Research Article Ganoderma tsugae Extract Inhibits Growth...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 219472 12 pageshttpdxdoiorg1011552013219472

Research ArticleGanoderma tsugae Extract Inhibits Growth ofHER2-Overexpressing Cancer Cells via Modulation ofHER2PI3KAkt Signaling Pathway

Han-Peng Kuo1 Shih-Chung Hsu2 Chien-Chih Ou3 Jhy-Wei Li4 Hsiu-Hsueh Tseng5

Tzu-Chao Chuang6 Jah-Yao Liu7 Shih-Jung Chen8 Muh-Hwan Su9 Yung-Chi Cheng10

Wei-Yuan Chou11 and Ming-Ching Kao111

1 Department of Biological Science and Technology College of Life Sciences China Medical University 91 Hsueh-Shih RoadTaichung 40402 Taiwan

2 Kang-Ning Junior College of Medical Care and Management Taipei 11486 Taiwan3Oncology New Drug Division SynCore Bio Taipei 11070 Taiwan4Department of Pathology Da-Chien General Hospital Miaoli 36052 Taiwan5 Graduate Institute of Life Sciences National Defense Medical Center Taipei 11490 Taiwan6Department of Chemistry Tamkang University Tamsui New Taipei 25137 Taiwan7Department of Obstetrics amp Gynecology Tri-Service General Hospital Taipei 11490 Taiwan8 Luo-Gui-Ying Fungi Agriculture Farm Taoyuan 33043 Taiwan9 Sinphar Group Headquarter Sinphar Group Yilan 26944 Taiwan10Department of Pharmacology Yale University School of Medicine New Haven CT 06520-8066 USA11Department of Biochemistry National Defense Medical Center Taipei 11490 Taiwan

Correspondence should be addressed to Ming-Ching Kao mckaomailcmuedutw

Received 11 December 2012 Accepted 25 February 2013

Academic Editor Chris J Branford-White

Copyright copy 2013 Han-Peng Kuo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Ganoderma also known as Lingzhi or Reishi has been used for medicinal purposes in Asian countries for centuries It is amedicinal fungus with a variety of biological properties including immunomodulatory and antitumor activities In this study weinvestigated themolecularmechanisms by whichGanoderma tsugae (GT) one of themost common species ofGanoderma inhibitsthe proliferation of HER2-overexpressing cancer cells Here we show that a quality assured extract of GT (GTE) inhibited thegrowth of HER2-overexpressing cancer cells in vitro and in vivo and enhanced the growth-inhibitory effect of antitumor drugs (egtaxol and cisplatin) in these cells We also demonstrate that GTE induced cell cycle arrest by interfering with the HER2PI3KAktsignaling pathway Furthermore GTE curtailed the expression of the HER2 protein by modulating the transcriptional activity ofthe HER2 gene and the stabilitydegradation of the HER2 protein In conclusion this study suggests that GTE may be a usefuladjuvant therapeutic agent in the treatment of cancer cells that highly express HER2

1 Introduction

Human epidermal growth factor receptor 2 (HER2) is a 185-kDa transmembrane receptor tyrosine kinase (RTK) belong-ing to the epidermal growth factor receptor (EGFR) familywhich contains four homologous members EGFRHER1HER2 HER3 and HER4 Ligand stimulation induces dimer-ization of the HER receptor (homo- or heterodimer) which

leads to self-phosphorylation (except for HER3) on tyrosineresidues localized to the C-terminal domain of HER recep-tors Then the phosphorylated HER receptors (activatedform) activate a variety of downstream signaling pathwayssuch as the phosphatidylinositol-3-kinase (PI3K)Akt andthe Rasmitogen-activated protein kinase (MAPK) pathwayswhich in turn promote cell proliferation survival andmetas-tasis [1]

2 Evidence-Based Complementary and Alternative Medicine

Aberrant upregulation ofHER2 is found in approximately25ndash30 of breast cancers [2] and in 6ndash50 of ovarian cancers[3] Patients with HER2-positive cancer have a high riskfor diminished effectiveness of cancer treatments increasedcancer metastasis and poor clinical outcomes [4] Thereforeinhibition of HER2 expression or its kinase activitymay be aneffective approach for the treatment of HER2-overexpressingcancers In fact a number of HER2-targeting agents includ-ing monoclonal antibodies (eg trastuzumab) and small-molecule tyrosine kinase inhibitors (eg lapatinib) havebeen developed for the treatment of cancers with HER2-overexpression [1] However there is still a need for noveltherapies to treatHER2-overexpressing cancers For exampletraditional Chinese medicine (TCM) and botanical productsare currently considered to be safer and may be used asalternative therapeutic agents for treatment of cancers thatoverexpress HER2 [5 6]

Ganoderma (also known as Lingzhi) has a long history ofuse in folkmedicines in Asian countriesGanoderma lucidum(GL) andGanoderma sinense (GS) listed in Chinese Pharma-copoeia (2010 edition) [7 8] are two of the most commonspecies of Ganoderma and have been used for medicinalpurposes in China for centuries The biological activitiesof GL and GS particularly their immunomodulatory andantitumor properties have been well documented [9] Inaddition Ganoderma tsugae (GT) another well-cultivatedspecies ofGanoderma has been shown to havemany biologi-cal and pharmacological properties such as antiautoantibodyformation [10] antifibrosis [11] antiinflammation [12] andantioxidation characteristics [13] A number of reports showthat GT has growth-inhibitory effects in a variety of humancancer cells such as MDA-MB-231 and MCF-7 breast cancercells [14] COLO 205 colorectal cancer cells [15] A431 epider-moid carcinoma cells [16] Hep3B hepatoma cells [17] andH23 and H2303 lung adenocarcinoma cells [18] AlthoughGT has antitumor activity in many human cancer cells themechanisms that underlie its growth-inhibitory effect onHER2-overexpressing cancer cells remain unclear

In this study we produced a quality assured extract of GT(GTE) and characterized its antitumor effects and relevantmolecular mechanisms in HER2-overexpressing cancer cellsin vitro and in vivo Our results show that GTE inhibits cancercell growth and induces cell cycle arrest via modulation ofthe HER2PI3KAkt signaling pathway We also show thatcombining GTE with taxol or cisplatin significantly slowsthe growth of HER2-overexpressing cancer cells indicatinga potential use of GTE in the treatment of cancers thatoverexpress HER2

2 Materials and Methods

21 Cell Culture Humanovarian carcinoma cell lines SKOV-3 (HER2high) and OVCAR-3 (HER2low) and breast carci-noma cell lines SKBR-3 (HER2high) and BT-474 (HER2high)were obtained from the American Type Culture Collection(ATCC Manassas VA USA) The MCF-7HER2 (HER2high)human breast carcinoma cell line (MCF-7 of an HER2-transfected stable line) was kindly provided by Dr M C

Hung (Department of Molecular and Cellular OncologyUniversity of TexasMDAndersonCancerCenterHoustonTX USA) The MDA-MB-435HER2 (HER2high) humanmelanoma cell line (MDA-MB-435 of an HER2-transfectedstable line) was kindly provided by Dr T D Way (Depart-ment of Biological Science and Technology China MedicalUniversity Taichung Taiwan) All cells were cultured inDMEMF12 medium (Gibco BRL Grand Island NY USA)supplemented with 10 fetal bovine serum (FBS) in ahumidified atmosphere of 5 CO

2at 37∘C

22 Chemicals andAntibodies The thiazolyl blue tetrazoliumbromide (MTT) cycloheximide (CHX) and N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL) were obtained fromSigma-Aldrich (St Louis MO USA) Antibodies againstcyclins D1 and E p21 p27 phospho-Akt (Ser308) Akt1 andubiquitin (Ub) were purchased from Santa Cruz Biotech-nology Inc (Santa Cruz CA USA) Antibodies againstphospho-PI3K PI3K phospho-Erk 12 and Erk 12 were pur-chased from Cell Signaling Technology Inc (Beverly MAUSA) Antibodies against phospho-HER2 (Ab-18) HER2(Ab-3) 120573-actin and Ki-67 (Clone MIB-1) were purchasedfrom Neomarkers Inc (Fremont CA USA) Calbiochem(San Diego CA USA) Chemicon International Inc (Temec-ula CA USA) and Dakocytomation Inc (Carpinteria CAUSA) respectively Taxol (paclitaxel) was purchased fromBristol-Myers Squibb (Wallingford CT USA) and cisplatinwas purchased from Pharmacia amp Upjohn SpA (Via RobertKoch 12 Milan Italy)

23 Preparation of Ganoderma tsugae Extracts Ganodermatsugae (GT) was kindly provided by the Luo-Gui-Ying FungiAgriculture Farm (with a registered name of Tien-ShenLingzhi) Taoyuan Taiwan The extract of GT (GTE) wasprepared as described previously [15] Briefly the powder ofthe GT fruiting body (5 g) was soaked in 999 methanol(200mL) mixed and shaken for 24 h on a rotating shakerAfter centrifugation the supernatant was poured through fil-ter paper (Whatman cat no 1001-110) and the residues wereextracted with methanol two additional times as mentionedabove The filtrates were collected together and subjectedto concentration under reduced pressure (ie evaporated todryness under reduced pressure) to produce a brown gel-likeGT extract (GTE) The yield was approximately 30 TheGTE was then prepared as a stock solution with methanolsolvent (100mgmL) and stored at minus80∘C until use Foranimal experiments the dry GTE was redissolved in ethanoland diluted with a suspension solution (745 corn oil16 PEG-400 4 Tween-80 4 Cremophor EL and 15Ethanol vv) to a concentration of 10mgmL

24 Quality Control of GTEs via Bioresponse FingerprintingThe quality of the GTEs was assessed as described previously[18 19] Briefly the genomic bioresponse to the GTEs wasdetermined in SKOV-3 cells treated with 05mgmL of GTEThe total RNA was extracted from the GTE-treated cellscleaned with a commercial kit (Qiagene RNA extractionkit cat no 75144) and then used to obtain transcription

Evidence-Based Complementary and Alternative Medicine 3

profiles in GeneChip hybridization studies using Affymetrixtechnology The changes in the individual gene expressionlevels obtained by the GeneChip experiments were measuredby Affymetrix MAS 50 software A statistical pattern com-parisonmethod from the PhytomicsQC platform PhytomicsSimilarity Index (PSI) was applied to determine the batch-to-batch similarity of the botanical products In generalclinically similar batches have a PSI more than 095

25 Cell Proliferation Assay Cell viability was determinedusing an MTT assay as previously described [6] Briefly cellswere seeded at a density of 6000 cellswell into 96-well platesand incubated overnight in a medium containing 10 FBSAfter the cells adhered to the plate various doses of GTEwere added to the cells and then the cultures were incu-bated at 37∘C for 72 h After incubation with MTT reagent(05mgmL) for 4 h the relative viable cell numbers weredirectly proportional to the production of formazan crystalssolubilized by DMSOThe final solution was measured usinga spectrophotometer at a wavelength of 545 nm against areference wavelength of 690 nm

26 Soft Agar Colony Formation Assay The effect of GTEon the potential for anchorage-independent growth wasdetermined by soft agar colony formation assay as describedpreviously [20] with slight modifications The cells (2 times104 cellswell) were seeded in 6-well plates containing 07base agar 035 top agar and exposed to different concentra-tions of GTE or an equal volume of DMEMF12 twiceweekand incubated at 37∘C for 3 weeks Colonies were stainedwith MTT reagent (5mgmL) and then photographed usinga phase contrast microscope (100X) equipped with a CCDcamera

27 Flow Cytometric Analysis For the analysis of the cellcycle the phase distribution was detected by flow cytometryas described previously [6] In brief cells were incubatedwith GTE or the vehicle for 24 h and then fixed with ice-cold 70 ethanol overnight at 4∘C Prior to analysis the cellswere washed twice with PBS buffer and then incubated withpropidium iodide (PI) solution (50120583gmL PI in PBS with 1Tween-20 and 10 120583g RNase) for approximately 30min in thedark at room temperature The DNA content was measuredusing flow cytometry (BD FACS Canto) The FCS Expressv20 software was used to analyze the results from the flowcytometric experiment

28 Reporter Gene Assay Cells were cotransfected withpHER2-luc (a HER2 promoter-driven luciferase gene plas-mid construct) and pCMV-120573-gal plasmids for 6 h and thenincubated with GTE or the vehicle for 24 h The HER2promoter and 120573-galactosidase (120573-gal) gene activity assayswere performed as previously described [21] The relativelight units of luciferase activity were normalized to 120573-galactivity

29 Semiquantitative Reverse Transcriptase-Polymerase ChainReaction (RT-PCR) Total RNA was isolated using TRIzol

solution (Invitrogen San Diego CA USA) Twomicrogramsof total RNA were used for first-strand cDNA synthesisThe appropriate primers (HER2 sense 51015840-CAATGGAGA-CCCGCTGAAC-31015840 HER2 antisense 51015840-CAGTGCGCG-TCAGGCTCT-31015840 glyceraldehyde-3-phosphate dehydroge-nase (GAPDH) sense 51015840-ACCACAGTCCATGCCATCAC-31015840 GAPDH antisense 51015840-TCCACCACCCTGTTGCTGTA-31015840) were used to perform the polymerase chain reaction (for 1cycle at 94∘C for 5min 32 cycles of 94∘C for 15 s 56∘C for 30 sand 72∘C for 1min with a final extension at 72∘C for 5min)The PCR products were separated by electrophoresis on a12 agarose gel and detected by ethidium bromide (EtBr)staining

210 Immunoprecipitation and Western Blotting Proteinswere extracted from the cells by the addition of lysis buffer(20mM Hepes buffer pH 70 10mM KCl 2mM MgCl

2

05NP-40 and protease inhibitors) Following cell lysis theextracts were centrifuged at 16000timesg for 10min at 4∘C Theprotein content of the supernatant was measured using theBio-Rad protein assay kit Immunoprecipitation was carriedout as previously described [22] with a slight modificationBriefly 300 120583g of total protein was incubated with anti-HER2 antibody overnight at 4∘C followed by protein AGPLUS-Agarose (Santa Cruz) for 3 h at 4∘C The precipitateswere resolved using sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and then transferred ontoa polyvinylidene fluoride (PVDF) membrane For Westernblotting as described previously [22] total protein (40120583g)was loaded to the gel and blotted onto the PVDF membraneThe membranes were blocked using 5 nonfat milk intris-buffered saline with Tween-20 (TBST) for 1 h at roomtemperature After blocking the PVDF membranes wereincubated with primary antibodies for 1 h at room temper-ature followed by an HRP-conjugated secondary antibodyThe reactive signals were visualized using the EnhancedChemiluminescence Kit (Amersham Biosciences ArlingtonHeights IL USA) The bands were scanned and quantifiedusing the ImageJ software

211 Animal Experiments The animal experiments wereperformed as described previously [15] with slight modifi-cations Briefly 5 times 106 SKOV-3 cells were subcutaneouslyimplanted into the flank region of female BALBc nude mice(BALBcAnNCg-Foxn1119899119906CrlNarl) In total 19 mice wereused for this experiment the tumor-implanted mice weretreated with GTE (119899 = 12) or with the vehicle (119899 = 7)respectively The GTE-treated mice were fed with GTE dailyat a dose of 200mgkg (119899 = 5) or 1000mgkg (119899 =7) body weight this dosing schedule was initiated whenthe developing tumor was approximately 50ndash100mm3 involume (approximately 2-3 weeks after the cancer cellswere implanted) The tumor volume and body weight weremonitored daily The mice were sacrificed for pathologyexaminations when the tumor volume exceeded 1000mm3The tumors were then completely excised from the subcu-taneous tissue and weighed Biochemical and hematologicalparameters were used to evaluate potential drug toxicity


212 Immunohistochemical (IHC) Staining SKOV-3 xenog-rafted tumors and the surrounding tissues were excisedfixed in formalin embedded in paraffin cut in 4-120583m serialsections and then placed onto glass slides The tumor tissue-coated slides were then dewaxed with xylene and graduallyhydrated with graded alcohols After antigen retrieval wasachieved by pressure-cooking in 10mM citrate buffer (pH60) for 6min immunostaining for Ki-67 HER2 and cyclinD1 (1 150 dilution) was then performed as described previ-ously [23]

213 Statistical Analysis All data are presented as themean plusmnSD from three independent experiments Statistical analysiswas performed by one-way ANOVA Differences betweentreatment groups were analyzed for significance by multi-ple comparisons using analysis of variance lowast119875 lt 005 andlowastlowast119875 lt 001 versus the vehicle-treated control group

3 Results

31 Quality Control of GTE Using Bioresponse FingerprintAnalysis The quality of TCMs are potentially influencedby many factors such as the growth conditions and pro-cessing procedures [24] To assess the quality of the GTEthe bioresponse fingerprints were analyzed by the patterncomparison method from the PhytomicsQC platform [19]which showed highly concordant biological profiles for GTEs(GTE1 GTE2 and GTE3) and extracted from three batchesof GT acting on SKOV-3 cells with a PSI value more than095 (See Supplementary Figure S1A available online athttpdxdoiorg1011552013219472) Under this PSI value376 genes with specifically altered expression (149 upregula-tions and 227 downregulations)were observed as bioresponsefingerprints of GTEs (See Supplementary Figure S1B) Theseresults suggest that theGTpowder products used in this studywere stable consistent and of high quality

32 GTE Inhibits Proliferation of HER2-Overexpressing Can-cer Cells To determine whether GTE inhibits the growthof HER2-overexpressing cancer cells we first evaluatedthe impact of GTE on cell proliferation using the MTTassay As shown in Figure 1(a) the treatment of SKOV-3cells (HER2high) with various concentrations of GTE (01ndash1mgmL) for 24ndash72 h resulted in significant dose- and time-dependent suppressive effects on the proliferation of SKOV-3 cells accounting for a 0ndash56 reduction at 24 h a 13ndash95reduction at 48 h and a 24ndash98 reduction at 72 h Moreoverthe trypan blue exclusion assay also clearly demonstratedthat the GTE exhibited growth suppression effect at dosesof 01ndash05mgmL while a less cytotoxic effect at 10mgmLon SKOV-3 cells (Figure 1(b)) Similar antiproliferative effectsof GTE were also observed in other HER2-overexpressingcancer cells for example BT-474 and SKBR-3 (Supplemen-tary Figures S2A and S2B) In addition we assessed theinfluence of GTE on the potential for anchorage-independentgrowth a hallmark of malignant cancer cells using the softagar colony formation assayWe found that GTE dramatically

reduced anchorage-independent growth of SKOV-3 cells in adose-dependent manner (Figure 1(c)) These results suggestthat GTE is capable of inhibiting the proliferation of HER2-overexpressing cancer cells

Resistance to chemotherapeutic agents (such as taxol andcisplatin) is a major problem in the treatment of cancersthat overexpress HER2 [25 26] We therefore examinedwhether GTE could enhance the growth-inhibitory effects ofanticancer drugs on SKOV-3 cells by incubating the cells withboth anticancer agents and GTE As shown in Figure 1(d)GTE significantly enhanced the growth-inhibitory effectsof taxol and cisplatin on SKOV-3 cells We found that theproliferation of SKOV-3 cells was reduced by 30 45 and37 in cells exposed to GTE (025mgmL) taxol (10 ngmL)and cisplatin (10 120583gmL) alone respectively However theproliferation of SKOV-3 cells was reduced by 73 and 77in cells exposed to GTE combined with taxol and cisplatinrespectively Similarly we also found that GTE could increasethe chemotherapeutic efficacy of anticancer drugs againstother HER2-overexpressing cancer cell lines for exampleMDA-MB-453HER2 (Supplementary Figures S3A and S3B)These findings suggest that GTE can chemosensitize HER2-overexpressing cancer cells to anticancer drugs (eg taxoland cisplatin)

33 GTE InducesG1 PhaseArrest byModulating the Expressionof Cell Cycle Regulatory Proteins As mentioned above weobserved a growth-inhibitory influence of GTE on SKOV-3cells (Figures 1(a)ndash1(c)) To determine if the antiproliferativeproperty of GTE was due to the disruption of cell cycle flowcytometry was used to analyze the cell cycle change in SKOV-3 cells As illustrated in Figure 2(a) treatment of SKOV-3cells with GTE resulted in a distinct increase (approximately24) in the number of G1 phase cells at a concentration of05mgmL GTE This increase in the number of cells in theG1 phase was accompanied by a concordant decrease in thenumber of cells in the S and G2M phases Similar GTE-mediated cell cycle distribution patterns were observed inBT-474 (HER2high) cells (Supplementary Figure S4A) Thesefindings suggest that GTE inhibits the growth of HER2-overexpressing cancer cells by modulating the progression ofthe cell cycle

Different cell cycle regulators such as cyclins cyclin-dependent kinases (CDKs) and CDK inhibitors (CKIs) areinvolved inmultiple cellular pathways that tightly regulate theprogression of the cell cycle [27] To elucidate the molecularmechanisms of GTE-induced cell cycle arrest we assessed theimpact of GTE on the expression of cell cycle regulators Wedemonstrated that after GTE treatment the protein levels ofcyclins D1 and Ewere downregulated while the protein levelsof p21 and p27 were upregulated in SKOV-3 cells (Figures2(b) and 2(c)) Similarly GTE also dramatically affected theexpression of cell cycle regulators (eg cyclins D1 and E) intwo more HER2-overexpressing cancer cell lines that is BT-474 (Supplementary Figure S4B) and SKBR-3 cells (data notshown) These results suggest that GTE inhibits cell growthby regulating the expression of cell cycle regulators in HER2-overexpressing cancer cells


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Figure 1 Effect of GTE on cell proliferation in HER2-overexpressing cancer cells (a) SKOV-3 cells were treated with 05methanol (vehiclecontrol) or various concentrations of GTE (01 025 05 075 and 1mgmL) for 72 h Cell proliferation was measured using theMTT assay asdescribed in Section 2 (b) SKOV-3 cells were treated with either vehicle control or GTE (01 05 or 10mgmL) for 05 1 2 and 3 days Cellnumbers were determined using trypan blue staining The parental SKOV-3 cells were not treated with vehicle (05 methanol) or GTE (c)SKOV-3 cells were treated with different doses of GTE (025 and 05mgmL) twice a week for 3 weeks in the soft agar colony formation assayas described in Section 2 (d) SKOV-3 cells were treated with various concentrations of taxol (1 10 100 and 1000 ngmL) or cisplatin (25 510 and 20 120583gmL) with or without GTE (025mgmL) for 72 h Cell proliferation was determined by MTT assay The results are expressed asthe mean plusmn SD of three independent experiments lowast119875 lt 005 lowastlowast119875 lt 001

34 GTE Inhibits HER2PI3KAkt Signaling Cascades Basedon the results mentioned above there was a significantgrowth-inhibitory effect of GTE on HER2-overexpressingcancer cells (Figure 1) We next explored whether the inhi-bition of proliferation was caused by regulating the expres-sion of HER2 protein As shown in Figures 3(a) and 3(b)

treatment of SKOV-3 cells with GTE resulted in a markeddose- and time-dependent decrease in HER2 protein levelsSimilarly GTE also decreased the protein expression ofHER2in other HER2high cell lines such as SKBR-3 BT-474 andMCF-7HER2 (Figure 3(d) Supplementary Figure S5A) andanHER2low cell line OVCAR-3 (Supplementary Figure S5B)


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Figure 2 Effect of GTE on cell cycle distribution in HER2-overexpressing cancer cells (a) SKOV-3 cells were treated with vehicle control(05 methanol) or various concentrations of GTE (0 025 and 05mgmL) for 24 h Cell cycle distribution was analyzed by flow cytometryas described in Section 2 The parental SKOV-3 cells were not treated with vehicle (05 methanol) or GTE (b) SKOV-3 cells were treatedwith various concentrations of GTE (0 025 and 05mgmL) for 16 h and 24 h The expression of G1 phase regulators was determined byWestern blotting as described in Section 2 (c) A histogram showing the relative protein levels from (b) Data are presented as the mean plusmn SDof three independent experiments lowast119875 lt 005 and lowastlowast119875 lt 001 versus the vehicle-treated control group

The HER2 signaling pathway is known to be associatedwith cell proliferation therefore we tested the impact ofGTE on two main downstream pathways of HER2 thePI3KAkt and RasMAPK signaling cascades [1] As shownin Figure 3(c) GTE exhibited inhibitory effects on phospho-HER2 phospho-PI3K and phospho-Aktwithout a noticeablereduction in phospho-Erk 12 in SKOV-3 cells MoreoverGTE showed similar effects on phospho-HER2 and phospho-Akt in other HER2-overexpressing cell lines for example

SKBR-3 and BT-474 (Figure 3d)) These data clearly indicatethat GTE exerts inhibitory effects on the HER2PI3KAktsignaling cascades in cancer cells with HER2-overexpression

35 GTE Downregulates HER2 Protein Expression by Modu-lating the Gene Expression and Protein Stability of HER2 Asmentioned above our results showed a dramatic inhibitoryinfluence of GTE on the expression of HER2 protein in


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Figure 3 Effect of GTE on HER2PI3KAkt and RasMAPK signaling cascades in HER2-overexpressing cancer cells (a) SKOV-3 cells weretreated with various concentrations of GTE (0 01 025 05 075 and 1mgmL) for 24 h The expression of HER2 protein was measured byWestern blotting (b) SKOV-3 cells were treated with 05mgmL GTE for 6 12 24 48 and 72 h The protein level of HER2 was determinedby Western blotting (c) Treatment of SKOV-3 cells with GTE (0 025 or 05mgmL) for 24 h inhibited HER2PI3KAkt but not HER2Erksignaling (d) Treatment of SKBR-3 and BT-474 cells with GTE (0 025 05mgmL) for 24 h led to inhibition of the HER2Akt but not theHER2Erk signaling pathway The results are expressed as the mean plusmn SD of three independent experiments lowast119875 lt 005 lowastlowast119875 lt 001

HER2-overexpressing cancer cells (Figure 3) To determinethe underlying molecular mechanisms of the GTE-mediateddownregulation of HER2 we tested the effect of GTEon the transcriptional activity of HER2 gene The expres-sion of HER2 mRNA was distinctly decreased in SKOV-3 (Figure 4(a)) and BT-474 (Supplementary Figure S6A)cells exposed to 025 and 05mgmL of GTE for 24 h asdetermined byRT-PCR Furthermore the reporter gene assayindicated that GTE decreased the HER2 promoter activityin a dose-dependent manner in SKOV-3 cells (Figure 4(b))Consistent with the decreased expression of HER2 proteinboth themRNA level and the promoter activity of HER2weredownregulated by GTE Taken together we conclude thatGTE depletes the protein levels of HER2 via modulation ofthe HER2 gene activity

Because an overall decrease in protein stability couldalso be responsible for the reduced HER2 protein levelswe examined the effect of GTE on HER2 protein stabilityand found that the half-life of HER2 was clearly shortenedby GTE treatment in SKOV-3 (Figure 4(c)) and BT-474(Supplementary Figure S6B) cells In general proteins suchas HER2 are tagged with polyubiquitin and then degraded bythe ubiquitin-proteasome system (UPS) We tested whetherthe GTE-mediated HER2 protein stability was due to the

activation of the UPS As shown in Figure 4(d) the amountof polyubiquitinated HER2 (HER2-Ub

(n)) protein was signif-icantly increased in SKOV-3 cells exposed to 05mgmLGTEfor 24 or 48 h In addition the treatment of SKOV-3 cellswith LLnL a proteasome inhibitor effectively prevented theGTE-mediated degradation of HER2 protein (Figure 4(e))These observations suggest that the curtailment of HER2 byGTE may also occur through the induction of HER2 proteininstabilitydegradation

36 GTE Inhibits the Growth of SKOV-3 Xenografted Tumorsby Modulating HER2 Protein To determine the potentialfor anticancer effects of GTE in vivo we used xenograftedtumor-bearing nude mice After the volume of the SKOV-3 xenografted tumors reached approximately 50ndash100mm3the mice were orally (po) administered either GTE (200and 1000mgkgday) or vehicle for 31 days As illus-trated in Figure 5(a) the nude mice treated with 200 or1000mgkgday of GTE exhibited a marked inhibition inthe growth of SKOV-3-implanted tumors relative to thatof the control group There was no significant alterationin the body weights of the nude mice with or with-out GTE treatment indicating GTE had no apparent toxicity(Figure 5(b)) In addition in comparison to the vehicle



GTE (mgmL)

Fold

002040608

112 SKOV-3

HER2

GAPDHSKOV-3

GTE (mgmL)

P 0 025 05

P 0 025 05

101 1 032 024

(a)

800006000040000

900060003000

0

GTE (mgmL)

lowastlowast

lowastlowast

SKOV-3

P 0 025 05

RLU

120573-g

al

(b)

HER2

Actin

SKOV-3

0

02

04

06

08

1

12

Rela

tive i

nten

sity

Trendline (CHX)

48 24 12 0 12 24 48 (h)

032 083 08 1 084 045 008

CHX

CHX

CHX + GTE

0 12 24 36 48Time after CHX treatment (h)

CHX + GTE

Trendline (CHX + GTE)

(c)

GTE (05 mgmL)

1701301007055

40

35

25

IP HER2IB Ub

IB HER2

IB actin

(kD

a)

SKOV-3

0 12 24 48 (h)

(d)

HER2

Actin

SKOV-3

002040608

11214

Fold

P C GTE

GTE+

LLnL

P C GTE GTE+

LLnL

092 1 074 11

lowast

(e)

Figure 4 Effect of GTE on the gene expression and protein stability of HER2 (a) SKOV-3 cells were treated with GTE (025 or 05mgmL)or the vehicle for 24 h The mRNA level of HER2 was measured by semiquantitative RT-PCR as described in Section 2 (b) SKOV-3 cellswere transfected with a luciferase gene plasmid construct driven by HER2 promoter (pHER2-luc) for 6 h and then treated with variousconcentrations of GTE (0 025 and 05mgmL) for 24 hThe activity of HER2 promoter was measured by a reporter gene assay as describedin Section 2 The relative light units (RLU) of luciferase activity were normalized against 120573-gal activity (c) SKOV-3 cells were pretreatedwith 20120583gmL of cycloheximide (CHX) for 30min and then treated with GTE (05mgmL) or the vehicle for 12 24 and 48 h Stability ofHER2 was determined by measuring the proteinrsquos half-life (d) SKOV-3 cells were treated with GTE (05mgmL) for 12 24 and 48 h Todetect polyubiquitinated HER2 (HER2-Ub

(n)) HER2 was immunoprecipitated and subjected to Western blot analysis using an antibody toubiquitin The total protein levels of HER2 and actin in the whole-cell extracts were also detected by Western blotting (e) SKOV-3 cellswere pretreated with proteasome inhibitor (LLnL) or the vehicle for 30min and then treated with GTE (05mgmL) for 24 h The proteinlevel of HER2 was measured by Western blotting P parental SKOV-3 cells C vehicle control Data are presented as the mean plusmn SD of threeindependent experiments lowast119875 lt 005 and lowastlowast119875 lt 001 versus the vehicle-treated control group

controls the expression of Ki-67 protein a proliferationmarker was significantly decreased in GTE-treated tumors(Figure 5(c)) indicating that GTE inhibited cell proliferationof SKOV-3 xenografted tumors in vivo

In our in vitro studies we showed that GTE inhib-ited cell proliferation and induced G1 cell cycle arrest inHER2-overexpressing cancer cells through the modulationof HER2 expression To determine the underlying molecularmechanisms of the GTE-mediated anticancer effect observed

in the SKOV-3 xenografted tumors tumor sections wereimmunostained for HER2 protein and cyclin D1 the firstcyclin that is activated during G1S phase progression Incomparison to the control group the staining intensitiesof HER2 and cyclin D1 were dramatically downregulatedin GTE-treated tumor cells (200mgkgday) (Figure 5(c))Together these data suggest that GTE inhibited tumor cellproliferation by inducing cell cycle arrest andmodulating theHER2 pathway in vitro and in vivo


0 5 10 15 20 25 30Days

Control (119899 = 7)200 mgkgday GTE (119899 = 5)1000 mgkgday GTE (119899 = 7)

0

400

800

1200

1600

Tum

or v

olum

e (m

m3)

(a)

25

20

15

10

Body

wei

ght (

g)

0 5 10 15 20 25 30Days


(b)

Control

GTE

Ki-67 Cyclin D1HER2

(c)

Figure 5 Effect of GTE on the growth of SKOV-3 xenografted tumors in vivo (a) Tumor growth rate was significantly slower in the GTE-treated group (200mgkgday 119899 = 5 or 1000mgkgday 119899 = 7) versus the control group (119899 = 7)The tumor volumes were estimated from thecalipermeasurements of three dimensions of the tumorThe estimated tumor volumeswere calculated as 119871times1198822times05 where119871 is themajor axisand119882 is tumor widthThe results are represented as themean plusmn SD (b)The body weight of nudemice was not significantly different betweenthe control and GTE-treated groups (c) Downregulation of Ki-67 HER2 and cyclin D1 expression by GTE in SKOV-3 xenografted tumors onnude miceThe IHC analysis was performed on SKOV-3-induced xenografted tumorsThe two representative specimens appear to show thatGTE-treatedmice (200mgkgday) have lower protein expression than vehicle controls for Ki-67 HER2 and cyclin D1 (400Xmagnification)

4 Discussion

HER2-overexpression is associatedwith a high risk for cancermetastasis and a poor response to antitumor therapies [4]Treatment with therapeutic agents that specifically targetcancer cells with HER2-overexpression such as lapatinib andtrastuzumab has improved clinical outcomes In additionto the anticancer agents a number of TCMs and botanicalproducts have been shown to be effective and useful adjuvantagents for the treatment of HER2-overexpressing cancer [56 26] Ganoderma tsugae (GT) one of the most commonspecies of Ganoderma cultivated in Taiwan has been shownto have antiproliferative effects on human cancer cells [15 1618] In this study we report for the first time that the extract ofGT (GTE) has a distinct growth-inhibitory effect on HER2-overexpressing cancer cells in vitro (Figures 1(a)ndash1(c)) and invivo (Figure 5(a))

Perturbation of cell cycle progression in cancer cells isa useful strategy to arrest cancer growth [28] Furthermore

cell cycle arrest also provides an occasion for cells to undergoeither repair or programmed cell death A number of TCMs(eg GT) exhibit marked growth-inhibitory effects on cancercells via disruption of cell cycle progression Previous reportsshow that GT inhibits cell proliferation by inducing cellcycle arrest in the G2M phase in Hep3B hepatoma andCOLO205 colorectal cancer cells [15 17] and in the S phasein H2303 lung adenocarcinoma cells [18] In this study ourin vitro results indicate that GTE treatment induces G1 phasearrest via modulation of cell cycle regulators (eg cyclinsD1 and E p21 and p27) in HER2-overexpressing SKOV-3ovarian cancer and BT-474 breast cancer cells (Figure 2 andSupplementary Figure S4) The varying effects of GTE on thecell cycle may be due to cell-type specificity andor resultfrom modulation of different signal transductions and cellcycle regulatory molecules

Two major therapeutic approaches to the treatment ofHER2-overexpressing cancers involve agents that curtailthe expression and activationphosphorylation of the HER2


receptor [29] In this study we demonstrate that GTEdownregulates both the level of HER2 and its phosphorylatedform in SKOV-3 BT-474 and SKBR-3 cells (Figure 3) Wesurmised that the inhibitory effect of GTE on the levels ofphospho-HER2may be due to its inhibition of the expressionof HER2 In agreement with this hypothesis we observeda significant decrease in the expression of HER2 mRNA(Figure 4(a)) and the activity of its promoter (Figure 4(b))following treatmentwithGTEMoreover we have establisheda number of HER2 promoter deletion constructs (F1 minus1067simminus103 F2minus871simminus103 F3minus495simminus103 and F4minus207simminus103) andfound that GTE interacts with the HER2 promoter in theminus871simminus495 region (unpublished data) Based on Genomatixsoftware predictions there are several putative transcriptionfactor binding sites located in this area such as T-cellfactor (TCF) forkhead-boxK2 (FOXK2) andGATA-bindingprotein 2 (GATA2) Therefore further studies are needed toclarify the molecular basis by which the transcription of theHER2 gene is regulated to ultimately aid in the developmentof better strategies for the treatment of cancers with HER2-overexpression

We also investigated the regulation of HER2 proteinstabilitydegradation as another possible explanation as tohow GTE controls HER2 protein expression We found thatthe half-life of theHER2protein is noticeably reduced byGTEin SKOV-3 (Figure 4(c)) and BT-474 cells (SupplementaryFigure S6B) This observation led us to hypothesize thatthe decreased stability of the HER2 protein may be dueto the induction of polyubiquitination of HER2 by GTE(Figure 4(d)) leading to its degradation by the proteasomecomplex We used LLnL a proteasome inhibitor to confirmthat the effect of GTE on the degradation of HER2 proteininvolves the activation of the ubiquitin-proteasome system(Figure 4(e)) Furthermore several molecules such as heatshock protein 90 (Hsp90) casitas B-lineage lymphoma (c-Cbl) and peptidyl-prolyl cistrans isomerase 1 (Pin1) arereported to be required for the maintenance of the stabilityand activation of HER2 [30ndash32] It would be worthwhileto determine if these molecules are involved in the GTE-induced degradationinstability of the HER2 protein

Generally cancer cells overexpressing HER2 respondpoorly to chemotherapeutic agents Suppression of the HER2pathway byHER2-targeting therapeutics potentiates the anti-cancer activity of chemotherapeutic agents in the treatment ofHER2-overexpressing cancers [25 33] A number of reportsshow that the combined usage of some extracts from TCMs(eg coptis rhizome and glycyrrhizae radix) with antitumoragents results in synergistic growth inhibition in cancer cells[34 35] It has also been reported that combining anticanceragents with GTE slows the growth rate of cancer cells [15 18]Herein we demonstrate for the first time that the combinedusage of GTE with taxol (Figure 1(d)) cisplatin (Supplemen-tary Figure S3) or doxorubicin (data not shown) results insynergistic growth inhibition of HER2-overexpressing cancercells These results indicate that GTE may be a promisingadjuvant therapeutic agent in the treatment of cancers withHER2-overexpression

In conclusion we provide a schematic presentation ofpossible molecular mechanisms in vitro and in vivo for the

Posttranslationalmodification

Cell proliferation

HER2 gene

HER2 mRNA

HER2 protein

Degradation

HER2

GTE Transcription

Translation

Proteasome pathway

HER2PI3KAktpathway

Figure 6 A schematic model of the GTE-mediated antiproliferativeeffect on HER2-overexpressing cancer cells Ligand stimulationinduces the activation of the HER2 receptor which in turn activatesthe PI3KAkt signaling pathway and then promotes cell growth andsurvival After GTE treatment the proliferation is inhibited becauseof an induction of cell cycle arrest The GTE-mediated growthrepression coincides with a reduction in the transcriptional activityofHER2 gene and an induction in the degradation of HER2 proteinleading to a downregulation of the HER2PI3KAkt pathway

inhibitory effects of GTE on the proliferation of HER2-overexpressing cancer cells (Figure 6) Our results indicatethat GTE induces G1 cell cycle arrest via regulation ofthe HER2PI3KAkt signaling pathway thereby leading toa reduction in the growth of cancer cells overexpressingHER2 Our data also demonstrate that the depletion of HER2protein by GTE involves an inhibition in the transcriptionalactivity of theHER2 gene and an increase in the proteasome-dependent degradation of the HER2 protein In additionwe have also shown that a combination of GTE with anti-cancer drugs (eg taxol cisplatin and doxorubicin) exertssynergistic growth-inhibitory effect onHER2-overexpressingcancer cells Taken together our findings suggest that GTEmay be a useful and effective adjuvant therapeutic agent forthe treatment of cancers that highly express HER2

Authorsrsquo Contribution

C-C Ou and J-W Li contributed equally to this study

Acknowledgments

This work was supported by Grants from the NationalScience Council (NSC100-2313-B-039-005-MY3) and partlyfrom the China Medical University (CMU) (CMU95-296)Taiwan The authors also wish to thank members of theMedical Research Core Facilities Center (Office of Researchamp Development CMU Taichung Taiwan) for their excellenttechnical support

References

[1] J Baselga and S M Swain ldquoNovel anticancer targets revisitingERBB2 and discovering ERBB3rdquo Nature Reviews Cancer vol 9no 7 pp 463ndash475 2009


[2] A CWolffM E HHammond J N Schwartz et al ldquoAmericanSociety of Clinical OncologyCollege of American Pathologistsguideline recommendations for human epidermal growth fac-tor receptor 2 testing in breast cancerrdquoArchives of Pathology andLaboratory Medicine vol 131 no 1 pp 18ndash43 2007

[3] E Verri P Guglielmini M Puntoni et al ldquoHER2neu onco-protein overexpression in epithelial ovarian cancer evaluationof its prevalence and prognostic significance clinical studyrdquoOncology vol 68 no 2-3 pp 154ndash161 2005

[4] D Yu andM C Hung ldquoOverexpression of ErbB2 in cancer andErbB2-targeting strategiesrdquo Oncogene vol 19 no 53 pp 6115ndash6121 2000

[5] J H Ju M J Jeon W Yang K M Lee H S Seo and I ShinldquoInduction of apoptotic cell death by Pharbitis nil extract inHER2-overexpressing MCF-7 cellsrdquo Journal of Ethnopharma-cology vol 133 no 1 pp 126ndash131 2011

[6] H P Kuo T C Chuang M H Yeh et al ldquoGrowth suppressionof HER2-overexpressing breast cancer cells by berberine viamodulation of the HER2PI3KAkt signaling pathwayrdquo Journalof Agricultural and Food Chemistry vol 59 no 15 pp 8216ndash8224 2011

[7] National Commission of Chinese Pharmacopoeia Pharma-copoeia of Peoples Republic of China ChinaMedical Science andTechnology Press Beijing China 2010

[8] J Da W Y Wu J J Hou et al ldquoComparison of two officinalChinese Pharmacopoeia species ofGanoderma based on chem-ical research with multiple technologies and chemometricsanalysisrdquo Journal of Chromatography A vol 1222 pp 59ndash702012

[9] X Zhou J Lin Y Yin et al ldquoGanodermataceae natural prod-ucts and their related pharmacological functionsrdquo The Ameri-can Journal of ChineseMedicine vol 35 no 4 pp 559ndash574 2007

[10] N S Lai R H Lin R S Lai U C Kun and S C Leu ldquoPreven-tion of autoantibody formation and prolonged survival in NewZealand BlackNew Zealand White F1 mice with an ancientChinese herb Ganoderma tsugaerdquo Lupus vol 10 no 7 pp 461ndash465 2001

[11] Y W Wu K D Chen and W C Lin ldquoEffect of Ganodermatsugae on chronically carbon tetrachloride-intoxicated ratsrdquoTheAmerican Journal of Chinese Medicine vol 32 no 6 pp 841ndash850 2004

[12] H H Ko C F Hung J P Wang and C N Lin ldquoAntiinflamma-tory triterpenoids and steroids from Ganoderma lucidum andG tsugaerdquo Phytochemistry vol 69 no 1 pp 234ndash239 2008

[13] J L Mau H C Lin and C C Chen ldquoAntioxidant properties ofseveralmedicinalmushroomsrdquo Journal of Agricultural and FoodChemistry vol 50 no 21 pp 6072ndash6077 2002

[14] G G L Yue K P Fung G M K Tse P C Leung and C BS Lau ldquoComparative studies of various Ganoderma speciesand their different parts with regard to their antitumor andimmunomodulating activities in vitrordquo Journal of Alternativeand Complementary Medicine vol 12 no 8 pp 777ndash789 2006

[15] S C Hsu C C Ou J W Li et al ldquoGanoderma tsugae extractsinhibit colorectal cancer cell growth via G

2M cell cycle arrestrdquo

Journal of Ethnopharmacology vol 120 no 3 pp 394ndash401 2008[16] S C Hsu C C Ou T C Chuang et al ldquoGanoderma tsugae

extract inhibits expression of epidermal growth factor receptorand angiogenesis in human epidermoid carcinoma cells in vitroand in vivordquo Cancer Letters vol 281 no 1 pp 108ndash116 2009

[17] K H Gan Y F Fann S H Hsu K W Kuo and C N LinldquoMediation of the cytotoxicity of lanostanoids and steroids of

Ganoderma tsugae through apoptosis and cell cyclerdquo Journal ofNatural Products vol 61 no 4 pp 485ndash487 1998

[18] Y H Yu H P Kuo H H Hsieh et al ldquoGanoderma tsugaeinduces S phase arrest and apoptosis in doxorubicin-resistantlung adenocarcinoma H230 3 cells via modulation of thePI3KAkt signaling pathwayrdquo Evidence-Based Complementaryand Alternative Medicine vol 2012 Article ID 371286 13 pages2012

[19] R Tilton A A Paiva J Q Guan et al ldquoA comprehensive plat-form for quality control of botanical drugs (PhytomicsQC) acase study of Huangqin Tang (HQT) and PHY906rdquo ChineseMedicine vol 5 article 30 2010

[20] B Hu H M An K P Shen et al ldquoPolygonum cuspidatumextract induces anoikis in hepatocarcinoma cells associatedwith generation of reactive oxygen species and downregulationof focal adhesion kinaserdquo Evidence-Based Complementary andAlternativeMedicine vol 2012 Article ID 607675 9 pages 2012

[21] T C Chuang S C Hsu Y T Cheng et al ldquoMagnolol down-regulates HER2 gene expression leading to inhibition of HER2-mediated metastatic potential in ovarian cancer cellsrdquo CancerLetters vol 311 no 1 pp 11ndash19 2011

[22] H P Kuo T C Chuang S C Tsai et al ldquoBerberine anisoquinoline alkaloid inhibits the metastatic potential of breastcancer cells via Akt pathway modulationrdquo Journal of Agricul-tural and Food Chemistry vol 60 no 38 pp 9649ndash9658 2012

[23] J W Li T C Chuang A H Yang C K Hsu and M C KaoldquoClinicopathological relevance ofHER2neu and a related gene-protein cubic regression correlation in colorectal adenocarcino-mas in Taiwanrdquo International Journal of Oncology vol 26 no 4pp 933ndash943 2005

[24] B Boh M Berovic J Zhang and L Zhi-Bin ldquoGanodermalucidum and its pharmaceutically active compoundsrdquo Biotech-nology Annual Review vol 13 pp 265ndash301 2007

[25] D Yu B Liu M Tan J Li S S Wang and M C HungldquoOverexpression of c-erbB-2neu in breast cancer cells confersincreased resistance to Taxol via mdr-1-independent mecha-nismsrdquo Oncogene vol 13 no 6 pp 1359ndash1365 1996

[26] L Y Shiu C H Liang Y S Huang H M Sheu and K WKuo ldquoDownregulation of HER2neu receptor by solamargineenhances anticancer drug-mediated cytotoxicity in breast can-cer cells with high-expressing HER2neurdquo Cell Biology andToxicology vol 24 no 1 pp 1ndash10 2008

[27] X Grana and E P Reddy ldquoCell cycle control in mammaliancells role of cyclins cyclin dependent kinases (CDKs) growthsuppressor genes and cyclin-dependent kinase inhibitors(CKIs)rdquo Oncogene vol 11 no 2 pp 211ndash219 1995

[28] K Collins T Jacks and N P Pavletich ldquoThe cell cycle andcancerrdquo Proceedings of the National Academy of Sciences UnitedStates of America vol 94 no 7 pp 2776ndash2778 1997

[29] K Imai and A Takaoka ldquoComparing antibody and small-molecule therapies for cancerrdquo Nature Reviews Cancer vol 6no 9 pp 714ndash727 2006

[30] S Chandarlapaty A Sawai Q Ye et al ldquoSNX2112 a syntheticheat shock protein 90 inhibitor has potent antitumor activ-ity against HER kinasemdashdependent cancersrdquo Clinical CancerResearch vol 14 no 1 pp 240ndash248 2008

[31] L N Klapper H Waterman M Sela and Y Yarden ldquoTumor-inhibitory antibodies to HER-2ErbB-2may act by recruiting c-Cbl and enhancing ubiquitination of HER-2rdquo Cancer Researchvol 60 no 13 pp 3384ndash3388 2000


[32] P B Lam L N Burga B P Wu E W Hofstatter K P Lu andG MWulf ldquoProlyl isomerase Pin1 is highly expressed in Her2-positive breast cancer and regulates erbB2 protein stabilityrdquoMolecular Cancer vol 7 article 91 2008

[33] D W Miles W H Harris C E Gillett et al ldquoEffect of c-erbB2 and estrogen receptor status on survival of womenwith primary breast cancer treated with adjuvant cyclophos-phamidemethotrexatefluorouracilrdquo International Journal ofCancer vol 84 no 4 pp 354ndash359 1999

[34] J Liu C He K Zhou et al ldquoCoptis extracts enhance theanticancer effect of estrogen receptor antagonists on humanbreast cancer cellsrdquo Biochemical and Biophysical Research Com-munications vol 378 no 2 pp 174ndash178 2009

[35] K Takara S Horibe Y Obata E Yoshikawa N Ohnishi andT Yokoyama ldquoEffects of 19 herbal extracts on the sensitivity topaclitaxel or 5-fluorouracil in HeLa cellsrdquo Biological and Phar-maceutical Bulletin vol 28 no 1 pp 138ndash142 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Aberrant upregulation ofHER2 is found in approximately25ndash30 of breast cancers [2] and in 6ndash50 of ovarian cancers[3] Patients with HER2-positive cancer have a high riskfor diminished effectiveness of cancer treatments increasedcancer metastasis and poor clinical outcomes [4] Thereforeinhibition of HER2 expression or its kinase activitymay be aneffective approach for the treatment of HER2-overexpressingcancers In fact a number of HER2-targeting agents includ-ing monoclonal antibodies (eg trastuzumab) and small-molecule tyrosine kinase inhibitors (eg lapatinib) havebeen developed for the treatment of cancers with HER2-overexpression [1] However there is still a need for noveltherapies to treatHER2-overexpressing cancers For exampletraditional Chinese medicine (TCM) and botanical productsare currently considered to be safer and may be used asalternative therapeutic agents for treatment of cancers thatoverexpress HER2 [5 6]

Ganoderma (also known as Lingzhi) has a long history ofuse in folkmedicines in Asian countriesGanoderma lucidum(GL) andGanoderma sinense (GS) listed in Chinese Pharma-copoeia (2010 edition) [7 8] are two of the most commonspecies of Ganoderma and have been used for medicinalpurposes in China for centuries The biological activitiesof GL and GS particularly their immunomodulatory andantitumor properties have been well documented [9] Inaddition Ganoderma tsugae (GT) another well-cultivatedspecies ofGanoderma has been shown to havemany biologi-cal and pharmacological properties such as antiautoantibodyformation [10] antifibrosis [11] antiinflammation [12] andantioxidation characteristics [13] A number of reports showthat GT has growth-inhibitory effects in a variety of humancancer cells such as MDA-MB-231 and MCF-7 breast cancercells [14] COLO 205 colorectal cancer cells [15] A431 epider-moid carcinoma cells [16] Hep3B hepatoma cells [17] andH23 and H2303 lung adenocarcinoma cells [18] AlthoughGT has antitumor activity in many human cancer cells themechanisms that underlie its growth-inhibitory effect onHER2-overexpressing cancer cells remain unclear

In this study we produced a quality assured extract of GT(GTE) and characterized its antitumor effects and relevantmolecular mechanisms in HER2-overexpressing cancer cellsin vitro and in vivo Our results show that GTE inhibits cancercell growth and induces cell cycle arrest via modulation ofthe HER2PI3KAkt signaling pathway We also show thatcombining GTE with taxol or cisplatin significantly slowsthe growth of HER2-overexpressing cancer cells indicatinga potential use of GTE in the treatment of cancers thatoverexpress HER2

2 Materials and Methods

21 Cell Culture Humanovarian carcinoma cell lines SKOV-3 (HER2high) and OVCAR-3 (HER2low) and breast carci-noma cell lines SKBR-3 (HER2high) and BT-474 (HER2high)were obtained from the American Type Culture Collection(ATCC Manassas VA USA) The MCF-7HER2 (HER2high)human breast carcinoma cell line (MCF-7 of an HER2-transfected stable line) was kindly provided by Dr M C

Hung (Department of Molecular and Cellular OncologyUniversity of TexasMDAndersonCancerCenterHoustonTX USA) The MDA-MB-435HER2 (HER2high) humanmelanoma cell line (MDA-MB-435 of an HER2-transfectedstable line) was kindly provided by Dr T D Way (Depart-ment of Biological Science and Technology China MedicalUniversity Taichung Taiwan) All cells were cultured inDMEMF12 medium (Gibco BRL Grand Island NY USA)supplemented with 10 fetal bovine serum (FBS) in ahumidified atmosphere of 5 CO

2at 37∘C

22 Chemicals andAntibodies The thiazolyl blue tetrazoliumbromide (MTT) cycloheximide (CHX) and N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL) were obtained fromSigma-Aldrich (St Louis MO USA) Antibodies againstcyclins D1 and E p21 p27 phospho-Akt (Ser308) Akt1 andubiquitin (Ub) were purchased from Santa Cruz Biotech-nology Inc (Santa Cruz CA USA) Antibodies againstphospho-PI3K PI3K phospho-Erk 12 and Erk 12 were pur-chased from Cell Signaling Technology Inc (Beverly MAUSA) Antibodies against phospho-HER2 (Ab-18) HER2(Ab-3) 120573-actin and Ki-67 (Clone MIB-1) were purchasedfrom Neomarkers Inc (Fremont CA USA) Calbiochem(San Diego CA USA) Chemicon International Inc (Temec-ula CA USA) and Dakocytomation Inc (Carpinteria CAUSA) respectively Taxol (paclitaxel) was purchased fromBristol-Myers Squibb (Wallingford CT USA) and cisplatinwas purchased from Pharmacia amp Upjohn SpA (Via RobertKoch 12 Milan Italy)

23 Preparation of Ganoderma tsugae Extracts Ganodermatsugae (GT) was kindly provided by the Luo-Gui-Ying FungiAgriculture Farm (with a registered name of Tien-ShenLingzhi) Taoyuan Taiwan The extract of GT (GTE) wasprepared as described previously [15] Briefly the powder ofthe GT fruiting body (5 g) was soaked in 999 methanol(200mL) mixed and shaken for 24 h on a rotating shakerAfter centrifugation the supernatant was poured through fil-ter paper (Whatman cat no 1001-110) and the residues wereextracted with methanol two additional times as mentionedabove The filtrates were collected together and subjectedto concentration under reduced pressure (ie evaporated todryness under reduced pressure) to produce a brown gel-likeGT extract (GTE) The yield was approximately 30 TheGTE was then prepared as a stock solution with methanolsolvent (100mgmL) and stored at minus80∘C until use Foranimal experiments the dry GTE was redissolved in ethanoland diluted with a suspension solution (745 corn oil16 PEG-400 4 Tween-80 4 Cremophor EL and 15Ethanol vv) to a concentration of 10mgmL

24 Quality Control of GTEs via Bioresponse FingerprintingThe quality of the GTEs was assessed as described previously[18 19] Briefly the genomic bioresponse to the GTEs wasdetermined in SKOV-3 cells treated with 05mgmL of GTEThe total RNA was extracted from the GTE-treated cellscleaned with a commercial kit (Qiagene RNA extractionkit cat no 75144) and then used to obtain transcription










2






3 Results








SKOV-3



0

20

40

60

80

100

120

Relat

ive p

rolif

erat

ion

()

24 h 48 h 72 h


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

(a)

0

1

2

3

4

5

6

7

05 1 15 2 25 3Days

Cel

l num

ber (times106)

SKOV-3

01 mgmL GTE


ParentalControl

(b)

025 mgmL 05 mgmL

ControlParental

SKOV-3

(c)

0

20

40

60

80

100

120

140

160

0

20

40

60

80

100

120

140

160

0 1 10 100 1000


Taxol (ngmL)0 25 5 10 20



lowast

lowastlowast

lowastlowastlowast

Relat

ive p

rolif

erat

ion

()

Relat

ive p

rolif

erat

ion

()

SKOV-3 SKOV-3

(d)





250

200

150

100

50

00 200 400 600 800 1000

Parental

Sub-G1 033G1 4045S 3301G2M 2621

PI

Cou

nt

250

200

150

100

50

00 200 400 600 800 1000

PIC

ount

Sub-G1 05G1 449S 2952G2M 2508

Control

250

200

150

100

50

00 200 400 600 800 1000

PI

Cou

nt

Sub-G1 068G1 4803S 3258G2M 1871

025 mgmL

250

200

150

100

50

00 200 400 600 800 1000

PI

Cou

ntSub-G1 102G1 6892S 1899G2M 1107

05 mgmL

(a)

1 202 791

1 261 441

1

1 139

242

282

298

1 052 046 1 035 036

1 078 087 1 094 059

16 h 24 h0 025 05 0 025 05

Actin

p21

p27

Cyclin E

Cyclin D1

GTE (mgmL)

SKOV-3

(b)

Cyclin D1 Cyclin E


p27 p21

002040608

11214

002040608

11214

Fold

Fold

Fold

Fold

01234567

02468

1012

16 h 24 h16 h 24 h

16 h 24 h 16 h 24 h

(c)






002040608

11214

Fold

0 01 025 05 075 1

0 01 025 05 075 1

GTE (mgmL)

GTE (mgmL)

HER2

Actin1 09 061047038011

lowast

lowastlowast

lowast

SKOV-3

SKOV-3

(a)

002040608

11214

Fold


1 104 071 057 051 043

0 6 12 24 48 72

0 6 12 24 48 72

(h)

SKOV-3

SKOV-3

GTE (05 mgmL)

HER2

Actin

lowast lowastlowast

lowastlowast

(b)

1 062 026

0 025 05 0 025 05

1 054 019

1 071 009

1 061 043

1 059 042

1 09 062

1 102 083

1 097 093

p-HER2

p-H

ER2


HER2

HER

2p-Erk 12

p-Er

k 1

2

Erk 12

Erk

12Actin

p-PI3K

p-PI

3K

PI3K

PI3K

p-Akt

p-A

kt

Akt

AktActin 0

02040608

11214

Fold

Control025 mgmL

05 mgmL


lowastlowast

lowastlowast

lowast

lowast lowastlowast

SKOV-3 SKOV-3

(c)

1 065 018

1 101 089

1 128 114

1 101 105

1 052 015

1 068 072

1 161 171

1 091 103

1 083 042 1 078 056

1 041 013

0 025 05 0 025 05

1 036 003

SKBR-3 BT-474

p-HER2


HER2

p-Erk 12

Erk 12

p-Akt

Akt

Actin

(d)









GTE (mgmL)

Fold

002040608

112 SKOV-3

HER2

GAPDHSKOV-3

GTE (mgmL)

P 0 025 05

P 0 025 05

101 1 032 024

(a)

800006000040000

900060003000

0

GTE (mgmL)

lowastlowast

lowastlowast

SKOV-3

P 0 025 05

RLU

120573-g

al

(b)

HER2

Actin

SKOV-3

0

02

04

06

08

1

12

Rela

tive i

nten

sity

Trendline (CHX)

48 24 12 0 12 24 48 (h)

032 083 08 1 084 045 008

CHX

CHX

CHX + GTE


CHX + GTE


(c)

GTE (05 mgmL)

1701301007055

40

35

25

IP HER2IB Ub

IB HER2

IB actin

(kD

a)

SKOV-3

0 12 24 48 (h)

(d)

HER2

Actin

SKOV-3

002040608

11214

Fold

P C GTE

GTE+

LLnL

P C GTE GTE+

LLnL

092 1 074 11

lowast

(e)







0 5 10 15 20 25 30Days


0

400

800

1200

1600

Tum

or v

olum

e (m

m3)

(a)

25

20

15

10

Body

wei

ght (

g)

0 5 10 15 20 25 30Days


(b)

Control

GTE

Ki-67 Cyclin D1HER2

(c)


4 Discussion











Cell proliferation

HER2 gene

HER2 mRNA

HER2 protein

Degradation

HER2

GTE Transcription

Translation

Proteasome pathway

HER2PI3KAktpathway





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























2






3 Results








SKOV-3



0

20

40

60

80

100

120

Relat

ive p

rolif

erat

ion

()

24 h 48 h 72 h


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

(a)

0

1

2

3

4

5

6

7

05 1 15 2 25 3Days

Cel

l num

ber (times106)

SKOV-3

01 mgmL GTE


ParentalControl

(b)

025 mgmL 05 mgmL

ControlParental

SKOV-3

(c)

0

20

40

60

80

100

120

140

160

0

20

40

60

80

100

120

140

160

0 1 10 100 1000


Taxol (ngmL)0 25 5 10 20



lowast

lowastlowast

lowastlowastlowast

Relat

ive p

rolif

erat

ion

()

Relat

ive p

rolif

erat

ion

()

SKOV-3 SKOV-3

(d)





250

200

150

100

50

00 200 400 600 800 1000

Parental

Sub-G1 033G1 4045S 3301G2M 2621

PI

Cou

nt

250

200

150

100

50

00 200 400 600 800 1000

PIC

ount

Sub-G1 05G1 449S 2952G2M 2508

Control

250

200

150

100

50

00 200 400 600 800 1000

PI

Cou

nt

Sub-G1 068G1 4803S 3258G2M 1871

025 mgmL

250

200

150

100

50

00 200 400 600 800 1000

PI

Cou

ntSub-G1 102G1 6892S 1899G2M 1107

05 mgmL

(a)

1 202 791

1 261 441

1

1 139

242

282

298

1 052 046 1 035 036

1 078 087 1 094 059

16 h 24 h0 025 05 0 025 05

Actin

p21

p27

Cyclin E

Cyclin D1

GTE (mgmL)

SKOV-3

(b)

Cyclin D1 Cyclin E


p27 p21

002040608

11214

002040608

11214

Fold

Fold

Fold

Fold

01234567

02468

1012

16 h 24 h16 h 24 h

16 h 24 h 16 h 24 h

(c)






002040608

11214

Fold

0 01 025 05 075 1

0 01 025 05 075 1

GTE (mgmL)

GTE (mgmL)

HER2

Actin1 09 061047038011

lowast

lowastlowast

lowast

SKOV-3

SKOV-3

(a)

002040608

11214

Fold


1 104 071 057 051 043

0 6 12 24 48 72

0 6 12 24 48 72

(h)

SKOV-3

SKOV-3

GTE (05 mgmL)

HER2

Actin

lowast lowastlowast

lowastlowast

(b)

1 062 026

0 025 05 0 025 05

1 054 019

1 071 009

1 061 043

1 059 042

1 09 062

1 102 083

1 097 093

p-HER2

p-H

ER2


HER2

HER

2p-Erk 12

p-Er

k 1

2

Erk 12

Erk

12Actin

p-PI3K

p-PI

3K

PI3K

PI3K

p-Akt

p-A

kt

Akt

AktActin 0

02040608

11214

Fold

Control025 mgmL

05 mgmL


lowastlowast

lowastlowast

lowast

lowast lowastlowast

SKOV-3 SKOV-3

(c)

1 065 018

1 101 089

1 128 114

1 101 105

1 052 015

1 068 072

1 161 171

1 091 103

1 083 042 1 078 056

1 041 013

0 025 05 0 025 05

1 036 003

SKBR-3 BT-474

p-HER2


HER2

p-Erk 12

Erk 12

p-Akt

Akt

Actin

(d)









GTE (mgmL)

Fold

002040608

112 SKOV-3

HER2

GAPDHSKOV-3

GTE (mgmL)

P 0 025 05

P 0 025 05

101 1 032 024

(a)

800006000040000

900060003000

0

GTE (mgmL)

lowastlowast

lowastlowast

SKOV-3

P 0 025 05

RLU

120573-g

al

(b)

HER2

Actin

SKOV-3

0

02

04

06

08

1

12

Rela

tive i

nten

sity

Trendline (CHX)

48 24 12 0 12 24 48 (h)

032 083 08 1 084 045 008

CHX

CHX

CHX + GTE


CHX + GTE


(c)

GTE (05 mgmL)

1701301007055

40

35

25

IP HER2IB Ub

IB HER2

IB actin

(kD

a)

SKOV-3

0 12 24 48 (h)

(d)

HER2

Actin

SKOV-3

002040608

11214

Fold

P C GTE

GTE+

LLnL

P C GTE GTE+

LLnL

092 1 074 11

lowast

(e)







0 5 10 15 20 25 30Days


0

400

800

1200

1600

Tum

or v

olum

e (m

m3)

(a)

25

20

15

10

Body

wei

ght (

g)

0 5 10 15 20 25 30Days


(b)

Control

GTE

Ki-67 Cyclin D1HER2

(c)


4 Discussion











Cell proliferation

HER2 gene

HER2 mRNA

HER2 protein

Degradation

HER2

GTE Transcription

Translation

Proteasome pathway

HER2PI3KAktpathway





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















3 Results








SKOV-3



0

20

40

60

80

100

120

Relat

ive p

rolif

erat

ion

()

24 h 48 h 72 h


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

(a)

0

1

2

3

4

5

6

7

05 1 15 2 25 3Days

Cel

l num

ber (times106)

SKOV-3

01 mgmL GTE


ParentalControl

(b)

025 mgmL 05 mgmL

ControlParental

SKOV-3

(c)

0

20

40

60

80

100

120

140

160

0

20

40

60

80

100

120

140

160

0 1 10 100 1000


Taxol (ngmL)0 25 5 10 20



lowast

lowastlowast

lowastlowastlowast

Relat

ive p

rolif

erat

ion

()

Relat

ive p

rolif

erat

ion

()

SKOV-3 SKOV-3

(d)





250

200

150

100

50

00 200 400 600 800 1000

Parental

Sub-G1 033G1 4045S 3301G2M 2621

PI

Cou

nt

250

200

150

100

50

00 200 400 600 800 1000

PIC

ount

Sub-G1 05G1 449S 2952G2M 2508

Control

250

200

150

100

50

00 200 400 600 800 1000

PI

Cou

nt

Sub-G1 068G1 4803S 3258G2M 1871

025 mgmL

250

200

150

100

50

00 200 400 600 800 1000

PI

Cou

ntSub-G1 102G1 6892S 1899G2M 1107

05 mgmL

(a)

1 202 791

1 261 441

1

1 139

242

282

298

1 052 046 1 035 036

1 078 087 1 094 059

16 h 24 h0 025 05 0 025 05

Actin

p21

p27

Cyclin E

Cyclin D1

GTE (mgmL)

SKOV-3

(b)

Cyclin D1 Cyclin E


p27 p21

002040608

11214

002040608

11214

Fold

Fold

Fold

Fold

01234567

02468

1012

16 h 24 h16 h 24 h

16 h 24 h 16 h 24 h

(c)






002040608

11214

Fold

0 01 025 05 075 1

0 01 025 05 075 1

GTE (mgmL)

GTE (mgmL)

HER2

Actin1 09 061047038011

lowast

lowastlowast

lowast

SKOV-3

SKOV-3

(a)

002040608

11214

Fold


1 104 071 057 051 043

0 6 12 24 48 72

0 6 12 24 48 72

(h)

SKOV-3

SKOV-3

GTE (05 mgmL)

HER2

Actin

lowast lowastlowast

lowastlowast

(b)

1 062 026

0 025 05 0 025 05

1 054 019

1 071 009

1 061 043

1 059 042

1 09 062

1 102 083

1 097 093

p-HER2

p-H

ER2


HER2

HER

2p-Erk 12

p-Er

k 1

2

Erk 12

Erk

12Actin

p-PI3K

p-PI

3K

PI3K

PI3K

p-Akt

p-A

kt

Akt

AktActin 0

02040608

11214

Fold

Control025 mgmL

05 mgmL


lowastlowast

lowastlowast

lowast

lowast lowastlowast

SKOV-3 SKOV-3

(c)

1 065 018

1 101 089

1 128 114

1 101 105

1 052 015

1 068 072

1 161 171

1 091 103

1 083 042 1 078 056

1 041 013

0 025 05 0 025 05

1 036 003

SKBR-3 BT-474

p-HER2


HER2

p-Erk 12

Erk 12

p-Akt

Akt

Actin

(d)









GTE (mgmL)

Fold

002040608

112 SKOV-3

HER2

GAPDHSKOV-3

GTE (mgmL)

P 0 025 05

P 0 025 05

101 1 032 024

(a)

800006000040000

900060003000

0

GTE (mgmL)

lowastlowast

lowastlowast

SKOV-3

P 0 025 05

RLU

120573-g

al

(b)

HER2

Actin

SKOV-3

0

02

04

06

08

1

12

Rela

tive i

nten

sity

Trendline (CHX)

48 24 12 0 12 24 48 (h)

032 083 08 1 084 045 008

CHX

CHX

CHX + GTE


CHX + GTE


(c)

GTE (05 mgmL)

1701301007055

40

35

25

IP HER2IB Ub

IB HER2

IB actin

(kD

a)

SKOV-3

0 12 24 48 (h)

(d)

HER2

Actin

SKOV-3

002040608

11214

Fold

P C GTE

GTE+

LLnL

P C GTE GTE+

LLnL

092 1 074 11

lowast

(e)







0 5 10 15 20 25 30Days


0

400

800

1200

1600

Tum

or v

olum

e (m

m3)

(a)

25

20

15

10

Body

wei

ght (

g)

0 5 10 15 20 25 30Days


(b)

Control

GTE

Ki-67 Cyclin D1HER2

(c)


4 Discussion











Cell proliferation

HER2 gene

HER2 mRNA

HER2 protein

Degradation

HER2

GTE Transcription

Translation

Proteasome pathway

HER2PI3KAktpathway





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SKOV-3



0

20

40

60

80

100

120

Relat

ive p

rolif

erat

ion

()

24 h 48 h 72 h


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

(a)

0

1

2

3

4

5

6

7

05 1 15 2 25 3Days

Cel

l num

ber (times106)

SKOV-3

01 mgmL GTE


ParentalControl

(b)

025 mgmL 05 mgmL

ControlParental

SKOV-3

(c)

0

20

40

60

80

100

120

140

160

0

20

40

60

80

100

120

140

160

0 1 10 100 1000


Taxol (ngmL)0 25 5 10 20



lowast

lowastlowast

lowastlowastlowast

Relat

ive p

rolif

erat

ion

()

Relat

ive p

rolif

erat

ion

()

SKOV-3 SKOV-3

(d)





250

200

150

100

50

00 200 400 600 800 1000

Parental

Sub-G1 033G1 4045S 3301G2M 2621

PI

Cou

nt

250

200

150

100

50

00 200 400 600 800 1000

PIC

ount

Sub-G1 05G1 449S 2952G2M 2508

Control

250

200

150

100

50

00 200 400 600 800 1000

PI

Cou

nt

Sub-G1 068G1 4803S 3258G2M 1871

025 mgmL

250

200

150

100

50

00 200 400 600 800 1000

PI

Cou

ntSub-G1 102G1 6892S 1899G2M 1107

05 mgmL

(a)

1 202 791

1 261 441

1

1 139

242

282

298

1 052 046 1 035 036

1 078 087 1 094 059

16 h 24 h0 025 05 0 025 05

Actin

p21

p27

Cyclin E

Cyclin D1

GTE (mgmL)

SKOV-3

(b)

Cyclin D1 Cyclin E


p27 p21

002040608

11214

002040608

11214

Fold

Fold

Fold

Fold

01234567

02468

1012

16 h 24 h16 h 24 h

16 h 24 h 16 h 24 h

(c)






002040608

11214

Fold

0 01 025 05 075 1

0 01 025 05 075 1

GTE (mgmL)

GTE (mgmL)

HER2

Actin1 09 061047038011

lowast

lowastlowast

lowast

SKOV-3

SKOV-3

(a)

002040608

11214

Fold


1 104 071 057 051 043

0 6 12 24 48 72

0 6 12 24 48 72

(h)

SKOV-3

SKOV-3

GTE (05 mgmL)

HER2

Actin

lowast lowastlowast

lowastlowast

(b)

1 062 026

0 025 05 0 025 05

1 054 019

1 071 009

1 061 043

1 059 042

1 09 062

1 102 083

1 097 093

p-HER2

p-H

ER2


HER2

HER

2p-Erk 12

p-Er

k 1

2

Erk 12

Erk

12Actin

p-PI3K

p-PI

3K

PI3K

PI3K

p-Akt

p-A

kt

Akt

AktActin 0

02040608

11214

Fold

Control025 mgmL

05 mgmL


lowastlowast

lowastlowast

lowast

lowast lowastlowast

SKOV-3 SKOV-3

(c)

1 065 018

1 101 089

1 128 114

1 101 105

1 052 015

1 068 072

1 161 171

1 091 103

1 083 042 1 078 056

1 041 013

0 025 05 0 025 05

1 036 003

SKBR-3 BT-474

p-HER2


HER2

p-Erk 12

Erk 12

p-Akt

Akt

Actin

(d)









GTE (mgmL)

Fold

002040608

112 SKOV-3

HER2

GAPDHSKOV-3

GTE (mgmL)

P 0 025 05

P 0 025 05

101 1 032 024

(a)

800006000040000

900060003000

0

GTE (mgmL)

lowastlowast

lowastlowast

SKOV-3

P 0 025 05

RLU

120573-g

al

(b)

HER2

Actin

SKOV-3

0

02

04

06

08

1

12

Rela

tive i

nten

sity

Trendline (CHX)

48 24 12 0 12 24 48 (h)

032 083 08 1 084 045 008

CHX

CHX

CHX + GTE


CHX + GTE


(c)

GTE (05 mgmL)

1701301007055

40

35

25

IP HER2IB Ub

IB HER2

IB actin

(kD

a)

SKOV-3

0 12 24 48 (h)

(d)

HER2

Actin

SKOV-3

002040608

11214

Fold

P C GTE

GTE+

LLnL

P C GTE GTE+

LLnL

092 1 074 11

lowast

(e)







0 5 10 15 20 25 30Days


0

400

800

1200

1600

Tum

or v

olum

e (m

m3)

(a)

25

20

15

10

Body

wei

ght (

g)

0 5 10 15 20 25 30Days


(b)

Control

GTE

Ki-67 Cyclin D1HER2

(c)


4 Discussion











Cell proliferation

HER2 gene

HER2 mRNA

HER2 protein

Degradation

HER2

GTE Transcription

Translation

Proteasome pathway

HER2PI3KAktpathway





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















250

200

150

100

50

00 200 400 600 800 1000

Parental

Sub-G1 033G1 4045S 3301G2M 2621

PI

Cou

nt

250

200

150

100

50

00 200 400 600 800 1000

PIC

ount

Sub-G1 05G1 449S 2952G2M 2508

Control

250

200

150

100

50

00 200 400 600 800 1000

PI

Cou

nt

Sub-G1 068G1 4803S 3258G2M 1871

025 mgmL

250

200

150

100

50

00 200 400 600 800 1000

PI

Cou

ntSub-G1 102G1 6892S 1899G2M 1107

05 mgmL

(a)

1 202 791

1 261 441

1

1 139

242

282

298

1 052 046 1 035 036

1 078 087 1 094 059

16 h 24 h0 025 05 0 025 05

Actin

p21

p27

Cyclin E

Cyclin D1

GTE (mgmL)

SKOV-3

(b)

Cyclin D1 Cyclin E


p27 p21

002040608

11214

002040608

11214

Fold

Fold

Fold

Fold

01234567

02468

1012

16 h 24 h16 h 24 h

16 h 24 h 16 h 24 h

(c)






002040608

11214

Fold

0 01 025 05 075 1

0 01 025 05 075 1

GTE (mgmL)

GTE (mgmL)

HER2

Actin1 09 061047038011

lowast

lowastlowast

lowast

SKOV-3

SKOV-3

(a)

002040608

11214

Fold


1 104 071 057 051 043

0 6 12 24 48 72

0 6 12 24 48 72

(h)

SKOV-3

SKOV-3

GTE (05 mgmL)

HER2

Actin

lowast lowastlowast

lowastlowast

(b)

1 062 026

0 025 05 0 025 05

1 054 019

1 071 009

1 061 043

1 059 042

1 09 062

1 102 083

1 097 093

p-HER2

p-H

ER2


HER2

HER

2p-Erk 12

p-Er

k 1

2

Erk 12

Erk

12Actin

p-PI3K

p-PI

3K

PI3K

PI3K

p-Akt

p-A

kt

Akt

AktActin 0

02040608

11214

Fold

Control025 mgmL

05 mgmL


lowastlowast

lowastlowast

lowast

lowast lowastlowast

SKOV-3 SKOV-3

(c)

1 065 018

1 101 089

1 128 114

1 101 105

1 052 015

1 068 072

1 161 171

1 091 103

1 083 042 1 078 056

1 041 013

0 025 05 0 025 05

1 036 003

SKBR-3 BT-474

p-HER2


HER2

p-Erk 12

Erk 12

p-Akt

Akt

Actin

(d)









GTE (mgmL)

Fold

002040608

112 SKOV-3

HER2

GAPDHSKOV-3

GTE (mgmL)

P 0 025 05

P 0 025 05

101 1 032 024

(a)

800006000040000

900060003000

0

GTE (mgmL)

lowastlowast

lowastlowast

SKOV-3

P 0 025 05

RLU

120573-g

al

(b)

HER2

Actin

SKOV-3

0

02

04

06

08

1

12

Rela

tive i

nten

sity

Trendline (CHX)

48 24 12 0 12 24 48 (h)

032 083 08 1 084 045 008

CHX

CHX

CHX + GTE


CHX + GTE


(c)

GTE (05 mgmL)

1701301007055

40

35

25

IP HER2IB Ub

IB HER2

IB actin

(kD

a)

SKOV-3

0 12 24 48 (h)

(d)

HER2

Actin

SKOV-3

002040608

11214

Fold

P C GTE

GTE+

LLnL

P C GTE GTE+

LLnL

092 1 074 11

lowast

(e)







0 5 10 15 20 25 30Days


0

400

800

1200

1600

Tum

or v

olum

e (m

m3)

(a)

25

20

15

10

Body

wei

ght (

g)

0 5 10 15 20 25 30Days


(b)

Control

GTE

Ki-67 Cyclin D1HER2

(c)


4 Discussion











Cell proliferation

HER2 gene

HER2 mRNA

HER2 protein

Degradation

HER2

GTE Transcription

Translation

Proteasome pathway

HER2PI3KAktpathway





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















002040608

11214

Fold

0 01 025 05 075 1

0 01 025 05 075 1

GTE (mgmL)

GTE (mgmL)

HER2

Actin1 09 061047038011

lowast

lowastlowast

lowast

SKOV-3

SKOV-3

(a)

002040608

11214

Fold


1 104 071 057 051 043

0 6 12 24 48 72

0 6 12 24 48 72

(h)

SKOV-3

SKOV-3

GTE (05 mgmL)

HER2

Actin

lowast lowastlowast

lowastlowast

(b)

1 062 026

0 025 05 0 025 05

1 054 019

1 071 009

1 061 043

1 059 042

1 09 062

1 102 083

1 097 093

p-HER2

p-H

ER2


HER2

HER

2p-Erk 12

p-Er

k 1

2

Erk 12

Erk

12Actin

p-PI3K

p-PI

3K

PI3K

PI3K

p-Akt

p-A

kt

Akt

AktActin 0

02040608

11214

Fold

Control025 mgmL

05 mgmL


lowastlowast

lowastlowast

lowast

lowast lowastlowast

SKOV-3 SKOV-3

(c)

1 065 018

1 101 089

1 128 114

1 101 105

1 052 015

1 068 072

1 161 171

1 091 103

1 083 042 1 078 056

1 041 013

0 025 05 0 025 05

1 036 003

SKBR-3 BT-474

p-HER2


HER2

p-Erk 12

Erk 12

p-Akt

Akt

Actin

(d)









GTE (mgmL)

Fold

002040608

112 SKOV-3

HER2

GAPDHSKOV-3

GTE (mgmL)

P 0 025 05

P 0 025 05

101 1 032 024

(a)

800006000040000

900060003000

0

GTE (mgmL)

lowastlowast

lowastlowast

SKOV-3

P 0 025 05

RLU

120573-g

al

(b)

HER2

Actin

SKOV-3

0

02

04

06

08

1

12

Rela

tive i

nten

sity

Trendline (CHX)

48 24 12 0 12 24 48 (h)

032 083 08 1 084 045 008

CHX

CHX

CHX + GTE


CHX + GTE


(c)

GTE (05 mgmL)

1701301007055

40

35

25

IP HER2IB Ub

IB HER2

IB actin

(kD

a)

SKOV-3

0 12 24 48 (h)

(d)

HER2

Actin

SKOV-3

002040608

11214

Fold

P C GTE

GTE+

LLnL

P C GTE GTE+

LLnL

092 1 074 11

lowast

(e)







0 5 10 15 20 25 30Days


0

400

800

1200

1600

Tum

or v

olum

e (m

m3)

(a)

25

20

15

10

Body

wei

ght (

g)

0 5 10 15 20 25 30Days


(b)

Control

GTE

Ki-67 Cyclin D1HER2

(c)


4 Discussion











Cell proliferation

HER2 gene

HER2 mRNA

HER2 protein

Degradation

HER2

GTE Transcription

Translation

Proteasome pathway

HER2PI3KAktpathway





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















GTE (mgmL)

Fold

002040608

112 SKOV-3

HER2

GAPDHSKOV-3

GTE (mgmL)

P 0 025 05

P 0 025 05

101 1 032 024

(a)

800006000040000

900060003000

0

GTE (mgmL)

lowastlowast

lowastlowast

SKOV-3

P 0 025 05

RLU

120573-g

al

(b)

HER2

Actin

SKOV-3

0

02

04

06

08

1

12

Rela

tive i

nten

sity

Trendline (CHX)

48 24 12 0 12 24 48 (h)

032 083 08 1 084 045 008

CHX

CHX

CHX + GTE


CHX + GTE


(c)

GTE (05 mgmL)

1701301007055

40

35

25

IP HER2IB Ub

IB HER2

IB actin

(kD

a)

SKOV-3

0 12 24 48 (h)

(d)

HER2

Actin

SKOV-3

002040608

11214

Fold

P C GTE

GTE+

LLnL

P C GTE GTE+

LLnL

092 1 074 11

lowast

(e)







0 5 10 15 20 25 30Days


0

400

800

1200

1600

Tum

or v

olum

e (m

m3)

(a)

25

20

15

10

Body

wei

ght (

g)

0 5 10 15 20 25 30Days


(b)

Control

GTE

Ki-67 Cyclin D1HER2

(c)


4 Discussion











Cell proliferation

HER2 gene

HER2 mRNA

HER2 protein

Degradation

HER2

GTE Transcription

Translation

Proteasome pathway

HER2PI3KAktpathway





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 5 10 15 20 25 30Days


0

400

800

1200

1600

Tum

or v

olum

e (m

m3)

(a)

25

20

15

10

Body

wei

ght (

g)

0 5 10 15 20 25 30Days


(b)

Control

GTE

Ki-67 Cyclin D1HER2

(c)


4 Discussion











Cell proliferation

HER2 gene

HER2 mRNA

HER2 protein

Degradation

HER2

GTE Transcription

Translation

Proteasome pathway

HER2PI3KAktpathway





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Cell proliferation

HER2 gene

HER2 mRNA

HER2 protein

Degradation

HER2

GTE Transcription

Translation

Proteasome pathway

HER2PI3KAktpathway





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Ganoderma tsugae Extract Inhibits Growth...

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