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© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Carcinogenesis  vol.35 no.3 pp.683–691, 2014doi:10.1093/carcin/bgt365

 Advance Access publication November 26, 2013

[6]-Shogaol inhibits growth and induces apoptosis of non-small cell lung cancer cellsby directly regulating Akt1/2

Myoung Ok Kim1,2,†, Mee-Hyun Lee1,3,†, Naomi Oi1,†,Sung-Hyun Kim1,2,†, Ki Beom Bae1, Zunnan Huang1,4,Dong Joon Kim1,5, Kanamata Reddy1, Sung-Young Lee1,6,

Si Jun Park

2

, Jae Young Kim

2

, Hua Xie

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, JoydebKumar Kundu8, Zae Young Ryoo2, Ann M. Bode1,Young-Joon Surh3 and Zigang Dong1,6,*

1The Hormel Institute, University of Minnesota, MN 55912, USA,2Kyungpook National University, School of Animal BT Science, Centerfor Laboratory Animal Resources, Department of Biochemistry, Schoolof Dentistry, Daegu 702-701, South Korea, 6WCU Department ofMolecular Medicine and Biopharmaceutical Sciences, Graduate Schoolof Convergence Science and Technology and College of Pharmacy,Seoul National University, Seoul 151-742, South Korea and 8College ofPharmacy, Keimyung University, Daegu 704-701, South Korea

3Present address: Integrated Research Institute of PharmaceuticalSciences, College of Pharmacy, The Catholic University of Korea,43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, South Korea4Present address: China-America Cancer Research Institute, GuangdongMedical College, Dongguan, Guangdong 523808, PR China5Present address: Medical Genomics Research Center, Korea ResearchInstitute of Bioscience and Biotechnology, Daejeon 306-809, South Korea7Present address: State Key Laboratory of Drug Research, ShanghaiInstitute of Materia Medica, Chinese Academy of Sciences, Shanghai201203, PR China

*To whom correspondence should be addressed. The Hormel Institute,University of Minnesota, 801 16th Avenue NE, Austin, MN 55912.Tel: +1 507 437 9600; Fax: +1 507 437 9606;Email: zgdong@hi.umn.edu

Non-small cell lung cancer (NSCLC) is the leading cause ofcancer mortality worldwide. Despite progress in developingchemotherapeutics for the treatment of NSCLC, primary andsecondary resistance limits therapeutic success. NSCLC cellsexhibit multiple mutations in the epidermal growth factorreceptor (EGFR), which cause aberrant activation of diverse

cell signaling pathways. Therefore, suppression of the inap-propriate amplification of EGFR downstream signaling cas-cades is considered to be a rational therapeutic and preventivestrategy for the management of NSCLC. Our initial moleculartarget–oriented virtual screening revealed that the ginger com-ponents, including [6]-shogaol, [6]-paradol and [6]-gingerol,seem to be potential candidates for the prevention and treat-ment of NSCLC. Among the compounds, [6]-shogaol showedthe greatest inhibitory effects on the NSCLC cell prolifera-tion and anchorage-independent growth. [6]-Shogaol inducedcell cycle arrest (G1 or G2/M) and apoptosis. Furthermore,[6]-shogaol inhibited Akt kinase activity, a downstream media-tor of EGFR signaling, by binding with an allosteric site of Akt.In NCI-H1650 lung cancer cells, [6]-shogaol reduced the con-stitutive phosphorylation of signal transducer and activator of

transcription-3 (STAT3) and decreased the expression of cyclinD1/3, which are target proteins in the Akt signaling pathway.The induction of apoptosis in NCI-H1650 cells by [6]-shogaolcorresponded with the cleavage of caspase-3 and caspase-7.Moreover, intraperitoneal administration of [6]-shogaol inhib-ited the growth of NCI-H1650 cells as tumor xenografts in nudemice. [6]-Shogaol suppressed the expression of Ki-67, cyclin D1

and phosphorylated Akt and STAT3 and increased terminadeoxynucleotidyl transferase-mediated dUTP nick end labeling-positivity in xenograft tumors. The current study clearlyindicates that [6]-shogaol can be exploited for the prevention

and/or treatment of NSCLC.

Introduction

The amplification of certain intracellular signaling pathways comprising various kinases and transcription factors has been implicated in the promotion and progression of cancer (1,2). Thereforetargeted inhibition of one or more components of an oncogenicsignaling cascade is considered to be a rational strategy to prevencancer. Numerous dietary phytochemicals have been reported toimpede multiple abnormally activated signal transduction pathways, thereby preventing cancer (1,2). Ginger ( Zingiber officinale

 Roscoe), a common condiment, has long been used as a component of oriental medicine. The major pungent constituents o

ginger include gingerols, shogaols and paradols. The anti-inflammatory and chemopreventive activities of these ginger constituents have been extensively investigated in different experimentamodel systems. Multiple mechanisms, including antioxidant, antiinflammatory, antiproliferative, antiangiogenic, anti-invasive andantimetastatic activities, have been attributed to the anticanceeffects of these ginger polyphenols (3,4). However, a comparative analysis of their efficacy is limited. Moreover, the underlyingmechanisms of chemoprevention with ginger polyphenols have nobeen completely elucidated.

Human non-small cell lung cancer (NSCLC) is the leading causeof cancer mortality worldwide (5,6). Because of the lack of earlydiagnostic procedures and the increasing rate of primary and secondary resistance to conventional chemotherapies, the search for amolecular target-based chemopreventive agent is a timely need to

reduce the incidence and mortality from NSCLC. Because mutations in epidermal growth factor receptor (EGFR), which result inthe amplification of diverse intracellular signaling pathways, havebeen implicated in the pathogenesis of NSCLC, targeting signatransduction cascades downstream of EGFR would be a rationaapproach to develop novel chemopreventive agents against NSCLCOne of the most extensively studied EGFR downstream signal cascades is the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. Thaberrant activation of PI3-K is associated with increases in cell proliferation and inhibition of apoptosis, thereby contributing to tumogrowth (7–9). Akt, which is constitutively activated in NSCLC, is adownstream effector of PI3-K. Moreover, EGFR-mediated signalalso cross talk with other cell signaling pathways, especially thoseenhancing cell survival. One such EGFR downstream target is signatransducer and activator of transcription-3 (STAT3), which is over

expressed in human NSCLC specimens (10). Thus, STAT3 and itregulated gene products are important molecular targets for chemoprevention of NSCLC.

On the basis of molecular target-based virtual screening of phytochemicals, we identified several ingredients of ginger, including[6]-shogaol, [6]-paradol and [6]-gingerol, as potential candidates fothe prevention and therapy of NSCLC. Comparative analysis of theseginger polyphenols revealed that [6]-shogaol is the most potent in suppressing the proliferation of NSCLC cells. We elucidated the underlying molecular mechanisms of anticancer activity of [6]-shogaol inNSCLC cells. In this study, we report that [6]-shogaol induced celcycle arrest and apoptosis in NSCLC cells and attenuated the in vivoxenograft tumor growth of these cells by blocking the Akt and STAT3signaling pathways.

Abbreviations:  EGFR, epidermal growth factor receptor; NSCLC, non-smallcell lung cancer; PI3-K, phosphatidylinositol 3-kinase; STAT3, signal trans-ducer and activator of transcription-3; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling.

†These authors contributed equally to this work.

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Materials and methods

 Reagents

[6]-Shogaol (purity > 96%) and [6]-paradol (purity > 98%) were synthesizedby slight modification of the processes described earlier (Supplementary Materials  and Methods, available at Carcinogenesis  Online) (11–13) andwere analyzed and authenticated by high-performance liquid chromatography.[6]-Gingerol (purity> 95%) was purchased from Dalton Chemical Laboratories(Toronto, Canada). Human recombinant proteins for kinase assays were pur-chased from Millipore (Temecula, CA). Antibodies to detect phosphorylated

Akt (pAkt, Ser473), total Akt, phosphorylated STAT3 (pSTAT3, Ser705 orSer727), total STAT3, cyclin D1 and cyclin D3 were purchased from CellSignaling Technology (Beverly, MA). The antibody against β-actin was pur-chased from Santa Cruz Biotechnology (Santa Cruz, CA). The small-hairpinRNA (shRNA) constructs against  Akt1 and  Akt2 were from the BioMedicalGenomics Center at the University of Minnesota (Minneapolis, MN).

Cell culture and transfection

Human NSCLC cell lines (NCI-H1650, NCI-H520 and NCI-H1975) and HEK293T cells were purchased from American Type Culture Collection (ATCC,Manassas, VA). Cells were cultured in RPMI-1640 containing penicillin (100units/ml), streptomycin (100 g/ml), sodium pyruvate (1 mM) and 10% fetalbovine serum (FBS, Gemini Bio-Products, Calabasas, CA) and maintainedat 5% CO2 and 37°C in a humidified atmosphere. Cytogenetically tested andauthenticated frozen cells were thawed and maintained for about 2 months.HEK 293T cells were cultured in MEM with 10% FBS. For knocking downthe expression of Akt1/2 in NCI-H1650 cells or overexpressing Akt1/2 in

NIH-3T3 or HEK 293T cells, transfection was performed with  pLKO.1-mock , shRNA-Akt1 or shRNA-Akt2 or pBabe-mock , CA-Akt1 or CA-Akt2 DNAplasmids together with packaging vectors,  pMD2.0G  and  psPAX   (AddgeneInc., Cambridge, MA) using the jetPEI poly transfection reagent (Polyplus-transfection SAS, Saint Quentin Yvelines, France) following the manufactur-er’s protocols. The transfection medium was changed at 4 h after transfectionand then cells were cultured for 36 h. The viral particles were harvested byfiltration using a 0.45 mm syringe filter and then infected into NCI-H1650 orNIH-3T3 cells together with 8 g/ml of polybrene (Millipore) for 24 h. Thecell culture media were replaced with fresh media and cultured for an addi-tional 24 h. After selection with puromycin (1 g/ml) for 48 h, the selected cellswere used for an anchorage-independent cell growth assay.

 In vitro kinase assay

The kinase assay was performed according to the instructions provided byMillipore. In brief, the reaction was conducted in the presence of 10 Ci of[γ-32P] ATP and each compound in 40 l of reaction buffer [20 mM HEPES

(pH 7.4), 10 mM MgCl2, 10 mM MnCl2 and 1 mM dithiothreitol]. After incuba-tion at room temperature for 30 min, the reaction was stopped by adding 10 lof protein loading buffer, and the mixture was separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis. Each experiment was repeatedtwice, and the relative amounts of incorporated radioactivity were assessedby autoradiography.

Computational modeling

The crystal structure of Akt1 (PDB id 3O96) (14) used as the receptor struc-ture was downloaded from the Protein Data Bank (15). The coordinates of[6]-shogaol were downloaded from the PubChem compound database (http:// pubchem.ncbi.nlm.nih.gov). Before ligand–protein docking, the raw PDBstructure was converted into an all-atom, fully prepared receptor modelstructure using the Protein Preparation Wizard module (16). The original 2Dstructure of [6]-shogaol was changed to 3D conformers using ConfGen (17).Protein–ligand docking was performed using the high-performance hierarchi-cal docking algorithm, Glide (18,19). The final binding structural model ofAkt1-[6]-shogaol was generated from Schrödinger Induced Fit Docking (20),which merges the predictive power of Prime with the docking and scoringcapabilities of Glide for accommodating the possible protein conformationalchange upon ligand binding.

Cell proliferation assay

For the proliferation assay, cells were seeded (1 × 103 cells per well) in 96-wellplates and incubated for 24 h and then treated with the indicated concentrationsof [6]-shogaol, [6]-paradol or [6]-gingerol and harvested at 12, 24, 48, 72 or96 h. Cell proliferation was measured by MTS assay (21). To assess anchor-age-independent growth, cells (8 × 103 cells per well) suspended in completemedium were added to 0.3% agar with 0, 10 or 20 M [6]-shogaol, [6]-paradolor [6]-gingerol in a top layer over a base layer of 0.5% agar with 0, 10 or20 M [6]-shogaol, [6]-paradol or [6]-gingerol. The cultures were maintainedat 37°C in a 5% CO2 incubator for 3 weeks and then colonies were counted

under a microscope using the Image-Pro Plus software (v.6.2) program (MediaCybernetics, Rockville, MD).

Cell cycle and apoptosis analyses

Cells were plated in 100 mm plates and treated with 0, 10 or 20 M [6]-shogaolfor 24 or 48 h. Cells were then fixed in 70% ethanol and stored at -20°C for24 h. After staining with annexin V for apoptosis or propidium iodide for cellcycle analysis, cells were analyzed by a BD FACSCalibur Flow Cytometer(BD Biosciences, San Jose, CA).

 In vitro and ex vivo pull-down assay

Recombinant human Akt1, Akt2 (200 ng) kinase or a NCI-H1650 cell lysate(500 g) was incubated with [6]-shogaol-conjugated Sepharose 4B orSepharose 4B beads only as a control (50 l; 50% slurry) in reaction buffer(50 mM Tris-HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol,0.01% NP-40 and 2 mg/ml bovine serum albumin). After incubation with gen-tle rocking overnight at 4°C, the beads were washed five times with buffer(50 mM Tris-HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol and0.01% NP-40), and the binding was visualized by Western blotting.

Western blot analysis

The total cellular protein extracts were prepared according to the proceduredescribed previously (21). Proteins were separated by sodium dodecyl sul-fate–polyacrylamide gel electrophoresis and transferred to polyvinylidenedifluoride membranes in 20 mM Tris-HCl (pH 8.0) containing 150 mM gly-cine and 20% (vol/vol) methanol. The membranes were blocked with 5%non-fat dry milk in 1× Tris-buffered saline containing 0.05% Tween 20and incubated with antibodies against pAkt (Ser473), pSTAT3 (Ser705 or

Ser727), cyclin D1, cyclin D3 or β-actin. Blots were washed three timesin 1× Tris-buffered saline containing 0.05% Tween 20 buffer, followed bythe incubation with the appropriate horseradish peroxidase-linked IgG. Thespecific proteins in the blots were visualized using an enhanced chemilumi-nescence detection system.

 Xenograft mouse model

Female BALB/c (nu/nu) mice, 6 weeks old, were purchased from CharlesRiver Laboratories (USA) and housed in a light/dark cycle of 12/12 h andfed with rodent chow and water ad libitum. All animal works were reviewedand approved by the KyungPook National University Ethics Research Board,Daegu, South Korea. NCI-H1650 cells (3 × 106 cells in 200 l of phosphate-buffered saline) were injected subcutaneous on the right hind flank. After6 days of implantation, two groups (n = 8 per group) were given [6]-shogaolat 10 or 40 mg/kg body weight (dissolved in 5% dimethyl sulfoxide and 10%Tween-20 in 1× phosphate-buffered saline) intraperitoneally three timesa week for three consecutive weeks. The third group received vehicle only.

Tumor volume (length × width × depth × 0.52) and body weights were meas-ured three times a week. Xenograft tumors were frozen in liquid nitrogen orfixed in 10% formalin and then embedded in paraffin.

 Immunohistochemical analysis

Tumor tissues embedded in paraffin from mice were subjected to hematoxylinand eosin staining and immunohistochemistry. Tumor tissues were de-par-affinized and hydrated and then permeabilized with 0.5% Triton X-100 in 1×phosphate-buffered saline for 10 min. They were then hybridized with Ki-67(1:100), cyclin D1 (1:100), pAkt (Ser473, 1:40) or pSTAT3 (Ser727, 1:400) asthe primary antibody and biotin-conjugated goat anti-rabbit or mouse IgG wasused as the secondary antibody. Xenograft tissue samples were also subjectedto terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay. All sections were observed by microscope and analyzed usingthe Image-Pro Plus software (v. 6.2) program.

Statistical analysis

All quantitative results are expressed as mean values ± SD. Statistically signifi-cant differences were obtained using the Student’s t -test or one-way analysisof variance. A value of P < 0.05 was considered to be statistically significant.

Results

 Effects of [6]-shogaol, [6]-gingerol and [6]-paradol on the proliferation of NSCLC cells

We first examined the effects of [6]-shogaol, [6]-paradol or [6]-gin-gerol (Figure 1A–C) on the viability of several NSCLC (NCI-H1650,NCI-H520 and NCI-H1975) cell lines. [6]-Shogaol showed the mostsignificant effect on viability of NCI-H1650 (Figure 2A), NCI-H520(Supplementary Figure 1A, available at Carcinogenesis Online) andNCI-H1975 (Supplementary Figure 1B, available at Carcinogenesis 

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[6]-Shogaol is an allosteric inhibitor of Akt in NSCLC

Online) cells compared with [6]-paradol or [6]-gingerol. Notably,20 M [6]-shogaol reduced the viability of NCI-H1650 cells by 73.4,77.9 and 92.6% at 48, 72, and 96 h, respectively. In contrast, even though20 M [6]-paradol decreased viability of NCI-H1650 cells at 72 and96 h, it failed to affect the viability of NCI-H520 and NCI-H1975 cells.[6]-Gingerol caused a moderate decrease in viability of NCI-H1650and NCI-H1975 cells, but did not affect NCI-H520 cells. [6]-Shogaolalso inhibited anchorage-independent growth of NCI-H1650 cells by

77.4 and 81.9% (Figure 2B) and suppressed the growth of NCI-H520cells by 74.2 and 89.3% at 10 and 20 M, respectively (SupplementaryFigure 1C, available at Carcinogenesis  Online). Although [6]-para-dol inhibited anchorage-independent growth of NCI-H1650 cells by41.2% at a concentration of 20 M (Figure 2B), it failed to affect thegrowth of NCI-H520 cells (Supplementary Figure 1C, available atCarcinogenesis Online). [6]-Gingerol had no effect on the anchorage-independent growth of NCI-H1650 (Figure 2B) or NCI-H520 cells(Supplementary Figure 1C, available at Carcinogenesis Online). Onthe basis of these findings, we selected [6]-shogaol for further experi-ments to elucidate the molecular mechanisms of its anticancer effectsagainst NSCLC cells. [6]-Shogaol also induced apoptosis in NCI-H1650 cells by 24.2 and 27.2% at concentrations of 10 and 20 M,respectively (Figure 2C). Although [6]-shogaol reduced the viability ofNCI-H520 cells, it failed to induce apoptosis (Supplementary Figure 

1D, available at Carcinogenesis Online). We therefore examined theeffect of [6]-shogaol on cell cycle progression in NCI-H1650 and NCI-H520 cells. Analysis of cell cycle distribution revealed that [6]-shogaolinduced G1 phase arrest in NCI-H1650 cells (Figure 2D) and G2/Mphase arrest in NCI-H520 cells (Supplementary Figure 1E, available atCarcinogenesis Online).

 Akt1/2 is a potential target of [6]-shogaol

Because the amplification of PI3-K/Akt-mediated signaling is associ-ated with NSCLC pathogenesis (22), we attempted to identify a directtarget of [6]-shogaol among the major components of the PI3-K/ Akt signaling pathway. Results indicated that the kinase activitiesof Akt1 and Akt2 (Figure 3A) were markedly decreased by treat-ment with [6]-shogaol. However, [6]-shogaol had no effect on the

kinase activities of PI3-K (Supplementary Figure 2A, available aCarcinogenesis Online) or mTOR (Supplementary Figure 2B, available at Carcinogenesis Online), which are upstream and downstreamsignaling molecules of Akt, respectively. The in vitro and ex vivo pulldown assay results revealed a direct binding between [6]-shogaol andAkt1 or Akt2 (Figure 3B). These findings were confirmed by incubating[6]-shogaol with either recombinant active Akt1 or Akt2 in vitro or withan NCI-H1650 cell lysate ex vivo. To determine whether [6]-shogaointeracts with the ATP binding pocket of Akt, we performed an ATP

competitive pull-down assay using recombinant active Akt1 or Akt2Results indicated that the interaction of [6]-shogaol with Akt1 or Akt2in the presence of ATP (1, 10 or 100 M) was not affected, suggesting an ATP-independent binding mode of [6]-shogaol with Akt1 oAkt2 (Figure 3C) and indicated that [6]-shogaol might interact withAkt1/2 at a site(s) other than the ATP-binding pocket. To identify theexact Akt binding site(s) for [6]-shogaol, we transfected DNA plasmidcontaining the constitutively active form of Akt  or a kinase active sitedead dominant negative (DN) form of Akt  into HEK 293 cells, and thecell lysate was pulled-down with [6]-shogaol-conjugated beads. Theresults showed that [6]-shogaol was bound to both active or DN-Ak(Figure 3D). The hierarchical docking algorithm Glide (18,19) andSchrödinger-Maestro v9.2 (20) software program was then used fodocking experiments to assess the possible binding mode between Aktand [6]-shogaol (Figure 3E). To capture the ligand-induced conforma

tional changes in the receptor active site, we performed flexible-ligandflexible-protein docking using IFD (Induced Fit Docking) Module (18)The binding pose of [6]-shogaol-Akt1 obtained from the IFD dockingstudy (Figure 3E, left panel) suggests that [6]-shogaol binds to a generally characterized “PH-in” conformation of Akt1. The Akt bindingsite for [6]-shogaol is located underneath the activation loop of Akt1Its binding to Akt1 is in the allosteric binding site at the lower interface between the N- and C-lobes of the kinase domain. [6]-Shogaoforms two hydrogen bonds with Ser205 and thus its activity might bedependent upon the presence of the Ser205 residue in Akt (Figure 3Eright panel). [6]-Shogaol also formed strong hydrophobic interactionwith several amino acid residues, including Leu210, Ile290, Leu275Leu261, Tyr272 and Leu264, from the kinase domain of Akt1. In addition, hydrophobic contacts were observed between the phenyl ring o[6]-shogaol and Trp80 in the PH domain of Akt1. As a result, the existence of the PH domain in the kinase assay could enhance the inhibition

of [6]-shogaol against Akt1. These computational results indicate tha[6]-shogaol, as a type-III kinase inhibitor, may elicit ATP noncompetitive inhibitory effects on the Akt1 kinase activity.

[6]-Shogaol fails to affect anchorage-independent growth of stable Akt-silenced NCI-H1650 cells

We constructed lentiviral particles containing shRNA-control oshRNA-Akt by transfecting an shRNA-control or shRNA-Akt1/2 plasmid with packaging DNA,  pMD2.0G and psPAX   and then infectingthese particles into NCI-H1650 cells. Cells stably expressing shcontrol or sh-Akt1/2 were selected by puromycin. Treatment with[6]-shogaol significantly inhibited anchorage-independent colony formation in sh-control RNA expressing cells (Figure 4A). Interestinglytreatment with [6]-shogaol failed to further reduce the colony numberin Akt1/2-silenced cells (Figure 4A). Furthermore, after transfectinwith mock vector or constitutive activated (CA)-Akt in NIH-3T3 cellswe performed an anchorage-independent colony formation assayThe results revealed that the mock vector could not induce colonyformation but transfection with CA-Akt significantly increased thecolony numbers (Figure 4B), which were decreased by treatment with[6]-shogaol in a concentration-dependent manner (Figure 4B). Thesefindings suggest that Akt is an essential target protein of [6]-shogaoto suppress the growth of NCI-H1650 NSCLC cells.

[6]-Shogaol inhibits the expression of Akt downstream signalingmolecules and induces markers of apoptosis

Because [6]-shogaol showed no effect on the kinase activities oPI3-K or mTOR, which are up- and downstream signaling molecule

Fig. 1. Chemical structures of major active components in ginger.(A) [6]-shogaol; (B) [6]-paradol; and (C) [6]-gingerol.

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in the Akt pathway, we examined the effect of [6]-shogaol on theactivation of STAT3, which is known to be regulated by Akt (23,24).[6]-Shogaol attenuated the constitutive phosphorylation of STAT3 in

NCI-H1650 cells (Figure 5A). Furthermore, the expression of STAT3target gene products, including cyclin D1, cyclin D3 and c-Myc, wasdecreased by treatment with [6]-shogaol in a concentration-dependentmanner (Figure 5B). To further elucidate the molecular mechanismsof [6]-shogaol-induced apoptosis in NCI-H1650 cells, we examinedthe effect of [6]-shogaol on several apoptotic markers. Results indi-cated that [6]-shogaol induced the cleavage of pro-caspase-3 and -7,resulting in increased cleavage of poly (ADP-ribose) polymerase(Figure 5C).

[6]-Shogaol inhibits NCI-H1650 lung cancer cell growth in a xenograft mouse model

We then examined the effect of [6]-shogaol on the growth of NCI-H1650 cells as xenografts in athymic mice in vivo. The average growth

of xenograft tumors was significantly retarded by treatment with[6]-shogaol (Figure 6A). Compared with the vehicle-treated group,the average tumor volume was significantly reduced by treatment

with [6]-shogaol (10 or 40 mg/kg body weight). Although the aver-age tumor volume was 393.4 mm3  in vehicle-treated group, tumorsin mice treated with [6]-shogaol at doses of 10 and 40 mg/kg bodyweight showed an average volume of 274.7 and 140.8 mm3, respec-tively. Treatment with [6]-shogaol did not cause any change in bodyweight (Figure 6B). Immunohistochemical analysis revealed that[6]-shogaol significantly inhibited the expression of Ki-67, which is acell proliferation biomarker (56.2% at 10 mg/kg, 93.8% at 40 mg/kg;P < 0.01, Figure 6C-a). Compared with the vehicle control group, thegroup treated with [6]-shogaol showed a marked increase in TUNEL-positive cells (2.1-fold at 10 mg/kg and 2.7- fold at 40 mg/kg; P  <0.01, Figure 6C-b). Furthermore, we detected the expression of Akt-target proteins in tumor samples. The expression of phosphorylatedAkt (Ser473) was significantly decreased by treatment of [6]-shogaol

Fig. 2. [6]-Shogaol inhibits the growth of NSCLC cells. (A) The effects of [6]-shogaol, [6]-paradol and [6]-gingerol on the proliferation of NCI-H1650 lungcancer cells were assessed at 12, 24, 48, 72 and 96 h by MTS assay. The asterisk (*) indicates a significant (P < 0.01) decrease in proliferation compared withuntreated control. (B) The effects of [6]-shogaol, [6]-paradol and [6]-gingerol on anchorage-independent growth of NCI-H1650 lung cancer cells were evaluated.The asterisks (*P < 0.05, **P < 0.01) indicate a significant decrease in colony formation with ginger compound treatment compared with untreated control. Theeffects of [6]-shogaol on (C) induction of apoptosis and (D) cell cycle distribution was assessed in NCI-H1650 lung cancer cells. Cells were treated with 0, 10or 20 M of [6]-shogaol and then incubated for 24 h (cell cycle analysis) or 48 h (annexin-V staining assay). The asterisks (*P < 0.05, **P < 0.01) indicate asignificant difference between untreated control and treated cells. Data are represented as means ± SD of values from triplicate samples and similar results wereobtained from three independent experiments.

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Fig. 3. Identification of potential targets of [6]-shogaol and predicted binding model. (A) The effect of [6]-shogaol on Akt1 and Akt2 kinase activity (CB;Coomassie blue staining). (B) The binding of [6]-shogaol to active Akt1, Akt2 or to Akt1/2 in NCI-H1650 cell lysates. (C) The binding of [6]-shogaol to activeAkt1 or Akt2 in the presence of ATP. (D) The binding of [6]-shogaol to Akt in lysates from cells expressing constitutively active Akt or kinase domain dead Akt(DN-Akt). (E) Computational docking model of binding between [6]-shogaol and Akt1; the binding pose of [6]-shogaol inside the allosteric binding site of Akt1(left); the interaction between [6]-shogaol and several residues in the binding site (right). [6]-Shogaol forms two hydrogen bonds with Ser205 (i.e., for clarity,only side chains of the protein residues, except for Ser205, are shown). In addition, the protein residues, including Leu210, Ile290, Leu275, Leu261, Tyr272 andLeu264, show strong hydrophobic interactions with the long chain of the CH2-groups of [6]-shogaol. Note: the α-helices are drawn as cylinders and the β-strandsas arrows. [6]-Shogaol is shown in stick model and protein residues are shown in line model. The figures were generated using Maestro (20).

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Fig. 5. The effects of [6]-shogaol on the expression of proteins involved in cell proliferation in NCI-H1650 cells. Cells were treated with [6]-shogaol at 0, 10 and20 M for 12 h and then harvested and proteins were extracted and subjected to Western blot analysis. (A) Inhibitory effects of [6]-shogaol on the constitutiveexpression of pSTAT3 (Ser705) or pSTAT3 (Ser727). (B) Effects of [6]-shogaol on the expression of cyclin D1, cyclin D3 and c-Myc, which are target proteins ofSTAT3. (C) Immunoblots showing the effects of [6]-shogaol (treated at concentrations of 10 or 20 M for 24 h) on the expression of cleaved poly (ADP-ribose)polymerase, cleaved caspase-3 and cleaved caspase-7. Representative blots from three independent experiments are shown.

Fig. 4. Akt is a molecular target of [6]-shogoal. (A) The effect of [6]-shogaol (0, 10, and 20 M) on the anchorage-independent growth of NCI-H1650 cellsexpressing shRNA-control or shRNA-Akt, (B) The effect of [6]-shogaol on the anchorage-independent growth of NIH-3T3 cells expressing mock or Akt. Data

are represented as means ± SD of values from triplicate samples and similar results were obtained from three independent experiments.

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at 20 mg/kg body weight (52.4%; P < 0.01, Figure 6C-c). [6]-Shogaol

reduced the expression of phosphorylated STAT3 (Ser727) in xeno-graft tumors by 51.9% (P < 0.01) and 42.5% (P < 0.05) at doses of 10and 40 mg/kg body weight, respectively (Figure 6C-d). Moreover, theexpression of cyclin D1 was also markedly attenuated by 79.1% (P <0.01) and 90.0 % (P < 0.01) upon treatment with 10 or 40 mg/kg bodyweight of [6]-shogaol, respectively (Figure 6C-e).

Discussion

Lung cancer is a leading cause of mortality throughout the world. Thenumber of deaths due to lung cancer has increased approximately4.3% between 1999 and 2008. According to a report from the NationalCancer Institute, deaths from small cell lung cancer and NSCLC areestimated to be 159,480 in 2013 in the United States. At the same

time, more than 200,000 new cases of lung cancer are expected to

be diagnosed during the same time period. Although NSCLC spreadmore slowly than small cell lung cancer, it is more common than smalcell lung cancer. Moreover, the majority of NSCLC is diagnosed onlywhen it has metastasized. Thus, adopting an appropriate preventionstrategy could reduce the incidence and mortality from NSCLC.

A wide variety of dietary phytochemicals have been reported topossess chemopreventive and/or chemotherapeutic activities. Gingera common spice and a component of traditional medicine, containa number of antioxidant, anti-inflammatory and anti-cancer components such as shogaols, paradols and gingerols (3,4). Despite substantial progress in understanding the molecular mechanisms of theanticancer effects of these ginger polyphenols (3,4,25–29), theieffects on NSCLC have been poorly investigated. In the currenstudy, we examined the effects of selected ginger polyphenols on

Fig. 6. [6]-Shogaol suppresses NCI-H1650 NSCLC tumor growth in a xenograft mouse model. (A) NCI-H1650 cells were inoculated in athymic nude micefor the development of tumor xenografts and mice were treated with either vehicle or [6]-shogaol (10 or 40 mg/kg body weight, intraperitoneal) as describedin Materials and methods. The average tumor volume in vehicle-treated control mice (n = 8) and [6]-shogaol-treated mice (n = 8) was plotted over 24 daysafter tumor cell injection. The asterisks indicate a significant inhibition by [6]-shogaol on tumor growth (*P < 0.05; **P < 0.01). (B) Effect of [6]-shogaol onmouse body weight. Body weights from treated or untreated groups of mice were measured 3× a week. (C) Quantification of hematoxylin and eosin stainingand immunohistochemical analysis of tumor tissues. [6]-Shogaol-treated or untreated mice were euthanized and tumor tissues were harvested. Sections of tumortissues were formalin-fixed and paraffin-embeded, and then stained with hematoxylin and eosin or the indicated antibodies. Expression of Ki-67, TUNEL, pAkt,pSTAT3 and cyclin D1 was visualized with a light microscope. Stained cells were counted from five separate areas on the slide and an average of three sampleswas calculated per group. Data are expressed as mean percent of control ± SD The asterisk indicates a significant decrease in Ki-67 (a), pAkt (c), pSTAT3 (d) andcyclin D1 (e). For the TUNEL staining, the asterisk indicates a significant increase (b) in positive cells.

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the growth of three different NSCLC cell lines and elucidated theunderlying mechanisms explaining the anticancer effects of the mostpotent compound. Our study revealed that [6]-shogaol is more potentthan [6]-paradol or [6]-gingerol in reducing cell viability and inhib-iting anchorage-independent growth of NSCLC cells. Our findingsare in good agreement with other studies reporting that [6]-shogaolis more potent than [6]-gingerol in preventing chemically inducedskin cancer (30) and possesses the most potent antioxidant and anti-inflammatory properties (25). The reason that [6]-shogaol is more

active than [6]-gingerol or [6]-paradol might be attributed to thepresence of an α,β-unsaturated carbonyl moiety, which is absent in[6]-gingerol or [6]-paradol. [6]-Shogaol has been reported to inhibitproliferation and induce apoptosis in various cancer cells, includ-ing colon cancer, hepatocellular carcinoma, breast cancer, cervicalcancer and oral cancer (29,31–36). Hung et al.  (37) reported that[6]-shogaol induced autophagic cell death in human lung cancer(A549) cells at a concentration of 80 M. In our study, we found that[6]-shogaol at a concentration of 20 M induced cell cycle arrest atG1 phase in NCI-H1650 cells and at G2/M phase in NCI-H520 cells.These findings suggest a cell type-specific antiproliferative effect of[6]-shogaol.

To elucidate the molecular mechanisms of the antiproliferativeactivity of [6]-shogaol in NCI-H1650 cells, we first predicted theidentity of potential kinase targets of the compound directly from

ligand–protein computational docking (data not shown) and thenperformed kinase assays. Our finding that [6]-shogaol inhibited thecatalytic activities of Akt1 and Akt2 is in good agreement with a studyby Hung et al., who demonstrated that this compound inhibited theAkt/mTOR pathway in A549 cells (37). However, in contrast to theirstudy, we noticed that [6]-shogaol failed to affect the kinase activ-ity of PI3-K or mTOR in NCI-H1650 cells. Moreover, [6]-shogaoldid not affect the activities of other kinases, such as EGFR, cyclic-AMP-activated kinase-α  (AMPKα), glycogen synthase kinase-3β  (GSK3β), cyclin-dependent kinase-2 (CDK2), check kinase-1(CHK1), casein kinase-1 (CK1), c-Jun-N-terminal kinase-1/2(JNK1/2), extracellular signal-regulated kinase-1/2 (ERK1/2) andp38α  (Supplementary Table 1, available at Carcinogenesis  Online).The finding that [6]-shogaol inhibited Akt activation in NCI-H1650cells is well supported by several previous studies demonstrating theinhibitory effect of [6]-shogaol on Akt phosphorylation and/or activ-

ity in other cancer models (28,30,38). Our new evidence showed that[6]-shogaol directly interacted with Akt1/2 and diminished Akt1/2activities. In addition, shRNA-based silencing of Akt significantlyattenuated anchorage-independent growth of NCI-H1650 cells, sug-gesting Akt as a key signaling molecule in regulating the survival andproliferation of NSCLC cells. Although treatment with [6]-shogaolfailed to reduce colony formation in Akt-silenced cells, the compoundsignificantly attenuated the anchorage-independent growth of Akt-overexpressing cells. These findings suggest that Akt is a bona fide target of [6]-shogaol in suppressing the proliferation and inducingapoptosis in NCI-H1650 cells.

Because [6]-shogaol failed to affect the activity of mTOR, an Aktdownstream target, and many other kinases involved in cell prolifera-tion (Supplementary Table 1, available at Carcinogenesis Online), andthe compound inhibited expression of cell proliferation-related proteins

cyclin D1 and D3, we examined the effect of [6]-shogaol on the phos-phorylation of STAT3, a transcriptional regulator of cyclin D1 and D3.Because Akt-mediated signaling is a cell survival pathway and Akt actsas an upstream kinase of STAT3 (23,24), the [6]-shogaol-induced cas-pase cleavage and diminished phosphorylation of STAT3 may resultfrom its inhibitory effect on Akt activation. These findings were fur-ther supported by an in vivo xenograft study. Our study revealed thattreatment with [6]-shogaol significantly decreased the tumor xenograftgrowth of NCI-H1650 cells, which was associated with decreased cellproliferation and increased apoptosis as evidenced by reduced Ki-67-positive cells and an increased number of TUNEL-positive cells in the[6]-shogaol-treated xenograft tumors. Moreover, the immunohisto-chemical analysis of these tumors showed a significant inhibition ofcyclin D1 expression and decreased phosphorylation of Akt and STAT3.

The activation of p53 is one of the well-established mechanismsfor apoptosis induction and growth arrest. Liu et al. demonstratedthat a steam-distilled extract of ginger induced apoptosis in humanendometrial cancer cells through activation of p53. However,shogaols were absent in this ginger extract (25). A p53-independentmechanism of apoptosis induction by [6]-shogaol has been reported(39). According to this study, [6]-shogaol induced apoptosis inMahlavu cells, a p53-mutant human hepatoma cell line, through thegeneration of reactive oxygen species and subsequent depletion of

glutathione.In conclusion, our study demonstrates that among the ginger poly-

phenols, [6]-shogaol is the most potent in suppressing the growth ofNSCLC cells and the compound inhibits proliferation and inducesapoptosis in NCI-H1650 cells by suppressing Akt signaling throughthe direct targeting of Akt1 and Akt2.

Supplementary material

Supplementary Table 1  and Figures 1  and 2 can be found at http:// carcin.oxfordjournals.org/ 

Funding

National Institute of Health (CA120388, CA166011, CA172457, R37

CA081064 and ES016548); Next-Generation BioGreen 21 ProgramRural Development Administration, Republic of Korea (PJ009560 toM.O.K.).

Acknowledgements

We thank Dr. Tae-Gyu Lim, Do Young Lim, Soouk Kang, and Todd Schusterfor supporting experiments. We thank Nicki Brickman at The Hormel Institute,University of Minnesota, for assistance in submitting our manuscript.

Conflict of Interest Statement : None declared.

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 Received May, 30, 2013; revised September 27, 2013;accepted October 18, 2013

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