Carcinogenesis vol.35 no.3 pp.

683691, 2014
doi:10.1093/carcin/bgt365
Advance Access publication November 26, 2013

[6]-Shogaol inhibits growth and induces apoptosis of non-small cell lung cancer cells
by directly regulatingAkt1/2
Myoung OkKim1,2,, Mee-HyunLee1,3,, NaomiOi1,,
Sung-HyunKim1,2,, Ki BeomBae1, ZunnanHuang1,4,
Dong JoonKim1,5, KanamataReddy1, Sung-YoungLee1,6,
Si JunPark2, Jae YoungKim2, HuaXie1,7, Joydeb
KumarKundu8, Zae YoungRyoo2, Ann M.Bode1,
Young-JoonSurh3 and ZigangDong1,6,*
1

The Hormel Institute, University of Minnesota, MN 55912, USA,

Kyungpook National University, School of Animal BT Science, Center
for Laboratory Animal Resources, Department of Biochemistry, School
of Dentistry, Daegu 702-701, South Korea, 6WCU Department of
Molecular Medicine and Biopharmaceutical Sciences, Graduate School
of Convergence Science and Technology and College of Pharmacy,
Seoul National University, Seoul 151-742, South Korea and 8College of
Pharmacy, Keimyung University, Daegu 704-701, South Korea
2

Present address: Integrated Research Institute of Pharmaceutical

Sciences, College of Pharmacy, The Catholic University of Korea,
43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, South Korea
4
Present address: China-America Cancer Research Institute, Guangdong
Medical College, Dongguan, Guangdong 523808, PR China
5
Present address: Medical Genomics Research Center, Korea Research
Institute of Bioscience and Biotechnology, Daejeon 306-809, South Korea
7
Present address: State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
201203, 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 of

cancer mortality worldwide. Despite progress in developing
chemotherapeutics for the treatment of NSCLC, primary and
secondary resistance limits therapeutic success. NSCLC cells
exhibit multiple mutations in the epidermal growth factor
receptor (EGFR), which cause aberrant activation of diverse
cell signaling pathways. Therefore, suppression of the inappropriate amplification of EGFR downstream signaling cascades is considered to be a rational therapeutic and preventive
strategy for the management of NSCLC. Our initial molecular
targetoriented virtual screening revealed that the ginger components, including [6]-shogaol, [6]-paradol and [6]-gingerol,
seem to be potential candidates for the prevention and treatment of NSCLC. Among the compounds, [6]-shogaol showed
the greatest inhibitory effects on the NSCLC cell proliferation and anchorage-independent growth. [6]-Shogaol induced
cell cycle arrest (G1 or G2/M) and apoptosis. Furthermore,
[6]-shogaol inhibited Akt kinase activity, a downstream mediator of EGFR signaling, by binding with an allosteric site of Akt.
In NCI-H1650 lung cancer cells, [6]-shogaol reduced the constitutive phosphorylation of signal transducer and activator of
transcription-3 (STAT3) and decreased the expression of cyclin
D1/3, which are target proteins in the Akt signaling pathway.
The induction of apoptosis in NCI-H1650 cells by [6]-shogaol
corresponded with the cleavage of caspase-3 and caspase-7.
Moreover, intraperitoneal administration of [6]-shogaol inhibited the growth of NCI-H1650 cells as tumor xenografts in nude
mice. [6]-Shogaol suppressed the expression of Ki-67, cyclin D1
Abbreviations: EGFR, epidermal growth factor receptor; NSCLC, non-small
cell lung cancer; PI3-K, phosphatidylinositol 3-kinase; STAT3, signal transducer and activator of transcription-3; TUNEL, terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling.

These authors contributed equally to this work.

and phosphorylated Akt and STAT3 and increased terminal

deoxynucleotidyl transferase-mediated dUTP nick end labeling-positivity in xenograft tumors. The current study clearly
indicates 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). Therefore,
targeted inhibition of one or more components of an oncogenic
signaling cascade is considered to be a rational strategy to prevent
cancer. Numerous dietary phytochemicals have been reported to
impede 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 of
ginger include gingerols, shogaols and paradols. The anti-inflammatory and chemopreventive activities of these ginger constituents have been extensively investigated in different experimental
model systems. Multiple mechanisms, including antioxidant, antiinflammatory, antiproliferative, antiangiogenic, anti-invasive and
antimetastatic activities, have been attributed to the anticancer
effects of these ginger polyphenols (3,4). However, a comparative analysis of their efficacy is limited. Moreover, the underlying
mechanisms of chemoprevention with ginger polyphenols have not
been completely elucidated.
Human non-small cell lung cancer (NSCLC) is the leading cause
of cancer mortality worldwide (5,6). Because of the lack of early
diagnostic procedures and the increasing rate of primary and secondary resistance to conventional chemotherapies, the search for a
molecular 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 in
the amplification of diverse intracellular signaling pathways, have
been implicated in the pathogenesis of NSCLC, targeting signal
transduction cascades downstream of EGFR would be a rational
approach to develop novel chemopreventive agents against NSCLC.
One of the most extensively studied EGFR downstream signal cascades is the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. The
aberrant activation of PI3-K is associated with increases in cell proliferation and inhibition of apoptosis, thereby contributing to tumor
growth (79). Akt, which is constitutively activated in NSCLC, is a
downstream effector of PI3-K. Moreover, EGFR-mediated signals
also cross talk with other cell signaling pathways, especially those
enhancing cell survival. One such EGFR downstream target is signal
transducer and activator of transcription-3 (STAT3), which is overexpressed in human NSCLC specimens (10). Thus, STAT3 and its
regulated gene products are important molecular targets for chemoprevention ofNSCLC.
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 for
the prevention and therapy of NSCLC. Comparative analysis of these
ginger 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 in
NSCLC cells. In this study, we report that [6]-shogaol induced cell
cycle arrest and apoptosis in NSCLC cells and attenuated the in vivo
xenograft tumor growth of these cells by blocking the Akt and STAT3
signaling pathways.

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M.O.Kim etal.

Materials and methods

Reagents
[6]-Shogaol (purity > 96%) and [6]-paradol (purity > 98%) were synthesized
by slight modification of the processes described earlier (Supplementary
Materials and Methods, available at Carcinogenesis Online) (1113) and
were 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 purchased from Millipore (Temecula, CA). Antibodies to detect phosphorylated
Akt (pAkt, Ser473), total Akt, phosphorylated STAT3 (pSTAT3, Ser705 or
Ser727), total STAT3, cyclin D1 and cyclin D3 were purchased from Cell
Signaling Technology (Beverly, MA). The antibody against -actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The small-hairpin
RNA (shRNA) constructs against Akt1 and Akt2 were from the BioMedical
Genomics Center at the University of Minnesota (Minneapolis, MN).
Cell culture and transfection
Human NSCLC cell lines (NCI-H1650, NCI-H520 and NCI-H1975) and HEK
293T cells were purchased from American Type Culture Collection (ATCC,
Manassas, VA). Cells were cultured in RPMI-1640 containing penicillin (100
units/ml), streptomycin (100g/ml), sodium pyruvate (1mM) and 10% fetal
bovine serum (FBS, Gemini Bio-Products, Calabasas, CA) and maintained
at 5% CO2 and 37C in a humidified atmosphere. Cytogenetically tested and
authenticated frozen cells were thawed and maintained for about 2 months.
HEK 293T cells were cultured in MEM with 10% FBS. For knocking down
the expression of Akt1/2 in NCI-H1650 cells or overexpressing Akt1/2 in
NIH-3T3 or HEK 293T cells, transfection was performed with pLKO.1mock, shRNA-Akt1 or shRNA-Akt2 or pBabe-mock, CA-Akt1 or CA-Akt2 DNA
plasmids together with packaging vectors, pMD2.0G and psPAX (Addgene
Inc., Cambridge, MA) using the jetPEI poly transfection reagent (Polyplustransfection SAS, Saint Quentin Yvelines, France) following the manufacturers protocols. The transfection medium was changed at 4h after transfection
and then cells were cultured for 36h. The viral particles were harvested by
filtration using a 0.45 mm syringe filter and then infected into NCI-H1650 or
NIH-3T3 cells together with 8g/ml of polybrene (Millipore) for 24h. The
cell culture media were replaced with fresh media and cultured for an additional 24h. After selection with puromycin (1g/ml) for 48h, the selected cells
were used for an anchorage-independent cell growth assay.
In vitro kinaseassay
The kinase assay was performed according to the instructions provided by
Millipore. In brief, the reaction was conducted in the presence of 10Ci of
[-32P] ATP and each compound in 40l of reaction buffer [20mM HEPES
(pH 7.4), 10mM MgCl2, 10mM MnCl2 and 1mM dithiothreitol]. After incubation at room temperature for 30min, the reaction was stopped by adding 10l
of protein loading buffer, and the mixture was separated by sodium dodecyl
sulfatepolyacrylamide gel electrophoresis. Each experiment was repeated
twice, and the relative amounts of incorporated radioactivity were assessed
by autoradiography.
Computational modeling
The crystal structure of Akt1 (PDB id 3O96) (14) used as the receptor structure 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 ligandprotein docking, the raw PDB
structure was converted into an all-atom, fully prepared receptor model
structure using the Protein Preparation Wizard module (16). The original 2D
structure of [6]-shogaol was changed to 3D conformers using ConfGen (17).
Proteinligand docking was performed using the high-performance hierarchical docking algorithm, Glide (18,19). The final binding structural model of
Akt1-[6]-shogaol was generated from Schrdinger Induced Fit Docking (20),
which merges the predictive power of Prime with the docking and scoring
capabilities of Glide for accommodating the possible protein conformational
change upon ligand binding.
Cell proliferationassay
For the proliferation assay, cells were seeded (1103 cells per well) in 96-well
plates and incubated for 24h and then treated with the indicated concentrations
of [6]-shogaol, [6]-paradol or [6]-gingerol and harvested at 12, 24, 48, 72 or
96h. Cell proliferation was measured by MTS assay (21). To assess anchorage-independent growth, cells (8103 cells per well) suspended in complete
medium were added to 0.3% agar with 0, 10 or 20M [6]-shogaol, [6]-paradol
or [6]-gingerol in a top layer over a base layer of 0.5% agar with 0, 10 or
20M [6]-shogaol, [6]-paradol or [6]-gingerol. The cultures were maintained
at 37C in a 5% CO2 incubator for 3 weeks and then colonies were counted

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under a microscope using the Image-Pro Plus software (v.6.2) program (Media
Cybernetics, Rockville, MD).
Cell cycle and apoptosis analyses
Cells were plated in 100 mm plates and treated with 0, 10 or 20M [6]-shogaol
for 24 or 48h. Cells were then fixed in 70% ethanol and stored at -20C for
24h. After staining with annexin V for apoptosis or propidium iodide for cell
cycle analysis, cells were analyzed by a BD FACSCalibur Flow Cytometer
(BD Biosciences, San Jose, CA).
In vitro and ex vivo pull-downassay
Recombinant human Akt1, Akt2 (200ng) kinase or a NCI-H1650 cell lysate
(500 g) was incubated with [6]-shogaol-conjugated Sepharose 4B or
Sepharose 4B beads only as a control (50l; 50% slurry) in reaction buffer
(50mM Tris-HCl pH 7.5, 5mM EDTA, 150mM NaCl, 1mM dithiothreitol,
0.01% NP-40 and 2mg/ml bovine serum albumin). After incubation with gentle rocking overnight at 4C, the beads were washed five times with buffer
(50mM Tris-HCl pH 7.5, 5mM EDTA, 150mM NaCl, 1mM dithiothreitol and
0.01% NP-40), and the binding was visualized by Western blotting.
Western blot analysis
The total cellular protein extracts were prepared according to the procedure
described previously (21). Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to polyvinylidene
difluoride membranes in 20mM Tris-HCl (pH 8.0) containing 150mM glycine and 20% (vol/vol) methanol. The membranes were blocked with 5%
non-fat dry milk in 1 Tris-buffered saline containing 0.05% Tween 20
and incubated with antibodies against pAkt (Ser473), pSTAT3 (Ser705 or
Ser727), cyclin D1, cyclin D3 or -actin. Blots were washed three times
in 1 Tris-buffered saline containing 0.05% Tween 20 buffer, followed by
the incubation with the appropriate horseradish peroxidase-linked IgG. The
specific proteins in the blots were visualized using an enhanced chemiluminescence detection system.
Xenograft mousemodel
Female BALB/c (nu/nu) mice, 6 weeks old, were purchased from Charles
River Laboratories (USA) and housed in a light/dark cycle of 12/12h and
fed with rodent chow and water ad libitum. All animal works were reviewed
and approved by the KyungPook National University Ethics Research Board,
Daegu, South Korea. NCI-H1650 cells (3106 cells in 200l of phosphatebuffered saline) were injected subcutaneous on the right hind flank. After
6days of implantation, two groups (n=8 per group) were given [6]-shogaol
at 10 or 40mg/kg body weight (dissolved in 5% dimethyl sulfoxide and 10%
Tween-20 in 1 phosphate-buffered saline) intraperitoneally three times
a week for three consecutive weeks. The third group received vehicle only.
Tumor volume (length width depth 0.52) and body weights were measured three times a week. Xenograft tumors were frozen in liquid nitrogen or
fixed in 10% formalin and then embedded in paraffin.
Immunohistochemical analysis
Tumor tissues embedded in paraffin from mice were subjected to hematoxylin
and eosin staining and immunohistochemistry. Tumor tissues were de-paraffinized and hydrated and then permeabilized with 0.5% Triton X-100 in 1
phosphate-buffered saline for 10min. They were then hybridized with Ki-67
(1:100), cyclin D1 (1:100), pAkt (Ser473, 1:40) or pSTAT3 (Ser727, 1:400) as
the primary antibody and biotin-conjugated goat anti-rabbit or mouse IgG was
used as the secondary antibody. Xenograft tissue samples were also subjected
to terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
(TUNEL) assay. All sections were observed by microscope and analyzed using
the Image-Pro Plus software (v. 6.2) program.
Statistical analysis
All quantitative results are expressed as mean values SD. Statistically significant differences were obtained using the Students t-test or one-way analysis
of variance. Avalue 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]-gingerol (Figure1AC) on the viability of several NSCLC (NCI-H1650,
NCI-H520 and NCI-H1975) cell lines. [6]-Shogaol showed the most
significant effect on viability of NCI-H1650 (Figure2A), NCI-H520
(Supplementary Figure 1A, available at Carcinogenesis Online) and
NCI-H1975 (Supplementary Figure 1B, available at Carcinogenesis

[6]-Shogaol is an allosteric inhibitor of Akt inNSCLC

Fig.1. Chemical structures of major active components in ginger.

(A) [6]-shogaol; (B) [6]-paradol; and (C) [6]-gingerol.

Online) cells compared with [6]-paradol or [6]-gingerol. Notably,

20M [6]-shogaol reduced the viability of NCI-H1650 cells by 73.4,
77.9 and 92.6% at 48, 72, and 96h, respectively. In contrast, even though
20M [6]-paradol decreased viability of NCI-H1650 cells at 72 and
96h, it failed to affect the viability of NCI-H520 and NCI-H1975 cells.
[6]-Gingerol caused a moderate decrease in viability of NCI-H1650
and NCI-H1975 cells, but did not affect NCI-H520 cells. [6]-Shogaol
also inhibited anchorage-independent growth of NCI-H1650 cells by
77.4 and 81.9% (Figure2B) and suppressed the growth of NCI-H520
cells by 74.2 and 89.3% at 10 and 20M, respectively (Supplementary
Figure 1C, available at Carcinogenesis Online). Although [6]-paradol inhibited anchorage-independent growth of NCI-H1650 cells by
41.2% at a concentration of 20M (Figure2B), it failed to affect the
growth of NCI-H520 cells (Supplementary Figure 1C, available at
Carcinogenesis Online). [6]-Gingerol had no effect on the anchorageindependent growth of NCI-H1650 (Figure 2B) or NCI-H520 cells
(Supplementary Figure 1C, available at Carcinogenesis Online). On
the basis of these findings, we selected [6]-shogaol for further experiments to elucidate the molecular mechanisms of its anticancer effects
against NSCLC cells. [6]-Shogaol also induced apoptosis in NCIH1650 cells by 24.2 and 27.2% at concentrations of 10 and 20M,
respectively (Figure2C). Although [6]-shogaol reduced the viability of
NCI-H520 cells, it failed to induce apoptosis (Supplementary Figure
1D, available at Carcinogenesis Online). We therefore examined the
effect of [6]-shogaol on cell cycle progression in NCI-H1650 and NCIH520 cells. Analysis of cell cycle distribution revealed that [6]-shogaol
induced G1 phase arrest in NCI-H1650 cells (Figure 2D) and G2/M
phase arrest in NCI-H520 cells (Supplementary Figure 1E, available at
Carcinogenesis Online).
Akt1/2 is a potential target of [6]-shogaol
Because the amplification of PI3-K/Akt-mediated signaling is associated with NSCLC pathogenesis (22), we attempted to identify a direct
target of [6]-shogaol among the major components of the PI3-K/
Akt signaling pathway. Results indicated that the kinase activities
of Akt1 and Akt2 (Figure 3A) were markedly decreased by treatment with [6]-shogaol. However, [6]-shogaol had no effect on the

kinase activities of PI3-K (Supplementary Figure 2A, available at

Carcinogenesis Online) or mTOR (Supplementary Figure 2B, available at Carcinogenesis Online), which are upstream and downstream
signaling molecules of Akt, respectively. The in vitro and ex vivo pulldown assay results revealed a direct binding between [6]-shogaol and
Akt1 or Akt2 (Figure3B). These findings were confirmed by incubating
[6]-shogaol with either recombinant active Akt1 or Akt2 in vitro or with
an NCI-H1650 cell lysate ex vivo. To determine whether [6]-shogaol
interacts with the ATP binding pocket of Akt, we performed an ATPcompetitive pull-down assay using recombinant active Akt1 or Akt2.
Results indicated that the interaction of [6]-shogaol with Akt1 or Akt2
in the presence of ATP (1, 10 or 100M) was not affected, suggesting an ATP-independent binding mode of [6]-shogaol with Akt1 or
Akt2 (Figure 3C) and indicated that [6]-shogaol might interact with
Akt1/2 at a site(s) other than the ATP-binding pocket. To identify the
exact Akt binding site(s) for [6]-shogaol, we transfected DNA plasmids
containing the constitutively active form of Akt or a kinase active site
dead dominant negative (DN) form of Akt into HEK 293 cells, and the
cell lysate was pulled-down with [6]-shogaol-conjugated beads. The
results showed that [6]-shogaol was bound to both active or DN-Akt
(Figure 3D). The hierarchical docking algorithm Glide (18,19) and
Schrdinger-Maestro v9.2 (20) software program was then used for
docking experiments to assess the possible binding mode between Akt1
and [6]-shogaol (Figure3E). To capture the ligand-induced conformational changes in the receptor active site, we performed flexible-ligand
flexible-protein docking using IFD (Induced Fit Docking) Module (18).
The binding pose of [6]-shogaol-Akt1 obtained from the IFD docking
study (Figure3E, left panel) suggests that [6]-shogaol binds to a generally characterized PH-in conformation of Akt1. The Akt binding
site for [6]-shogaol is located underneath the activation loop of Akt1.
Its binding to Akt1 is in the allosteric binding site at the lower interface between the N- and C-lobes of the kinase domain. [6]-Shogaol
forms two hydrogen bonds with Ser205 and thus its activity might be
dependent upon the presence of the Ser205 residue in Akt (Figure3E,
right panel). [6]-Shogaol also formed strong hydrophobic interactions
with several amino acid residues, including Leu210, Ile290, Leu275,
Leu261, Tyr272 and Leu264, from the kinase domain of Akt1. In addition, hydrophobic contacts were observed between the phenyl ring of
[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 that
[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-H1650cells
We constructed lentiviral particles containing shRNA-control or
shRNA-Akt by transfecting an shRNA-control or shRNA-Akt1/2 plasmid with packaging DNA, pMD2.0G and psPAX and then infecting
these 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 (Figure4A). Interestingly,
treatment with [6]-shogaol failed to further reduce the colony numbers
in Akt1/2-silenced cells (Figure4A). Furthermore, after transfecting
with mock vector or constitutive activated (CA)-Akt in NIH-3T3 cells,
we performed an anchorage-independent colony formation assay.
The results revealed that the mock vector could not induce colony
formation but transfection with CA-Akt significantly increased the
colony numbers (Figure4B), which were decreased by treatment with
[6]-shogaol in a concentration-dependent manner (Figure4B). These
findings suggest that Akt is an essential target protein of [6]-shogaol
to suppress the growth of NCI-H1650 NSCLCcells.
[6]-Shogaol inhibits the expression of Akt downstream signaling
molecules and induces markers of apoptosis
Because [6]-shogaol showed no effect on the kinase activities of
PI3-K or mTOR, which are up- and downstream signaling molecules

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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 lung
cancer cells were assessed at 12, 24, 48, 72 and 96h by MTS assay. The asterisk (*) indicates a significant (P < 0.01) decrease in proliferation compared with
untreated 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. The
effects 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, 10
or 20M of [6]-shogaol and then incubated for 24h (cell cycle analysis) or 48h (annexin-V staining assay). The asterisks (*P<0.05, **P<0.01) indicate a
significant difference between untreated control and treated cells. Data are represented as means SD of values from triplicate samples and similar results were
obtained from three independent experiments.

in the Akt pathway, we examined the effect of [6]-shogaol on the

activation of STAT3, which is known to be regulated by Akt (23,24).
[6]-Shogaol attenuated the constitutive phosphorylation of STAT3 in
NCI-H1650 cells (Figure5A). Furthermore, the expression of STAT3
target gene products, including cyclin D1, cyclin D3 and c-Myc, was
decreased by treatment with [6]-shogaol in a concentration-dependent
manner (Figure5B). To further elucidate the molecular mechanisms
of [6]-shogaol-induced apoptosis in NCI-H1650 cells, we examined
the effect of [6]-shogaol on several apoptotic markers. Results indicated that [6]-shogaol induced the cleavage of pro-caspase-3 and -7,
resulting in increased cleavage of poly (ADP-ribose) polymerase
(Figure5C).
[6]-Shogaol inhibits NCI-H1650 lung cancer cell growth in a
xenograft mousemodel
We then examined the effect of [6]-shogaol on the growth of NCIH1650 cells as xenografts in athymic mice in vivo. The average growth

686

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 40mg/kg body weight). Although the average tumor volume was 393.4mm3 in vehicle-treated group, tumors
in mice treated with [6]-shogaol at doses of 10 and 40mg/kg body
weight showed an average volume of 274.7 and 140.8mm3, respectively. Treatment with [6]-shogaol did not cause any change in body
weight (Figure 6B). Immunohistochemical analysis revealed that
[6]-shogaol significantly inhibited the expression of Ki-67, which is a
cell proliferation biomarker (56.2% at 10mg/kg, 93.8% at 40mg/kg;
P < 0.01, Figure6C-a). Compared with the vehicle control group, the
group treated with [6]-shogaol showed a marked increase in TUNELpositive cells (2.1-fold at 10mg/kg and 2.7- fold at 40mg/kg; P <
0.01, Figure6C-b). Furthermore, we detected the expression of Akttarget proteins in tumor samples. The expression of phosphorylated
Akt (Ser473) was significantly decreased by treatment of [6]-shogaol

[6]-Shogaol is an allosteric inhibitor of Akt inNSCLC

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 active
Akt1 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 and
Leu264, show strong hydrophobic interactions with the long chain of the CH2-groups of [6]-shogaol. Note: the -helices are drawn as cylinders and the -strands
as 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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M.O.Kim etal.

Fig.4. Akt is a molecular target of [6]-shogoal. (A) The effect of [6]-shogaol (0, 10, and 20M) on the anchorage-independent growth of NCI-H1650 cells
expressing 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.

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 and
20M for 12h and then harvested and proteins were extracted and subjected to Western blot analysis. (A) Inhibitory effects of [6]-shogaol on the constitutive
expression 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 of
STAT3. (C) Immunoblots showing the effects of [6]-shogaol (treated at concentrations of 10 or 20M for 24h) on the expression of cleaved poly (ADP-ribose)
polymerase, cleaved caspase-3 and cleaved caspase-7. Representative blots from three independent experiments are shown.

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[6]-Shogaol is an allosteric inhibitor of Akt inNSCLC

Fig.6. [6]-Shogaol suppresses NCI-H1650 NSCLC tumor growth in a xenograft mouse model. (A) NCI-H1650 cells were inoculated in athymic nude mice
for the development of tumor xenografts and mice were treated with either vehicle or [6]-shogaol (10 or 40mg/kg body weight, intraperitoneal) as described
in Materials and methods. The average tumor volume in vehicle-treated control mice (n=8) and [6]-shogaol-treated mice (n=8) was plotted over 24days
after 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 on
mouse body weight. Body weights from treated or untreated groups of mice were measured 3 a week. (C) Quantification of hematoxylin and eosin staining
and immunohistochemical analysis of tumor tissues. [6]-Shogaol-treated or untreated mice were euthanized and tumor tissues were harvested. Sections of tumor
tissues 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 samples
was 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) and
cyclin D1 (e). For the TUNEL staining, the asterisk indicates a significant increase (b) in positive cells.

at 20mg/kg body weight (52.4%; P < 0.01, Figure6C-c). [6]-Shogaol

reduced the expression of phosphorylated STAT3 (Ser727) in xenograft tumors by 51.9% (P < 0.01) and 42.5% (P < 0.05) at doses of 10
and 40mg/kg body weight, respectively (Figure6C-d). Moreover, the
expression of cyclin D1 was also markedly attenuated by 79.1% (P <
0.01) and 90.0 % (P < 0.01) upon treatment with 10 or 40mg/kg body
weight of [6]-shogaol, respectively (Figure6C-e).
Discussion
Lung cancer is a leading cause of mortality throughout the world. The
number of deaths due to lung cancer has increased approximately
4.3% between 1999 and 2008. According to a report from the National
Cancer Institute, deaths from small cell lung cancer and NSCLC are
estimated 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 spreads
more slowly than small cell lung cancer, it is more common than small
cell lung cancer. Moreover, the majority of NSCLC is diagnosed only
when it has metastasized. Thus, adopting an appropriate prevention
strategy could reduce the incidence and mortality fromNSCLC.
A wide variety of dietary phytochemicals have been reported to
possess chemopreventive and/or chemotherapeutic activities. Ginger,
a common spice and a component of traditional medicine, contains
a 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 the
anticancer effects of these ginger polyphenols (3,4,2529), their
effects on NSCLC have been poorly investigated. In the current
study, we examined the effects of selected ginger polyphenols on

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M.O.Kim etal.

the growth of three different NSCLC cell lines and elucidated the
underlying mechanisms explaining the anticancer effects of the most
potent compound. Our study revealed that [6]-shogaol is more potent
than [6]-paradol or [6]-gingerol in reducing cell viability and inhibiting anchorage-independent growth of NSCLC cells. Our findings
are in good agreement with other studies reporting that [6]-shogaol
is more potent than [6]-gingerol in preventing chemically induced
skin cancer (30) and possesses the most potent antioxidant and antiinflammatory properties (25). The reason that [6]-shogaol is more
active than [6]-gingerol or [6]-paradol might be attributed to the
presence of an ,-unsaturated carbonyl moiety, which is absent in
[6]-gingerol or [6]-paradol. [6]-Shogaol has been reported to inhibit
proliferation and induce apoptosis in various cancer cells, including colon cancer, hepatocellular carcinoma, breast cancer, cervical
cancer and oral cancer (29,3136). Hung et al. (37) reported that
[6]-shogaol induced autophagic cell death in human lung cancer
(A549) cells at a concentration of 80M. In our study, we found that
[6]-shogaol at a concentration of 20M induced cell cycle arrest at
G1 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 antiproliferative
activity of [6]-shogaol in NCI-H1650 cells, we first predicted the
identity of potential kinase targets of the compound directly from
ligandprotein computational docking (data not shown) and then
performed kinase assays. Our finding that [6]-shogaol inhibited the
catalytic activities of Akt1 and Akt2 is in good agreement with a study
by Hung etal., who demonstrated that this compound inhibited the
Akt/mTOR pathway in A549 cells (37). However, in contrast to their
study, we noticed that [6]-shogaol failed to affect the kinase activity of PI3-K or mTOR in NCI-H1650 cells. Moreover, [6]-shogaol
did not affect the activities of other kinases, such as EGFR, cyclicAMP-activated kinase- (AMPK), glycogen synthase kinase3 (GSK3), 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) and
p38 (Supplementary Table 1, available at Carcinogenesis Online).
The finding that [6]-shogaol inhibited Akt activation in NCI-H1650
cells is well supported by several previous studies demonstrating the
inhibitory effect of [6]-shogaol on Akt phosphorylation and/or activity in other cancer models (28,30,38). Our new evidence showed that
[6]-shogaol directly interacted with Akt1/2 and diminished Akt1/2
activities. In addition, shRNA-based silencing of Akt significantly
attenuated anchorage-independent growth of NCI-H1650 cells, suggesting Akt as a key signaling molecule in regulating the survival and
proliferation of NSCLC cells. Although treatment with [6]-shogaol
failed to reduce colony formation in Akt-silenced cells, the compound
significantly attenuated the anchorage-independent growth of Aktoverexpressing cells. These findings suggest that Akt is a bona fide
target of [6]-shogaol in suppressing the proliferation and inducing
apoptosis in NCI-H1650cells.
Because [6]-shogaol failed to affect the activity of mTOR, an Akt
downstream target, and many other kinases involved in cell proliferation (Supplementary Table1, available at Carcinogenesis Online), and
the compound inhibited expression of cell proliferation-related proteins
cyclin D1 and D3, we examined the effect of [6]-shogaol on the phosphorylation of STAT3, a transcriptional regulator of cyclin D1 and D3.
Because Akt-mediated signaling is a cell survival pathway and Akt acts
as an upstream kinase of STAT3 (23,24), the [6]-shogaol-induced caspase cleavage and diminished phosphorylation of STAT3 may result
from its inhibitory effect on Akt activation. These findings were further supported by an in vivo xenograft study. Our study revealed that
treatment with [6]-shogaol significantly decreased the tumor xenograft
growth of NCI-H1650 cells, which was associated with decreased cell
proliferation and increased apoptosis as evidenced by reduced Ki-67positive cells and an increased number of TUNEL-positive cells in the
[6]-shogaol-treated xenograft tumors. Moreover, the immunohistochemical analysis of these tumors showed a significant inhibition of
cyclin D1 expression and decreased phosphorylation of Akt andSTAT3.

690

The activation of p53 is one of the well-established mechanisms

for apoptosis induction and growth arrest. Liu etal. demonstrated
that a steam-distilled extract of ginger induced apoptosis in human
endometrial cancer cells through activation of p53. However,
shogaols were absent in this ginger extract (25). Ap53-independent
mechanism of apoptosis induction by [6]-shogaol has been reported
(39). According to this study, [6]-shogaol induced apoptosis in
Mahlavu cells, a p53-mutant human hepatoma cell line, through the
generation of reactive oxygen species and subsequent depletion of
glutathione.
In conclusion, our study demonstrates that among the ginger polyphenols, [6]-shogaol is the most potent in suppressing the growth of
NSCLC cells and the compound inhibits proliferation and induces
apoptosis in NCI-H1650 cells by suppressing Akt signaling through
the 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 Program
Rural Development Administration, Republic of Korea (PJ009560 to
M.O.K.).
Acknowledgements
We thank Dr. Tae-Gyu Lim, Do Young Lim, Soouk Kang, and Todd Schuster
for 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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