All animal experiments were approved by the Ethics Committee of The First Affiliated Hospital of Fujian Medical University (Fujian, China). A total of 156 male BALB/C nude mice, (age, 5–6 weeks; weight, 16–20 g) were provided by the Shanghai Laboratory Animal Research Center (Shanghai, China). The mice were maintained in individual cages at a controlled temperature (22±2°C) and humidity (55%), under 12/12 h light/dark cycles, with free access to food and water. To develop xenograft tumors, Eca-109 cells transfected with LKB1 overexpression plasmid or empty vector were harvested, washed with PBS and implanted into the hind limb of the BALB/C nude mice (2×106 cells/0.1 ml). When xenograft tumors reached a mean diameter of 5 mm, the ‘radiation group’ animals were irradiated every 4 days with a dose of 12 Gy radiation in three fractions. Tumor growth was measured every 2 days by a caliper until day 25, and the tumor volume (mm3) was calculated according to the formula: Tumor volume (mm3) = length (mm) × width (mm²)/2.

Eca-109 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco's modified Eagle's medium (Hyclone; GE Healthcare, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 µg/ml penicillin at 37°C in a humidified atmosphere containing 5% CO₂. Compound C was purchased from EMD Millipore (Billerica, MA, USA) and used at a concentration of 20 µM.

The LKB1-pcDNA3.1 plasmid was synthesized by Shenzhen Zhonghong Boyuan Biological Technology Co., Ltd. (Shenzhen, China). A total of 1 µg plasmid DNA was transfected into the Eca-109 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Cell viability was determined via an MTT assay. The cells were plated in 96-well plates at a density of 5×104 cells/well. Following incubation for 1, 2, 3 and 4 days, 5 µl of MTT solution (1 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to each well, and the plates were incubated for an additional 4 h. Finally, dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) was added to dissolve the formazan crystals and the absorbance was measured at a wavelength of 570 nm.

Eca-109 cells were seeded into six-wells plates, at a density of 500 cells/well. Following overnight culture at 37°C, the cells were exposed to radiation at 0, 2, 4, 6 and 8 Gy with an average dose rate of 100 cGy/min. Following incubation at 37°C in an environment containing 5% CO₂ for 10 days, the colonies were fixed in 4% formaldehyde at room temperature for 30 min, and stained with crystal violet at room temperature for 2 h. Stained cells were observed under a microscope (TS100; Nikon Corporation, Tokyo, Japan) and colonies containing >50 cells were counted as clonogenic survivors.

The Annexin V-FITC and Propidium Iodide (PI) Apoptosis assay kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was used to determine the percentage of cells undergoing apoptosis, according to the manufacturer's protocol. Briefly, cells were harvested 8 h post-irradiation at a dose of 8 Gy. Following washing with PBS, 1×106 cells were dual-stained with 10 µl PI and 5 µl Alexa Fluor 488-Annexin V at room temperature for 15 min, and subsequently analyzed by flow cytometry (BD FACSCanto II flow cytometer; BD Biosciences, Franklin Lakes, NJ, USA) using the BD FACSCanto™ Clinical software version 2.1 (BD Biosciences).

Cells were lysed in a radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) at room temperature for 5 min. Cell lysates were centrifuged at 13,000 × g for 5 min at room temperature, and the supernatants were harvested. Protein concentrations were quantified using a BCA kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of extracted protein samples (5 µg) were separated by 12% SDS-PAGE and transferred onto polyvinylidence difluoride membranes (EMD Millipore). The membranes were blocked with 3% bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China) at 4°C overnight, and the blots were probed with rabbit polyclonal antibodies against LKB1 (catalog no. ab79355; 1:500), rabbit polyclonal to active caspase-3 (catalog no. ab2302; 1:200), mouse monoclonal to cleaved poly(ADP-ribose) polymerase (PARP; catalog no. ab13907; 1:200), rabbit polyclonal to microtubule-associated protein 1 light chain 3 α/β (LC3 α/β; catalog no. ab128025; 1:800) (all from Abcam, Cambridge, MA, USA), rabbit monoclonal to phosphorylated (p)-AMP-activated protein kinase (AMPK) α (Thr172; catalog no. 4188; 1:400), and mouse monoclonal to beclin-1 (catalog no. 4122; 1:400) (both from Cell Signaling Technology, Inc., Danvers, MA, USA) and mouse monoclonal to GAPDH (catalog no. ab8245; 1:2,000; Abcam) at 37°C for 2 h. Following washing with Tris buffered saline Tween-20, the membranes were incubated with goat anti-mouse horseradish peroxidase-conjugated immunoglobulin (Ig) G (catalog no. sc-2005; 1:5,000) or goat anti-rabbit horseradish peroxidase-conjugated IgG (catalog no. sc-2004; 1:5,000) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) secondary antibodies at 37°C for 1 h, and protein bands were visualized by a SuperSignal West Femto kit (Pierce; Thermo Fisher Scientific, Inc.). Blots were semi-quantified by densitometric analysis using ImageJ software version 1.46 (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis was performed using SPSS statistical software, version 19.0 (IBM SPSS, Armonk, NY, USA). Data are presented as the mean ± standard deviation of 3 independent experiments. The statistical significance of the differences between groups was assessed using unpaired Student's t-test for pair-wise comparisons or one-way analysis of variance followed by a post hoc least significant difference test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of radiation treatment on LKB1 expression, the Eca-109 cells were subjected to irradiation from 1–8 Gy 10 times, and then western blot analysis was performed to examine LKB1 expression level in the Eca-109 cells. The results demonstrated that LKB1 protein expression increased in the irradiated cells compared with the untreated cells (Fig. 1).

To determine the effect of LKB1 on the modulation of Eca-109 cell radiosensitivity, Eca-109 cells were transfected with the LKB1-pcDNA3.1 plasmid to overexpress LKB1, and the pcDNA3.1 vector was transfected as the control. As demonstrated in Fig. 2A, the LKB1 protein level was significantly increased in LKB1-pcDNA3.1-transfected cells compared with the control.

Following irradiation at a dose of 8 Gy for 24 h, an MTT assay was performed and the results demonstrated that the cell viability was significantly increased in the LKB1-pcDNA3.1 group compared with the control group (Fig. 2B). The results of the colony formation assay demonstrated that the LKB1-pcDNA3.1-transfected cells exhibited an increased survival fraction compared with control cells (Fig. 2C).

To investigate if LKB1 affected the radiosensitivity of esophageal cancer in vivo, xenograft tumors with a high level of LKB1 expression were established in BALB/c nude mice and exposed to irradiation. Xenograft tumors transduced with empty vector were used as a control. It was observed that the tumor volume was significantly increased in LKB1-overexpressed tumors compared with that of the control tumors, when treated with radiation (Fig. 3).

Subsequently, the present study explored the role of LKB1 in apoptosis and autophagy of Eca-109 cells following radiation. Following exposure to radiation at 8 Gy for 24 h, the cells were harvested for flow cytometry and western blot assays. The results of the flow cytometry assay demonstrated that the cell apoptosis rate was significantly downregulated in LKB1-pcDNA3.1-transfected cells compared with the control cells (Fig. 4A). In addition, the expression of apoptotic proteins, including the active caspase-3 and cleaved PARP-1 were markedly reduced in LKB1-pcDNA3.1-transfected cells compared with control cells. The expression levels of autophagy-associated proteins (beclin-1 and LC3α/β) were revealed to be significantly increased in LKB1-pcDNA3.1-transfected cells compared with control cells (Fig. 4B).

To further examine the molecular mechanism underlying the effect of LKB1 on radiosensitivity of Eca-109 cells, the cells were transfected with the LKB1-pcDNA3.1 plasmid and/or treated with compound C, an AMPK inhibitor, and then subjected to irradiation. The activation of AMPK was measured by AMPKα phosphorylation on Thr172. The western blot analysis demonstrated that the expression level of p-AMPKα (Thr172) in LKB1-pcDNA3.1-transfected cells was significantly upregulated compared with pcDNA3.1-transfected cells, following radiation treatment. Compound C effectively inhibited p-AMPKα (Thr172) protein expression (Fig. 5A). As presented in e. 5B and C, following irradiation treatment, the cell viability and survival fraction were significantly increased in LKB1-pcDNA3.1-transfected cells compared with control cells. However, following inhibition of AMPK, the increased cell viability and survival fraction induced by LKB1 was inhibited.