibits constilutively active NF-KB by suppressing Aktkinase activity, and the effect of Ganoderma lucidum on Aktactivity is reversible.

Ganoderma lucidum Downregulates theExpression of Cyclin Dl and cdk4

Because Akt kinase controls the activation of NF-KB (23)and because NF-KB regulates the expression of cyclin Dl(16), we hypothesized that cell cycle arrest at GO/Gl by

212

Ganoderma lucidum is the result of down regulation of theexpression of cyclin Dl. The cell extracts fromMDA-MB-231 cells treated with Ganoderma lucidum weresubjected to Western blot analysis with anti-cyclin Dl anti-body. As shown in Fig. 5A, Ganoderma lucidum markedlydecreased the expression of cyclin Dl in a time-responsemanner. Because cyclin Dl regulates the activity ofcyclin-dependent protein kinase cdk4, which controls the G1cell cycle checkpoint (24), we investigated the effect ofGanoderma lucidum on cdk4 kinase. As expected,Ganoderma lucidum also inhibits expression of cdk4 (Fig.

Nutrition and Cancer 2004

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Vol. 49, No. 2 213

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5B), suggesting that cell cycle arrest at GO/GI byGanodermu lucidum is a result ot the inhibition ot the cell cy-cle regulatory proteins cyclin Dl and cdk4.

Discussion

The wide consumption of mushroom Ganodermalucidum and its medicinal benefits bave been reported sinceancient times, and references to its use were described in theearliest written Chinese "Classic of Materia Medica" around25-220 AD (25). Different biologically active compounds,such as poiysaccharides. triterpenes. proteins, and lipids,were isolated from Ganoderma lucidum (7). However,Ganoderma lucidum is usually consumed in the form of di-etary supplements, powder, and tea (26). We have previouslyreported that fruiting body or spores of Ganoderma lucidumsuppressed constitutively active transcription factors AP-1and NF-KB. which resulted in the inhibition of secretion ofuPA and inhibition of cell niotility of highly invasive cancercells (8). In tbe present study, we used Ganoderma lucidumin the form of a dietary supplement containing powdered ex-tract (20:1) and spores with 13.5% poiysaccharides and 6%triterpenes. It has been suggested that isolated poiysaccha-rides and triterpenes have anticancer effects through differentmechanisms, including by stimulation of the immune re-sponse (poiysaccharides) or by the cytotoxic effects againstcancer cells (triterpenes: for review see ref 27). The use ofGanoderma lucidum containing botb poiysaccharides andtriterpenes is in agreement with the practice of herbal medi-cine, and the use of the whole product can also decrease pos-sible toxic effects of the isolated components. AlthoughGanoderma lucidum inhibits cell proliferation, this effect isnot caused by cytotoxicity because the same dose (0-1.0mg/ml) did not suppress tbe viability of breast cancer cells(data not shown).

Here, we show that Ganoderma lucidum inhibits thegrowth of highly invasive MDA-MB-231 breast cancer cellsby ceil cycle arrest at the GO/G I phase. Our data suggest thatthe arrest at the Gl cell cycle checkpoint by Ganodermalucidum is a result of tbe inhibition of Akt kinase activity,with subsequent inbibition of transcription factor NF-KB,

followed by downregulation of the expression of cyclin Dland cdk4 kinase.

Serine-threonine kinase Akt. also known as protein kinaseB (PKB). consists of a family of highly conserved kinasesAktl- Akt2. and Akt3 (28.29). Akt3 expression was signifi-cantly upregulated in estrogen receptor-negative (ERa-nega-tive) primary breast cancer tumors, suggesting that Akt3 maycontribute to the more aggressive clinical phenotype (30).The enzymatic activity of Akt3 was also significantly ele-vated in ERa-negative. highly invasive MDA-MB-231 breastcancer cells, further confirming tbe role of Akt as an onco-gene responsible for the survival and growth of cancer cells(30). Therefore. Akt is a suitable target for inhibiting highlyinvasive and metastatic cancers. Our data demonstrate thatGanodenna lucidum downregulates the expression of Akt

and suppresses the activity of Akt by inhibitingphosphoryiation of Akt at Ser^^\ which is the regulatorypbosphorylation site responsible for the activity of Akt. Al-though Akt activity can be suppressed by specific syntheticinhibitors of PI3K such as wortmannin and LY294002, herewe show the inhibition of Akt with tbe mushroom extractfrom Ganodenna lucidum. Interestingly, other natural prod-ucts, such as epigallocatecbin-3-galate (EGCG), a major bio-logically active component of green tea, and genistein, anisoflavonoid from soy beans, inhibited Akt in MDA-MB-231breast cancer cells (31. 32). However, EGCG inhibited TGF-a-dependent pbosphorylation of Akt at Ser '̂'-', whereas thesame treatment did not affect the levels of Akt expression(31). On the other hand, genistein inhibited both tbe kinaseactivity of Akt as well as the expression of total Akt andphosphorylated Akt at Ser*" (32). Considering all of thesefindings, we can conclude that Ganoderma lucidumdownregulates the expression of constitutively active Aktand inhibits phosphoryiation of Akt at Sei-^''l

In the present study, we also demonstrated that Gano-derma lucidum suppresses the activity of NF-KB in MDA-MB-231 cells. The suppression of NF-KB activity by Gano-derma lucidum is probably modulated through the inhibitionof Akt kinase, which controls the activity of NF-KB byphosphoryiation of IKB kinase (IKK) and liberation ofNF-KB by degradation of IKB (14, 15). Our data are consis-tent with studies by Masuda et al.. which demonstrate that thesuppression of Akt by EGCG results in the inhibition of con-stitutively active and TNF-a-induced NF-KB in a reportergene assay (31). Furthermore, Gong et al. show thatgenistein, which inhibits Akt, also inhibits the DNA-bindingactivity of NF-KB in unstimulated or EGF-stimulatedMDA-MB-231 cells (32). Alternatively, the effect of Gano-derma lucidum on the activity of NF-KB can be caused by tbeinhibition of PKC. and we have also recently demonstratedthat the PKC inhibitor bisindolylamelimide I suppressesNF-KB activity in MDA-MB-231 cells (33). Finally, recentstudies demonstrate that poiysaccharides isolated fromGanoderma lucidum inhibit all ox an-induced activation ofNF-KB in pancreas (34) and that the herbal mixture PC-SPES, which also contains Ganoderma lucidum, suppressesIipopolysaccharide-induced NF-KB in macrophages (35).

As we demonstrated previously, Ganoderma lucidum in-hibits cell proliferation and induces cell cycle arrest atGO/G I. As we expected, this effect is a result of tbe inhibitionof cyclin Dl, the expression of which is controlled by NF-KB(16). Subsequently, inhibition of cyclin Dl suppressed cdk4kinase, which resulted in cell cycle arrest at GO/Gl and theinbibition of proliferation. In addition to inducing cell cyclearrest, extended exposure of Ganoderma lucidum also in-duced apoptosis in MDA-MB-231 cells (not shown). Experi-ments are in progress to characterize tbe mechanism(s) bywhich Ganoderma lucidum induces apoptosis in breast can-cer cells. A recent study suggests that EGF-induced NF-KB

activation is a major pathway for the proliferation ofMDA-MB-231 cells, and the inbibition of NF-KB suppressedregulatory proteins of Gl/S cell cycle progression, such as

214 Nutrition and Cancer 2004

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cyclin Dl and pRb (11). In addition to controlling cell prolif-eration, NF-KB also controls tbe expression of proteins re-sponsible for metastasis and angiogenesis of cancers (19);we have recently demonstrated that Ganoderma lucidum in-hibits NF-KB-dependent expression of uPA and uPAR. whichresulted in the inhibition of the metastatic behavior of highlyinvasive breast cancer cells (8,9).

Cell cycle arrest at GO/Gl phase and inbibition of cyclinDl by the alcohol extract oi Ganoderma lucidum was also re-ported by Hu et al. (36). However, this effect was observed inbreast cancer MCF-7 cells (36), cancer cells tbat do not con-tain constitutively active NF-KB and are not as invasive as thehighly metastatic MDA-MB-231 cells (17). We have alsofound that Ganoderma lucidum inhibits proliferation of tbenormal breast cells MCFIOA (not shown), cells that also donot bave constitutively active NF-KB, nor do theyoverexpress cyclin Dl (37.38). suggesting that Ganodermalucidum inhibits cell proliferation by multiple mechanisms.Furthermore, etbanol extracts of Ganoderma lucidum inhib-ited growth and induced cell cycle arrest at the GO/Gl phaseof human cervical cancer HeLa cells (39). Alternatively, Linet al. (40) recently described the inbibition of growtb and G2cell cycle arrest by Ganoderma lucidum in buman bepatomacells, which were linked to the downregulation of expressionof cyclin B, and we found that Ganoderma lucidum arrestedPC-3 prostate cancer cells at the G2/M phase independentlyof the inhibition of NF-KB (41). All of these findings togethersuggest tbat Ganoderma lucidum can inhibit tbe proliferationof normal and cancer cells by specific mecbanism(s), whichare different in various cell lines.

In summary, our data demonstrate tbat Ganodermalucidum inhibited the growth of human breast cancer cells bycell cycle arrest at the GO/G 1 phase. The biological effects ofGanoderma lucidum on MDA-MB-231 cells are mediated bytbe inhibition of pbosphorylation and downregulation of ex-pression of Akt kinase, wbicb results in the suppression ofNF-KB activity following the downregulation of cyclin Dland cdk4 expression. The study presented demonstrates bowthe edible mushroom Ganodenna lucidum inhibits tbegrowth of bighly invasive and metastatic breast cancer cellson tbe molecular level and could validate its possible use fortbe prevention and/or treatment of breast cancer.

Acknowledgments and Notes

We thank Dr. Karen Spear lor editing the niiinuscript. This work wassupponed by a grani Imm the Showaller Foundation to D. Sliva. Addresscorrespondence to D. Siiva. Cancer Research Laboratory, Methodist Re-search Institute. 1800 N Capitol Ave. E504, Indianapolis, IN 46202. Phone:(317) %2-573l. FAX: (317) 962-9369. E-mail: [email protected].

Submiited 11 February 2004; aeeepted in final form 15 June 2004.

References

1. Jemal A, Tiwari RC, Murray T, Ghalbor A, Samuels A, et al.; Cancerstatistics. CA Cancer J Clin 54. 8-29, 2004,

2. Bowcock AM (ed.): Breast Cancer: Molecular Genetics. Palho-fjenexis. and Therapeutics. Totowa. NJ: Humana Press, 1999.

3. American Institute lor Cancer Research: Food. Nutrition and (he Pre-vention of Cancer: A Global Perspective. Washington DC. 1997.

4. Go VLW, Wong DA. Resnick MS. and Hebcr D: Evaluation of botani-cals and dietary supplements therapy in cancer palients. J Nitlr 13t.179S-IS0S. 2001.

5. Stamets P: Growing Gourmet and Medicinal Mushrooms. Berkeley,CA: Ten Speed Press, 2(X)0.

6. Yun TK: Update from Asia. Asia studies on cancer chemoprevention.AnnNYAcadSci%%9. 157-192, 1999,

7. Gao Y and Zhou S; Cancer prevention and treatment by Gunoderma. amushroom with medical properties. Food Rev hu 19, 275-325, 2(X)3.

8. Sliva D, Labarrere C, Slivova V. Sedlak M. Lloyd FP Jr, et al.:Ganodermu lucidum suppresses motility of highly invasive breast andprostate cancer cells. Biotheni Biophys Res Commun 298, 603-612,2002.

9. Slivova V. Valachovicova T, Jiang J, and Sliva D: Ganoderma luciduminhibits invasiveness ot" breast cancer cell. J Cancer Integr Med 2,25-30, 2004.

10. WoodburnJR: The epidermal growth factor receptor and its inhibitionin cancer Iherapy. Pharmacol Ther 82, 241-250, 1999.

11. Biswas DK, Cruz AP. Gansberger E, and Pardee AB: Epidermalgrowth factor-induced nuclear factor kB activation: a major pathwayof cell-cycle progression in estrogen-receptor negative breast cancercells. Pr<,c NatI Acad Sci USA 97, 8,^42-8547, 2000.

! 2. Burgering BM and Coffer PJ: Protein kinase B (c-Akt) in phosphatidy-linositol-3-OH kinase signal transdueiion. Nature i76,599-602, 1995.

13. Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, et al,:Mechanism of activation of protein kinase B by insulin and IGF-1.EA/S071S, 6541-6551, 1996.

14. Ozes ON, Mayo LD, Guslin JA, Pfeffer SR, Pfeffer LM, et af: NF-KBactivation requires the Akt serine-lhreonone kinase. Nature 401,82-85, 1999.

15. Romashkova JA and Makarov SS: NF-KB is a target of AKT inanti-apoptotic PDGF signaling. Nature 401, 86-90. 1999.

16. Guttridge DC, Albanese C, Reuther JY, Pestell RG, and Baldwin ASJr: NF-KB conirols cell growth and differentiation throughtranscriptional regulation of cyclin DI, Mol Cell Biol 19, 5785-5799,1999,

17. Stiva D, Rizzo MT, and English D: Phosphatidylinositol 3-kinase andNF-KB regulate motility of invasive MDA-MB-231 human breast can-cer cells by the secretion of urokinase-type plasminogen activator(uPA). J Biol Chem 277, 3150-3157, 2002,

18. Nakatani K, Thompson DA, Barthel A, Sakaue H, Liu W, et al.:Up-regulation of Akt3 in estrogen receptor deficient breast cancersand andrngen-independent prostate cancer lines. J Biol Chem 274,21528-21532, 1999,

19. Orlowski RZand Baldwin Jr AS: NF-kappaB as a therapeutic target incancer. Trends Mot Med 8, 385-389, 2002.

20. Sliva D, Harvey K, Mason R. Lloyd Jr F, and English D: Effect ofphosphatidie acid on human breast cancer cells exposed todoxorubicin. Cancer Invest 19, 781-788, 2001.

21. Sliva D, Sedlak M, Slivova V. Valachovicova T, Lloyd FP Jr, et al,: Bio-logic activity of Ganoderma lucidum for the inhibition of highly inva-sive breast and prostate cancer cells, J Ahem Complement Med 9,491^97,2003,

22. Downward J: Lipid-regulaled kinases: some common themes at last,Sf/cHCf 279, 673-674, 1998.

23. Madrid LV, Wang CY, Guttridge DC, Schotteiius AJ. Baldwin AS Jr, etal,: Akt suppresses apoptosis by stimulating the transaetivation poten-tial of the RelA/p65 subunit of NF-kappaB, Mol Celt Biol 20, 1626-1638, 2000,

24. Diehl JA; Cycling to cancer with cyclin Dl, Cancer Biol Ther \.22^231.2002.

25. Wachtel-Galor S, Szeio YT, Tomlinson B, and Benzie IFF:Ganodenna lucidum ("Lingzhi"): acute and short-term biomarker re-sponse to supplementation, Int J Food Sci Nulr 55, 75-83, 2004.

Vol. 49, No. 2 215

Page 8: Ganoderma lucidum Suppresses Growth of Breast Cancer Cells ... · We recently reported that Ganoderma lucidum inhibits the invasiveness of breast cancer cells by suppressing ceil

26. Sliva D: Ganoderma lucidum (Reishi) in cancer treatment. Integ Can-cer T/ie/-2. 358-364, 2(H)3.

27. Chiu SW, Wang ZM, Leung TM, and Moore D: Nutritional value ofGanodenna extract and assessment of its genotoxicity using comet as-says of mouse lymphocytes. Food Chem Toxicol 3S, 173-178, 2000,

28. Brodbeck D, Cron P, and Hemmings BA: A human protein kinase Bywith regulatory phosphoryiation sites in the activation loop and in theC-terminal hydropbobic domain, 7 Biol Chem 274,9133-9136, 1999.

29. Niikatani K, Sakaue H, Thompson DA, Weigel RJ, and Roth RA: Iden-tification of a human Akt3 (protein kinase B y) which contains the reg-ulatory serine phosphoryiation site. Biochem Biophys Res Commun257,906-910. 1999.

30. Nakatani K. Thompson DA, Barthel A, Sakaue H, Liu W, et a!.:Up-reguliition of Akt3 in estrogen receptor-deficient breast cancersand androgen-independent prostate eancer lines. J Biol Chem 274,21528-215.32. 1999,

31. Masuda M, Suzui M, Lim JTE. Deguchi A, Soh JW, et al,:Epigallocatechin-3-gallate decreases VF.GF produclion in head andneck and breast carcinoma cells by inhibiting EGFR-related pathwaysof signal transduction. J Exper Ther Oncol 2, 350-359, 2002,

32. Gong L, Li Y, Nedeljkovic-Kurepa A, and Sarkar FH: Inactivation ofNF-KB by genisteir is mediated via Akt signaling pathway in breastcancer cells. Oncogene 22. 4702^709, 2003,

33. Sliva D, English D, Lyons D, and Lloyd Jr FP: Protein kinase C in-duces motility of breast cancers by upregulating secretion ofurokinase-type plasminogen activator (uPA) through the activation ofAP-1 and NF-KB. Biochem Biophys Res Commun 290,552-557,2002,

34, Zhang HN, He JH, Yuan L, and Lin ZB: In vitro and in vivo protectiveeffects of Ganoderma lucidum poiysaccharides on alloxan-inducedpancreatic islets damage. Life Sci 7X 2307-2319, 2003.

35, Ikezoe T, Yang Y, Heber D, Taguchi H, and Koeffler HP: PC-SPES: apotent inhibitor of nuclear factor-KB rescues mice fromlipopolysaccharide-induced septic shcxjk. Mol Pharnacot 64,1521-1529,2003.

36. Hu H, Ahn NS, Yang X. Lee YS, and Kang KS: Ganoderma lucidumextract induces cell cycle arrest and apoptosis in MCF-7 human breastcancer cell, Int J Cancer 102, 250-253, 2002,

37. Surh Y-J, Na H-K, Lee J-Y. and Keum YS: Molecular mechanisms un-derlying anti-tumor promoting activities of heat-processed Panax gin-seng C.A, Meyer. J Korean Med Sci 16(Suppl). S38-S41, 2001.

38. Zhou Q. Wulfkuhle J. Ouatas T, Fukushima P, Stetler-Stevenson M, etal.: Cyclin Dl overcxpression is a model of human breastpremalignancy: preferential stimulation of anhorage-independent butnot anchorage-dependent growth is associated with increased edk2 ac-tivity. Breast Cancer Res Treat 59, 27-39, 2000,

39. Zhu HS, Yang XL, Wang LB, Zhao DX, and Chen L: Effects of ex-tracts from sporoderm-broken spores of Ganoderma lucidum on HeLacells. Cell Biol Toxicol 16, 201-206, 20(K),

40, Lin SB, Li CH, Lee SS, and Kan LS: Triterpene-enriched extracts fromGanoderma lucidum inhibit growth of hepatoma cells via suppressingprotein kinase C, activating mitogen-activated protein kinases andG2-phase cell cycle arrest. Life Sci 72. 2381-2390, 2003,

41, Jiang J, Stivova V, Valachovicova T. Harvey K, and Sliva D:Ganoderma lucidum inhibits proliferation and induces apoptosis inhuman prostate cancer cells PC-3, hu J Oncol 24: 1093-1099. 2004.

216 Nutrition and Cancer 2004

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