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Inhibition of TrxR2 suppressed NSCLC cell proliferation, metabolism and induced cell apoptosis through decreasing antioxidant activity
Accepted Manuscript Inhibition of TrxR2 suppressed NSCLC cell proliferation, metabolism and induced cell apoptosis through decreasing antioxidant activity
Lina Bu, Wei Li, Zongjuan Ming, Jie Shi, Ping Fang, Shuanying Yang PII: DOI: Reference:
S0024-3205(17)30156-X doi: 10.1016/j.lfs.2017.04.008 LFS 15179
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
5 February 2017 30 March 2017 12 April 2017
Please cite this article as: Lina Bu, Wei Li, Zongjuan Ming, Jie Shi, Ping Fang, Shuanying Yang , Inhibition of TrxR2 suppressed NSCLC cell proliferation, metabolism and induced cell apoptosis through decreasing antioxidant activity. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi: 10.1016/j.lfs.2017.04.008
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Inhibition of TrxR2 suppressed NSCLC cell proliferation, metabolism and induced cell apoptosis through decreasing antioxidant activity Lina Bu1, Wei Li1, Zongjuan Ming1, Jie Shi1, Ping Fang1, Shuanying Yang1,* 1
Department of Respiratory Medicine, the Second Affiliated Hospital of Xi’an Jiaotong University,
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*Corresponding author: Shuanying Yang
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Xi’an, 710003, Shaanxi, China
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Email: [email protected]
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Abstract Aims: This study aims to analyze the effect of thioredoxin reductase 2 (TrxR2) on lung cancer cell proliferation, apoptosis, invasion and migration in vitro. Main methods: Real-time PCR was used to measure the expression of TrxR2 in NSCLC tumor tissues.
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After pAd-TrxR2 or shRNA-TrxR2 was transfected into A549 or NCI-H1299 cells, the cell
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proliferation was measured by CCK-8 method; cell apoptosis was measured by flow cytometry; cell
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invasion and migration was measured by Transwell method. The production of ROS was measured by DCFH-DA method; the activity of SOD, CAT and GSH-Px was measured by relative ELISA kit.
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Key findings: The results showed that TrxR2 was up-regulated in NSCLC tumor tissues. Inhibition of
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TrxR2 suppressed NSCLC cell proliferation and induced apoptosis, and inhibited cell invasion and migration. However, overexpression of TrxR2 showed the opposite effect. Furthermore, when cells
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were transfected with shRNA-TrxR2, the production of ROS was significantly increased, and SOD,
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CAT and GSH-Px activity was decreased. Conversely, pAd-TrxR2 transfection showed the opposite effect.
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Significance: Taken together, our results suggest that TrxR2 acts as an oncogenic gene in the context of
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lung cancer progression. The inhibition of TrxR2 suppressed lung cancer cell proliferation, invasion and migration and induced cell apoptosis by inducing ROS production and decreasing antioxidant activity. TrxR2 may be a potential target for NSCLC treatment. Key words: TrxR2; lung cancer; proliferation; apoptosis; metabolism; antioxidant activity
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1. Introduction Lung cancer is a leading cause of cancer death worldwide[1, 2]. It is classified into two main histological groups: non-small-cell lung cancer (NSCLC, 85%) and small-cell lung cancer (SCLC, 15%)[3]. The improvements in the treatment of lung cancer have been achieved by the combination
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therapy, such as surgical resection, chemotherapy, radiotherapy, and biological target therapy[4, 5].
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However, the 5-year survival rate for lung cancer is only 15%[6, 7]. Therefore, it is urgent to find a
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novel target and discover the changes at the molecular level for the treatment of lung cancer. Thioredoxin reductase 2 (TrxR2) is a selenium (Se) containing protein[8]. It is necessary to
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maintain thioredoxin 2 in its reduced state by using electrons from NADPH. TrxR2 is mainly expressed
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in mitochondria and play an important role in protecting cells from oxidative damage[9]. It is reported that TrxR2 is involved in many cancers, such as prostate cancer[10], osteosarcoma[11], colon and
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rectal cancer[12]. Whereas the effect of TrxR2 in the progression of lung cancer and its underlying
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mechanism remain unknown. Since increasing studies have reported that oxidant stress is involved in the progression of lung cancer; for example, lung tumor cells are able to grow in a highly oxidative
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environment, which is believed to contribute to tumor progression and metastasis[13]. Gupta et al.
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reported that oxidative stress influenced the treatment efficacy and survival of lung cancer patients[14]. This study aims to analyze the effect of TrxR2 on NSCLC cell proliferation, apoptosis, metastasis and antioxidant activity, and also seeks to elucidate the underlying mechanism. 2. Materials and methods 2.1 Lung tissue samples Informed consent was obtained from every patient in the study. NSCLC tumor tissue samples and adjacent lung tissues were collected from 20 chemotherapy native patients who underwent thoracic 3
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surgery. After surgery, all samples were immediately frozen in liquid nitrogen and stored at -80°C. All samples were obtained the informed consent of each patient. 2.2 Cell lines and culture NSCLC cell lines, including A549 and NCI-H1299, were obtained from the Type Culture collection of
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the Chinese Academy of Sciences and cultured according to the supplier’s recommendations. All cells
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were cultured in RPMI 1640 medium (supplemented with 10% fetal bovine serum with 100 U/mL
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penicillin and 100 mg/mL streptomycin included). All cells were maintained in a humidified environment containing 5% CO2 at 37°C.
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2.3 RNA extraction and Real-time PCR
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Total RNA of lung tumor tissues and adjacent lung tissues were isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA). The first-strand cDNA was synthesized using PrimeScript RT Master Mix
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(TaKaRa, Da Lian, China). The Real-time PCR was performed using SYBR Premix Ex TaqTM Kit
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(TaKaRa). All the primers used in this study were synthesized by Sangon Biotech (Shang Hai, China). Each individual sample was run in triplicate wells and conducted in the ABI7500 Real-time PCR
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system (Applied Biosystems, Carlsbad, CA). The reactions were as follows: 95°C for 5 min, 40 cycles
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at 95°C for 10 s, and 60°C for 60 s. The relative expression of the genes tested were calculated using 2-△△CT method. β-actin was selected as the internal controls. Primers used in this study were: β-actin: 5’-GAA ATC GTG CGT GAC ATT AA-3’, 5’-AAG GAA GGC TGG AAG AGT G-3’; TrxR2: 5’-GCT GGG CCT GC ACTT CCT-3’; 5’-CTG TCT GCA TCA CCT GTG CAT-3’. 2.4 Western blot Total protein was extracted from lung tissue samples using the Total Protein Extraction Kit (Phygene Life Sciences, Shanghai, China). The 50 μg of protein was separated by SDS-PAGE and transferred 4
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onto PVDF membrane (Millipore, Darmstadt, Germany). Then the membranes were probed with primary antibodies specific for TrxR2 (1: 1000 dilution, Abcam, Cambridge, UK). After incubation at 4°C overnight, the appropriate HRP-conjugate secondary antibody was added for 1 h incubation at room temperature. The protein bands were visualized by Super Signal West Pico Chemiluminescent
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Substrate (Pierce, Rockford, IL, USA), and the membranes were subjected to X-ray autoradiography.
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Bind intensities were determined with Quantity One software (Bio-Rad, Hercules, CA, USA).
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2.5 Transfection
The recombinant adenovirus vector that overexpressed TrxR2 (pAd-TrxR2) was obtained from
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Genomeditech Co., Ltd (Shanghai). The lentiviral plasmids that contained shRNA specific for human
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TrxR2 (shRNA-TrxR2) were also obtained from Genomeditech(Shanghai) Co., Ltd. One day prior to transfection, the A549 and NCI-H1299 cells were seeded on six-well plates at a density of 3 × 105 cells
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per well. When cells were grown to 80% confluency, transfection were performed according to the
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instruction of LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA). The cells were divided to the following groups: Control group (no transfection group), shRNA control transfection group,
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shRNA-TrxR2 (2 mg/mL) transfection group, pAd-vector transfection group and pAd-TrxR2 (2
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mg/mL) transfection group. Each treatment condition was tested in three wells. 2.6 Cell Counting Kit-8 (CCK-8) assay Cell proliferation was assayed using the CCK-8 method. The transfected cells were plated in 96-well plates at a density of 4000 cells per well. After 24, 48 and 72 h, 20 μL of CCK-8 was added for incubation. Finally, each well was measured spectrophotometrically at 450 nm. 2.7 Cell apoptosis assay Cell apoptosis was evaluated by flow cytometry using the annexin V-FITC apoptosis detection kit 5
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(Sigma, Chemical Co., St. Louis, MO, USA). The transfected cells were plated in 24-well plates at a density of 1 × 104 per well and cultured for 48 h. The cells were harvested and washed with PBS, then re-suspended in 500 μL of binding buffer. Then cells were stained with 5 μL of Annexin V-FITC and 5 μL propidium iodide at 4°C for 30 min. Finally, the cells were analyzed by flow cytometry (BD
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FACSCalibur, BD Bioscience, San Diego, CA, USA).
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2.8 Cell cycle analysis
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After cells were transfected for 48 h, the cells were harvested, washed with PBS and fixed by 70% ethanol. The fixed cells added with 100 μg/mL RNase A and were stained with PI (50 μg/mL) for 30
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min. Finally, cell cycle distribution was analyzed using FACSCalibur (Becton-Dickinson, Franklin
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Lakes, NJ). 2.9 Cell invasion
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The Transwell assay was used to quantify cell invasion. The transfected cells were plated in the upper
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chamber of Transwell assay inserts (BD Bioscience, Franklin Lakes, NJ, USA) containing 500 μL of serum-free 1640 medium. The lower chambers were filled with 1640 medium containing 10% FBS.
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After 48 h, the cells on the filter surface were fixed with methanol for 10 min, stained with 0.1%
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crystal violet, and photographed. Invasion was assessed by counting the number of stained cell nuclei from five random fields per filter for each group. 2.10 Cell migration
The transfected cells were seeded in the top chambers (8 mm pores, Millipore, Billerica, MA). The lower chambers were filled with 1640 medium containing 10% FBS. After 48 h, the cells on the filter surface were fixed with methanol for 10 min, stained with 0.1% crystal violet. Migration was assessed by counting the number of stained cell nuclei from 5 random fields per filter in each group. 6
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2.11 Measurement of intracellular ROS levels The relative changes of ROS was measured by DCFH-DA method. After cells were transfected for 48 h, the cells were incubated with 10 μmol/L od DCFH-DA for 30 min. After cells were washed for 3 times, cells were subjected to fluorescence analyzer (BioTek, USA) with excitation/emission set at 488/525
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nm. The values were presented as percentage of relative fluorescence compared with control group.
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2.12 ELISA
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The activity of SOD, CAT and GSH-Px was measured by using the relative ELISA Kit (Sangon Biotech, Shanghai, China). Briefly, the cells were collected and centrifuged at 1000 g for 10 min, the
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supernatant was collected and was measured according to the instructions.
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2.13 Statistical analysis
Data were expressed as mean ± SD and considered significant at P < 0.05. Statistical analysis was
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performed using a student’s unpaired t test (SPSS release 19.0; SPSS Inc.,).
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3. Results
3.1 TrxR2 was up-regulated in NSCLC tumor tissues
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To investigate the role of TrxR2 in the progression of lung cancer, we first measured the expression of
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TrxR2 in NSCLC tumor tissues. The results of Real-time PCR and western blot showed that TrxR2 was significantly up-regulated in NSCLC tumor tissues compared with adjacent lung tissues (Figure 1A, B). 3.2 Inhibition of TrxR2 inhibited A549 and NCI-H1299 cell proliferation and induced cell apoptosis, whereas overexpression of TrxR2 showed the opposite effects To further analyze the effect of TrxR2 on NSCLC cell proliferation and apoptosis, A549 and YTMLC-90 cells were transfected with shRNA-TrxR2 and pAd-TrxR2, respectively. The result of western blot showed that after A549 and NCI-H1299 cells were transfected with shRNA-TrXR2 or 7
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pAd-TrxR2, the expression of TrxR2 was significantly decreased or increased, respectively (Figure 2A, B); The CCK-8 assay showed that the cell proliferation of A549 and NCI-H1299 was significantly suppressed when cells were transfected with shRNA-TrxR2 (Figure 2C, D). Conversely, the cell proliferation of A549 and NCI-H1299 was dramatically increased when cells were transfected with
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pAd-TrxR2 (Figure 2E, F). The results of flow cytometry showed that shRNA-TrxR2 significantly
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induced A549 and NCI-H1299 cell apoptosis, and the rate of cell apoptosis increased by nearly 1-fold
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when compared with the control group, respectively (Figure 2G). When cells were transfected with pAd-TrxR2, the cell apoptosis of A549 and NCI-H1299 was significantly decreased (Figure 2H). In
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addition, we also measured the effect of TrxR2 on the cell cycle. The results showed that inhibition of
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TrxR2 induced A549 and NCI-H1299 cell cycle arrest at G1/S phase (Table 1, 2, 3, 4).These results indicate that the inhibition of TrxR2 inhibited A549 and NCI-H1299 cell proliferation and induced cell
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apoptosis, whereas the overexpression of TrxR2 had the opposite effects.
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3.3 Inhibition of TrxR2 suppressed A549 and NCI-H1299 cell invasion and migration, whereas overexpression of TrxR2 showed the opposite effects
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To further analyze the effect of TrxR2 on the metastasis of NSCLC cells, Transwell assays were used to
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measure cell invasion and migration after cells were transfected with shRNA-TrxR2 or pAd-TrxR2. The results showed that when A549 and NCI-H1299 cells were transfected with shRNA-TrxR2, the cell invasion and migration was significantly inhibited compared with control group (Figure 3A, C). Whereas when cells were transfected with pAd-TrxR2, the cell invasion and migration were notably increased compared with control group (Figure 3B, D). These results suggested that the inhibition of TrxR2 suppressed NSCLC cell metastasis, whereas the overexpression of TrxR2 induced NSCLC cell metastasis. 8
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3.4 Inhibition of TrxR2 decreased the antioxidant activities, whereas overexpression of TrxR2 showed the opposite effects. Because TrxR2 plays an important role in protecting cells from oxidative damage, we also analyzed the effect of TrxR2 on antioxidant activities. The results showed that when cells were transfected with
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shRNA-TrxR2, the production of ROS was dramatically increased (Figure 4A). Moreover, the activity
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of SOD, CAT and GSH-Px were notably decreased (Figure 4B, C, D,). However, when cells were
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transfected with pAd-TrxR2, ROS production was significantly decreased (Figure 4E); SOD, CAT and GSH-Px activity levels were significantly increased (Figure 4F, G, H). These results indicate that the
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inhibition of TrxR2 decreased antioxidant activities, whereas overexpression of TrxR2 had the opposite
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effects. 4 Discussion
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As lung carcinogenesis is a chronic and a multiple-step process in which accumulation of genetic and
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epigenetic alternations are involved, to achieve a more effective treatment of lung cancer is essential[15]. Our study proves that TrxR2 was upregulated in NSCLC tumor tissues and TrxR2 could
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potentially function as an oncogenic gene in lung cancer progression.
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An increasing number of studies have verified that TrxR2 was highly expressed in various cancers and promoted cancer progression. For example, TrxR2 was upregulated and may be involved in the formation and development of hepatocellular carcinomas[16]. Lincoln et al. reported that TrxR was upregulated in tumors which had a high proliferation capacity, a low apoptosis rate and an elevated metastatic, such as breast cancer, thyroid, prostate and colorectal carcinoma[17]. Ylermi et al. reported that the Trx and TrxR were highly expressed in lung carcinomas[18]. Because TrxR2 transfers electrons from NADPH/H+ to Trx2[19] and was overexpressed in cancer cells, the molecule may act as a 9
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proto-oncogene in cancer cells by increasing cell proliferation and decreasing apoptosis[20]. Therefore, TrxR2 may regulate the progression of lung cancer. In this study, we also identified that TrxR2 was overexpressed in NSCLC tumor tissues, inhibition of TrxR2 not only suppressed NSCLC cell proliferation, and induced cell apoptosis and induced cell cycle arrest in G1/S phase in vitro.
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Furthermore, the inhibition of TrxR2 suppressed NSCLC cell invasion and migration, whereas the
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overexpression of TrxR2 reversed these effects. Therefore, TrxR2 promoted the progression of lung
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cancer.
The current understanding of the etiology of lung cancer indicates that oxidative stress is
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implicated in its pathogenesis[21]. Lung cancer cells exist in an elevated oxidative state, and increased
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the production of ROS[22]. Moreover, high levels of ROS may increase the activity of antioxidants in lung cancer patients. For example, previous study reported that lung cancer patients have increased the
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activity of CAT and ceruloplasmin in peripheral blood [23]. Kaynar et al. demonstrated that the activity
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of GSH, xanthine oxidase, catalase and Cu-Zn superoxide dismutase activities in the blood of patients with NSCLC was significantly increased[24]. Mitochondrial antioxidant enzymes, including
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manganese superoxide dismutase (MnSOD), glutathione-dependent peroxidase (GPX) and TrxR2,
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comprise a primary defense system in mitochondria and are essential for detoxification against ROS[25]. Therefore, the inhibition of antioxidant systems is an option for cancer intervention[26]. Our results proved that the level of ROS was significantly increased compared with control group. When cells were transfected with shRNA-TrxR2, SOD, CAT and GSH-Px activity levels was decreased and ROS levels was increased. Whereas pAd-TrxR2 transfection showed the opposite effects. Furthermore, incapacitating antioxidant defense systems by inducing ROS production was reported to be a novel
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strategy for cancer therapy[27-29], so the inhibition of TrxR2 could suppress NSCLC cell proliferation and induce cell apoptosis by inducing ROS production and decreasing antioxidant activity. 5 Conclusion Taken together, these results show that TrxR2 was overexpressed in NSCLC cells. The inhibition of
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TrxR2 suppressed NSCLC cell proliferation, invasion and migration, and induced cell apoptosis by
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inducing ROS production and decreasing antioxidant activity. TrxR2 may be a potential target for
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treating lung cancer.
Funding: This work was supported by Natural Science Basic Research Plan in Shaanxi Province of
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China-General Program (No. 2015JM8480) . The West Light Talent Culture Project of the Chinese
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Academy of Sciences (2014).
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Conflict of interest: The authors declare that they have no conflict of interest.
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Figure legends: Figure 1 TrxR2 was up-regulated in NSCLC tumor tissues. NSCLC tumor tissue samples and adjacent tissues were collected from 20 chemotherapy native patients who underwent thoracic surgery. A. The expression of TrxR2 in NSCLC tumor tissues was measured by Real-time PCR. B. The
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expression of TrxR2 in NSCLC tumor tissues was measured by western blot. *P < 0.05 vs. adjacent
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tissues.
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Figure 2 Inhibition of TrxR2 inhibited A549 and NCI-H1299 cell proliferation and induced cell apoptosis, whereas overexpression of TrxR2 showed the opposite effects. A. After A549 and
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NCI-H1299 cells were transfected with shRNA-TrxR2, the expression of TrxR2 was measured by
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western blot. B. After A549 and NCI-H1299 cells were transfected with pAd-TrxR2, the expression of TrxR2 was measured by western blot. C. After A549 cells were transfected with shRNA-TrxR2 for 24,
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48 and 72 h, cell proliferation was measured using the CCK-8 method. D. After NCI-H1299 cells were
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transfected with shRNA-TrxR2 for 24, 48 and 72 h, the cell proliferation was measured by CCK-8 method. C. The effect of pAd-TrxR2 on the proliferation of A549 cells. E. The effect of pAd-TrxR2 on
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the proliferation of NCI-H1299. F. After A549 and NCI-H1299 cells were transfected with
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shRNA-TrxR2 for 48 h, cell apoptosis was measured by flow cytometry. G. After A549 and NCI-H1299 cells were transfected with pAd-TrxR2 for 48 h, the cell apoptosis was measured by Flow cytometry. *P < 0.05 vs. control group. Figure 3 Inhibition of TrxR2 suppressed A549 and NCI-H1299 cell invasion and migration, whereas overexpression of TrxR2 showed the opposite effects. After A549 and NCI-H1299 cells were transfected with shRNA-TrxR2 for 48 h, A-B. the cell invasion was measured by Transwell method. C-D. the cell migration was measured by Transwell method. *P < 0.05 vs. control group. 16
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Figure 4 Inhibition of TrxR2 decreased antioxidant activities, whereas overexpression of TrxR2 showed the opposite effects. After cells were transfected with shRNA-TrxR2 or pAd-TrxR2 for 48 h, A. The production of ROS was measured by DCFH-DA method. B-H. The activity of SOD, CAT and
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GSH-Px was measured by ELISA. *P < 0.05 vs control group.
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Table 1 Effect of overexpression of TrxR2 on the cell cycle of A549 cells. S
G2/M
control
83.26%
12.15%
4.59%
pAd-control
80.25%
13.42%
6.33%
pAd-TrxR2
52.13%*
35.28%*
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12.59%*
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*P<0.05 vs control group
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Table 2 Effect of overexpression of TrxR2 on the cell cycle of NCI-H1299 cells. S
G2/M
control
80.14%
14.17%
5.69%
pAd-control
78.34%
15.39%
6.27%
pAd-TrxR2
56.47%*
32.06%*
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11.47%*
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*P<0.05 vs control group
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Table 3 Effect of inhibition of TrxR2 on the cell cycle of A549 cells S
G2/M
control
74.36%
14.55%
11.09%
shRNA-control
72.39%
16.23%
11.38%
shRNA-TrxR2
85.16%*
10.24%*
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4.60%*
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*P<0.05 vs control group
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Table 4 Effect of inhibition of TrxR2 on the cell cycle of NCI-H1299 cells S
G2/M
control
70.13%
15.22%
14.65%
shRNA-control
73.48%
14.82%
11.70%
shRNA-TrxR2
88.57%*
8.26%*
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3.17%*
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*P<0.05 vs control group
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Highlights 1. TrxR2 was up-regulated in lung cancer tissues. 2. The inhibition of TrxR2 suppressed lung cancer cell proliferation, invasion and migration and induced cell apoptosis by inducing ROS production and decreasing antioxidant activity. Whereas
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overexpression of TrxR2 had the opposite effect.
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3. TrxR2 acts as an oncogenic gene in the context of lung cancer progression.
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Lina Bu, Wei Li, Zongjuan Ming, Jie Shi, Ping Fang, Shuanying Yang PII: DOI: Reference:
S0024-3205(17)30156-X doi: 10.1016/j.lfs.2017.04.008 LFS 15179
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
5 February 2017 30 March 2017 12 April 2017
Please cite this article as: Lina Bu, Wei Li, Zongjuan Ming, Jie Shi, Ping Fang, Shuanying Yang , Inhibition of TrxR2 suppressed NSCLC cell proliferation, metabolism and induced cell apoptosis through decreasing antioxidant activity. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi: 10.1016/j.lfs.2017.04.008
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Inhibition of TrxR2 suppressed NSCLC cell proliferation, metabolism and induced cell apoptosis through decreasing antioxidant activity Lina Bu1, Wei Li1, Zongjuan Ming1, Jie Shi1, Ping Fang1, Shuanying Yang1,* 1
Department of Respiratory Medicine, the Second Affiliated Hospital of Xi’an Jiaotong University,
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*Corresponding author: Shuanying Yang
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Xi’an, 710003, Shaanxi, China
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Email: [email protected]
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Abstract Aims: This study aims to analyze the effect of thioredoxin reductase 2 (TrxR2) on lung cancer cell proliferation, apoptosis, invasion and migration in vitro. Main methods: Real-time PCR was used to measure the expression of TrxR2 in NSCLC tumor tissues.
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After pAd-TrxR2 or shRNA-TrxR2 was transfected into A549 or NCI-H1299 cells, the cell
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proliferation was measured by CCK-8 method; cell apoptosis was measured by flow cytometry; cell
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invasion and migration was measured by Transwell method. The production of ROS was measured by DCFH-DA method; the activity of SOD, CAT and GSH-Px was measured by relative ELISA kit.
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Key findings: The results showed that TrxR2 was up-regulated in NSCLC tumor tissues. Inhibition of
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TrxR2 suppressed NSCLC cell proliferation and induced apoptosis, and inhibited cell invasion and migration. However, overexpression of TrxR2 showed the opposite effect. Furthermore, when cells
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were transfected with shRNA-TrxR2, the production of ROS was significantly increased, and SOD,
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CAT and GSH-Px activity was decreased. Conversely, pAd-TrxR2 transfection showed the opposite effect.
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Significance: Taken together, our results suggest that TrxR2 acts as an oncogenic gene in the context of
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lung cancer progression. The inhibition of TrxR2 suppressed lung cancer cell proliferation, invasion and migration and induced cell apoptosis by inducing ROS production and decreasing antioxidant activity. TrxR2 may be a potential target for NSCLC treatment. Key words: TrxR2; lung cancer; proliferation; apoptosis; metabolism; antioxidant activity
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1. Introduction Lung cancer is a leading cause of cancer death worldwide[1, 2]. It is classified into two main histological groups: non-small-cell lung cancer (NSCLC, 85%) and small-cell lung cancer (SCLC, 15%)[3]. The improvements in the treatment of lung cancer have been achieved by the combination
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therapy, such as surgical resection, chemotherapy, radiotherapy, and biological target therapy[4, 5].
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However, the 5-year survival rate for lung cancer is only 15%[6, 7]. Therefore, it is urgent to find a
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novel target and discover the changes at the molecular level for the treatment of lung cancer. Thioredoxin reductase 2 (TrxR2) is a selenium (Se) containing protein[8]. It is necessary to
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maintain thioredoxin 2 in its reduced state by using electrons from NADPH. TrxR2 is mainly expressed
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in mitochondria and play an important role in protecting cells from oxidative damage[9]. It is reported that TrxR2 is involved in many cancers, such as prostate cancer[10], osteosarcoma[11], colon and
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rectal cancer[12]. Whereas the effect of TrxR2 in the progression of lung cancer and its underlying
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mechanism remain unknown. Since increasing studies have reported that oxidant stress is involved in the progression of lung cancer; for example, lung tumor cells are able to grow in a highly oxidative
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environment, which is believed to contribute to tumor progression and metastasis[13]. Gupta et al.
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reported that oxidative stress influenced the treatment efficacy and survival of lung cancer patients[14]. This study aims to analyze the effect of TrxR2 on NSCLC cell proliferation, apoptosis, metastasis and antioxidant activity, and also seeks to elucidate the underlying mechanism. 2. Materials and methods 2.1 Lung tissue samples Informed consent was obtained from every patient in the study. NSCLC tumor tissue samples and adjacent lung tissues were collected from 20 chemotherapy native patients who underwent thoracic 3
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surgery. After surgery, all samples were immediately frozen in liquid nitrogen and stored at -80°C. All samples were obtained the informed consent of each patient. 2.2 Cell lines and culture NSCLC cell lines, including A549 and NCI-H1299, were obtained from the Type Culture collection of
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the Chinese Academy of Sciences and cultured according to the supplier’s recommendations. All cells
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were cultured in RPMI 1640 medium (supplemented with 10% fetal bovine serum with 100 U/mL
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penicillin and 100 mg/mL streptomycin included). All cells were maintained in a humidified environment containing 5% CO2 at 37°C.
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2.3 RNA extraction and Real-time PCR
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Total RNA of lung tumor tissues and adjacent lung tissues were isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA). The first-strand cDNA was synthesized using PrimeScript RT Master Mix
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(TaKaRa, Da Lian, China). The Real-time PCR was performed using SYBR Premix Ex TaqTM Kit
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(TaKaRa). All the primers used in this study were synthesized by Sangon Biotech (Shang Hai, China). Each individual sample was run in triplicate wells and conducted in the ABI7500 Real-time PCR
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system (Applied Biosystems, Carlsbad, CA). The reactions were as follows: 95°C for 5 min, 40 cycles
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at 95°C for 10 s, and 60°C for 60 s. The relative expression of the genes tested were calculated using 2-△△CT method. β-actin was selected as the internal controls. Primers used in this study were: β-actin: 5’-GAA ATC GTG CGT GAC ATT AA-3’, 5’-AAG GAA GGC TGG AAG AGT G-3’; TrxR2: 5’-GCT GGG CCT GC ACTT CCT-3’; 5’-CTG TCT GCA TCA CCT GTG CAT-3’. 2.4 Western blot Total protein was extracted from lung tissue samples using the Total Protein Extraction Kit (Phygene Life Sciences, Shanghai, China). The 50 μg of protein was separated by SDS-PAGE and transferred 4
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onto PVDF membrane (Millipore, Darmstadt, Germany). Then the membranes were probed with primary antibodies specific for TrxR2 (1: 1000 dilution, Abcam, Cambridge, UK). After incubation at 4°C overnight, the appropriate HRP-conjugate secondary antibody was added for 1 h incubation at room temperature. The protein bands were visualized by Super Signal West Pico Chemiluminescent
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Substrate (Pierce, Rockford, IL, USA), and the membranes were subjected to X-ray autoradiography.
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Bind intensities were determined with Quantity One software (Bio-Rad, Hercules, CA, USA).
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2.5 Transfection
The recombinant adenovirus vector that overexpressed TrxR2 (pAd-TrxR2) was obtained from
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Genomeditech Co., Ltd (Shanghai). The lentiviral plasmids that contained shRNA specific for human
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TrxR2 (shRNA-TrxR2) were also obtained from Genomeditech(Shanghai) Co., Ltd. One day prior to transfection, the A549 and NCI-H1299 cells were seeded on six-well plates at a density of 3 × 105 cells
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per well. When cells were grown to 80% confluency, transfection were performed according to the
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instruction of LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA). The cells were divided to the following groups: Control group (no transfection group), shRNA control transfection group,
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shRNA-TrxR2 (2 mg/mL) transfection group, pAd-vector transfection group and pAd-TrxR2 (2
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mg/mL) transfection group. Each treatment condition was tested in three wells. 2.6 Cell Counting Kit-8 (CCK-8) assay Cell proliferation was assayed using the CCK-8 method. The transfected cells were plated in 96-well plates at a density of 4000 cells per well. After 24, 48 and 72 h, 20 μL of CCK-8 was added for incubation. Finally, each well was measured spectrophotometrically at 450 nm. 2.7 Cell apoptosis assay Cell apoptosis was evaluated by flow cytometry using the annexin V-FITC apoptosis detection kit 5
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(Sigma, Chemical Co., St. Louis, MO, USA). The transfected cells were plated in 24-well plates at a density of 1 × 104 per well and cultured for 48 h. The cells were harvested and washed with PBS, then re-suspended in 500 μL of binding buffer. Then cells were stained with 5 μL of Annexin V-FITC and 5 μL propidium iodide at 4°C for 30 min. Finally, the cells were analyzed by flow cytometry (BD
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FACSCalibur, BD Bioscience, San Diego, CA, USA).
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2.8 Cell cycle analysis
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After cells were transfected for 48 h, the cells were harvested, washed with PBS and fixed by 70% ethanol. The fixed cells added with 100 μg/mL RNase A and were stained with PI (50 μg/mL) for 30
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min. Finally, cell cycle distribution was analyzed using FACSCalibur (Becton-Dickinson, Franklin
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Lakes, NJ). 2.9 Cell invasion
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The Transwell assay was used to quantify cell invasion. The transfected cells were plated in the upper
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chamber of Transwell assay inserts (BD Bioscience, Franklin Lakes, NJ, USA) containing 500 μL of serum-free 1640 medium. The lower chambers were filled with 1640 medium containing 10% FBS.
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After 48 h, the cells on the filter surface were fixed with methanol for 10 min, stained with 0.1%
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crystal violet, and photographed. Invasion was assessed by counting the number of stained cell nuclei from five random fields per filter for each group. 2.10 Cell migration
The transfected cells were seeded in the top chambers (8 mm pores, Millipore, Billerica, MA). The lower chambers were filled with 1640 medium containing 10% FBS. After 48 h, the cells on the filter surface were fixed with methanol for 10 min, stained with 0.1% crystal violet. Migration was assessed by counting the number of stained cell nuclei from 5 random fields per filter in each group. 6
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2.11 Measurement of intracellular ROS levels The relative changes of ROS was measured by DCFH-DA method. After cells were transfected for 48 h, the cells were incubated with 10 μmol/L od DCFH-DA for 30 min. After cells were washed for 3 times, cells were subjected to fluorescence analyzer (BioTek, USA) with excitation/emission set at 488/525
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nm. The values were presented as percentage of relative fluorescence compared with control group.
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2.12 ELISA
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The activity of SOD, CAT and GSH-Px was measured by using the relative ELISA Kit (Sangon Biotech, Shanghai, China). Briefly, the cells were collected and centrifuged at 1000 g for 10 min, the
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supernatant was collected and was measured according to the instructions.
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2.13 Statistical analysis
Data were expressed as mean ± SD and considered significant at P < 0.05. Statistical analysis was
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performed using a student’s unpaired t test (SPSS release 19.0; SPSS Inc.,).
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3. Results
3.1 TrxR2 was up-regulated in NSCLC tumor tissues
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To investigate the role of TrxR2 in the progression of lung cancer, we first measured the expression of
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TrxR2 in NSCLC tumor tissues. The results of Real-time PCR and western blot showed that TrxR2 was significantly up-regulated in NSCLC tumor tissues compared with adjacent lung tissues (Figure 1A, B). 3.2 Inhibition of TrxR2 inhibited A549 and NCI-H1299 cell proliferation and induced cell apoptosis, whereas overexpression of TrxR2 showed the opposite effects To further analyze the effect of TrxR2 on NSCLC cell proliferation and apoptosis, A549 and YTMLC-90 cells were transfected with shRNA-TrxR2 and pAd-TrxR2, respectively. The result of western blot showed that after A549 and NCI-H1299 cells were transfected with shRNA-TrXR2 or 7
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pAd-TrxR2, the expression of TrxR2 was significantly decreased or increased, respectively (Figure 2A, B); The CCK-8 assay showed that the cell proliferation of A549 and NCI-H1299 was significantly suppressed when cells were transfected with shRNA-TrxR2 (Figure 2C, D). Conversely, the cell proliferation of A549 and NCI-H1299 was dramatically increased when cells were transfected with
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pAd-TrxR2 (Figure 2E, F). The results of flow cytometry showed that shRNA-TrxR2 significantly
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induced A549 and NCI-H1299 cell apoptosis, and the rate of cell apoptosis increased by nearly 1-fold
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when compared with the control group, respectively (Figure 2G). When cells were transfected with pAd-TrxR2, the cell apoptosis of A549 and NCI-H1299 was significantly decreased (Figure 2H). In
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addition, we also measured the effect of TrxR2 on the cell cycle. The results showed that inhibition of
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TrxR2 induced A549 and NCI-H1299 cell cycle arrest at G1/S phase (Table 1, 2, 3, 4).These results indicate that the inhibition of TrxR2 inhibited A549 and NCI-H1299 cell proliferation and induced cell
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apoptosis, whereas the overexpression of TrxR2 had the opposite effects.
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3.3 Inhibition of TrxR2 suppressed A549 and NCI-H1299 cell invasion and migration, whereas overexpression of TrxR2 showed the opposite effects
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To further analyze the effect of TrxR2 on the metastasis of NSCLC cells, Transwell assays were used to
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measure cell invasion and migration after cells were transfected with shRNA-TrxR2 or pAd-TrxR2. The results showed that when A549 and NCI-H1299 cells were transfected with shRNA-TrxR2, the cell invasion and migration was significantly inhibited compared with control group (Figure 3A, C). Whereas when cells were transfected with pAd-TrxR2, the cell invasion and migration were notably increased compared with control group (Figure 3B, D). These results suggested that the inhibition of TrxR2 suppressed NSCLC cell metastasis, whereas the overexpression of TrxR2 induced NSCLC cell metastasis. 8
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3.4 Inhibition of TrxR2 decreased the antioxidant activities, whereas overexpression of TrxR2 showed the opposite effects. Because TrxR2 plays an important role in protecting cells from oxidative damage, we also analyzed the effect of TrxR2 on antioxidant activities. The results showed that when cells were transfected with
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shRNA-TrxR2, the production of ROS was dramatically increased (Figure 4A). Moreover, the activity
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of SOD, CAT and GSH-Px were notably decreased (Figure 4B, C, D,). However, when cells were
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transfected with pAd-TrxR2, ROS production was significantly decreased (Figure 4E); SOD, CAT and GSH-Px activity levels were significantly increased (Figure 4F, G, H). These results indicate that the
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inhibition of TrxR2 decreased antioxidant activities, whereas overexpression of TrxR2 had the opposite
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effects. 4 Discussion
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As lung carcinogenesis is a chronic and a multiple-step process in which accumulation of genetic and
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epigenetic alternations are involved, to achieve a more effective treatment of lung cancer is essential[15]. Our study proves that TrxR2 was upregulated in NSCLC tumor tissues and TrxR2 could
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potentially function as an oncogenic gene in lung cancer progression.
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An increasing number of studies have verified that TrxR2 was highly expressed in various cancers and promoted cancer progression. For example, TrxR2 was upregulated and may be involved in the formation and development of hepatocellular carcinomas[16]. Lincoln et al. reported that TrxR was upregulated in tumors which had a high proliferation capacity, a low apoptosis rate and an elevated metastatic, such as breast cancer, thyroid, prostate and colorectal carcinoma[17]. Ylermi et al. reported that the Trx and TrxR were highly expressed in lung carcinomas[18]. Because TrxR2 transfers electrons from NADPH/H+ to Trx2[19] and was overexpressed in cancer cells, the molecule may act as a 9
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proto-oncogene in cancer cells by increasing cell proliferation and decreasing apoptosis[20]. Therefore, TrxR2 may regulate the progression of lung cancer. In this study, we also identified that TrxR2 was overexpressed in NSCLC tumor tissues, inhibition of TrxR2 not only suppressed NSCLC cell proliferation, and induced cell apoptosis and induced cell cycle arrest in G1/S phase in vitro.
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Furthermore, the inhibition of TrxR2 suppressed NSCLC cell invasion and migration, whereas the
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overexpression of TrxR2 reversed these effects. Therefore, TrxR2 promoted the progression of lung
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cancer.
The current understanding of the etiology of lung cancer indicates that oxidative stress is
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implicated in its pathogenesis[21]. Lung cancer cells exist in an elevated oxidative state, and increased
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the production of ROS[22]. Moreover, high levels of ROS may increase the activity of antioxidants in lung cancer patients. For example, previous study reported that lung cancer patients have increased the
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activity of CAT and ceruloplasmin in peripheral blood [23]. Kaynar et al. demonstrated that the activity
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of GSH, xanthine oxidase, catalase and Cu-Zn superoxide dismutase activities in the blood of patients with NSCLC was significantly increased[24]. Mitochondrial antioxidant enzymes, including
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manganese superoxide dismutase (MnSOD), glutathione-dependent peroxidase (GPX) and TrxR2,
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comprise a primary defense system in mitochondria and are essential for detoxification against ROS[25]. Therefore, the inhibition of antioxidant systems is an option for cancer intervention[26]. Our results proved that the level of ROS was significantly increased compared with control group. When cells were transfected with shRNA-TrxR2, SOD, CAT and GSH-Px activity levels was decreased and ROS levels was increased. Whereas pAd-TrxR2 transfection showed the opposite effects. Furthermore, incapacitating antioxidant defense systems by inducing ROS production was reported to be a novel
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strategy for cancer therapy[27-29], so the inhibition of TrxR2 could suppress NSCLC cell proliferation and induce cell apoptosis by inducing ROS production and decreasing antioxidant activity. 5 Conclusion Taken together, these results show that TrxR2 was overexpressed in NSCLC cells. The inhibition of
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TrxR2 suppressed NSCLC cell proliferation, invasion and migration, and induced cell apoptosis by
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inducing ROS production and decreasing antioxidant activity. TrxR2 may be a potential target for
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treating lung cancer.
Funding: This work was supported by Natural Science Basic Research Plan in Shaanxi Province of
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China-General Program (No. 2015JM8480) . The West Light Talent Culture Project of the Chinese
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Academy of Sciences (2014).
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Conflict of interest: The authors declare that they have no conflict of interest.
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Figure legends: Figure 1 TrxR2 was up-regulated in NSCLC tumor tissues. NSCLC tumor tissue samples and adjacent tissues were collected from 20 chemotherapy native patients who underwent thoracic surgery. A. The expression of TrxR2 in NSCLC tumor tissues was measured by Real-time PCR. B. The
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expression of TrxR2 in NSCLC tumor tissues was measured by western blot. *P < 0.05 vs. adjacent
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tissues.
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Figure 2 Inhibition of TrxR2 inhibited A549 and NCI-H1299 cell proliferation and induced cell apoptosis, whereas overexpression of TrxR2 showed the opposite effects. A. After A549 and
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NCI-H1299 cells were transfected with shRNA-TrxR2, the expression of TrxR2 was measured by
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western blot. B. After A549 and NCI-H1299 cells were transfected with pAd-TrxR2, the expression of TrxR2 was measured by western blot. C. After A549 cells were transfected with shRNA-TrxR2 for 24,
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48 and 72 h, cell proliferation was measured using the CCK-8 method. D. After NCI-H1299 cells were
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transfected with shRNA-TrxR2 for 24, 48 and 72 h, the cell proliferation was measured by CCK-8 method. C. The effect of pAd-TrxR2 on the proliferation of A549 cells. E. The effect of pAd-TrxR2 on
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the proliferation of NCI-H1299. F. After A549 and NCI-H1299 cells were transfected with
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shRNA-TrxR2 for 48 h, cell apoptosis was measured by flow cytometry. G. After A549 and NCI-H1299 cells were transfected with pAd-TrxR2 for 48 h, the cell apoptosis was measured by Flow cytometry. *P < 0.05 vs. control group. Figure 3 Inhibition of TrxR2 suppressed A549 and NCI-H1299 cell invasion and migration, whereas overexpression of TrxR2 showed the opposite effects. After A549 and NCI-H1299 cells were transfected with shRNA-TrxR2 for 48 h, A-B. the cell invasion was measured by Transwell method. C-D. the cell migration was measured by Transwell method. *P < 0.05 vs. control group. 16
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Figure 4 Inhibition of TrxR2 decreased antioxidant activities, whereas overexpression of TrxR2 showed the opposite effects. After cells were transfected with shRNA-TrxR2 or pAd-TrxR2 for 48 h, A. The production of ROS was measured by DCFH-DA method. B-H. The activity of SOD, CAT and
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GSH-Px was measured by ELISA. *P < 0.05 vs control group.
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Table 1 Effect of overexpression of TrxR2 on the cell cycle of A549 cells. S
G2/M
control
83.26%
12.15%
4.59%
pAd-control
80.25%
13.42%
6.33%
pAd-TrxR2
52.13%*
35.28%*
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12.59%*
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*P<0.05 vs control group
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Table 2 Effect of overexpression of TrxR2 on the cell cycle of NCI-H1299 cells. S
G2/M
control
80.14%
14.17%
5.69%
pAd-control
78.34%
15.39%
6.27%
pAd-TrxR2
56.47%*
32.06%*
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*P<0.05 vs control group
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Table 3 Effect of inhibition of TrxR2 on the cell cycle of A549 cells S
G2/M
control
74.36%
14.55%
11.09%
shRNA-control
72.39%
16.23%
11.38%
shRNA-TrxR2
85.16%*
10.24%*
PT
G1
RI
4.60%*
AC
CE
PT E
D
MA
NU
SC
*P<0.05 vs control group
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ACCEPTED MANUSCRIPT
Table 4 Effect of inhibition of TrxR2 on the cell cycle of NCI-H1299 cells S
G2/M
control
70.13%
15.22%
14.65%
shRNA-control
73.48%
14.82%
11.70%
shRNA-TrxR2
88.57%*
8.26%*
PT
G1
RI
3.17%*
AC
CE
PT E
D
MA
NU
SC
*P<0.05 vs control group
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ACCEPTED MANUSCRIPT
Highlights 1. TrxR2 was up-regulated in lung cancer tissues. 2. The inhibition of TrxR2 suppressed lung cancer cell proliferation, invasion and migration and induced cell apoptosis by inducing ROS production and decreasing antioxidant activity. Whereas
PT
overexpression of TrxR2 had the opposite effect.
AC
CE
PT E
D
MA
NU
SC
RI
3. TrxR2 acts as an oncogenic gene in the context of lung cancer progression.
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