J Huazhong Univ Sci Technol［Med Sci］ 34(5):761-767,2014 DOI 10.1007/s11596-014-1349-2 J Huazhong Univ Sci Technol［Med Sci］ 34(5):2014

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Hypoxia-induced Autophagy Contributes to Radioresistance via c-Jun-mediated Beclin1 Expression in Lung Cancer Cells* Yan-mei ZOU (邹燕梅)1, Guang-yuan HU (胡广原)1, Xue-qi ZHAO (赵雪琪)1, Tao LU (卢 涛)2, Feng ZHU (朱 峰)2, Shi-ying YU (于世英)1, Hua XIONG (熊 华)1# 1 Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 2 Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014

Summary: Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncology. The hypoxia-inducible factor (HIF)-1α plays a crucial role in radiosensitivity, but the detailed mechanisms remain elusive. A relationship has been suggested to exist between hypoxia and autophagy recently. In the current study, we studied the effect of hypoxia-induced autophagy on radioresistance in lung cancer cell lines. A549 and H1299 cells were cultured under normoxia or hypoxia, followed by irradiation at dosage ranging from 0 to 8 Gy. Clonogenic assay was performed to calculate surviving fraction. EGFP-LC3 plasmid was stably transfected into cells to monitor autophagic processes. Western blotting was used to evaluate the protein expression levels of HIF-1α, c-Jun, phosphorylated c-Jun, Beclin 1, LC3 and p62. The mRNA levels of Beclin 1 were detected by qRT-PCR. We found that under hypoxia, both A549 and H1299 cells were radio-resistant compared with normoxia. Hypoxia-induced elevated HIF-1α protein expression preferentially triggered autophagy, accompanied by LC3 induction, EGFP-LC3 puncta and p62 degradation. In the meantime, HIF-1α increased downstream c-Jun phosphorylation, which in turn upregulated Beclin 1 mRNA and protein expression. The upregulation of Beclin 1 expression, instead of HIF-1α, could be blocked by SP600125 (a specific inhibitor of c-Jun NH2terminal kinase), followed by suppression of autophagy. Under hypoxia, combined treatment of irradiation and chloroquine (a potent autophagy inhibitor) significantly decreased the survival potential of lung cancer cells in vitro and in vivo. In conclusion, hypoxia-induced autophagy through evaluating Beclin1 expression may be considered as a target to reverse the radioresistance in cancer cells. Key words: hypoxia; radioresistance; autophagy; Beclin 1

Lung cancer is one of the most common cancers in the world. It remains one of the leading causes for cancer mortality in China. Although radiotherapy displays favorable therapeutic outcomes for early stage cases, local failure is still the major issue for the poor survival and can be fatal. One of the most important reasons for local recurrence and relapse of lung cancer is treatment resistance, especially in radiotherapy[1]. To overcome this problem, it is critical to elucidate resistant mechanisms through which cancer cells survive and recur after radiotherapy. Evidence obtained from radiobiological studies revealed tumor radioresistance could be caused by a tumor-specific microenvironment, as well-known, hypoxia[2]. Hypoxia is recognized as a fundamental feature of solid tumors and plays a critical role in various physiologic processes, including cell proliferation, angiogenesis, tumor invasion, and metastasis[3]. Other than this, hypoxia could activate multiple downstream signaling pathways as well to impair many modalities of anticancer treatment, such as radiotherapy and chemotherapy[4, 5]. Yan-mei ZOU, E-mail: [email protected] # Corresponding author, E-mail: [email protected] * This project was supported by the National Natural Science Foundation of China (No. 81201779).

A key transcription factor, hypoxia-inducible factor 1 (HIF-1), was identified as pivotal to hypoxia-mediated radioresistance. HIF-1 is a heterodimer of an α subunit that is unstable in the presence of relatively high levels of oxygen (above 5%), and a β subunit that is not oxygen regulated[6]. Under hypoxia, stabilized and activated HIF1α induces various genes expression, leading to cellular radioresistant phenotype[7], yet the detailed mechanisms remain elusive. Autophagy is an evolutionarily conserved lysosomal degradation pathway whereby cells recycle excessive or damaged macromolecules and organelles under stresses[8]. Thus, it confers tolerance, limits damage, and sustains viability of cells under adverse conditions including extracellular stresses, nutrition deprivation, and importantly, hypoxia[9]. A series of signaling pathways are involved in autophagy induction in the context. Autophagy is enhanced in the presence of hypoxia via evaluating HIF-1α downstream target genes BNIP3 and BNIP3L[10]. In addition, a recent study[11] has shown that Beclin 1, an essential protein in autophagosome formation, was upregulated by c-Jun NH2-terminal kinase (JNK) pathway during anticancer agents-induced autophagy. Meanwhile, JNK signaling pathway could be activated primarily by exposure to hypoxia[12]. However,

762 the relationships among hypoxia, radioresistance and autophagy need to be elucidated. In this study, we employed lung cancer cell lines to monitor autophagy induction and examined its role in radioresistance under hypoxia. Furthermore, we also dissected the underlying mechanism for hypoxia-induced autophagy. 1 MATERIALS AND METHODS 1.1 Reagents and Drugs Chloroquine (CQ) and SP600125 were purchased from Sigma-Aldrich (USA). CQ was formulated in PBS, and SP600125 was dissolved in DMSO. RMPI-1640 and fetal bovine serum (FBS) were purchased from GIBCO Life Technologies Ltd. (USA). Lipofectamine 2000 was purchased from Invitrogen (USA). Primary rabbit antibodies against GAPDH, LC3, p62, HIF-1α, c-Jun, p-cJun and Beclin 1 were all from Cell Signaling (USA). 1.2 Cell Culture Human non-small-cell lung cancer cell lines A549 and H1299 were obtained from the Oncology Laboratory at Tongji Hospital of Tongji Medical College (China). Generally, these cells were incubated in RPMI medium supplemented with 5% FBS and 100 U/mL penicillin and 100 mg/mL streptomycin at 37ºC in a humidified atmosphere (5% CO2, 20% O2). For hypoxia, the cells were cultured in a humidified O2 control incubator (5% CO2, 1% O2) for indicated time. 1.3 Irradiation and Clonogenic Assay For irradiation, 2×105 cells were seeded into a sixwell plate and incubated for 36 h before the indicated dose of radiation. Five h after the indicated dose of radiation, the cells were treated with SP600125 (10 μmol/L) for 2 h, or CQ (25 μmol/L) for 6 h. For the clonogenic assay, 1×104 cells were seeded into six-well culture dishes for radiation or CQ treatment after irradiation by using a Gulmay Medical RS225 Research System (200 kV; 15 mA; dose rate: 3 Gy/min). After being incubated under normoxia or hypoxia for 7 to 10 days, colonies were washed by PBS, fixed with cold methanol and stained with hematoxylin. The percentage of colonies counted for each radiation dosage course was calculated by dividing the number of cultured test colonies by the number of the appropriate nonirradiated control colonies. The surviving fraction was calculated as the ratio of the plating efficiency of the treated cells to that of controls. 1.4 Viability Assay A549 and H1299 cells were separately seeded in 96-well plate (1.0×104/well, BD Falcon) and the plates were incubated overnight to allow cells to attach. At 12 h after incubation under normoxia or hypoxia, cells were irradiated with indicated dose. At 72 h after irradiation, MTT solution was added to each well, and the plates were incubated at 37ºC for 4 h. Then the culture medium was removed and 200 μL DMSO per well was added. The value was quantified by measurement of the absorbance (A) at 560 nm. The A reading for control cells was set as 100% and that for the treated cells was compared with the controls. 1.5 Fluorescence Microscopy/EGFP-LC3 Autophagy Assay When A549 and H1299 cells grew to 80% conflu-

J Huazhong Univ Sci Technol［Med Sci］ 34(5):2014

ence, they were transfected with pEGFP-LC3 plasmid (Addgen Co., USA) by lipofectamine 2000. Two days after transfection, cells were reseeded and incubated under normoxia or hypoxia for 12 h. Then the cells with EGFP-LC3 puncta were counted and photographed. Five randomly selected fields (40×) were photographed and the average percentage of cells per field containing more than 10 intracellular EGFP-LC3 puncta was calculated. 1.6 Immunoblotting Analysis Cells were lysed with sample loading buffer containing 125 mmol/L Tris-HCl, pH 6.8, 6% SDS, 10% glycerol, 10 mmol/L 2-mercaptoethanol, and protease inhibitor. After sonication, cell lysates were centrifuged at 13 000 g for 20 min at 4°C. Protein concentrations were determined by using a BCA protein assay kit (Pierce Chemical Co., USA). Whole cell-extracts (50 mg protein per lane) were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk in phosphate-buffered saline and then probed with the appropriate primary antibody in 5% non-fat milk overnight at 4ºC [anti-LC3 (1:1000), anti-p62 (1:1000), anti-Beclin 1 (1:1000), anti-HIF-1α (1:500), anti-p-c-Jun (1:1000), anti-c-Jun (1:200) and anti-GAPDH (1:2000)]. Subsequently, membranes were washed in phosphate-buffered saline with 0.3% Tween 20, and incubated with secondary antibody conjugated to horseradish peroxidase at room temperature for 1 h. Proteins were visualized by enhanced chemiluminescence reagents according to the manufacture’s instruction. Protein levels were quantified by the Image Quant analysis software (Quantity One; Bio-rad; USA). 1.7 Real-time PCR for mRNA Total RNA was extracted using Trizol kit (Qiagen, USA) according to manufacturer’s instruction. cDNA was synthesized from 2 μg of total RNA using Superscript Ⅲ First-Strand Synthesis kit with oligo dT primers (Invitrogen, USA). Quantitative real-time PCR was performed with GeneAmp PCR System 9700 (Applied Biosystems, USA) using SYBR Green Real-Time PCR Master Mix (Applied Biosystems, USA). The primers used in this study for PCR amplification were as follows: Beclin 1 (sense, 5′-AAGACAGAGC-GATGGTAG-3′; antisense, 5′-CTGGGCTGTGGTAAGTAA-3′); and GAPDH (sense, 5′-CGGGAAGCTTGTATCAATGG-3′; antisense, 5′-GGCAGTGATGGCATGGACTG-3′). The PCR conditions were as follows: 40 cycles of 95ºC for 15 s, 50ºC for 40 s and 72ºC for 40 s. The qRT-PCR results were normalized to GAPDH and analyzed as relative RNA levels of the cycle threshold (Ct) value. 1.8 Xenograft Treatment The A549 cells in the logarithmic phase were cultured, digested, and suspended to a final density of 5×106/mL. For xenograft tumors, 1×106 (in 200 μL of PBS) A549 cells were injected subcutaneously into the left forelimb of athymic nude mice. The mice were monitored for tumor formation to around 50–100 mm3 (day 0) in one week and randomly divided into four groups (n=6 each). For drug treatment, mice were given CQ (30 mg/kg in PBS) or vehicle (PBS) by intraperitoneal injection (i.p.) once a day for 3 weeks. Xenograft tumors were locally irradiated with dose of 1.25 Gy per fraction (twice a week) beginning on day 2 after the treatment with CQ or vehicle. Tumor volume was meas-

J Huazhong Univ Sci Technol［Med Sci］ 34(5):2014

ured once a week and calculated by the following formula: Tumor volume=(Length×Width2)/2. All animal procedures were performed in accordance with a protocol approved by Institutional Animal Ethics Committee, Tongji Medical College, Huazhong University of Science and Technology (China). 1.9 Statistical Analysis Each experiment was repeated at least three times. Statistical comparisons were analyzed by ANOVA (greater than 2 groups) or Student’s t test (2 groups only) with Graphad Prism (v.5). For subcutaneous tumor volume, initial comparison of 3 or more groups was done by the Kruskal-Wallis test (nonparametric alternative to ANOVA). Statistical significances were accepted at P