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. 2013 Nov 26;35(3):683–691. doi: 10.1093/carcin/bgt365

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

Myoung Ok Kim 1,2,†, Mee-Hyun Lee 1,3,†, Naomi Oi 1,†, Sung-Hyun Kim 1,2,†, Ki Beom Bae 1, Zunnan Huang 1,4, Dong Joon Kim 1,5, Kanamata Reddy 1, Sung-Young Lee 1,6, Si Jun Park 2, Jae Young Kim 2, Hua Xie 1,7, Joydeb Kumar Kundu 8, Zae Young Ryoo 2, Ann M Bode 1, Young-Joon Surh 3, Zigang Dong 1,6,*
PMCID: PMC3941745  PMID: 24282290

Summary

[6]-Shogaol, a component of ginger root, suppressed non-small cell lung cancer (NSCLC) cell growth mediated by EGFR signaling. It directly binds to Akt to suppress its kinase activity resulting in increased cancer cell death both ex vivo and in vivo.

Abstract

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 target–oriented 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 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, anti-inflammatory, 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 (7–9). 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 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 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.

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) (11–13) 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 β-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 (100 µg/ml), sodium pyruvate (1mM) and 10% fetal bovine serum (FBS, Gemini Bio-Products, Calabasas, CA) and maintained at 5% CO2 and 37°C 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.1-mock, 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 (Polyplus-transfection SAS, Saint Quentin Yvelines, France) following the manufacturer’s 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 8 µg/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 (1 µg/ml) for 48h, the selected cells were used for an anchorage-independent cell growth assay.

In vitro kinase assay

The kinase assay was performed according to the instructions provided by Millipore. In brief, the reaction was conducted in the presence of 10 µCi of [γ-32P] ATP and each compound in 40 µl 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 10 µl of protein loading buffer, and the mixture was separated by sodium dodecyl sulfate–polyacrylamide 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 ligand–protein 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). Protein–ligand 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 Schrödinger 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 proliferation assay

For the proliferation assay, cells were seeded (1×103 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 (8×103 cells per well) suspended in complete medium were added to 0.3% agar with 0, 10 or 20 µM [6]-shogaol, [6]-paradol or [6]-gingerol in a top layer over a base layer of 0.5% agar with 0, 10 or 20 µM [6]-shogaol, [6]-paradol or [6]-gingerol. The cultures were maintained at 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 (Media Cybernetics, Rockville, MD).

Cell cycle and apoptosis analyses

Cells were plated in 100 mm plates and treated with 0, 10 or 20 µM [6]-shogaol for 24 or 48h. Cells were then fixed in 70% ethanol and stored at -20°C 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-down assay

Recombinant human Akt1, Akt2 (200ng) kinase or a NCI-H1650 cell lysate (500 µg) was incubated with [6]-shogaol-conjugated Sepharose 4B or Sepharose 4B beads only as a control (50 µl; 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 4°C, 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 sulfate–polyacrylamide 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 β-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 mouse model

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 (3×106 cells in 200 µl of phosphate-buffered saline) were injected subcutaneous on the right hind flank. After 6 days 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 Student’s t-test or one-way analysis of 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]-gingerol (Figure 1A–C) 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 (Figure 2A), NCI-H520 (Supplementary Figure 1A, available at Carcinogenesis Online) and NCI-H1975 (Supplementary Figure 1B, available at Carcinogenesis Online) cells compared with [6]-paradol or [6]-gingerol. Notably, 20 µM [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 20 µM [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% (Figure 2B) and suppressed the growth of NCI-H520 cells by 74.2 and 89.3% at 10 and 20 µM, 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 20 µM (Figure 2B), it failed to affect the growth of NCI-H520 cells (Supplementary Figure 1C, available at Carcinogenesis 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). 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 NCI-H1650 cells by 24.2 and 27.2% at concentrations of 10 and 20 µM, respectively (Figure 2C). 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 NCI-H520 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).

Fig. 1.

Fig. 1.

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

Fig. 2.

Fig. 2.

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[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 20 µM 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.

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 pull-down assay results revealed a direct binding between [6]-shogaol and Akt1 or Akt2 (Figure 3B). 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 ATP-competitive 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 100 µM) 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 Schrödinger-Maestro v9.2 (20) software program was then used for docking experiments to assess the possible binding mode between Akt1 and [6]-shogaol (Figure 3E). 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 (Figure 3E, 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 (Figure 3E, 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.

Fig. 3.

Fig. 3.

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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 α-helices are drawn as cylinders and the β-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).

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

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 sh-control 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). Interestingly, treatment with [6]-shogaol failed to further reduce the colony numbers in Akt1/2-silenced cells (Figure 4A). 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 (Figure 4B), which were decreased by treatment with [6]-shogaol in a concentration-dependent manner (Figure 4B). These findings suggest that Akt is an essential target protein of [6]-shogaol to suppress the growth of NCI-H1650 NSCLC cells.

Fig. 4.

Fig. 4.

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Akt is a molecular target of [6]-shogoal. (A) The effect of [6]-shogaol (0, 10, and 20 µM) 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.

[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 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 (Figure 5A). 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 (Figure 5B). 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 (Figure 5C).

Fig. 5.

Fig. 5.

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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 20 µM 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 20 µM 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.

[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 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, Figure 6C-a). Compared with the vehicle control group, the group treated with [6]-shogaol showed a marked increase in TUNEL-positive cells (2.1-fold at 10mg/kg and 2.7- fold at 40mg/kg; P < 0.01, Figure 6C-b). Furthermore, we detected the expression of Akt-target proteins in tumor samples. The expression of phosphorylated Akt (Ser473) was significantly decreased by treatment of [6]-shogaol at 20mg/kg body weight (52.4%; P < 0.01, Figure 6C-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 (Figure 6C-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 (Figure 6C-e).

Fig. 6.

Fig. 6.

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[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 24 days 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.

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 from NSCLC.

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 (3,4,25–29), their effects on NSCLC have been poorly investigated. In the current study, we examined the effects of selected ginger polyphenols on 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 anti-inflammatory properties (25). The reason that [6]-shogaol is more active than [6]-gingerol or [6]-paradol might be attributed to the presence of an α,β-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 (29,31–36). Hung et al. (37) reported that [6]-shogaol induced autophagic cell death in human lung cancer (A549) cells at a concentration of 80 µM. In our study, we found that [6]-shogaol at a concentration of 20 µM 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 ligand–protein 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 et al., 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, 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) and p38α (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 Akt-overexpressing cells. These findings suggest that Akt is a bona fide target of [6]-shogaol in suppressing the proliferation and inducing apoptosis in NCI-H1650 cells.

Because [6]-shogaol failed to affect the activity of mTOR, an Akt downstream target, and many other kinases involved in cell proliferation (Supplementary Table 1, 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-67-positive 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 and STAT3.

The activation of p53 is one of the well-established mechanisms for apoptosis induction and growth arrest. Liu et al. 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). A p53-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.).

Supplementary Material

Supplementary Data
supp_35_3_683__index.html (1.6KB, html)

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.

Glossary

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.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data
supp_35_3_683__index.html (1.6KB, html)
supp_bgt365_FigS1.tif (564.7KB, tif)
supp_bgt365_Supplemental_Figure_Legends_R_092413.doc (29.5KB, doc)
supp_bgt365_Supplemental_materials_and_methods_R_092413.doc (41KB, doc)
supp_bgt365_Supplementary_Table1_R_092413.docx (17.6KB, docx)
supp_bgt365_FigS2.tif (641.9KB, tif)

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