1. Introduction

Lung cancer is the most frequently diagnosed malignant cancer

and the leading cause of cancer mortality worldwide.1,2 The two

types of lung cancer are non-small cell lung cancer (NSCLC), which

comprises approximately 85% of lung cancer cases, and small cell

lung cancer (SCLC).3 Although clinical advances in prevention and

therapeutics against lung cancer have progressed in recent years,

the survival rate is still less than 20%.4 The poor survival of lung

cancer patients is due to chemoresistance and the distant metastasis at the time of diagnosis.5 Therefore, novel approaches to

chemotherapy, targeted therapy, and prevention of metastasis are

urgently needed.

Signaling pathways that regulate cell growth, cell survival,

genomic instability, and angiogenesis are correlated with lung

cancer progression and metastatic potential.6 V-Akt murine thymoma viral oncogene homolog (AKT) is a serine/threonine kinase

that regulates cell survival, proliferation and many other biological

responses through directly inducing the phosphorylation of

downstream substrates.7 AKT isoforms (AKT1, AKT2 and AKT3)

have a high sequence homology in the catalytic domains, but

diverge in the hydrophobic motif (HM) domain and the pleckstrin

homology (PH) domain.8 It was recently discovered that the AKT

signaling pathway induces epithelialemesenchymal transition

(EMT) and migration through regulating E-cadherin expression.9

Dual-treatment with EGFR and AKT inhibitors was found to synergistically inhibit tumor growth and promote apoptosis in EGFRresistant NSCLC models.10

2.3. Western blotting

Proteins were measured by BCA kit (Solarbio) following the

manufacturer's suggested protocol. Proteins were separated by

SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ, USA). Membranes were then blocked with 5% nonfat dry milk (Solarbio) in

TBST (TBS with 1% Tween 20) at room temperature for 1 h. After

blocking, the membranes were washed three times with TBST

and incubated overnight with appropriate primary antibodies at

4 C. The next day, the membranes were washed with TBST three

times and then incubated with an appropriate horseradish

peroxidaseelinked secondary antibody for 1 h. The membranes

were washed three times with TBST and the immuno-reactive

proteins were detected by Thermo Scientific SuperSignal West

Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) using the ImageQuant LA S4000 system (GE Healthcare,

Piscataway, NJ, USA).

2.4. Cell proliferation assay

Cells were seeded (2  103 cells per well) in 96-well plates

with 100 ml complete growth medium and incubated for 24 h at

37 C. Cells were treated with various concentrations of 2,6-

DMBQ diluted in 100 ml of complete growth medium for 48 h.

After the incubation period, 20 ml of MTT solution (Solarbio)

were added to each well and the cells were incubated for an

additional 2 h at 37 C. The cell culture medium was then discarded and replaced with 150 ml of DMSO (Kermel, Tianjin,

China). Formazan crystals were dissolved by gentle agitation.

Cell proliferation was measured at 570 nm wavelength using a

Thermo Multiskan plate-reader (Thermo Fisher Scientific, Waltham, MA, USA). To determine the IC50value of all compounds

used within this study, we chose the 72 h MTT result to calculate

the half maximal inhibitory concentration using GraphPad Prism

5 (GraphPad Software, San Diego, CA, USA). We used the calculated IC50 value of AKT-I or SB203580 or GSK3b-I for cotreatment with 2,6-DMBQ.

2.5. Anchorage-independent cell growth

Cells (8  103 per well) suspended in complete growth medium

-RPMI 1640 supplemented with 10% FBS and 10 mg/ml gentamycin

(Solarbio) were added to 0.3% agar (Becton, Dickinson and Company, NJ, USA) with or without various concentrations of 2,6-DMBQ

in a top layer over a base layer of 0.6% agar with or without various

concentrations of 2,6-DMBQ. The culture dishes were maintained

at 37 C in a 5% CO2 incubator for 2 weeks. Colonies were photographed using a wide-field microscope and processed for analysis

with the Image-Pro Plus software (v.6) program (Media Cybernetics, Rockville, MD, USA).

2.6. Colony formation assay

Cells were seeded (500 cells per well) in 6-well plates with 2 ml

complete growth medium (RPMI 1640 supplemented with 10% FBS

and 1% antibiotic-antimycotic solution) and incubated for 24 h.

Cells were treated with different concentrations of 2,6-DMBQ in

2 ml of complete growth medium and incubated for 1 week. Cells

were stained with 0.5% Coomassie brilliant blue (Solarbio) for

20 min and then imaged.

2.7. Cell cycle analysis

Cells were seeded (8  104 cells per dish) into 60 mm culture

dishes. After incubation for 24 h, cells were treated with various

concentrations of 2,6-DMBQ for 24 h and harvested. Cells were then

washed with cold phosphate buffered saline (PBS) and fixed in 1 ml

70% cold ethanol. After rehydration, cells were permeabilized with

0.6% Triton X-100 (Solarbio) and digested with 100 mg/ml RNase A

(Solarbio) for 1 h. Cells were subsequently stained with 20 mg/ml

propidium iodide (Clontech, Palo Alto, CA, USA) for 15 min before

analysis by flow cytometry (Becton, Dickinson and Company,

Franklin Lakes, NJ, USA).

2.8. Wound healing assay

Cells were seeded (1  105 cells per well) in 6-well plates with

100 ml complete growth medium. After incubation for 24 h, the cells

were gently scratched using a plastic micro pipette tip and then

washed three times with PBS. Cells were treated with various

concentrations of 2,6-DMBQ for 24 h or 48 h. The 6-well plates

containing the scratch-wounds were photographed in three

different fields of each well. The average width of each scratchwounds was measured and calculated using ImageJ (National Institutes of Health, Bethesda, MD, USA).

2.9. Migration assay

The lower compartment of transwell chambers (Corning, Bedford, MA, USA) were coated with Matrigel (Becton, Dickinson and

Company, NJ, USA) and incubated for 30 min at room temperature

under sterile conditions. The lower compartment of each chamber

was then filled with 600 ml complete growth medium. Fifty thousand cells suspended in 200 ml complete growth medium with or

without 2,6-DMBQ were added to the upper compartment. The

chambers were incubated for 24 h at 37 C in a 5% CO2 atmosphere.

Migrated cells were fixed with methanol (Tianjin Zhiyuan Chemical

Reagent Co. LTD, Tianjin, China) and stained with hematoxylin

(Baso Diagnostics INC, Zhuhai, China) and eosin (Baso Diagnostics

INC) prior to imaging. Photos of the stained cells were analyzed

using the Image-Pro Plus software (v.6) program (Media

Cybernetics).

2.10. Statistical analysis

All quantitative results are indicated as mean values ± S.D.

Statistically significant differences were determined using the

Student's t test or by one-way ANOVA (p < 0.05). Statistical significance was determined using the Statistical Package for Social

Science (SPSS) 21.0 (Xishu software, Shanghai, China).