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ROS-dependent Atg4 upregulation mediated autophagy plays an important role in Cd-induced proliferation and invasion in A549 cells
Chemico-Biological Interactions 279 (2018) 136–144
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ROS-dependent Atg4 upregulation mediated autophagy plays an important role in Cd-induced proliferation and invasion in A549 cells
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Wei Lva,1, Linlin Suib,1, Xiaona Yana, Huaying Xiec, Liping Jianga, Chengyan Genga, Qiujuan Lia, Xiaofeng Yaoa, Ying Kongb,∗∗, Jun Caoa,∗ a
Department of Occupational and Environmental Health, Dalian Medical University, No. 9 W. Lvshun South Road, Dalian 116044, China Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China c Zhang Xisen General Clinic, No. 147, The New Century Star City, Dongcheng District, Dongguan, Guangdong Province 116023, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Cadmium Atg4 ROS Cell proliferation Autophagy Invasion
Cadmium (Cd) is a toxic heavy metal that is widely used in industry and agriculture. In this study the role of autophagy in Cd-induced proliferation, migration and invasion was investigated in A549 cells. Exposure to Cd (2 μM) significantly increased reactive oxygen species (ROS) production, induced autophagy and enhanced cell growth, migration and invasion in A549 cells. Western blot analysis showed that the expression of autophagyrelated proteins, LC3-II, Beclin-1 and Atg4 and invasion-related protein MMP-9 were upregulated in Cd-treated cells. N-acetyl cysteine (NAC) markedly prevented Cd-induced proliferation of A549 cells and the increasing protein level of LC3-II and Atg4. Blocking Atg4 expression by siRNA strongly reduced Beclin-1 and LC3-II protein expression and the number of autophagosome positive cells induced by Cd. Furthermore, Atg4 siRNA increased the number of cells at G0/G1 phase, reduced the number of S and G2/M phase cells, and inhibited Cd-induced cell growth significantly compared with that of Cd-treated Control siRNA cells. 3-MA pretreatment increased the percentage of G0/G1 phase cells, decreased S phase and G2/M phase percentage, and inhibited Cd-induced cell growth remarkably compared with that of only Cd-treated cells. Knocking down Atg4 reduced the number of cells that migrated and invaded through the Matrigel matrix significantly and led to a significant decrease of MMP-9 expression. In addition, in lung tissues of Cd-treated BALB/c mice, the increased expression of LC3-II, Beclin-1 and Atg4 were observed. Taken together, our results demonstrated that ROS-dependent Atg4-mediated autophagy plays an important role in Cd-induced cell growth, migration and invasion in A549 cells.
1. Introduction Cadmium (Cd) is an environmental pollutant distributed through nature sources, agriculture and industry. Occupational exposure to Cd usually occurs in workplaces via inhalation. For the general population exposure to the metal can result from cigarette smoke, contaminated water or food. In 1993, the International Agency for Cancer Research classified Cd as a type Ⅰ carcinogen [1]. Epidemiological data indicate that causal associations exist between Cd exposure and prostate and breast, and there is a strong correlation between Cd exposure and occurrence of lung cancer [2]. Growing evidence showed that Cd induced autophagy in many cell
lines and multiple signals were reported to be involved in Cd-induced autophagy. In MES-13 mesangial cells, Cd induces autophagy through elevation of Ca2+ and Ca2+ -dependent activation of ERK [3]. Cd-induced autophagy was mediated through ROS-dependent activation of glycogen synthase kinase-3β signaling pathway was also reported in MES-13 mesangial cells [4]. In skin epidermal cells, Cd induces autophagy through the activation of LKB1-AMPK signaling and the downregulation of mTOR mediated by ROS generation caused PARP activation and energy depletion [5]. Other findings suggest that Cd induces cytoprotective autophagy by activating class III PI3K/Beclin-1/Bcl-2 signaling pathways and the autophagy serves a positive function in the reduction of Cd-induced cytotoxicity in PC-12 cells [6].
Abbreviations: Cd, cadmium; 3-MA, 3-methyladenine; ROS, reactive oxygen species; NAC, N-acetyl cysteine; A549 cells, the human lung glandular cancer cells; siRNA, Small interfering RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; GFP, green fluorescent protein; AO, acridine orange; AVOs, acidic vesicular organelles; AVs, autophagy vesicles; DCFH-DA, 2′,7′-dichlorofluorescin diacetate ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y. Kong), [email protected] (J. Cao). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.cbi.2017.11.013 Received 21 August 2017; Received in revised form 30 October 2017; Accepted 21 November 2017 Available online 24 November 2017 0009-2797/ © 2017 Elsevier B.V. All rights reserved.
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4 °C. The pellets were post fixed in 2% osmium tetroxide for 2 h, dehydrated in a graded series of ethanol (20%, 40%, 60%, 70%, 80%, 90% and 100% every15 min once time). And then the pellets were embedded in araldite and sectioned at 70 nm thickness using an ultramicrotome. Ultrathin sections were stained with 2% uranyl acetate and 0.2% lead citrate. Images were observed by a Tecnai Spirit electron microscope.
Autophagy is an evolutionarily conserved catabolic process in which cytosolic components, organelles, and invading bacteria are sequestered within autophagosomes, and delivered to lysosomes for degradation. Autophagy is well recognized as a survival mechanism for maintaining cell viability during conditions of nutrient limitation. Meanwhile, extensive autophagy may promote cell death through selfdegradation of essential cellular constituents. Dysregulation of autophagy contributes to different pathological conditions, such as neurodegenerative diseases, aging and carcinogenesis [7,8]. Up to now, however, the effect of autophagy on influencing cell survival or death is controversial. Some recent studies of autophagy genes have addressed the role of autophagy in both cell death and survival. In our previous study, we found that exposure to Cd at 2 μM for 48 h significantly enhanced the growth of MRC-5 cells [9]. So, in this study, we attempted to validate the role of autophagy in Cd-induced cell proliferation and invasion in A549 cells.
2.5. Autophagic vesicles staining (GFP) The cellular autophagy level was further measured by fluorescence microscopy. A549 cells were seeded on glass plates and grown for 24 h in the typical cell culture media. The cells were pretreated with 1 mM 3MA for 2 h, or 100 nM rapamycin for 2 h, and then incubated with Cd for 48 h. After that, the cells were stained using the Cyto-ID Autophagy Detec-tion Kit (Enzo Life Sciences) according to the manufacturer's introduction [13]. In brief, the cells were incubated with Cyto-ID Green Autophagy Detection Reagent in PBS for 15 min at 37 °C in the dark. The cells were washed with 1 × assay buffer provided with Cyto-ID® Autophagy detection kit, and then the cells were examined using a fluorescence microscope (Olympus BX63) with a standard FITC filter set for using a standard FITC filter set for imaging the autophagic signal.
2. Materials and methods 2.1. Cell culture and treatment A549 cells, the human lung glandular cancer cells, were purchased from Cell Center for Peking Union Medical College (Peking, China), which cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), penicillin (100 UI/ml)-streptomycin (0.1 mg/ml) at 37 °C in a humidified atmosphere of 5% CO2. Cadmium chloride (CAS No. 10108-64-2; purity of 99%) was purchased from Sigma-Aldrich and 2.9 mg of cadmium chloride was dissolved in 1 ml distilled water to prepare a stock solution of 16 mM. For each experiment, A549 cells were treated with different concentrations of Cd.
2.6. Acridine orange (AO) staining assay for autophagy detection As a marker of autophagy, the volume of the cellular acidic compartment can be visualized after AO staining [14]. A549 Cells were seeded onto glass coverslips in 24-well plates, pretreated with 50 nM Atg4 siRNA or Control siRNA duplexes, for 12 h and exposed to various concentrations of Cd for 48 h, respectively. After treatment, media were discarded and the cells were washed twice in PBS. Then the A549 cells on the glass coverslips were stained with PBS containing 10 μg/ml AO for 15 min at 37 °C in the dark. After washing with PBS in twice, the fluorescent micrographs were taken to detect the autophagy of A549 cells.
2.2. MTT assay The proliferation of A549 cells treated by Cd was measured by MTT assay as described previously [10]. Briefly, A549 cells (1 × 105/ml) were seeded in 96-well plates and treated with Cd (0, 2, 4, 8, 16 and 32 μM) for 48 h. To determine the effect of NAC, 3-methyladenine (3MA) and Atg4 siRNA on Cd-induced cell growth, A549 cells were pretreated with 1.5 mM NAC for 1 h, 1 mM 3-MA for 2 h, or 50 nM Atg4 siRNA duplexes for 12 h respectively, and then treated with Cd for 48 h. After treatment, MTT solution was added, and the cells were incubated for 4 h at 37 °C. The supernatant was discarded, and insoluble purple formazan was dissolved by adding 100 μl DMSO to each well. The plate was gently agitated until the blue formazan crystals were fully dissolved. The absorbance at 570 nm was read using a Bio-Rad Microplate Reader, and the cell viability (%) was calculated using the following equation: (A570 of treated sample/A570 of control) × 100 [11].
2.7. Western blot analysis At the end of the designated treatments, the cells were washed twice with ice-cold PBS and completely lysed in the lysis buffer provided with a protein extraction kit (Keygen Biotech). The cell lysate was centrifuged at 16,000 × g and 4 °C for 5 min and the supernatants containing the total protein were isolated. The concentration of total protein was quantified using the Bio-Rad protein dye microassay according to the manufacturer's recommendations. SDS-polyacrylamide gel electrophoresis was performed, and the proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking, the filters were incubated with the antibody-containing solution over night at 4 °C, washed twice, and incubated with a blocking solution-coupled secondary antibody at 37 °C for 2 h.
2.3. Determination of ROS production The formation of intracellular ROS was measured by fluorescence microscopy after DCFH-DA staining [12]. Briefly, A549 cells were incubated with Cd (0 and 2 μM) at 37 °C for 24 h. To evaluate the protective effect of NAC, A549 cells were pretreated with NAC (1.5 mM) at 37 °C for 2 h, and then incubated with Cd at 37 °C for 24 h. After harvesting by trypsinisation, the cells were washed with PBS twice and stained with 5 μM DCFH-DA for 30 min at 37 °C in the dark, and then viewed under the fluorescence micro-scope (Olympus BX63). The intensity of DCF fluorescence was analyzed by Image-Pro Plus 6.0 soft ware.
2.8. Cell cycle analysis Cell cycle analysis was performed using a flow cytometer [9,15]. A549 cells were plated into culture flasks and serum starved for 24 h and then cultured in RPMI 1640 medium with 10% FCS and Cd for 48 h. To determine the involvement of autophagy in Cd-induced cell cycle progression, A549 cells were pretreated with 3-MA or Atg4 siRNA. Subsequently, cells (1.0 × 106) were suspended thoroughly in 0.5 ml of PBS, and fixed with 70% ethanol overnight at 4 °C. Fixed cells were washed with PBS, suspended with RNase A (100 μg/ml) at 37 °C for 30 min, and stained with propidium iodide (PI) at 4 °C in dark for 30 min. The cell cycle was analyzed with a flow cytometer.
2.4. Electronic microscopy After A549 cells were treated with different concentrations of Cd and incubated for 48 h at 37 °C, the cells were harvested and washed with PBS twice, then fixed with 2.5% glutaraldehyde at least for 2 h at 137
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Fig. 1. Effects of Cd on cell viability and autophagy in A549 cells. A: The cell viability was measured using the MTT assay. A549 cells were exposed to CdCl2 (0, 2, 4, 8, 16 and 32 μM) for 48 h. Each point represents mean ± SD of three independent experiments (∗∗P < 0.01 vs. Control). B, C, D: Effects of Cd on expression of LC3-II, Beclin-1 and Atg4 in A549 cells. A549 cells were incubated with CdCl2 (0, 2, 4 μM) for 48 h. Western blots were performed on the total protein of untreated and Cd-treated cells. β-actin was used as control. Relative expression of these proteins was expressed as a percentage of β-actin. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Control). E: Transmission electron microscopy was used for observing. Scale bar: 500 nm. F: Quantitation of autophagy vesicles (AVs) in Cd-treated A549 cells was done. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Control). G: Cd-treated A549 cells, with or without pretreatment of 1 mM 3-MA for 2 h or 100 nM rapamycin for 4 h, were stained with the Cyto-ID Autophagy Detection Kit and then autophagosomes were observed by fluorescence microscope. Scale bar: 500 μm. H: Quantitation of AVs in Cd-treated A549 cells was done. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Control, ##P < 0.01 vs. Cd alone).
Biosciences). Experiments were performed in triplicate.
2.9. Small interfering RNA transfection For transient transfection, the complete medium was converted to serum-free culture just before experiments when the confluence of A549 cells was 90%. The oligo nucleotides encoding Atg4 small interfering RNA (siRNA) were 5′-CCUAGAUUCUUCUGAUGUATT-3′and 5′-UACUUCAGAAGAAUCUAGGTT -3’. The oligo nucleotides encoding scramble siRNA were 5′-UUCUCCGAACGUG UCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3’. All siRNAs were synthesized by Shanghai GenePharma Co. Ltd (Shanghai, China). Transfection of siRNA was done according to the instructions of the manufacturer. 8 μl Atg4 specific siRNAs or Control siRNA were lightly added into 0.5 ml RPMI 1640 medium without fetal bovine serum. After 5 min, 8 μl siRNA-Mate Transfection Reagent was softly added. After another 15 min, put this 0.5 ml mixture into 3.5 ml of antibiotic-free medium. The cells were incubated with this media for 48 h, harvested and then processed for Western blot analysis. Atg4 protein levels were determined to assess the effects of RNA interference.
2.11. Animals and treatments To evaluate the effects of Cd-induced autophagy in vivo, the experiments were performed using male BALB/c mice (n = 40 and average body weight of 18–22 g) obtained from Dalian Medical University Animal Research Center. Animal work was carried out in compliance with the United States NIH Guide for the Care and Use of Laboratory Animals (National Research Council of the National Academies 2011), and was approved by the Institutional Animal Ethical Committee of Dalian Medical University. Animals were clinically healthy and kept under hygienic conditions in plastic cages in an environment maintained at 24 ± 2 °C, 55 ± 10% humidity and 12/12 h cycle of light and darkness for acclimatization. The animals had free access to standard pellet diet and water ad libitum. The 40 experimental mice were assigned into four equal groups (G1–G4), and given CdCl2 (0, 0.25, 0.5 or 1 mg/kg body weight respectively) by subcutaneous injection (S/C) daily for 6 weeks. The lung tissues of mice were used for Western blot to observe the expression of autophagy-related proteins, LC3-II, Beclin-1, Atg4.
2.10. Cell migration and invasion assays Cell migration was assayed in 24-well, 6.5-mm-internal-diameter transwell plates (8.0 μm pore size; Corning, USA). After treatment of Cd, serum-starved cells (2 × 105 cells/ml) were placed in the upper chambers, and the lower chamber was filled with media containing 10% FBS. Cells were allowed to migrate. After incubation for the indicated time, cells on the upper surfaces of the filters were removed by wiping with a clean cotton swab and migrated cells on the undersides of the filters were fixed with methanol, stained with 0.5% crystal violet. Then migrated cells were viewed and counted under an upright microscope (5 fields per chamber). For invasion, the cell invasion assay was essentially conducted as above, except that transwell filters, an 8 μm pore size insert pre-coated with Matrigel (1:4 dilution in media; BD
2.12. Statistical analysis All values were expressed as mean ± standard deviation calculated from three independent experiments. The data were analyzed with oneway analysis of variance (ANOVA) followed by Tukey multiple comparison post hoc tests (SPSS 17.0 software). P < 0.05 and P < 0.01 were taken as statistically significant.
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3. Results
3.3. Cd induced autophagy in A549 cells
3.1. Cd caused proliferation in A549 cells
The occurrence of autophagy by Cd treatment was detected by direct observation of the formation of autophagosomes using electron microscopy. As shown in Fig. 1E–F, the control cells exhibited normal nuclei with uniform and finely dispersed chromatin, whereas abundant autophagosomes in the cytoplasm were produced in Cd-exposed cells. The quantification of the AVs numbers per viable cell implied that Cd increased AVs number significantly compared to the control (P < 0.01). Cd-induced autophagy was further examined by staining with CytoID Green Autophagy Detection Reagent in A549 cells treated with 2 μM Cd for 48 h. As demonstrated in Fig. 1G–H, the number of autophagosome positive green puncta in Cd-treated cells significantly increased compared with the control (P < 0.01). However, the number of cells stained with Cyto-ID Green was reduced in the presence of 3-MA, an autophagy inhibitor, where the number of cells stained with Cyto-ID Green increased significantly by treatment with rapamycin. To confirm the effect of Cd-induced autophagy, we detected the expression of the autophagy-regulatory proteins including LC3, Beclin-1 and Atg4 in Cd-treated A549 cells. As shown in Fig. 1B–D, after Cd treatment, the conversion of LC3-I to LC3-II and the upregulation of beclin-1 and Atg4 were observed.
The proliferation of A549 cells in the presence of Cd was observed after incubation for 48 h (Fig. 1A). The results showed that Cd increased the growth of A549 cells at the concentrations of 2 and 4 μM (P < 0.01), and at the concentration of 2 μM, the growth of A549 cells increased the most significantly. In addition, Cd at the concentrations above 16 μM induced a marked dose-dependent decrease in cell growth suggesting that Cd had cytotoxicity at these levels. These results showed that Cd at low levels could induce A549 cell growth, but at high doses, Cd imposed strong toxicity. This result is consistent with the effects of Cd on MRC-5 cells [9].
3.2. Cd induced intracellular ROS formation in A549 cells To evaluate the generation of ROS in A549 cells treated with Cd, DCFH-DA fluorescence dye was employed. A significant increase of DCF fluorescence intensity was observed in cells treated with Cd at the concentration of 2 μM (P < 0.01). To certify the oxidative stress, NAC was used to reduce intracellular ROS formation. When the cells were pretreated with NAC for 2 h, the DCF fluorescence intensity in A549 cells was significantly decreased compared to that of only Cdtreated cells (P < 0.01). This result demonstrated that Cd had a strong effect on ROS production in A549 cells (Fig. 2A–B).
3.4. Role of ROS in Cd-induced cell growth and autophagy in A549 cells To evaluate the role of ROS in Cd-induced growth of A549 cells,
Fig. 2. The role of ROS in Cd-induced cell growth and autophagy in A549 cells. A: Fluorescence microscopy was used to detect ROS formation. A549 cells were pretreated with or without NAC (1.5 mM) for 2 h and then exposed to CdCl2 (2 μM) for 24 h. The level of ROS was analyzed using fluorescence microscopy. Scale bar: 500 μm. B: Each bar represents mean ± SD of three independent experiments (∗∗P < 0.01 vs. Control, ##P < 0.01 vs. Cd alone). C: NAC was used to evaluate the role of ROS in Cd-induced cell growth. The cell viability was measured using the MTT assay. Cells were pretreated with NAC at a concentration of 1.5 mM for 2 h and then treated with Cd (0, 2, 4, 8, 16 and 32 μM) for 48 h. Each point represents mean ± SD of three independent experiments (∗∗P < 0.01 vs. Control, ##P < 0.01 vs. Cd alone). D: To confirm whether ROS was involved in Cd-induced autophagy, the protein level of Atg4 and LC3 was analyzed by Western blot. β-actin was used as control. E, F: Relative expression of Atg4 and LC3 proteins were expressed as a percentage of β-actin. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Control, ##P < 0.01 vs. Cd alone). G: The effect of NAC on Cd-induced autophagolysosomes formation in A549 cells was measured by AO staining. Scale bar: 500 μm. H: Quantitation of formation of autophagolysosomes was done. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Control, ##P < 0.01 vs. Cd alone).
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Fig. 3. The involvement of Atg4 in Cd-induced autophay and cell growth in A549 cells. Cells were pretreated with Atg4 siRNA or Control siRNA, and then exposed to Cd for 48 h. A: Western blots were performed on the total protein of untreated and treated cells. B, C, D: β-actin was used as control. Relative expression of these proteins was expressed as a percentage of β-actin. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Si Control, ##P < 0.01 vs. Si Control+Cd). E: Formation of autophagolysosomes in A549 cells was measured by AO staining. Scale bar: 500 μm. F: Quantitation of formation of autophagolysosomes in was done. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Si Control, ##P < 0.01 vs. Si Control+Cd). G: To determine the role of Atg4 in Cd-induced cell growth, MTT assay was performed. Each point represents mean ± SD of three independent experiments (##P < 0.01 vs. Si Control+Cd). H: To determine the role of autophagy in Cd-induced cell growth, 3-MA was used and MTT assay was performed. Each point represents mean ± SD of three independent experiments (##P < 0.01 vs. Cd alone).
induced cell growth in A549 cells (P < 0.01). These two results demonstrated that autophagy play an important role in Cd-induced cell growth in A549 cells.
A549 cells were pretreated with NAC (Fig. 2C). After the 2 h pre-incubation with 1.5 mM NAC, there was a significant decrease in absorbance (P < 0.01) compared to only Cd-treated A549 cells. These data suggested that NAC is effective on Cd-induced growth in A549 cells and ROS may play an important role in Cd-induced A549 cell proliferation. In an attempt to determine the effect of ROS on Cd-induced autophagy in A549 cells, ROS scavenger NAC was used. As shown in Fig. 2D–F, when cells were pretreated with NAC, the conversion of LC3 and the upregulation of Atg4 induced by Cd-treatment were both decreased significantly as compared with only Cd-treated A549 cells (P < 0.01). Pretreatment with NAC blocking the ROS generation can obviously inhibited Cd-induced autophagy as demonstrated by inhibition of the formation of acidic vesicular organelles (AVOs) (Fig. 2G–H). These results demonstrated that ROS play an important role in Cd-induced autophagy.
3.7. Role of autophagy in Cd-induced cell cycle in A549 cells In our previous study, we found that Cd treatment markedly reduced the number of cells in G0/G1 phase, accompanied with an increase in the number of cells in S phase and G2/M phase compared to the control group at the time point of 48 h in MRC-5 cells (P < 0.01). In this study, the same results were obtained in Cd-treated A549 cells (Fig. 4A–B). To establish that Cd-induced autophagy plays a role in cell cycle change induced by Cd, first, 3-MA was employed to block Cdinduced autophagy. Cell cycle analysis was performed in triplicate and showed that 3-MA pretreatment increased the percentage of G0/G1 phase cells and decreased S phase and G2/M phase percentage compared with that of only Cd-treated cells (Fig. 4E–F), and the differences in the percentages are not significant. Then, Atg4 siRNA was also used to investigate the role of autophagy in Cd-induced cell cycle progression. As demonstrated in Fig. 4C–D, when Atg4 expression was inhibited by siRNA, the number of cells at G0/G1 phase was increased and the number of S and G2/M phase cells was reduced compared to that of Cd-treated Control siRNA cells. Furthermore, blocking Atg4 expression strongly reduced Cyclin-D1 and Cyclin-E expression induced by Cd (Fig. 4G–H). These results suggest that knocking down of Atg4 expression by siRNA blocked Cd-induced changes of cell cycle, indicating that Atg4-mediated autophagy might involved in Cd-induced cell cycle progression in A549 cells.
3.5. Role of Atg4 in Cd-induced autophagy in A549 cells In order to investigate the role of Atg4 in Cd-induced autophagy in A549 cells, the expression of Atg4 protein was inhibited by its siRNA. As illustrated in Fig. 3A–D, blocking Atg4 expression strongly reduced Beclin-1 and LC3-II protein expression induced by Cd. Furthermore, as shown in Fig. 3E–F, Atg4 siRNA decreased the number of autophagosome positive cells significantly compared to that of Cd-treated Control siRNA cultures (P < 0.01). Taken together, we conclude that Cd can induce autophagy in A549 cells involving induction of Atg4 protein. 3.6. Role of autophagy in Cd-induced proliferation in A549 cells In order to investigate the role of autophagy in Cd-induced proliferation of A549 cells, 3-MA, an autophagy inhibitor was used in MTT assay. Pretreatment with 1 mM 3-MA for 2 h significantly inhibited the Cd-induced cell viability of A549 cells (Fig. 3H). Atg4 siRNA was also used to block Cd-induced autophagy and MTT assay was performed. As shown in Fig. 3G, blocking Atg4 expression strongly inhibited Cd-
3.8. Role of autophagy in Cd-induced migration and invasion in A549 cells The role of autophagy in Cd-induced cell migration and invasion was further examined. As shown in Fig. 5A–B, the number of cells that migrated through transwell plate was significantly increased after the treatment of Cd, illustrating that Cd treatment enhanced cell migration 140
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Fig. 4. The involvement of autophay in Cd-induced cell cycle progression in A549 cells. A549 cells were pretreated with or without 3-MA, Atg4 siRNA or Control siRNA, and treated with Cd for 48 h, and then collected, fixed, stained with PI, and cell cycles were monitored by a flow cytometer. A, B: Effect of Cd on cell cycle of A549 cells. The population of each phase was calculated. C, D: The role of Atg4 in Cd-induced cell cycle change in A549 cells. The population of each phase was calculated. E, F: Effect of 3-MA on Cd-induced cell cycle change in A549 cells. The population of each phase was calculated. G: The role of Atg4 in Cd-induced expression of Cyclin-D1 and Cyclin-E in A549 cells. H: β-actin and GAPDH were used as control. Relative expressions were expressed as a percentage of β-actin and GAPDH. Each bar represents mean ± SD of three independent experiments (∗∗P < 0.01 vs. Si Control, ##P < 0.01 vs. Si Control+Cd).
cell growth, migration and invasion in A549 cells. The effect of Cd-induced autophagy has been detected in many different cell types, such as A549 cells [16], human prostate epithelial cells [17] and breast cancer cells [18]. In this study, we demonstrated that Cd induced autophagy in A549 cells the most significantly at the concentration of 2 μM, as proven by the appearance of autophagosomes, the increase of GFP-LC3 functa cells and the formation of LC3-II. Cd-induced autophagy was further supported by the observation that the protein level of Beclin-1 was increased after Cd treatment. Beclin-1 has an important role at every central step in autophagic pathways, from autophagosome formation to autophagosome maturation [19]. Consequently, our results support that Cd induces autophagy in A549 cells. Meanwhile, on the basis of Western blot analysis, in BALB/c mice's lung tissues, the protein levels of LC3-II and Beclin-1 were both increased significantly after Cd treatment. Atg4 is an enzyme that plays a major role in the processing of LC3. It cleaves LC3-I to produce LC3-II, and colocalizes with LC3-II in the membranes of autophagic vesicles [20]. Our previous results of gene microassay showed that Cd induced Atg4 gene expression significantly in A549 cells. In this study, using Western blot, we demonstrated that Cd could induce Atg4 protein in A549 cells. To determine the role of Atg4 in Cd-induced autophagy, Atg4 was knocked down in A549 cells by siRNA. Western blot results showed that blocking Atg4 expression strongly reduced expression of Beclin-1 and LC3-II protein induced by Cd. Furthermore, Atg4 siRNA decreased the number of autophagosome positive cells significantly compared to that of Cd-treated Control siRNA cells. These results revealed that Atg4 plays an important role in Cd-induced autophagy in A549 cells. ROS has been reported to participate in autophagy. A large body of data shows that starvation can induce the production of ROS and lead to autophagy. It has also been studied that ROS scavengers can block
in A549 cells (P < 0.01). To determine the role of autophagy in Cdinduced cell migration, Atg4 siRNA was employed to block Cd-induced autophagy. The number of cells that migrated through transwell plates was significantly decreased in Cd-treated Atg4 siRNA cells compared to that of Cd-treated Control siRNA cultures (P < 0.01). At the same time, Transwell chamber, pre-coated with Matrigel, was used to detect the effect of Cd-induced cell invasion. As shown in Fig. 5C–D, the number of invasion cells was obviously increased after the treatment of Cd (P < 0.01). Meanwhile, the number of cells that invaded through Transwell chamber was significantly decreased in Cd-treated Atg4 siRNA cells compared to that of Cd-treated Control siRNA cultures (P < 0.01). The effect of Cd on the expression of invasion-related factor, MMP-9 was examined by Western blot (Fig. 5E–F). The results revealed that Atg4 siRNA led to a significant decrease of MMP-9 expression in Cd-treated cells compared to that of Cd-treated Control siRNA groups (P < 0.01). These results suggest that Cd might promote cell migration and invasion by facilitating autophagy in A549 cells. 3.9. Cd induced expressions of autophagy-related proteins in lung tissues of mice After Cd treatments, the lung tissues of mice were used for Western blot. As shown in Fig. 6, the expressions of autophagy-related proteins, LC3-II, Beclin-1 and Atg4 were all increased significantly in Cd-treated mice compared with the control mice (P < 0.05 and P < 0.01). This result suggested that Cd induced autophagy in the lung tissues of mice. 4. Discussion In this study, for the first time, we demonstrated that ROS-dependent Atg4-mediated autophagy plays an important role in Cd-induced 141
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Fig. 5. The involvement of autophay in Cd-induced migration and invasion in A549 cells. Cells were pretreated with Atg4 siRNA or Control siRNA, and then exposed to Cd for 48 h. Transwell assay was used for observing migrative (A) and invasive (C) ability of A549 cells. Ability of migration (B) and invasion (D) of A549 cells were quantified by counting the number of migrating or invasive cells in ten randomly chosen high-power fields (n = 3). Scale bar: 500 μm. E: Western blots were performed on the total protein of A549 cells. F: Relative expression of MMP-9 was expressed as a percentage of β-actin. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Si Control, ##P < 0.01 vs. Si Control+Cd).
of LC3, and contributes to autophagosome formation [20]. However, Li et al. reported that N-Benzoyl-O-(N′-(1-benzyloxycarbonyl-4-piperidiylcarbonyl) -D-phenylalanyl)-D -phenylalaninol (BBP) induced the autophagic cell death through a JNK-dependent Atg4 upregulation involving ROS production in MCF-7 cells [26]. It remains unclear whether ROS-regulated Atg4 is a more general characteristic of autophagy signaling. In this study, to explore the role of oxidative stress in Cd-induced Atg4 expression and autophagy, NAC, an intracellular ROS inhibitor was employed. When A549 cells were pretreated with NAC, the upregulation of Atg4 and the conversion of LC3 induced by Cd-
starvation-induced autophagy, indicating that ROS can regulate autophagy [21]. Increasing evidence has revealed that massive ROS can modulate multiple autophagy process, including Atg4–Atg8/LC3, Beclin-1, PTEN, PI3K–Akt–mTOR, p53 and MAPK signaling [22,23]. ROS generated by mitochondria can directly modulate the cysteine protease Atg4, resulting in inhibition of Atg4 delipidating activity, whereas the initial processing of LC3 (priming) by Atg4 is not altered [24]. Increasing evidence showed that Atg4 is a direct target for oxidation by H2O2, because a cysteine residue was identified as critical for this regulation [25]. ROS-mediated inhibition of Atg4 promotes lipidation
Fig. 6. Effects of Cd on expressions of autophagy-related proteins in lung tissues of mice. Forty mice were divided into four groups, each group was given CdCl2 (0, 0.25, 0.5 or 1 mg/kg body weight respectively) by subcutaneous injection daily for 6 weeks. A: The lung tissues were used for Western blot to observe the expression of LC3II, Beclin-1, Atg4. B, C, D: Relative expression of these proteins was expressed as a percentage of GAPDH. Each bar represents mean ± SD from three independent experiments. (∗P < 0.05 vs. Control; ∗∗P < 0.01 vs. Control).
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prevention of Cd-resulted toxicities.
treatment were both decreased significantly as compared with only Cdtreated A549 cells (P < 0.01). Furthermore, pretreatment with NAC blocking the ROS generation can obviously inhibited Cd-induced autophagy as demonstrated by inhibition of the formation of AVOs. These data suggest that ROS play an important role in Cd-induced autophagy and Cd induced autophagy might be through ROS-initiated Atg4 pathway. Cd was reported to induce cytoprotective autophagy by delaying the occurrence of apoptosis and activating class III PI3K/Beclin-1/Bcl-2 signaling pathway in PC-12 cells [6]. In this study, the growth of A549 cells were increased after Cd treatment at the concentration of 2 μM, and NAC pretreatment significantly decreased Cd-induced cell growth. 3-MA, an autophagy inhibitor could also significantly inhibit the Cdinduced cell viability of A549 cells. Meanwhile, blocking Atg4 expression by siRNA strongly inhibited Cd-induced cell growth in A549 cells. These results demonstrated that autophagy dependent ROS-initiated Atg4 upregulation plays an important role in Cd-induced cell growth in A549 cells. It is well known that cell cycle checkpoints are crucial regulatory points in cell proliferation. There are growing evidences concerning the role of autophagy in cell cycle arrest and cellular proliferation [27]. Experiments have demonstrated that autophagy is active in the G1 and S phases of the cell cycle, while inhibited in mitosis [28]. Seung-il Choi found that treatment with bafilomycin A 1, the autophagy flux inhibitor, resulted in decreased expression of Cyclin A1, B1, D1, and p53 in corneal fibroblasts. Furthermore, a decrease in Cyclin A1, B1, and D1 expression was observed in Atg7 gene knockout cells [29]. In our previous study, the cell cycle analysis revealed that Cd-treatment markedly reduced the number of cells in G0/G1 phase with an increase in the number of cells in S phase and G2/M phase after 48 h, suggesting that Cd at 2 μM promotes cell cycle progression. So, in this study, the involvement of autophagy in Cd-induced cell cycle change was investigated. The results showed that 3-MA pretreatment increased the percentage of G0/G1 phase cells and decreased S phase and G2/M phase percentage compared with that of only Cd-treated cells. Then, Atg4 siRNA was also used to investigate the role of autophagy in Cdinduced cell cycle progression. When Atg4 expression was inhibited by siRNA, the number of cells at G0/G1 phase was increased and the number of S and G2/M phase cells was reduced in Cd-treated Atg4 siRNA cultures compared with that of Cd-treated Control siRNA cells. This result suggest that knocking down of Atg4 expression by siRNA blocked Cd-induced changes of cell cycle, indicating that Atg4-mediated autophagy might be involved in Cd-induced cell cycle progression in A549 cells. Accumulating evidence showed that autophagy plays a complex role in migration, invation and cancer metastasis, because reports have suggested both pro-metastatic and anti-metastatic roles of autophagy [30]. Ischemia/reperfusion was reported to induce the expression of iNOS, which subsequently increased the migration and apoptosis of HUVECs via autophagy [31]. PM 2.5 exposure induces ROS, which activates loc146880 expression, and then up-regulates autophagy and promotes the malignant of lung cancer cells [32]. In our experiments, Cd treatment enhanced both cell migration and invasion in A549 cells. Atg4 knockdown significantly reduced MMP-9 expression and the number of cells that migrated and invaded through the Matrigel matrix compared to that of Cd-treated Control siRNA cells. These results demonstrated that Cd might promote cell migration and invasion by facilitating autophagy in A549 cells. Taken together, in this study we demonstrated that Cd induced cell proliferation, migration and invasion in A549 cells, resulting in marked increases in ROS levels and autophagy. ROS serves as a molecular signal to increase the expression of Atg4, which enhances cell autophagy. Both Atg4 and autophagy could promote A549 cell proliferation, migration and invasion. These results suggested that Cd exposure could facilitate lung tumor formation and progression, and suppressing ROS formation, Atg4 expression or cell autophagy might have potential values in
Conflicts of interest The authors declare that there are no conflicts of interest in the present work. Acknowledgments This work was (2013E15SF140).
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Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2017.11.013. References [1] IARC, Cadmium and cadmium compounds, IARC Monogr. Eval. Carcinog. Risks Hum. 58 (1993) 119–237. [2] D. Luce, I. StuckerICARE study group, Investigation of occupational and environmental causes of respiratory cancers (ICARE): a multicenter, population-based casecontrol study in France, BMC Public Health 11 (2011) 928. [3] S.H. Wang, Y.L. Shih, W.C. Ko, Y.H. Wei, C.M. Shih, Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway, Cell Mol. Life Sci. 65 (2008) 3640–3652. [4] S.H. Wang, Y.L. Shih, T.C. Kuo, W.C. Ko, C.M. Shih, Cadmium toxicity toward autophagy through ROS-activated GSK-3beta in mesangial cells, Toxicol. Sci. 108 (2009) 124–131. [5] Y. Son, X. Wang, J.A. Hitron, Z. Zhang, S. Cheng, A. Budhraja, S. Ding, J. Lee, X. Shi, Cadmium induces autophagy through ROS- dependent activation of the LKB1AMPK signaling in skin epidermal cells, Toxicol. Appl. Pharmacol. 255 (3) (2011) 287–296. [6] Q. Wang, J. Zhu, K. Zhang, C. Jiang, Y. Wang, Y. Yuan, J. Bian, X. Liu, J. Gu, Z. Liu, Induction of cytoprotective autophagy in PC-12 cells by cadmium, Biochem. Biophysical Res. Commun. 438 (2013) 186–192. [7] J.H. Lee, A.V. Budanov, E.J. Park, R. Birse, T.E. Kim, G.A. Perkins, K. Ocorr, M.H. Ellisman, R. Bodmer, E. Bier, M. Karin, Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies, Science 327 (2010) 1223–1228. [8] Y. Kondo, T. Kanzawa, R. Sawaya, S. Kondo, The role of autophagy in cancer development and response to therapy, Nat. Rev. Cancer 5 (2005) 726–734. [9] H. Xie, J. Wang, L. Jiang, C. Geng, Q. Li, D. Mei, L. Zhao, J. Cao, ROS-dependent HMGA2 upregulation mediates Cd-induced proliferation in MRC-5 cells, Toxicol. Vitro 34 (2016) 146–152. [10] J. Cao, L. Jia, H.M. Zhou, Y. Liu, L.F. Zhong, Mitochondrial and nuclear DNA damage induced by curcumin in human hepatoma G2 cells, Toxicol. Sci. 91 (2006) 476–483. [11] W.K. Lee, M. Abouhamed, F. Thevenod, Caspase-dependent and -independent pathways for cadmium-induced apoptosis in cultured kidney proximal tubule cells, Am. J. Physiol. Ren. Physiol. 291 (2006) 823–832. [12] Y.J. Zhou, S.P. Zhang, C.W. Liu, Y.Q. Cai, The protection of selenium on ROS mediated-apoptosis by mitochondria dysfunction in cadmium-induced LLC-PK(1) cells, Toxicol Vitro 23 (2009) 288–294. [13] Anja K. Klappan, Stefanie Hones, Ioannis Mylonas, Ansgar Brȕning, Proteasome inhibition by quercetin triggers macroautophagy and blocks mTOR activity, Histochem Cell Biol. 137 (2011) 25–36. [14] Eduardo Cremonese Filippi-Chiela, Msrdja Manssur Bueno e Sliva, Marcos Paulo Thomé, Guido Lenz, Single-cell analysis challenges the connection between autophagy and senescence induced by DNA damage, Autophagy 7 (2015) 1099–1113. [15] Y. Pan, H. Fu, Q. Kong, Y. Xiao, Q. Shou, H. Chen, Y.H. Ke, M.L. Chen, Prevention of pulmonary fibrosis with salvianolic acid a by inducing fibroblast cell cycle arrest and promoting apoptosis, J. Ethnopharmacol. 155 (2014) 1589–1596. [16] K. Subhadip, S. Suman, B. Arindam, EGFR upregulates inflammatory and proliferative responses in human lung adenocarcinoma cell line (A549), induced by lower dose of cadmium chloride, Inhal. Toxicol. 23 (2011) 339–348. [17] S. Bakshi, X. Zhang, S. Godoy-Tundidor, R.Y. Cheng, M.A. Sartor, M. Medvedovic, S. Ho, Transcriptome analyses in normal prostate epithelial cells exposed to lowdose cadmium: oncogenic and immunomodulations involving the action of tumor necrosis factor, Environ. Health Perspect. 116 (2008) 769–776. [18] Z.X. Wei, X.L. Song, Z.A. Shaikh, Cadmium promotes the proliferation of triplenegative breast cancer cells through EGFR-mediated cell cycle regulation, Toxicol. Appl. Pharmacol. 289 (2015) 98–108. [19] R. Kang, H.J. Zeh, M.T. Lotze, D. Tang, The Beclin 1 network regulates autophagy and apoptosis, Cell Death Differ. 18 (2011) 571–580. [20] R. Scherz-Shouval, E. Shvets, E. Fass, H. Shorer, L. Gil, Z. Elazar, Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4, EMBO J. 26 (2007) 1749–1760. [21] S.H. Park, G.Y. Chi, H.S. Eom, G.Y. Kim, J.W. Hyun, W.J. Kim, S.J. Lee, Y.H. Yoo,
143
Chemico-Biological Interactions 279 (2018) 136–144
W. Lv et al.
[22]
[23]
[24]
[25]
[26]
[27] R.C. Wang, B. Levine, Autophagy in cellular growth control, FEBS Lett. 584 (2010) 1417–1426. [28] Y. Takahashi, D. Coppola, N. Matsushita, H.D. Cualing, M. Sun, Y. Sato, C. Liang, J.U. Jung, J.Q. Cheng, J.J. Mule, et al., Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis, Nat. Cell Biol. 9 (2007) 1142–1151. [29] S. Choi, S. Dadakhujaev, Y. Maeng, S. Ahn, T. Kimab, E.K. Kim, Disrupted cell cycle arrest and reduced proliferation in corneal fibroblasts from GCD2 patients: a potential role for altered autophagy flux, Biochem. Biophysical Res. Commun. 456 (2015) 288–293. [30] Z. Su, Z. Yang, Y. Xu, Y. Chen, Q. Yu, Apoptosis, autophagy, necroptosis, and cancer metastasis, Mol. Cancer 14 (2015) 1–14. [31] T. Zhu, Q. Yao, W. Wang, H. Yao, J. Chao, iNOS induces vascular endothelial cell migration and apoptosis via autophagy in ischemia/reperfusion injury, Cell Physiol. Biochem. 38 (2016) 1575–1588. [32] X. Deng, N. Feng, M. Zheng, X. Ye, H. Lin, X. Yu, Z. Gan, Z. Fang, H. Zhang, M. Gao, Z. Zheng, H. Yu, W. Ding, B. Qian, PM 2.5 exposure-induced autophagy is mediated by lncRNA loc146880 which also promotes the migration and invasion of lung cancer cells, Biochimica Biophysica Acta 1861 (2016) 112–125.
Y.H. Choi, Role of autophagy in apoptosis induction by methylene chloride extracts of Mori cortex in NCI-H460 human lung carcinoma cells, Int. J. Oncol. 40 (2012) 1929–1940. Z. Li, Y. Yang, M. Ming, B. Liu, Mitochondrial ROS generation for regulation of autophagic pathways in cancer, Biochem. Biophysical Res. Commun. 414 (2011) 5–8. X. Xie, L. Le, Y. Fan, L. Lv, J. Zhang, Autophagy is induced through the ROS-TP53DRAM1 pathway in response to mitochondrial protein synthesis inhibition, Autophagy 8 (2012) 1071–1084. Y. Li, Q. Luo, L. Yuan, C. Miao, X. Mu, W. Xiao, J. Li, T. Sun, E. Ma, JNK-dependent Atg4 upregulation mediates asperphenamate derivative BBP-induced autophagy in MCF-7 cells, Toxicol. Appl. Pharmacol. 263 (2012) 21–31. S. Qiao, M. Dennis, X. Song, D.D. Vadysirisack, D. Salunke, Z. Nash, Z. Yang, M. Liesa, J. Yoshioka, S. Matsuzawa, S. Orian, A REDD1/TXNIP pro-oxidant complex regulates ATG4B activity to control stress-induced autophagy and sustain exercise capacity, Nat. Commun. 6 (2015) 7014. Y. Li, Q. Luo, L. Yuan, C. Miao, X. Mu, W. Xiao, J. Li, T. Sun, E. Ma, JNK-dependent Atg4 upregulation mediates asperphenamate derivative BBP-induced autophagy in MCF-7 cells, Toxicol. Appl. Pharmacol. 263 (2012) 21–31.
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ROS-dependent Atg4 upregulation mediated autophagy plays an important role in Cd-induced proliferation and invasion in A549 cells
T
Wei Lva,1, Linlin Suib,1, Xiaona Yana, Huaying Xiec, Liping Jianga, Chengyan Genga, Qiujuan Lia, Xiaofeng Yaoa, Ying Kongb,∗∗, Jun Caoa,∗ a
Department of Occupational and Environmental Health, Dalian Medical University, No. 9 W. Lvshun South Road, Dalian 116044, China Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China c Zhang Xisen General Clinic, No. 147, The New Century Star City, Dongcheng District, Dongguan, Guangdong Province 116023, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Cadmium Atg4 ROS Cell proliferation Autophagy Invasion
Cadmium (Cd) is a toxic heavy metal that is widely used in industry and agriculture. In this study the role of autophagy in Cd-induced proliferation, migration and invasion was investigated in A549 cells. Exposure to Cd (2 μM) significantly increased reactive oxygen species (ROS) production, induced autophagy and enhanced cell growth, migration and invasion in A549 cells. Western blot analysis showed that the expression of autophagyrelated proteins, LC3-II, Beclin-1 and Atg4 and invasion-related protein MMP-9 were upregulated in Cd-treated cells. N-acetyl cysteine (NAC) markedly prevented Cd-induced proliferation of A549 cells and the increasing protein level of LC3-II and Atg4. Blocking Atg4 expression by siRNA strongly reduced Beclin-1 and LC3-II protein expression and the number of autophagosome positive cells induced by Cd. Furthermore, Atg4 siRNA increased the number of cells at G0/G1 phase, reduced the number of S and G2/M phase cells, and inhibited Cd-induced cell growth significantly compared with that of Cd-treated Control siRNA cells. 3-MA pretreatment increased the percentage of G0/G1 phase cells, decreased S phase and G2/M phase percentage, and inhibited Cd-induced cell growth remarkably compared with that of only Cd-treated cells. Knocking down Atg4 reduced the number of cells that migrated and invaded through the Matrigel matrix significantly and led to a significant decrease of MMP-9 expression. In addition, in lung tissues of Cd-treated BALB/c mice, the increased expression of LC3-II, Beclin-1 and Atg4 were observed. Taken together, our results demonstrated that ROS-dependent Atg4-mediated autophagy plays an important role in Cd-induced cell growth, migration and invasion in A549 cells.
1. Introduction Cadmium (Cd) is an environmental pollutant distributed through nature sources, agriculture and industry. Occupational exposure to Cd usually occurs in workplaces via inhalation. For the general population exposure to the metal can result from cigarette smoke, contaminated water or food. In 1993, the International Agency for Cancer Research classified Cd as a type Ⅰ carcinogen [1]. Epidemiological data indicate that causal associations exist between Cd exposure and prostate and breast, and there is a strong correlation between Cd exposure and occurrence of lung cancer [2]. Growing evidence showed that Cd induced autophagy in many cell
lines and multiple signals were reported to be involved in Cd-induced autophagy. In MES-13 mesangial cells, Cd induces autophagy through elevation of Ca2+ and Ca2+ -dependent activation of ERK [3]. Cd-induced autophagy was mediated through ROS-dependent activation of glycogen synthase kinase-3β signaling pathway was also reported in MES-13 mesangial cells [4]. In skin epidermal cells, Cd induces autophagy through the activation of LKB1-AMPK signaling and the downregulation of mTOR mediated by ROS generation caused PARP activation and energy depletion [5]. Other findings suggest that Cd induces cytoprotective autophagy by activating class III PI3K/Beclin-1/Bcl-2 signaling pathways and the autophagy serves a positive function in the reduction of Cd-induced cytotoxicity in PC-12 cells [6].
Abbreviations: Cd, cadmium; 3-MA, 3-methyladenine; ROS, reactive oxygen species; NAC, N-acetyl cysteine; A549 cells, the human lung glandular cancer cells; siRNA, Small interfering RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; GFP, green fluorescent protein; AO, acridine orange; AVOs, acidic vesicular organelles; AVs, autophagy vesicles; DCFH-DA, 2′,7′-dichlorofluorescin diacetate ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y. Kong), [email protected] (J. Cao). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.cbi.2017.11.013 Received 21 August 2017; Received in revised form 30 October 2017; Accepted 21 November 2017 Available online 24 November 2017 0009-2797/ © 2017 Elsevier B.V. All rights reserved.
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4 °C. The pellets were post fixed in 2% osmium tetroxide for 2 h, dehydrated in a graded series of ethanol (20%, 40%, 60%, 70%, 80%, 90% and 100% every15 min once time). And then the pellets were embedded in araldite and sectioned at 70 nm thickness using an ultramicrotome. Ultrathin sections were stained with 2% uranyl acetate and 0.2% lead citrate. Images were observed by a Tecnai Spirit electron microscope.
Autophagy is an evolutionarily conserved catabolic process in which cytosolic components, organelles, and invading bacteria are sequestered within autophagosomes, and delivered to lysosomes for degradation. Autophagy is well recognized as a survival mechanism for maintaining cell viability during conditions of nutrient limitation. Meanwhile, extensive autophagy may promote cell death through selfdegradation of essential cellular constituents. Dysregulation of autophagy contributes to different pathological conditions, such as neurodegenerative diseases, aging and carcinogenesis [7,8]. Up to now, however, the effect of autophagy on influencing cell survival or death is controversial. Some recent studies of autophagy genes have addressed the role of autophagy in both cell death and survival. In our previous study, we found that exposure to Cd at 2 μM for 48 h significantly enhanced the growth of MRC-5 cells [9]. So, in this study, we attempted to validate the role of autophagy in Cd-induced cell proliferation and invasion in A549 cells.
2.5. Autophagic vesicles staining (GFP) The cellular autophagy level was further measured by fluorescence microscopy. A549 cells were seeded on glass plates and grown for 24 h in the typical cell culture media. The cells were pretreated with 1 mM 3MA for 2 h, or 100 nM rapamycin for 2 h, and then incubated with Cd for 48 h. After that, the cells were stained using the Cyto-ID Autophagy Detec-tion Kit (Enzo Life Sciences) according to the manufacturer's introduction [13]. In brief, the cells were incubated with Cyto-ID Green Autophagy Detection Reagent in PBS for 15 min at 37 °C in the dark. The cells were washed with 1 × assay buffer provided with Cyto-ID® Autophagy detection kit, and then the cells were examined using a fluorescence microscope (Olympus BX63) with a standard FITC filter set for using a standard FITC filter set for imaging the autophagic signal.
2. Materials and methods 2.1. Cell culture and treatment A549 cells, the human lung glandular cancer cells, were purchased from Cell Center for Peking Union Medical College (Peking, China), which cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), penicillin (100 UI/ml)-streptomycin (0.1 mg/ml) at 37 °C in a humidified atmosphere of 5% CO2. Cadmium chloride (CAS No. 10108-64-2; purity of 99%) was purchased from Sigma-Aldrich and 2.9 mg of cadmium chloride was dissolved in 1 ml distilled water to prepare a stock solution of 16 mM. For each experiment, A549 cells were treated with different concentrations of Cd.
2.6. Acridine orange (AO) staining assay for autophagy detection As a marker of autophagy, the volume of the cellular acidic compartment can be visualized after AO staining [14]. A549 Cells were seeded onto glass coverslips in 24-well plates, pretreated with 50 nM Atg4 siRNA or Control siRNA duplexes, for 12 h and exposed to various concentrations of Cd for 48 h, respectively. After treatment, media were discarded and the cells were washed twice in PBS. Then the A549 cells on the glass coverslips were stained with PBS containing 10 μg/ml AO for 15 min at 37 °C in the dark. After washing with PBS in twice, the fluorescent micrographs were taken to detect the autophagy of A549 cells.
2.2. MTT assay The proliferation of A549 cells treated by Cd was measured by MTT assay as described previously [10]. Briefly, A549 cells (1 × 105/ml) were seeded in 96-well plates and treated with Cd (0, 2, 4, 8, 16 and 32 μM) for 48 h. To determine the effect of NAC, 3-methyladenine (3MA) and Atg4 siRNA on Cd-induced cell growth, A549 cells were pretreated with 1.5 mM NAC for 1 h, 1 mM 3-MA for 2 h, or 50 nM Atg4 siRNA duplexes for 12 h respectively, and then treated with Cd for 48 h. After treatment, MTT solution was added, and the cells were incubated for 4 h at 37 °C. The supernatant was discarded, and insoluble purple formazan was dissolved by adding 100 μl DMSO to each well. The plate was gently agitated until the blue formazan crystals were fully dissolved. The absorbance at 570 nm was read using a Bio-Rad Microplate Reader, and the cell viability (%) was calculated using the following equation: (A570 of treated sample/A570 of control) × 100 [11].
2.7. Western blot analysis At the end of the designated treatments, the cells were washed twice with ice-cold PBS and completely lysed in the lysis buffer provided with a protein extraction kit (Keygen Biotech). The cell lysate was centrifuged at 16,000 × g and 4 °C for 5 min and the supernatants containing the total protein were isolated. The concentration of total protein was quantified using the Bio-Rad protein dye microassay according to the manufacturer's recommendations. SDS-polyacrylamide gel electrophoresis was performed, and the proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking, the filters were incubated with the antibody-containing solution over night at 4 °C, washed twice, and incubated with a blocking solution-coupled secondary antibody at 37 °C for 2 h.
2.3. Determination of ROS production The formation of intracellular ROS was measured by fluorescence microscopy after DCFH-DA staining [12]. Briefly, A549 cells were incubated with Cd (0 and 2 μM) at 37 °C for 24 h. To evaluate the protective effect of NAC, A549 cells were pretreated with NAC (1.5 mM) at 37 °C for 2 h, and then incubated with Cd at 37 °C for 24 h. After harvesting by trypsinisation, the cells were washed with PBS twice and stained with 5 μM DCFH-DA for 30 min at 37 °C in the dark, and then viewed under the fluorescence micro-scope (Olympus BX63). The intensity of DCF fluorescence was analyzed by Image-Pro Plus 6.0 soft ware.
2.8. Cell cycle analysis Cell cycle analysis was performed using a flow cytometer [9,15]. A549 cells were plated into culture flasks and serum starved for 24 h and then cultured in RPMI 1640 medium with 10% FCS and Cd for 48 h. To determine the involvement of autophagy in Cd-induced cell cycle progression, A549 cells were pretreated with 3-MA or Atg4 siRNA. Subsequently, cells (1.0 × 106) were suspended thoroughly in 0.5 ml of PBS, and fixed with 70% ethanol overnight at 4 °C. Fixed cells were washed with PBS, suspended with RNase A (100 μg/ml) at 37 °C for 30 min, and stained with propidium iodide (PI) at 4 °C in dark for 30 min. The cell cycle was analyzed with a flow cytometer.
2.4. Electronic microscopy After A549 cells were treated with different concentrations of Cd and incubated for 48 h at 37 °C, the cells were harvested and washed with PBS twice, then fixed with 2.5% glutaraldehyde at least for 2 h at 137
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Fig. 1. Effects of Cd on cell viability and autophagy in A549 cells. A: The cell viability was measured using the MTT assay. A549 cells were exposed to CdCl2 (0, 2, 4, 8, 16 and 32 μM) for 48 h. Each point represents mean ± SD of three independent experiments (∗∗P < 0.01 vs. Control). B, C, D: Effects of Cd on expression of LC3-II, Beclin-1 and Atg4 in A549 cells. A549 cells were incubated with CdCl2 (0, 2, 4 μM) for 48 h. Western blots were performed on the total protein of untreated and Cd-treated cells. β-actin was used as control. Relative expression of these proteins was expressed as a percentage of β-actin. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Control). E: Transmission electron microscopy was used for observing. Scale bar: 500 nm. F: Quantitation of autophagy vesicles (AVs) in Cd-treated A549 cells was done. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Control). G: Cd-treated A549 cells, with or without pretreatment of 1 mM 3-MA for 2 h or 100 nM rapamycin for 4 h, were stained with the Cyto-ID Autophagy Detection Kit and then autophagosomes were observed by fluorescence microscope. Scale bar: 500 μm. H: Quantitation of AVs in Cd-treated A549 cells was done. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Control, ##P < 0.01 vs. Cd alone).
Biosciences). Experiments were performed in triplicate.
2.9. Small interfering RNA transfection For transient transfection, the complete medium was converted to serum-free culture just before experiments when the confluence of A549 cells was 90%. The oligo nucleotides encoding Atg4 small interfering RNA (siRNA) were 5′-CCUAGAUUCUUCUGAUGUATT-3′and 5′-UACUUCAGAAGAAUCUAGGTT -3’. The oligo nucleotides encoding scramble siRNA were 5′-UUCUCCGAACGUG UCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3’. All siRNAs were synthesized by Shanghai GenePharma Co. Ltd (Shanghai, China). Transfection of siRNA was done according to the instructions of the manufacturer. 8 μl Atg4 specific siRNAs or Control siRNA were lightly added into 0.5 ml RPMI 1640 medium without fetal bovine serum. After 5 min, 8 μl siRNA-Mate Transfection Reagent was softly added. After another 15 min, put this 0.5 ml mixture into 3.5 ml of antibiotic-free medium. The cells were incubated with this media for 48 h, harvested and then processed for Western blot analysis. Atg4 protein levels were determined to assess the effects of RNA interference.
2.11. Animals and treatments To evaluate the effects of Cd-induced autophagy in vivo, the experiments were performed using male BALB/c mice (n = 40 and average body weight of 18–22 g) obtained from Dalian Medical University Animal Research Center. Animal work was carried out in compliance with the United States NIH Guide for the Care and Use of Laboratory Animals (National Research Council of the National Academies 2011), and was approved by the Institutional Animal Ethical Committee of Dalian Medical University. Animals were clinically healthy and kept under hygienic conditions in plastic cages in an environment maintained at 24 ± 2 °C, 55 ± 10% humidity and 12/12 h cycle of light and darkness for acclimatization. The animals had free access to standard pellet diet and water ad libitum. The 40 experimental mice were assigned into four equal groups (G1–G4), and given CdCl2 (0, 0.25, 0.5 or 1 mg/kg body weight respectively) by subcutaneous injection (S/C) daily for 6 weeks. The lung tissues of mice were used for Western blot to observe the expression of autophagy-related proteins, LC3-II, Beclin-1, Atg4.
2.10. Cell migration and invasion assays Cell migration was assayed in 24-well, 6.5-mm-internal-diameter transwell plates (8.0 μm pore size; Corning, USA). After treatment of Cd, serum-starved cells (2 × 105 cells/ml) were placed in the upper chambers, and the lower chamber was filled with media containing 10% FBS. Cells were allowed to migrate. After incubation for the indicated time, cells on the upper surfaces of the filters were removed by wiping with a clean cotton swab and migrated cells on the undersides of the filters were fixed with methanol, stained with 0.5% crystal violet. Then migrated cells were viewed and counted under an upright microscope (5 fields per chamber). For invasion, the cell invasion assay was essentially conducted as above, except that transwell filters, an 8 μm pore size insert pre-coated with Matrigel (1:4 dilution in media; BD
2.12. Statistical analysis All values were expressed as mean ± standard deviation calculated from three independent experiments. The data were analyzed with oneway analysis of variance (ANOVA) followed by Tukey multiple comparison post hoc tests (SPSS 17.0 software). P < 0.05 and P < 0.01 were taken as statistically significant.
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3. Results
3.3. Cd induced autophagy in A549 cells
3.1. Cd caused proliferation in A549 cells
The occurrence of autophagy by Cd treatment was detected by direct observation of the formation of autophagosomes using electron microscopy. As shown in Fig. 1E–F, the control cells exhibited normal nuclei with uniform and finely dispersed chromatin, whereas abundant autophagosomes in the cytoplasm were produced in Cd-exposed cells. The quantification of the AVs numbers per viable cell implied that Cd increased AVs number significantly compared to the control (P < 0.01). Cd-induced autophagy was further examined by staining with CytoID Green Autophagy Detection Reagent in A549 cells treated with 2 μM Cd for 48 h. As demonstrated in Fig. 1G–H, the number of autophagosome positive green puncta in Cd-treated cells significantly increased compared with the control (P < 0.01). However, the number of cells stained with Cyto-ID Green was reduced in the presence of 3-MA, an autophagy inhibitor, where the number of cells stained with Cyto-ID Green increased significantly by treatment with rapamycin. To confirm the effect of Cd-induced autophagy, we detected the expression of the autophagy-regulatory proteins including LC3, Beclin-1 and Atg4 in Cd-treated A549 cells. As shown in Fig. 1B–D, after Cd treatment, the conversion of LC3-I to LC3-II and the upregulation of beclin-1 and Atg4 were observed.
The proliferation of A549 cells in the presence of Cd was observed after incubation for 48 h (Fig. 1A). The results showed that Cd increased the growth of A549 cells at the concentrations of 2 and 4 μM (P < 0.01), and at the concentration of 2 μM, the growth of A549 cells increased the most significantly. In addition, Cd at the concentrations above 16 μM induced a marked dose-dependent decrease in cell growth suggesting that Cd had cytotoxicity at these levels. These results showed that Cd at low levels could induce A549 cell growth, but at high doses, Cd imposed strong toxicity. This result is consistent with the effects of Cd on MRC-5 cells [9].
3.2. Cd induced intracellular ROS formation in A549 cells To evaluate the generation of ROS in A549 cells treated with Cd, DCFH-DA fluorescence dye was employed. A significant increase of DCF fluorescence intensity was observed in cells treated with Cd at the concentration of 2 μM (P < 0.01). To certify the oxidative stress, NAC was used to reduce intracellular ROS formation. When the cells were pretreated with NAC for 2 h, the DCF fluorescence intensity in A549 cells was significantly decreased compared to that of only Cdtreated cells (P < 0.01). This result demonstrated that Cd had a strong effect on ROS production in A549 cells (Fig. 2A–B).
3.4. Role of ROS in Cd-induced cell growth and autophagy in A549 cells To evaluate the role of ROS in Cd-induced growth of A549 cells,
Fig. 2. The role of ROS in Cd-induced cell growth and autophagy in A549 cells. A: Fluorescence microscopy was used to detect ROS formation. A549 cells were pretreated with or without NAC (1.5 mM) for 2 h and then exposed to CdCl2 (2 μM) for 24 h. The level of ROS was analyzed using fluorescence microscopy. Scale bar: 500 μm. B: Each bar represents mean ± SD of three independent experiments (∗∗P < 0.01 vs. Control, ##P < 0.01 vs. Cd alone). C: NAC was used to evaluate the role of ROS in Cd-induced cell growth. The cell viability was measured using the MTT assay. Cells were pretreated with NAC at a concentration of 1.5 mM for 2 h and then treated with Cd (0, 2, 4, 8, 16 and 32 μM) for 48 h. Each point represents mean ± SD of three independent experiments (∗∗P < 0.01 vs. Control, ##P < 0.01 vs. Cd alone). D: To confirm whether ROS was involved in Cd-induced autophagy, the protein level of Atg4 and LC3 was analyzed by Western blot. β-actin was used as control. E, F: Relative expression of Atg4 and LC3 proteins were expressed as a percentage of β-actin. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Control, ##P < 0.01 vs. Cd alone). G: The effect of NAC on Cd-induced autophagolysosomes formation in A549 cells was measured by AO staining. Scale bar: 500 μm. H: Quantitation of formation of autophagolysosomes was done. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Control, ##P < 0.01 vs. Cd alone).
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Fig. 3. The involvement of Atg4 in Cd-induced autophay and cell growth in A549 cells. Cells were pretreated with Atg4 siRNA or Control siRNA, and then exposed to Cd for 48 h. A: Western blots were performed on the total protein of untreated and treated cells. B, C, D: β-actin was used as control. Relative expression of these proteins was expressed as a percentage of β-actin. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Si Control, ##P < 0.01 vs. Si Control+Cd). E: Formation of autophagolysosomes in A549 cells was measured by AO staining. Scale bar: 500 μm. F: Quantitation of formation of autophagolysosomes in was done. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Si Control, ##P < 0.01 vs. Si Control+Cd). G: To determine the role of Atg4 in Cd-induced cell growth, MTT assay was performed. Each point represents mean ± SD of three independent experiments (##P < 0.01 vs. Si Control+Cd). H: To determine the role of autophagy in Cd-induced cell growth, 3-MA was used and MTT assay was performed. Each point represents mean ± SD of three independent experiments (##P < 0.01 vs. Cd alone).
induced cell growth in A549 cells (P < 0.01). These two results demonstrated that autophagy play an important role in Cd-induced cell growth in A549 cells.
A549 cells were pretreated with NAC (Fig. 2C). After the 2 h pre-incubation with 1.5 mM NAC, there was a significant decrease in absorbance (P < 0.01) compared to only Cd-treated A549 cells. These data suggested that NAC is effective on Cd-induced growth in A549 cells and ROS may play an important role in Cd-induced A549 cell proliferation. In an attempt to determine the effect of ROS on Cd-induced autophagy in A549 cells, ROS scavenger NAC was used. As shown in Fig. 2D–F, when cells were pretreated with NAC, the conversion of LC3 and the upregulation of Atg4 induced by Cd-treatment were both decreased significantly as compared with only Cd-treated A549 cells (P < 0.01). Pretreatment with NAC blocking the ROS generation can obviously inhibited Cd-induced autophagy as demonstrated by inhibition of the formation of acidic vesicular organelles (AVOs) (Fig. 2G–H). These results demonstrated that ROS play an important role in Cd-induced autophagy.
3.7. Role of autophagy in Cd-induced cell cycle in A549 cells In our previous study, we found that Cd treatment markedly reduced the number of cells in G0/G1 phase, accompanied with an increase in the number of cells in S phase and G2/M phase compared to the control group at the time point of 48 h in MRC-5 cells (P < 0.01). In this study, the same results were obtained in Cd-treated A549 cells (Fig. 4A–B). To establish that Cd-induced autophagy plays a role in cell cycle change induced by Cd, first, 3-MA was employed to block Cdinduced autophagy. Cell cycle analysis was performed in triplicate and showed that 3-MA pretreatment increased the percentage of G0/G1 phase cells and decreased S phase and G2/M phase percentage compared with that of only Cd-treated cells (Fig. 4E–F), and the differences in the percentages are not significant. Then, Atg4 siRNA was also used to investigate the role of autophagy in Cd-induced cell cycle progression. As demonstrated in Fig. 4C–D, when Atg4 expression was inhibited by siRNA, the number of cells at G0/G1 phase was increased and the number of S and G2/M phase cells was reduced compared to that of Cd-treated Control siRNA cells. Furthermore, blocking Atg4 expression strongly reduced Cyclin-D1 and Cyclin-E expression induced by Cd (Fig. 4G–H). These results suggest that knocking down of Atg4 expression by siRNA blocked Cd-induced changes of cell cycle, indicating that Atg4-mediated autophagy might involved in Cd-induced cell cycle progression in A549 cells.
3.5. Role of Atg4 in Cd-induced autophagy in A549 cells In order to investigate the role of Atg4 in Cd-induced autophagy in A549 cells, the expression of Atg4 protein was inhibited by its siRNA. As illustrated in Fig. 3A–D, blocking Atg4 expression strongly reduced Beclin-1 and LC3-II protein expression induced by Cd. Furthermore, as shown in Fig. 3E–F, Atg4 siRNA decreased the number of autophagosome positive cells significantly compared to that of Cd-treated Control siRNA cultures (P < 0.01). Taken together, we conclude that Cd can induce autophagy in A549 cells involving induction of Atg4 protein. 3.6. Role of autophagy in Cd-induced proliferation in A549 cells In order to investigate the role of autophagy in Cd-induced proliferation of A549 cells, 3-MA, an autophagy inhibitor was used in MTT assay. Pretreatment with 1 mM 3-MA for 2 h significantly inhibited the Cd-induced cell viability of A549 cells (Fig. 3H). Atg4 siRNA was also used to block Cd-induced autophagy and MTT assay was performed. As shown in Fig. 3G, blocking Atg4 expression strongly inhibited Cd-
3.8. Role of autophagy in Cd-induced migration and invasion in A549 cells The role of autophagy in Cd-induced cell migration and invasion was further examined. As shown in Fig. 5A–B, the number of cells that migrated through transwell plate was significantly increased after the treatment of Cd, illustrating that Cd treatment enhanced cell migration 140
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Fig. 4. The involvement of autophay in Cd-induced cell cycle progression in A549 cells. A549 cells were pretreated with or without 3-MA, Atg4 siRNA or Control siRNA, and treated with Cd for 48 h, and then collected, fixed, stained with PI, and cell cycles were monitored by a flow cytometer. A, B: Effect of Cd on cell cycle of A549 cells. The population of each phase was calculated. C, D: The role of Atg4 in Cd-induced cell cycle change in A549 cells. The population of each phase was calculated. E, F: Effect of 3-MA on Cd-induced cell cycle change in A549 cells. The population of each phase was calculated. G: The role of Atg4 in Cd-induced expression of Cyclin-D1 and Cyclin-E in A549 cells. H: β-actin and GAPDH were used as control. Relative expressions were expressed as a percentage of β-actin and GAPDH. Each bar represents mean ± SD of three independent experiments (∗∗P < 0.01 vs. Si Control, ##P < 0.01 vs. Si Control+Cd).
cell growth, migration and invasion in A549 cells. The effect of Cd-induced autophagy has been detected in many different cell types, such as A549 cells [16], human prostate epithelial cells [17] and breast cancer cells [18]. In this study, we demonstrated that Cd induced autophagy in A549 cells the most significantly at the concentration of 2 μM, as proven by the appearance of autophagosomes, the increase of GFP-LC3 functa cells and the formation of LC3-II. Cd-induced autophagy was further supported by the observation that the protein level of Beclin-1 was increased after Cd treatment. Beclin-1 has an important role at every central step in autophagic pathways, from autophagosome formation to autophagosome maturation [19]. Consequently, our results support that Cd induces autophagy in A549 cells. Meanwhile, on the basis of Western blot analysis, in BALB/c mice's lung tissues, the protein levels of LC3-II and Beclin-1 were both increased significantly after Cd treatment. Atg4 is an enzyme that plays a major role in the processing of LC3. It cleaves LC3-I to produce LC3-II, and colocalizes with LC3-II in the membranes of autophagic vesicles [20]. Our previous results of gene microassay showed that Cd induced Atg4 gene expression significantly in A549 cells. In this study, using Western blot, we demonstrated that Cd could induce Atg4 protein in A549 cells. To determine the role of Atg4 in Cd-induced autophagy, Atg4 was knocked down in A549 cells by siRNA. Western blot results showed that blocking Atg4 expression strongly reduced expression of Beclin-1 and LC3-II protein induced by Cd. Furthermore, Atg4 siRNA decreased the number of autophagosome positive cells significantly compared to that of Cd-treated Control siRNA cells. These results revealed that Atg4 plays an important role in Cd-induced autophagy in A549 cells. ROS has been reported to participate in autophagy. A large body of data shows that starvation can induce the production of ROS and lead to autophagy. It has also been studied that ROS scavengers can block
in A549 cells (P < 0.01). To determine the role of autophagy in Cdinduced cell migration, Atg4 siRNA was employed to block Cd-induced autophagy. The number of cells that migrated through transwell plates was significantly decreased in Cd-treated Atg4 siRNA cells compared to that of Cd-treated Control siRNA cultures (P < 0.01). At the same time, Transwell chamber, pre-coated with Matrigel, was used to detect the effect of Cd-induced cell invasion. As shown in Fig. 5C–D, the number of invasion cells was obviously increased after the treatment of Cd (P < 0.01). Meanwhile, the number of cells that invaded through Transwell chamber was significantly decreased in Cd-treated Atg4 siRNA cells compared to that of Cd-treated Control siRNA cultures (P < 0.01). The effect of Cd on the expression of invasion-related factor, MMP-9 was examined by Western blot (Fig. 5E–F). The results revealed that Atg4 siRNA led to a significant decrease of MMP-9 expression in Cd-treated cells compared to that of Cd-treated Control siRNA groups (P < 0.01). These results suggest that Cd might promote cell migration and invasion by facilitating autophagy in A549 cells. 3.9. Cd induced expressions of autophagy-related proteins in lung tissues of mice After Cd treatments, the lung tissues of mice were used for Western blot. As shown in Fig. 6, the expressions of autophagy-related proteins, LC3-II, Beclin-1 and Atg4 were all increased significantly in Cd-treated mice compared with the control mice (P < 0.05 and P < 0.01). This result suggested that Cd induced autophagy in the lung tissues of mice. 4. Discussion In this study, for the first time, we demonstrated that ROS-dependent Atg4-mediated autophagy plays an important role in Cd-induced 141
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Fig. 5. The involvement of autophay in Cd-induced migration and invasion in A549 cells. Cells were pretreated with Atg4 siRNA or Control siRNA, and then exposed to Cd for 48 h. Transwell assay was used for observing migrative (A) and invasive (C) ability of A549 cells. Ability of migration (B) and invasion (D) of A549 cells were quantified by counting the number of migrating or invasive cells in ten randomly chosen high-power fields (n = 3). Scale bar: 500 μm. E: Western blots were performed on the total protein of A549 cells. F: Relative expression of MMP-9 was expressed as a percentage of β-actin. Each bar represents mean ± SD from three independent experiments (∗∗P < 0.01 vs. Si Control, ##P < 0.01 vs. Si Control+Cd).
of LC3, and contributes to autophagosome formation [20]. However, Li et al. reported that N-Benzoyl-O-(N′-(1-benzyloxycarbonyl-4-piperidiylcarbonyl) -D-phenylalanyl)-D -phenylalaninol (BBP) induced the autophagic cell death through a JNK-dependent Atg4 upregulation involving ROS production in MCF-7 cells [26]. It remains unclear whether ROS-regulated Atg4 is a more general characteristic of autophagy signaling. In this study, to explore the role of oxidative stress in Cd-induced Atg4 expression and autophagy, NAC, an intracellular ROS inhibitor was employed. When A549 cells were pretreated with NAC, the upregulation of Atg4 and the conversion of LC3 induced by Cd-
starvation-induced autophagy, indicating that ROS can regulate autophagy [21]. Increasing evidence has revealed that massive ROS can modulate multiple autophagy process, including Atg4–Atg8/LC3, Beclin-1, PTEN, PI3K–Akt–mTOR, p53 and MAPK signaling [22,23]. ROS generated by mitochondria can directly modulate the cysteine protease Atg4, resulting in inhibition of Atg4 delipidating activity, whereas the initial processing of LC3 (priming) by Atg4 is not altered [24]. Increasing evidence showed that Atg4 is a direct target for oxidation by H2O2, because a cysteine residue was identified as critical for this regulation [25]. ROS-mediated inhibition of Atg4 promotes lipidation
Fig. 6. Effects of Cd on expressions of autophagy-related proteins in lung tissues of mice. Forty mice were divided into four groups, each group was given CdCl2 (0, 0.25, 0.5 or 1 mg/kg body weight respectively) by subcutaneous injection daily for 6 weeks. A: The lung tissues were used for Western blot to observe the expression of LC3II, Beclin-1, Atg4. B, C, D: Relative expression of these proteins was expressed as a percentage of GAPDH. Each bar represents mean ± SD from three independent experiments. (∗P < 0.05 vs. Control; ∗∗P < 0.01 vs. Control).
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prevention of Cd-resulted toxicities.
treatment were both decreased significantly as compared with only Cdtreated A549 cells (P < 0.01). Furthermore, pretreatment with NAC blocking the ROS generation can obviously inhibited Cd-induced autophagy as demonstrated by inhibition of the formation of AVOs. These data suggest that ROS play an important role in Cd-induced autophagy and Cd induced autophagy might be through ROS-initiated Atg4 pathway. Cd was reported to induce cytoprotective autophagy by delaying the occurrence of apoptosis and activating class III PI3K/Beclin-1/Bcl-2 signaling pathway in PC-12 cells [6]. In this study, the growth of A549 cells were increased after Cd treatment at the concentration of 2 μM, and NAC pretreatment significantly decreased Cd-induced cell growth. 3-MA, an autophagy inhibitor could also significantly inhibit the Cdinduced cell viability of A549 cells. Meanwhile, blocking Atg4 expression by siRNA strongly inhibited Cd-induced cell growth in A549 cells. These results demonstrated that autophagy dependent ROS-initiated Atg4 upregulation plays an important role in Cd-induced cell growth in A549 cells. It is well known that cell cycle checkpoints are crucial regulatory points in cell proliferation. There are growing evidences concerning the role of autophagy in cell cycle arrest and cellular proliferation [27]. Experiments have demonstrated that autophagy is active in the G1 and S phases of the cell cycle, while inhibited in mitosis [28]. Seung-il Choi found that treatment with bafilomycin A 1, the autophagy flux inhibitor, resulted in decreased expression of Cyclin A1, B1, D1, and p53 in corneal fibroblasts. Furthermore, a decrease in Cyclin A1, B1, and D1 expression was observed in Atg7 gene knockout cells [29]. In our previous study, the cell cycle analysis revealed that Cd-treatment markedly reduced the number of cells in G0/G1 phase with an increase in the number of cells in S phase and G2/M phase after 48 h, suggesting that Cd at 2 μM promotes cell cycle progression. So, in this study, the involvement of autophagy in Cd-induced cell cycle change was investigated. The results showed that 3-MA pretreatment increased the percentage of G0/G1 phase cells and decreased S phase and G2/M phase percentage compared with that of only Cd-treated cells. Then, Atg4 siRNA was also used to investigate the role of autophagy in Cdinduced cell cycle progression. When Atg4 expression was inhibited by siRNA, the number of cells at G0/G1 phase was increased and the number of S and G2/M phase cells was reduced in Cd-treated Atg4 siRNA cultures compared with that of Cd-treated Control siRNA cells. This result suggest that knocking down of Atg4 expression by siRNA blocked Cd-induced changes of cell cycle, indicating that Atg4-mediated autophagy might be involved in Cd-induced cell cycle progression in A549 cells. Accumulating evidence showed that autophagy plays a complex role in migration, invation and cancer metastasis, because reports have suggested both pro-metastatic and anti-metastatic roles of autophagy [30]. Ischemia/reperfusion was reported to induce the expression of iNOS, which subsequently increased the migration and apoptosis of HUVECs via autophagy [31]. PM 2.5 exposure induces ROS, which activates loc146880 expression, and then up-regulates autophagy and promotes the malignant of lung cancer cells [32]. In our experiments, Cd treatment enhanced both cell migration and invasion in A549 cells. Atg4 knockdown significantly reduced MMP-9 expression and the number of cells that migrated and invaded through the Matrigel matrix compared to that of Cd-treated Control siRNA cells. These results demonstrated that Cd might promote cell migration and invasion by facilitating autophagy in A549 cells. Taken together, in this study we demonstrated that Cd induced cell proliferation, migration and invasion in A549 cells, resulting in marked increases in ROS levels and autophagy. ROS serves as a molecular signal to increase the expression of Atg4, which enhances cell autophagy. Both Atg4 and autophagy could promote A549 cell proliferation, migration and invasion. These results suggested that Cd exposure could facilitate lung tumor formation and progression, and suppressing ROS formation, Atg4 expression or cell autophagy might have potential values in
Conflicts of interest The authors declare that there are no conflicts of interest in the present work. Acknowledgments This work was (2013E15SF140).
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Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2017.11.013. References [1] IARC, Cadmium and cadmium compounds, IARC Monogr. Eval. Carcinog. Risks Hum. 58 (1993) 119–237. [2] D. Luce, I. StuckerICARE study group, Investigation of occupational and environmental causes of respiratory cancers (ICARE): a multicenter, population-based casecontrol study in France, BMC Public Health 11 (2011) 928. [3] S.H. Wang, Y.L. Shih, W.C. Ko, Y.H. Wei, C.M. Shih, Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway, Cell Mol. Life Sci. 65 (2008) 3640–3652. [4] S.H. Wang, Y.L. Shih, T.C. Kuo, W.C. Ko, C.M. Shih, Cadmium toxicity toward autophagy through ROS-activated GSK-3beta in mesangial cells, Toxicol. Sci. 108 (2009) 124–131. [5] Y. Son, X. Wang, J.A. Hitron, Z. Zhang, S. Cheng, A. Budhraja, S. Ding, J. Lee, X. Shi, Cadmium induces autophagy through ROS- dependent activation of the LKB1AMPK signaling in skin epidermal cells, Toxicol. Appl. Pharmacol. 255 (3) (2011) 287–296. [6] Q. Wang, J. Zhu, K. Zhang, C. Jiang, Y. Wang, Y. Yuan, J. Bian, X. Liu, J. Gu, Z. Liu, Induction of cytoprotective autophagy in PC-12 cells by cadmium, Biochem. Biophysical Res. Commun. 438 (2013) 186–192. [7] J.H. Lee, A.V. Budanov, E.J. Park, R. Birse, T.E. Kim, G.A. Perkins, K. Ocorr, M.H. Ellisman, R. Bodmer, E. Bier, M. Karin, Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies, Science 327 (2010) 1223–1228. [8] Y. Kondo, T. Kanzawa, R. Sawaya, S. Kondo, The role of autophagy in cancer development and response to therapy, Nat. Rev. Cancer 5 (2005) 726–734. [9] H. Xie, J. Wang, L. Jiang, C. Geng, Q. Li, D. Mei, L. Zhao, J. Cao, ROS-dependent HMGA2 upregulation mediates Cd-induced proliferation in MRC-5 cells, Toxicol. Vitro 34 (2016) 146–152. [10] J. Cao, L. Jia, H.M. Zhou, Y. Liu, L.F. Zhong, Mitochondrial and nuclear DNA damage induced by curcumin in human hepatoma G2 cells, Toxicol. Sci. 91 (2006) 476–483. [11] W.K. Lee, M. Abouhamed, F. Thevenod, Caspase-dependent and -independent pathways for cadmium-induced apoptosis in cultured kidney proximal tubule cells, Am. J. Physiol. Ren. Physiol. 291 (2006) 823–832. [12] Y.J. Zhou, S.P. Zhang, C.W. Liu, Y.Q. Cai, The protection of selenium on ROS mediated-apoptosis by mitochondria dysfunction in cadmium-induced LLC-PK(1) cells, Toxicol Vitro 23 (2009) 288–294. [13] Anja K. Klappan, Stefanie Hones, Ioannis Mylonas, Ansgar Brȕning, Proteasome inhibition by quercetin triggers macroautophagy and blocks mTOR activity, Histochem Cell Biol. 137 (2011) 25–36. [14] Eduardo Cremonese Filippi-Chiela, Msrdja Manssur Bueno e Sliva, Marcos Paulo Thomé, Guido Lenz, Single-cell analysis challenges the connection between autophagy and senescence induced by DNA damage, Autophagy 7 (2015) 1099–1113. [15] Y. Pan, H. Fu, Q. Kong, Y. Xiao, Q. Shou, H. Chen, Y.H. Ke, M.L. Chen, Prevention of pulmonary fibrosis with salvianolic acid a by inducing fibroblast cell cycle arrest and promoting apoptosis, J. Ethnopharmacol. 155 (2014) 1589–1596. [16] K. Subhadip, S. Suman, B. Arindam, EGFR upregulates inflammatory and proliferative responses in human lung adenocarcinoma cell line (A549), induced by lower dose of cadmium chloride, Inhal. Toxicol. 23 (2011) 339–348. [17] S. Bakshi, X. Zhang, S. Godoy-Tundidor, R.Y. Cheng, M.A. Sartor, M. Medvedovic, S. Ho, Transcriptome analyses in normal prostate epithelial cells exposed to lowdose cadmium: oncogenic and immunomodulations involving the action of tumor necrosis factor, Environ. Health Perspect. 116 (2008) 769–776. [18] Z.X. Wei, X.L. Song, Z.A. Shaikh, Cadmium promotes the proliferation of triplenegative breast cancer cells through EGFR-mediated cell cycle regulation, Toxicol. Appl. Pharmacol. 289 (2015) 98–108. [19] R. Kang, H.J. Zeh, M.T. Lotze, D. Tang, The Beclin 1 network regulates autophagy and apoptosis, Cell Death Differ. 18 (2011) 571–580. [20] R. Scherz-Shouval, E. Shvets, E. Fass, H. Shorer, L. Gil, Z. Elazar, Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4, EMBO J. 26 (2007) 1749–1760. [21] S.H. Park, G.Y. Chi, H.S. Eom, G.Y. Kim, J.W. Hyun, W.J. Kim, S.J. Lee, Y.H. Yoo,
143
Chemico-Biological Interactions 279 (2018) 136–144
W. Lv et al.
[22]
[23]
[24]
[25]
[26]
[27] R.C. Wang, B. Levine, Autophagy in cellular growth control, FEBS Lett. 584 (2010) 1417–1426. [28] Y. Takahashi, D. Coppola, N. Matsushita, H.D. Cualing, M. Sun, Y. Sato, C. Liang, J.U. Jung, J.Q. Cheng, J.J. Mule, et al., Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis, Nat. Cell Biol. 9 (2007) 1142–1151. [29] S. Choi, S. Dadakhujaev, Y. Maeng, S. Ahn, T. Kimab, E.K. Kim, Disrupted cell cycle arrest and reduced proliferation in corneal fibroblasts from GCD2 patients: a potential role for altered autophagy flux, Biochem. Biophysical Res. Commun. 456 (2015) 288–293. [30] Z. Su, Z. Yang, Y. Xu, Y. Chen, Q. Yu, Apoptosis, autophagy, necroptosis, and cancer metastasis, Mol. Cancer 14 (2015) 1–14. [31] T. Zhu, Q. Yao, W. Wang, H. Yao, J. Chao, iNOS induces vascular endothelial cell migration and apoptosis via autophagy in ischemia/reperfusion injury, Cell Physiol. Biochem. 38 (2016) 1575–1588. [32] X. Deng, N. Feng, M. Zheng, X. Ye, H. Lin, X. Yu, Z. Gan, Z. Fang, H. Zhang, M. Gao, Z. Zheng, H. Yu, W. Ding, B. Qian, PM 2.5 exposure-induced autophagy is mediated by lncRNA loc146880 which also promotes the migration and invasion of lung cancer cells, Biochimica Biophysica Acta 1861 (2016) 112–125.
Y.H. Choi, Role of autophagy in apoptosis induction by methylene chloride extracts of Mori cortex in NCI-H460 human lung carcinoma cells, Int. J. Oncol. 40 (2012) 1929–1940. Z. Li, Y. Yang, M. Ming, B. Liu, Mitochondrial ROS generation for regulation of autophagic pathways in cancer, Biochem. Biophysical Res. Commun. 414 (2011) 5–8. X. Xie, L. Le, Y. Fan, L. Lv, J. Zhang, Autophagy is induced through the ROS-TP53DRAM1 pathway in response to mitochondrial protein synthesis inhibition, Autophagy 8 (2012) 1071–1084. Y. Li, Q. Luo, L. Yuan, C. Miao, X. Mu, W. Xiao, J. Li, T. Sun, E. Ma, JNK-dependent Atg4 upregulation mediates asperphenamate derivative BBP-induced autophagy in MCF-7 cells, Toxicol. Appl. Pharmacol. 263 (2012) 21–31. S. Qiao, M. Dennis, X. Song, D.D. Vadysirisack, D. Salunke, Z. Nash, Z. Yang, M. Liesa, J. Yoshioka, S. Matsuzawa, S. Orian, A REDD1/TXNIP pro-oxidant complex regulates ATG4B activity to control stress-induced autophagy and sustain exercise capacity, Nat. Commun. 6 (2015) 7014. Y. Li, Q. Luo, L. Yuan, C. Miao, X. Mu, W. Xiao, J. Li, T. Sun, E. Ma, JNK-dependent Atg4 upregulation mediates asperphenamate derivative BBP-induced autophagy in MCF-7 cells, Toxicol. Appl. Pharmacol. 263 (2012) 21–31.
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