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].

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′-CCGGCCCGGATCATTCACCCTATTGC

TCGAGCAATAGGGTGAATGA.

TCCGGGTTTTTG-3′; #4, 5′-CCGGGAACCAATTA

TACCCGTTCTTCTCGAGAA.

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. 

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.