Background
Gastric cancer (GC) is a cancer of the digestive tract that remains one of the common malignant cancers worldwide [
1,
2]. Specifically, it is the third leading cause of cancer-related mortality and the second frequently diagnosed cancer in the world [
3]. Although clinical advances have been made in the fields of surgery, radiotherapy, and chemotherapy, the five-year survival rate of gastric cancer patients is approximately 15 to 35% [
4]. Additionally, many types of targeted therapies, including inhibition of tyrosine kinase (TK) and receptor tyrosine kinase (RTK), are currently being used as treatment options for GC; however, they have shown only minimal efficacy [
5,
6]. Therefore, identification of novel therapeutic targets and inhibitors are important for improving the survival rate of gastric cancer patients.
Mammalian target of rapamycin (mTOR) plays a central role in cell proliferation, cell motility, cell survival, cellular metabolism and protein synthesis [
7]. mTOR is a serine/threonine protein kinase that is activated by various growth factors, cellular energy, cell stress and amino acids [
8]. mTOR is classified structurally and functionally in two complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which share common subunits, such as mTOR, the mammalian lethal with SEC13 protein 8 (mLST8), telomere maintenance 2 (Tel2) and Tel2-interacting protein 1 (Tti1) [
9]. mTORC1 contains the regulatory-associated protein of mTOR (RAPTOR), which is a scaffolding protein in the mTORC1 assembly, and mTORC2 contains the rapamycin-insensitive companion of mTOR (RICTOR) [
10]. AKT phosphorylates Ser2448 of mTOR in addition to tuberous sclerosis complex 2 (TSC2) thereby resulting in activation of mTOR kinase activity [
11]. Additionally, mTOR is auto-phosphorylated at Ser2481 which is located in a hydrophobic region near the conserved carboxyl-terminal and required for FRAP kinase activity [
12]. The complex in which it participates dictates the substrate specificity of mTOR. The mTORC1 substrate S6 Kinase 1 (S6K1) associates with mRNAs and regulates both mRNA translation initiation and progression, thus enhancing protein synthesis [
13]. S6K1 is a serine/threonine protein kinase that is necessary for cell growth and G1 cell cycle progression [
14]. In contrast, mTORC2 phosphorylates and activates v-Akt murine thymoma viral oncogene homolog (AKT) which regulates cell growth, cell survival and cell cycle progression [
13]. AKT is a serine/threonine kinase that belongs to the AGC family of kinases [
15], and regulates many cellular functions, including proliferation, survival, epithelial mesenchymal transition (EMT), and metabolism; additionally, AKT directly phosphorylates a wide range of downstream substrates [
16]. mTOR is dysregulated in various cancers due to its direct mutation, mutations of mTOR components and mutation of upstream genes including oncogenes and tumor suppressor genes [
17]. mTOR and AKT are overexpressed in GC cells and the mTOR pathway is activated in 60% of GC patients [
18]. Currently, mTOR inhibitors have been investigated in preclinical studies and clinical trials of GC [
19]. mTOR inhibitors have been tested in many clinical trials in the context of other cancers, but they achieved only modest efficacy applied as monotherapies in cancer treatments due to resistance mechanisms [
20,
21]. Therefore, combined therapies with mTOR inhibitors and other target inhibitors are under investigation in preclinical and clinical trials in various cancers [
22]. Thus, novel therapeutic strategies with mTOR inhibitor should be further investigated.
Fermented wheat germ extract possesses preventive and therapeutic functions in various cancer cells [
23,
24]. 2,6-Dimethoxy-1,4-benzoquinone (2,6-DMBQ), a derivative of fermented wheat germ extract, is found in sourdough fermentation of wheat germ and other fermented foods. However, the anticancer activity of 2,6-DMBQ and its molecular mechanism(s) against gastric cancer have not been investigated. In the present study, we report that 2,6-DMBQ is a novel mTOR inhibitor that reduces gastric cancer growth in vitro and in vivo.
Methods
Reagents and antibodies
2,6-DMBQ was purchased from Shanghai Chemic Industry (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Tianjin Kemai Chemical Reagent Company (Tianjin, China). AZD8055 was purchased from Selleckchem (Houston, TX, USA) and CMPD101 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Biological Industries (Cromwell, CT, USA). MEM/EBSS medium was purchased from GE Healthcare (Logan, UT, USA). Active mTOR recombinant protein for kinase assay was purchased from ThermoFisher (Shanghai, China). Inactive p70S6K recombinant protein for in vitro kinase assay was purchased from SignalChem (Richmond, BC, Canada). The antibody to detect β-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and all the other antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).
Cell lines
AGS, HGC27, NCI-N87 and SNU-1 gastric cancer cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). JB6 mouse epithelial cells were purchased from American Type Culture Collection (Manassas, VA, USA). Enough frozen vials were available for each cell line to ensure that all cell-based experiments were conducted on cells that had been authenticated and in culture for a maximum of 8 weeks. AGS, NCI-N87 and SNU-1 cells were cultured in Roswell Park Memorial Institute medium 1640 (RPMI1640) medium with 10% FBS and 1% penicillin–streptomycin. HGC27 cells were cultured in Minimum Essential Medium with Earle’s Balanced Salts (MEM/EBSS) supplemented with 1% non-essential amino acid (NEAA), 10% FBS and 1% antibiotic-antimycotic. The JB6 cells were cultured in MEM supplemented with 5% FBS and 1% penicillin–streptomycin. All cells were maintained at 37 °C in a 5% CO2 humidified incubator.
Cell proliferation assay
AGS (1.2 × 103 cells per well) or HGC27 (2.0 × 103 cells per well) cells were seeded in 96-well plates with 100 μl complete growth medium (10% FBS) and incubated for 24 h. Cells were treated with various concentrations of 2,6-DMBQ (dissolved in DMSO) or vehicle (DMSO) in 100 μl of complete growth medium. After incubation for 48 h, 20 μl of the MTT solution (Solarbio, Beijing, China) were added to each well. After incubation for 2 h at 37 °C in a 5% CO2 incubator, the cell culture medium was removed. Subsequently, 150 μl of DMSO was added to each well and the crystal formation was dissolved. Absorbance was measured at 570 nm using the Thermo Multiskan plate-reader (Thermo Fisher Scientific, Waltham, MA, USA).
Anchorage-independent cell growth assay
Cells (8 × 103 cells per well) suspended in complete growth medium supplemented with 10% FBS were added to 0.3% agar with different concentrations of 2,6-DMBQ (dissolved in DMSO) or vehicle (DMSO) in a top layer over a base layer of 0.6% agar with or without different concentrations of 2,6-DMBQ. The cultures were maintained at 37 °C in a 5% CO2 incubator for 2 weeks and then colonies were imaged under a microscope and quantified using the Image-Pro Plus software (v.6) program (Media Cybernetics, Rockville, MD, USA).
Western blot analysis
Cells were lysed in radio-immunoprecipitation assay buffer (RIPA) buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 1 mM EDTA, 1 × protease inhibitor solution), and incubated on ice for 1 h. The soluble cell lysates were collected by centrifugation at 10000 g for 10 min. Proteins were separated by SDS/PAGE and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ, USA). Membranes were blocked with 5% nonfat dry milk at room temperature for 1 h and incubated with appropriate primary antibodies at 4 °C for overnight. The next day the membranes were washed with TBST, followed by 1 h incubation with 1:5000 dilution of horseradish peroxidase–linked secondary antibody. The immuno-reactive proteins were detected by chemiluminescence reagent (Amersham Biosciences Corp) using the ImageQuant LA S4000 system (GE Healthcare, Piscataway, NJ, USA).
In vitro ATP assay for mTOR kinase activity
To determine mTOR kinase activity, an ATP assay was carried out using the ADP-Glo Kinase Assay Kit, in accordance with the manufacturer’s instructions (Promega, Madison, WI, USA). The active recombinant mTOR (50 ng) protein was mixed with different concentrations of 2,6-DMBQ, AZD8055 (dissolved in DMSO) as a mTOR inhibitor, or vehicle (DMSO) in reaction buffer (Cell Signaling Technology) and incubated at room temperature for 15 min. The inactive p70S6K recombinant protein (100 ng) and ATP were added and the mixtures were incubated at 30 °C for 30 min. The fluorescence of each sample was measured at excitation and emission wavelengths of 530 nm and 590 nm, respectively.
Cell cycle analysis
AGS (6 × 104 cells per dish) or HGC27 (7 × 104 cells per dish) cells were plated into 60-mm culture dishes and incubated for 24 h. Cells were synchronized by serum starvation for 24 h and treated with serum and 2,6-DMBQ (dissolved in DMSO) or vehicle (DMSO) for 24 h in 10% serum and medium. Cells were collected by trypsinization and washed with phosphate-buffered saline (PBS) and then fixed in 1000 μl of 70% cold ethanol. After rehydration, cells were incubated in RNase (100 μg/mL) and stained with propidium iodide (PI; 20 μg/mL). PI staining was accomplished following the manufacturer’s instructions (Clontech, Palo Alto, CA) and the cells were analyzed by flow cytometry.
Apoptosis assay
Cells were plated into 6 well plates (5 × 104 cells per well). After incubation for 24 h, cells were treated with different doses of 2,6-DMBQ (dissolved in DMSO) or vehicle (DMSO) for 48 h in 10% serum-containing medium. Cells were collected by trypsinization and washed with PBS. Cells were subsequently stained with Annexin V (BioLegend, San Diego, CA) and propidium iodide before apoptosis was analyzed by flow cytometry.
Lentiviral infection
Short hairpin RNA sequences against mTOR were designed (#3, 5′-CCGGCCCGGATCATTCACCCTATTGCTCGAGCAATAGGGTGAATGA.
TCCGGGTTTTTG-3′; #4, 5′-CCGGGAACCAATTATACCCGTTCTTCTCGAGAA.
GAACGGGTATAATTGGTTCTTTTTG-3′) and cloned into the lentiviral vector (pLKO.1-mTOR). The lentiviral packaging vectors (pMD2.0G and psPAX) were purchased from Addgene Inc. (Cambridge, MA, USA). To prepare mTOR viral particles, each viral vector and package vectors were transfected into HEK293T cells by using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) following the manufacturer’s suggested protocol. After incubation for 48 h, viral particles were harvested by filtration using a 0.45 mm sodium acetate syringe filter. The virus-containing media was combined with 8 μg/ml of polybrene (Millipore, Billerica, MA, USA) before being used to infect AGS or HGC27 cells. After incubation for 24 h, cells were selected with puromycin (1 μg/ml) for 48 h. The selected cells were used for experiments.
Patient-derived xenograft gastric tumor growth assay and ethics statement
To examine the effect of 2, 6-DMBQ on patient-derived gastric tumor growth, female mice (Vital River Labs, Beijing, China) with severe combined immunodeficiency (SCID; 6–9 weeks old) were maintained under “specific pathogen-free” conditions based on the guidelines established by Zhengzhou University Institutional Animal Care and Use Committee (Zhengzhou, China). Human tumor specimens of gastric cancer tissue were obtained from the Affiliated Cancer Hospital in Zhengzhou University. The gastric cancer patients did not receive any chemotherapy or radiotherapy prior to surgery. Tissue histology was confirmed by a pathologist. Prior written informed consent was obtained from patients. Mice were anesthetized by 0.4% pentobarbital sodium (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). Mice were pierced to the back of neck using a blunt puncher and then treated with Penicillin-Streptomycin (80,000 U/ml) in the affected area. Gastric cancer tissues composed of normal, cancerous stromal and tumor were cut into pieces (3–4 mm3) and implanted into the back of the neck of 3 individual mice. After the 3rd generation of human gastric cancer tissue growth, tissues were again cut into pieces and implanted into mice. Mice were divided into 2 groups of 7 animals as follows: 1) vehicle (10% DMSO and 20% tween 80) group and 2) 80 mg 2,6-DMBQ/kg of body weight in vehicle (10% DMSO and 20% tween 80) were administered by oral gavage once a day Monday through Friday. Tumor volume was calculated from measurements of 2 diameters of the individual tumor base using the following formula: tumor volume (mm3) = (length ×width× height× 0.52). Mice were monitored until tumors reached 1.5cm3 total volume, at which time mice were euthanized and tumors, liver, kidney, and spleen extracted.
Hematoxylin-eosin staining and immunohistochemistry
The liver, spleen, kidney, and tumor tissues from mice were embedded in paraffin blocks and used for hematoxylin and eosin (H&E) staining or immunohistochemistry (IHC). For H&E staining, the tissue sections were deparaffinized, hydrated and stained with H&E and then dehydrated. For IHC, tumor tissue sections were deparaffinized and hydrated. After antigen retrieval with 10 mM citrate acid and blocking with 5% BSA, the tumor tissue sections were hybridized with a primary antibody (Ki-67, 1:100; Thermo Fisher Scientific) for 18 h at 4 °C and then an HRP-conjugated goat anti-rabbit or mouse IgG antibody (ZSGB-BIO, Beijing, China) was added and incubated for 30 min. Tissue sections were developed with 3, 3′-diaminobenzidine (ZSGB-BIO) for 10 s and then counterstained with hematoxylin for 1 min. All sections were observed by microscope and analyzed using the Image-Pro Plus software (v. 6) program.
In vivo toxicity assay
Female mice (SCID; 6–9 weeks old) were maintained under “specific pathogen-free” conditions based on the guidelines established by Zhengzhou University Institutional Animal Care and Use Committee. Mice were divided into 4 groups as follows: 1) vehicle group (n = 4); 2) 20 mg 2,6-DMBQ/kg of body weight in vehicle (n = 4); 3) 50 mg 2,6-DMBQ/kg of body weight in vehicle (n = 4); and 4) 80 mg 2,6-DMBQ/kg of body weight in vehicle (n = 4). 2,6-DMBQ or vehicle (10% DMSO in 20% tween 80) was orally administered for 2 weeks. Blood samples from each group of mice were collected in heparin-treated tubes. The AST or ALT activity from serum was measured at 510 nm.
Statistical analysis
All quantitative results are expressed as mean ± S.D. or ± S. E values. Significant differences were compared using the Student’s t-test or one-way analysis of variance (ANOVA). Differences with a p < 0.05 were considered to be statistically significant. The statistical package for social science for Windows (IBM, Inc. Armonk, NY, USA) was used to calculate the p-value to determine statistical significance.
Discussion
Dietary intake of quinones have been reported to show cancer prevention through inhibitory effects on cell proliferation and tumor development [
26]. 2,6-DMBQ is a benzoquinone compound that is isolated from sourdough fermentation of wheat germ. Recently, 2,6-DMBQ has been reported to possess cancer prevention properties against TPA-induced skin carcinogenesis [
27]. However, the molecular targets of 2,6-DMBQ and its potential therapeutic effect have not been investigated in cancer. In this study, we report that 2,6-DMBQ reduces the growth of gastric cancer by targeting mTOR in vitro and in vivo.
The results of signaling pathway (Fig.
3a, b) and in vitro kinase assay (Fig.
3c) strongly support that 2,6-DMBQ is a potent mTOR protein kinase inhibitor and can reduce mTOR signaling pathway in gastric cancer cells. Additionally, the results of our cancer-related kinase screening showed that 2,6-DMBQ at 10 μM reduced about 30% of the PKCα activity (Supplemental Fig.
2). Therefore, we next examined whether 2,6-DMBQ could affect growth of gastric cancer cells through targeting PKCα. We first investigated the effect of PKCα inhibitor (CMPD101) on growth of gastric cancer cells. The results showed that PKCα inhibitor significantly reduced growth of gastric cancer cells (Supplemental Fig.
7a, b). We next assessed the effect of 2,6-DMBQ combined with PKCα inhibitor on gastric cancer cell growth. Cells were treated with PKCα inhibitor combined with or without 2,6-DMBQ. Results indicated that PKCα inhibitor-treated cells were not resistant to 2,6-DMBQ’s effect on cell growth compared to 2,6-DMBQ-treated cells (Supplemental Fig.
7c, d). Therefore, we suggest that it is highly likely that 2,6-DMBQ preferentially targets mTOR as opposed to PKCα. However, the result of the anticancer effect upon treatment with 2,6-DMBQ in cells with low mTOR expression (
shmTOR #4) suggested that 20 μM of 2,6-DMBQ still reduced cell growth (Fig.
5a, b). It is possible there are other molecular targets of 2,6-DMBQ. Therefore, additional studies are planned to further characterize 2,6-DMBQ in identifying additional potential molecular targets.
mTOR signaling plays an important role in G1 to S phase cell cycle transition through regulation of cyclin D1 and c-myc expression [
28], and inhibition of mTOR activity by an mTOR inhibitor induced G1 phase cell cycle arrest [
29]. Based on the results of cell cycle and cell cycle marker proteins (Fig.
1d, e), we suggest that the reduction of mTOR activity by 2,6-DMBQ treatment may induce G1 phase cell cycle arrest and reduce the expression of cyclin D1 and cyclin D3.
Although many anticancer reagents have shown favorable tumor responses in preclinical studies, only 5% of anticancer drugs developed have been approved by the Food and Drug Administration (FDA) [
30,
31]. This is due to a number of reasons, including the development of resistance conferred by tumor heterogeneity as well as human stromal microenvironmental conditions [
32]. Therefore, to overcome low clinical efficacy, researchers established the patient-derived xenograft (PDX) model to screen potential candidate drugs [
33]. We first investigated the antitumor effects of 2,6-DMBQ on gastric cancer PDX models and the results showed that 2,6-DMBQ significantly reduced gastric tumor growth by inhibiting the mTOR/p70S6K signaling pathway (Fig.
6a, d).
Previously, phosphorylated mTOR was found to be significantly over-expressed and correlated with various clinical and pathologic parameters in patients with gastric cancer [
34,
35]. Additionally, the mTOR signaling pathway is positively correlated with Ki-67 expression [
36‐
38] and rapamycin was found to inhibit Ki-67 expression in patients with glioblastoma [
39]. Therefore, we examined whether 2,6-DMBQ could reduce the expression of Ki-67 in gastric cancer PDX tissues. We found that the expression of Ki-67, phosphorylated mTOR and phosphorylated p70S6K was significantly decreased in the 2,6-DMBQ-treated group compared to the vehicle-treated group (Fig.
6c, d). Therefore, reducing mTOR signaling by an inhibitor could provide antineoplastic effects for treatment of gastric cancer.
In conclusion, our findings demonstrate that 2,6-DMBQ is a potent mTOR inhibitor that reduces growth of gastric cancer. These findings could be useful for treating gastric cancers.
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