p62 signaling in apoptosis resistance and its role in cadmium-induced carcinogenesis.

Cell Biology: Nrf2/p62 Signaling in Apoptosis Resistance and Its Role in Cadmium-induced Carcinogenesis

J. Biol. Chem. 2014, 289:28660-28675. doi: 10.1074/jbc.M114.595496 originally published online August 25, 2014

Access the most updated version of this article at doi: 10.1074/jbc.M114.595496 Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites. Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 42 references, 14 of which can be accessed free at http://www.jbc.org/content/289/41/28660.full.html#ref-list-1

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Young-Ok Son, Poyil Pratheeshkumar, Ram Vinod Roy, John Andrew Hitron, Lei Wang, Zhuo Zhang and Xianglin Shi

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 41, pp. 28660 –28675, October 10, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Nrf2/p62 Signaling in Apoptosis Resistance and Its Role in Cadmium-induced Carcinogenesis* Received for publication, July 9, 2014, and in revised form, August 20, 2014 Published, JBC Papers in Press, August 25, 2014, DOI 10.1074/jbc.M114.595496

Young-Ok Son‡§1, Poyil Pratheeshkumar‡§1, Ram Vinod Roy‡§, John Andrew Hitron§, Lei Wang‡§, Zhuo Zhang§, and Xianglin Shi‡§2 From the ‡Center for Research on Environmental Disease and the §Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, Kentucky 40536-0305

The cadmium-transformed human lung bronchial epithelial BEAS-2B cells exhibit a property of apoptosis resistance as compared with normal non-transformed BEAS-2B cells. The level of basal reactive oxygen species (ROS) is extremely low in transformed cells in correlation with elevated expressions of both antioxidant enzymes (catalase, SOD1, and SOD2) and antiapoptotic proteins (Bcl-2/Bcl-xL). Moreover, Nrf2 and p62 are highly expressed in these transformed cells. The knockdown of Nrf2 or p62 by siRNA enhances ROS levels and cadmium-induced apoptosis. The binding activities of Nrf2 on the antioxidant response element promoter regions of p62/Bcl-2/Bcl-xL were dramatically increased in the cadmium-exposed transformed cells. Cadmium exposure increased the formation of LC3-II and the frequency of GFP-LC3 punctal cells in nontransformed BEAS-2B cells, whereas these increases are not shown in transformed cells, an indication of autophagy deficiency of transformed cells. Furthermore, the expression levels of Nrf2 and p62 are dramatically increased during chronic long term exposure to cadmium in the BEAS-2B cells as well as antiapoptotic proteins and antioxidant enzymes. These proteins are overexpressed in the tumor tissues derived from xenograft mouse models. Moreover, the colony growth is significantly attenuated in the transformed cells by siRNA transfection specific for Nrf2 or p62. Taken together, this study demonstrates that cadmium-transformed cells have acquired autophagy deficiency, leading to constitutive p62 and Nrf2 overexpression. These overexpressions up-regulate the antioxidant proteins catalase and SOD and the antiapoptotic proteins Bcl-2 and Bcl-xL. The final consequences are decrease in ROS generation, apoptotic resistance, and increased cell survival, proliferation, and tumorigenesis.

* This work was supported, in whole or in part, by National Institutes of Health 1 2

Grants R01 ES015518, R01 CA119028, R01 ES015375, and R01 CA116697. Both authors contributed equally to this work. To whom correspondence should be addressed. Tel.: 859-257-4054; Fax: 859-323-1059; E-mail: [email protected].

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The p62/SQSTM1 (sequestosome 1) protein is a multifunction, ubiquitin-binding adapter protein that serves multiple cellular functions for autophagy, apoptosis, ROS3 signaling, and cancer (1, 2). p62 is one of the selective substrates for autophagy and has been found to accumulate in autophagy-deficient cells (3). The accumulation of p62 due to autophagy defects promotes cell survival and tumorigenesis through an activation of nuclear factor ␬B (NF-␬B) (3). Elevated expression of p62 has been reported in human lung cancers and transformed cells induced by H-Ras (2). The p62 directly interacts with the Nrf2binding site of Keap1 (kelch-like ECH-associated protein 1), which results in constitutive activation of Nrf2 (nuclear factor erythroid 2-related factor) (4). Thus, p62 accumulation activates Nrf2 and Nrf2 target gene expression (5–7). These reports suggest that up-regulation of p62 is due to autophagy dysfunction that leads to transcriptional activation of the Nrf2-dependent genes, including antioxidant enzyme genes. Further, Nrf2 regulates p62 expression through direct binding to the antioxidant response element (ARE) binding motif of the p62 promoter (7), suggesting a positive feedback of these two proteins. Moreover, a recent study has shown that Nrf2 up-regulates antiapoptotic proteins Bcl-2 and Bcl-xL through binding of ARE on the promoter of these genes, which enhances cell survival (8, 9). Apoptosis is well described as programmed cell death, and it involves activation of proteases. It is characterized by typical morphological and biochemical hallmarks, including cell shrinkage, membrane blebbing, nuclear condensation, and

3

The abbreviations used are: ROS, reactive oxygen species; ARE, antioxidant response element; NF-␬B, nuclear factor ␬B; LC3, microtuble-associated protein 1 light chain 3; ABT-263, 4-(4-((4⬘-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1⬘-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-morpholino-1(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl) benzamide; SOD, superoxide dismutase; CAT, catalase; 3AT, 3-amino-1,2,4triazole; 2ME, 2-methoxyestradiol; CdT, cadmium-induced transformed BEAS2B; PI, propidium iodide; ESR, electron spin resonance; DMPO, 5,5-dimethyl-1pyrroline-1-oxide; MTT, 3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide; CM-H2DCFDA, 5-(and 6)-chloromethyl-2⬘,7⬘-dichlorodihydrofluorescein diacetate; Z, benzyloxycarbonyl; ANOVA, analysis of variance; BisTris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane.

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Background: Cadmium-transformed cells have a property of apoptosis resistance. Results: Cadmium-transformed cells express high antioxidant enzymes and antiapoptotic proteins. Conclusion: The constitutive p62 and Nrf2 expressions of transformed cells result in a decrease in ROS generation, apoptosis resistance, and tumorigenesis. Significance: Constitutive expression of Nrf2/p62 is important in cadmium carcinogenesis and its possible prevention using these proteins.

Nrf2/p62 Signaling in Cadmium Carcinogenesis

MATERIALS AND METHODS Chemicals and Laboratory Wares—Unless specified otherwise, all chemicals and laboratory wares were purchased from Sigma and Falcon Labware (BD Biosciences), respectively. DulbecOCTOBER 10, 2014 • VOLUME 289 • NUMBER 41

co’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), gentamicin, Geneticin, and L-glutamine were purchased from Invitrogen. Bcl-2 family protein inhibitor ABT-263 (4-(4-((4⬘chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1⬘-biphenyl]-2-yl)methyl) piperazin-1-yl)-N-((4-((4-morpholino-1-(phenylthio)butan-2-yl) amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide) was purchased from Active Biochemicals Co., Ltd. (Maplewood, NJ). SOD1 inhibitor LCS-1 was purchased from Calbiochem. Catalase inhibitor 3-amino-1,2,4-triazole (3AT), Mn-SOD inhibitor 2-methoxyestradiol (2ME), and pancaspase inhibitor Z-VAD(Ome)-fluoromethyl ketone were purchased from Santa Cruz Biotechnology, Inc. Wortmannin and bafilomycin A1 were from Cell Signaling (Beverly, MA). Cell Culture and Treatment—The human lung bronchial epithelial cell line BEAS-2B was obtained from the American Type Culture Collection (Manassas, VA). BEAS-2B cells were exposed continuously to cadmium chloride (CdCl2) (0, 0.125, 0.25, 0.5, 1, and 2 ␮M) for 5 months. Cells were subcultured twice per week. After 5 months of cadmium exposure, the soft agar colony formation assay was performed. Transformed cells were considered to be anchorage-independent growth colonies formed in semisolid soft agar. The viable transformed cells were recovered and used for further study. Transformation ability and tumorigenicity of transformed cells were confirmed by soft agar and xenograft assay, respectively. Cadmium-induced transformed BEAS-2B (CdT) cells and their parents’ non-transformed BEAS-2B cells were maintained in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin and then processed in further experiments. Plasmids and Transfection—The overexpression of catalase, SOD1, and SOD2 in BEAS-2B cells has been described previously (21). The mCherry-EGFP-LC3B plasmid was purchased from Addgene (Cambridge, MA), and GFP-LC3 plasmid has been described previously (24). Transfections were performed using LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s protocol. Measurement of Cell Proliferation, Viability, and Cytotoxicity—For the cell proliferation assay, 0.2 ⫻ 106 non-transformed BEAS-2B cells or CdT cells were seeded on 10-cm cell culture dishes for 7 days. Each day, the total cell number was counted using a Z2 Coulter威 particle count and size analyzer (Beckman Coulter). 3-(4,5-Dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT) was used for evaluating a cell’s viability. Catalase (500 units/ml), SOD (100 units/ml), ABT263 (10 ␮M), 3AT (10 mM), LCS-1 (10 ␮M), 2ME (1 ␮M), Z-VAD (20 ␮M), wortmannin (100 nM), and bafiolmycin A1 (100 nM) were added into the cultures in the presence or absence of 10 ␮M cadmium. After a 24-h incubation, 5 ␮l of an MTT solution (5 mg/ml in PBS as stock solution) was added into each well of a 96-well plate and incubated for another 4 h at 37 °C. The MTT-reducing activity of cells was measured by treating them with acidic isopropyl alcohol prior to reading at 570 nm with an EL800 microplate reader (BioTek威, Winooski, VT). In addition, the trypan blue exclusion assay was conducted to measure cytotoxicity. After cells were harvested, they were stained with 0.4% trypan blue, and viable cells were counted using a TC20TM automated cell counter (Bio-Rad). JOURNAL OF BIOLOGICAL CHEMISTRY

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nuclear DNA fragmentation (10). Another mechanism of cell death is autophagy, which is a self-eating process in which supernumerary, damaged, or aged organelles are sequestered within autophagosomes and are finally delivered to lysosomes for bulk degradation (11). Autophagy serves as a stress adaptation that helps protective mechanisms in certain circumstances, but it induces cell death in other cellular environments (11, 12). Usually, upstream signals of apoptosis and autophagy are sharing common pathways. This leads to the fact that similar stimuli can induce either apoptosis or autophagy as well as mixed phenotypes of apoptosis and autophagy (11). On the other hand, apoptosis and autophagy play a critical role in carcinogenesis. In general, apoptosis serves as a natural barrier to tumor development (13). The apoptotic machinery is controlled by pro- and antiapoptotic members of the Bcl-2 family (13). Cancer cells have a variety of strategies to escape apoptosis, such as the loss of the p53 tumor suppressor and an increase in expression of antiapoptotic proteins, such as Bcl-2 and Bcl-xL (14, 15). Presumably, apoptosis-avoiding mechanisms are important for cancer cell survival and development. Autophagy is also involved in tumor development. However, autophagy is a double-edged sword. For example, autophagy has a protective role in regard to cancer cells under nutrient starvation, radiotherapy, and certain drugs (16). Meanwhile, inhibiting autophagy increases cancer development in certain circumstances (17). Further, impaired autophagy is found in various tumor cells (18). Until now, the molecular mechanisms underlying the role of autophagy during carcinogenesis have not been well understood, and the molecular mechanisms behind the cross-talk between autophagy and apoptosis during carcinogenesis are still unclear. Cadmium is a toxic heavy metal and human carcinogen. The food chain, cigarette smoke, and the cadmium mining industry are the main sources of cadmium exposure to humans (19). Cadmium induces apoptosis or transformation, depending on exposure concentrations and times (20, 21). Cadmium also induces autophagy in the mesangial cells (22, 23) and in the skin’s epidermal cells (24). Interestingly, cadmium-induced apoptosis, autophagy, or transformation is caused by ROS (20, 21, 24). Although ROS, autophagy, apoptosis, and cell transformation are important players in the mechanism of cadmium carcinogenesis, most studies are focused on the events leading to cell transformation, which can be considered the first stage of cadmium carcinogenesis. There are few studies that have investigated events following cell transformation, the secondary stage of cadmium carcinogenesis. The present study will test our hypothesis that cadmium-transformed cells have an acquired autophagy deficiency, leading to constitutive p62 and Nrf2 overexpression. These overexpressions up-regulate the antioxidant proteins catalase and SOD and the antiapoptotic proteins Bcl-2 and Bcl-xL. These up-regulations result in a decrease in ROS generation, apoptosis resistance, and the promotion of cell proliferation, survival, and tumorigenesis.



night incubation, cultures were treated with CM-H2DCFDA (5 ␮M) for 30 min. After washing two times with PBS, DCF fluorescence was measured using a Spectramax GEMINIXPS fluorescence microplate reader (Molecular Devices, Sunnyvale, CA). In addition, cells (0.5 ⫻ 106 cells/well) were seeded into 60-mm culture dishes and, after overnight incubation, were exposed to CM-H2DCFDA at a final concentration of 5 ␮M for 30 min and processed for flow cytometric analysis. Small Interfering RNA Transfection—Silencer predesigned small interference RNA (siRNA) for human p62 (siRNA ID s16960), Nrf2 (siRNA ID s9491), and control siRNA (AM4611) were obtained from Ambion (Austin, TX) and used to inhibit p62 and Nrf2 protein. The coding strand of p62 siRNA was 5⬘-GGAGCACGGAGGGAAAAGAtt-3⬘; the coding strand of Nrf2 siRNA was 5⬘-GAAUGGUCCUAAAACACCAtt-3⬘. Normal BEAS-2B cells and CdT cells were seeded in 96- or 6-well culture plates and transfected with 50 nM siRNA duplexes using LipofectamineTM RNAi MAX (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours after transfection, the cells were harvested, and cellular levels of proteins specific for the siRNA transfection were checked by immunoblotting. Anchorage-independent Colony Growth Assays—Anchorageindependent growth is one of the hallmarks of cell transformation, and the soft agar colony formation assay is a common method for anchorage-independent growth of the transformed cells (18). The soft agar assay was performed as described previously (21). Briefly, 3 ml of 0.5% agar in DMEM supplemented with 10% FBS was spread onto each well of a 6-well culture plate. A suspension (1 ml) containing BEAS-2B cells or CdT cells (1 ⫻ 104) was mixed with 2 ml of 0.5% agarDMEM and layered on the top of the 0.5% agar layer. The plates were incubated at 37 °C in 5% CO2 for 1 month, and colonies larger than 50 ␮m in diameter were counted under a light microscope. Chromatin Immunoprecipitation (ChIP) Assay—ChIP assay was performed using a PierceTM agarose ChIP kit (Thermo Scientific, Rockford, IL). Briefly, 90% confluent non-transformed BEAS-2B cells and transformed cells were treated with or without cadmium (10 ␮M) for 6 h. DNA and proteins were crosslinked by incubating cells with 1% formaldehyde for 10 min at room temperature. Excess formaldehyde was quenched with glycine for 5 min. Cells were lysed, and nuclei were digested using micrococcal nuclease. Sheared chromatin was diluted and immunoprecipitated with 2 ␮g of anti-Nrf2 or control IgG antibody. DNA-protein complexes were eluted from the protein A/G-agarose beads using a spin column and were reverse cross-linked by incubating with NaCl at 65 °C. The relative Nrf2 binding to the ARE regions of the p62, Bcl-2, and Bcl-xL was analyzed by the MyiQTM single-color real-time PCR detection system (Bio-Rad) with SYBR Green PCR master mix. General PCR amplification was also performed by Mastercycler威 thermal cyclers (Eppendorf, Foster City, CA). Statistical Analysis—All of the data are expressed as means ⫾ S.E. One-way analysis of variance (ANOVA) using IBM SPSS Statistics 21 was used for the multiple comparisons. A value of p ⬍ 0.05 was considered statistically significant. VOLUME 289 • NUMBER 41 • OCTOBER 10, 2014


FITC-Annexin V/Propidium Iodide (PI) Staining—The apoptosis assay was performed as described previously (20). The scatter parameters of the cells (20,000 cells/experiment) were analyzed using a FACSCalibur威 system. The early apoptotic population in the lower right quadrant (low PI and high FITC signals) and late apoptotic population in the upper right quadrant (high PI and high FITC signals) were considered apoptotic cells. Western Blot Analysis—Western blotting was performed as described previously (20). Monoclonal antibodies specific for ␤-actin (SC-47778) and Nrf2 (SC-365949) were purchased from Santa Cruz Biotechnology, Inc. Caspase-8 (804-242C100) was from ALEXIS (Atlanta, GA), and rabbit monoclonal anti-catalase (NB100-79910) was from Novus Biologicals (Littleton, CO). Anti-Cu/Zn-SOD (07-403) and anti-Mn-SOD (06-984) were purchased from Millipore (Temecula, CA). Mouse anti-GAPDH monoclonal antibody (A00839) was from GeneScript (Piscataway, NJ). Anti-p62/SQSTM1 antibody (P0067) and monoclonal anti-actin antibody produced in mice (A4700) were from Sigma-Aldrich. Bcl-xL (catalog no. 2762), Bcl-2 (catalog no. 2876), cleaved caspase-3 (Asp-175) (catalog no. 9661), and cleaved caspase-7 (Asp-198) (catalog no. 9491) polyclonal antibodies were purchased from Cell Signaling (Beverly, MA). The polyclonal antibody specific to LC3 (PD014) and a monoclonal antibody specific to poly(ADP-ribose) polymerase (SA-249) were purchased from BML (Woburn, MA) and Biomol (Plymouth Meeting, PA), respectively. Secondary antibodies and enhanced chemiluminescence substrate were from Pierce. The blots were exposed to Hyperfilm (Amersham Biosciences), and bands were quantified with ImageJ densitometry software (National Institutes of Health, Bethesda, MD). GFP-LC3 and mCherry-EGFP-LC3 Punctal Formation Assays—GFP-LC3 punctal cells were quantified as described elsewhere (24). The number of red-positive (mCherry⫹/GFP⫺) and yellow-positive (mCherry⫹/GFP⫹) puncta from 25 cells was scored using the ImageJ software. Briefly, non-transformed BEAS-2B cells and CdT cells were transfected with GFP-LC3 or mCherry-EGFP-LC3 plasmid, and then cells were divided on coverslips plated in 6-well plates (0.2 ⫻ 106/coverslip). Cells were exposed to cadmium (10 ␮M) with or without various inhibitors for 24 h and fixed in ice-cold methanol. Fluorescence-positive cells were counted under a fluorescence microscope (Carl Zeiss). Measurement of Cellular ROS Levels—An electron spin resonance (ESR) assay was performed using a Bruker EMX spectrometer (Bruker Instruments, Billerica, MA) and a flat cell assembly, as described previously (25). Normal BEAS-2B cells and CdT cells (1 ⫻ 106 cells) were cultured overnight, harvested, and mixed with DMPO (50 mM). The Acquisit program was used for data acquisition and analysis (Bruker Instruments). For fluorescence microscope image analysis, the cells (2 ⫻ 104 cells) were seeded onto a glass coverslide in the bottom of a 24-well plate overnight. The cells were exposed to CM-H2DCFDA (5 ␮M) for 30 min. Cells were washed with PBS, mounted, and observed under a fluorescence microscope (Carl Zeiss). To determine the fluorescence intensity of the 2⬘,7⬘dichlorodihydrofluorescein diacetate signal, cells (10,000 cells/ well) were seeded into a 96-well culture plate, and after over-


RESULTS Cadmium-transformed BEAS-2B Cells Have a High Proliferative Potential and Cell Death Resistance—Cadmium treatment showed cytotoxicity in both non-transformed BEAS-2B cells and CdT cells. However, CdT cells showed less cytotoxicity when cells were exposed to cadmium compared with nontransformed cells. Morphological study showed that many of the cadmium-exposed non-transformed BEAS-2B cells had changed to a round shape and shrunk, but in the CdT cells, only a few were found to have a round shape and to have shrunk (Fig. 1A). MTT-reducing activity transparently showed that the CdT cells show less cytotoxic effects against cadmium than nontransformed BEAS-2B cells (Fig. 1B). The optical density of MTT revealed that the cell viability significantly differs between the CdT cells and the non-transformed cells at each cadmium concentration (Fig. 1C). Additionally, the CdT cells had a higher proliferative potential than the non-transformed BEAS-2B cells (Fig. 1D). The difference in the proliferative rate started after 2 days of culture and increased definitely after 4 days of culture. Cadmium-transformed BEAS-2B Cells Have a Property of Apoptosis Resistance—To investigate the cell death resistance mechanisms of the transformed cells, various apoptosis detection assays were performed. Annexin V/PI staining shows that ⬃55 and 40% of cell populations were apoptotic cells in the cadmium-exposed normal BEAS-2B cells and the CdT cells, respectively (Fig. 2A). Cadmium-induced apoptosis was dosedependent in both normal and transformed BEAS-2B cells, but the CdT cells showed significantly less induced apoptosis than in non-transformed BEAS-2B cells for all concentrations (Fig. 2B). In addition, the cleaved poly(ADP-ribose) polymerase, OCTOBER 10, 2014 • VOLUME 289 • NUMBER 41

caspase-3/7, and the decreased procaspase-8 were dose-dependent in both the non-transformed and the CdT cells (Fig. 2C). These changes were much higher in the non-transformed BEAS-2B cells than in the transformed cells (Fig. 2C). Further study revealed that cadmium-induced cell death occurred mainly through caspase-dependent apoptosis. This is because cadmium-induced cell death and apoptosis by annexin V/PI staining assay were significantly attenuated in the treatment with the pancaspase inhibitor, Z-VAD-fluoromethyl ketone (Fig. 2, D and E). Cadmium-transformed BEAS-2B Cells Have a Low ROS Level and Express High Antioxidant Enzymes—The intracellular ROS level is a crucial factor in determining cell fate upon exposure to stimuli, in particular, stimuli such as oxidative stress or toxic substance (26). To investigate whether the intracellular ROS level differs between non-transformed BEAS-2B cells and CdT cells, levels were measured using various methods. First, ESR spin trapping was used to detect the basal level of ROS in cells. The spectrum recorded from a mixture containing the cells with DMPO (Fig. 3A) shows that the normal BEAS-2B cells generated a 1:2:2:1 quartet ESR signal. The ESR signal intensity was extremely low in the CdT cells. Further, we stained the cells with CM-H2DCFDA to measure intracellular ROS levels and analyzed fluorescence intensity using flow cytometry (Fig. 3B), fluorescence microscopy (Fig. 3C), and a fluorescence microplate reader (Fig. 3D). The fluorescence intensity for ROS levels in CdT cell populations was significantly lower than in nontransformed BEAS-2B cells. Additionally, we investigated the antioxidant enzyme level by Western blotting. The main antioxidant enzymes, such as catalase, SOD1, and SOD2, were more highly expressed in the transformed cells than in nonJOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 1. Cadmium-transformed BEAS-2B cells have a cell death resistance and high proliferative potential. CdT cells and their parent non-transformed BEAS-2B cells were exposed to increasing concentrations (0 –10 ␮M) of cadmium for 24 h. The cell morphology was visualized by microscopy (A), and the MTT assay result is presented as a photograph (B) and graph (C). For the proliferation assay, the cadmium-transformed BEAS-2B cells and non-transformed cells (0.2 ⫻ 106) were seeded on 10-cm culture dishes for 7 days. The total cell number was counted each day using a cell counter. The results were shown as the mean ⫾ S.E. (error bars) of three independent experiments. **, p ⬍ 0.01; ***, p ⬍ 0.001, significant differences between the experiments (ANOVA, Scheffe’s test).


transformed cells (Fig. 3E). When the cells were exposed to cadmium, the expression level of antioxidant enzymes was reduced in a dose-dependent manner in normal and transformed BEAS-2B cells (Fig. 3F). However, the expression levels were maintained for 24 h at the highest concentration of cadmium (10 ␮M) in the CdT cells, whereas a low amount of antioxidant enzymes was detected in the non-transformed cells. Moreover, pharmacological depletion of ROS scavengers by adding 3AT (catalase inhibitor), LCS-1 (SOD1 inhibitor), and 2ME (SOD2 inhibitor) accelerated cadmium-induced decrease of cell viability (Fig. 3G). Especially, cell viability was markedly reduced by SOD inhibitors in CdT cells. These results suggest that the transformed BEAS-2B cells contain low ROS levels due to high expression of antioxidant enzymes. Decreased ROS generation contributes to the apoptosis resistance of CdT cells. This enhanced defense mechanism, when compared with the non-transformed cells, may contribute to cell death resistance. High Expression of Bcl-2 and Bcl-xL Is Involved in Cell Death Resistance Mechanisms—Proteins in the Bcl-2 family are well known apoptosis regulators. To investigate whether Bcl-2 and Bcl-xL were involved in cell death resistance in CdT cells, we analyzed expression levels of Bcl-2 and Bcl-xL. As expected, the basal levels of Bcl-2 and Bcl-xL were higher in the CdT cells than in the non-transformed cells (Fig. 4A). The decrease of the rate of Bcl-2 and Bcl-xL expression by cadmium was much less in CdT cells in a dose and time course experiment (Fig. 4, B and


C). Depletion of Bcl-2 and Bcl-xL expression by adding ABT263, a Bcl-2/Bcl-xL inhibitor, resulted in the expression of Bcl-2 and Bcl-xL being inhibited completely in the cadmium-exposed normal and transformed cells (Fig. 4D); the viability drop down in cadmium-exposed transformed cells had a rate similar to that in the non-transformed cells (Fig. 4E). These results show high expression of Bcl-2 and Bcl-xL in CdT cells involved in cell death resistance mechanisms. Nrf2 Controls Cell Death Resistance in Cadmium-transformed BEAS-2B Cells—Nrf2 is a transcription factor that regulates the critically important cellular defense mechanisms serving in response to oxidative stimuli and toxic substances, including cadmium (27). To investigate whether Nrf2 is involved in cell death resistance in the CdT cells, we performed a functional analysis. The basal level of Nrf2 is higher in CdT cells than in non-transformed cells (Fig. 5A). When the cells were exposed to cadmium, the expression level of Nrf2 slightly increased at 1 and 5 ␮M cadmium, but the level reduced at 10 ␮M of cadmium in normal BEAS-2B cells (Fig. 5B). In the transformed cells, the expression levels of Nrf2 increased at 10 ␮M cadmium, although the levels slightly decreased when compared with low concentrations of cadmium (1 or 5 ␮M) (Fig. 5B). In a time course experiment, Nrf2 expression slightly increased until 12 h and decreased in the normal BEAS-2B cells when the cells were exposed to 10 ␮M cadmium (Fig. 5C, top). However, VOLUME 289 • NUMBER 41 • OCTOBER 10, 2014


FIGURE 2. Cadmium-transformed BEAS-2B cells have an apoptosis resistance against cadmium. The cadmium-transformed BEAS-2B cells and nontransformed cells were exposed to cadmium (0 –10 ␮M). After a 24-h exposure, cells were analyzed by a flow cytometer after staining with annexin V and PI (A). The early (annexin V⫹/PI⫺) and late apoptotic (annexin V⫹/PI⫹) cells were calculated and presented in B. Protein lysates were analyzed by a NuPAGE BisTris electrophoresis system, and the levels of poly(ADP-ribose) polymerase, caspase-8, and cleaved caspase-3 and -7 were determined by Western blot analysis (C). In addition, cells were incubated with cadmium (10 ␮M) for 24 h in the presence and absence of the pancaspase inhibitor (Z-VAD, 20 ␮M). Thereafter, total death cells (D) and apoptotic cells (E) were determined by annexin V/PI staining assay. Values are the means ⫾ S.E. (error bars) of triplicate experiments. *, p ⬍ 0.05; **, p ⬍ 0.01; ***, p ⬍ 0.001 represent significant difference between the experiments (ANOVA, Scheffe’s test). GAPDH was used as a loading control.


the Nrf2 expression levels increased in a time-dependent manner in the transformed cells (Fig. 5C, bottom). To further study the role of Nrf2 in cell death resistance, we depleted Nrf2 expression by siRNA transfection. The basal level and cadmium-induced Nrf2 expression levels were attenuated by the Nrf2 siRNA transfection in the both cell lines (Fig. 5D). The cadmium-mediated apoptosis (Fig. 5, E and F) and cell death (Fig. 5G) were much accelerated by the blockage of Nrf2 expression in cadmium-transformed cells. These results strongly suggest that Nrf2 plays a critical role in the cytoprotective function against cadmium. p62 Is an Important Mediator for the Cell Survival Mechanism in Cadmium-transformed BEAS-2B Cells—p62 is a stress response protein, induced by oxidants, metals, and proteasomal inhibitors (2, 4, 28). Furthermore, it has been reported that p62 is required for cell survival (2) and is an Nrf2 target gene (7). To investigate whether p62 is involved in cell survival mechanisms, we analyzed the expression levels of p62. The basal levels of p62 were dramatically overexpressed in the transformed BEAS-2B cells when compared with the non-transformed cells (Fig. 6A). The expression level of p62 was slightly increased at 1 ␮M cadOCTOBER 10, 2014 • VOLUME 289 • NUMBER 41

mium but was reduced at high concentrations of cadmium in the normal BEAS-2B cells (Fig. 6B). However, the p62 levels increased dramatically in a dose-dependent manner in the transformed cells (Fig. 6B). The increase in p62 expression level by cadmium started at 1 h and was reduced after 6 h of exposure in the non-transformed BEAS-2B cells (Fig. 6C, top). However, the increase in p62 levels by cadmium in the transformed cells was sustained until 24 h (Fig. 6C, bottom). To further investigate the role of p62 in cell survival function, we knocked down the p62 level by siRNA transfection specific for p62 in the cells. The basal level and cadmium-induced p62 levels were reduced by siRNA p62 (Fig. 6D). The blockage of p62 significantly accelerated apoptosis induced by cadmium (Fig. 6, E and F) as well as cell death (Fig. 6G). These results indicate that the p62 is an important factor for cell survival mechanisms in the transformed cells. p62 and Nrf2 Down-regulate the Intracellular ROS Level of Cadmium-transformed BEAS-2B Cells—Nrf2 and p62 regulate intracellular ROS levels and sensitivity of apoptotic cell death under oxidative stress (2, 29). To investigate whether the high expression of Nrf2 and p62 are related to low concentration of JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 3. Cadmium-transformed BEAS-2B cells have a low level of ROS due to high expression of antioxidant enzymes. To measure a basal level of ROS, the cell suspensions were prepared from cadmium-transformed BEAS-2B cells or normal cells; afterward, an ESR spectra was recorded. The generation of a 1:2:2:1 quartet ESR signal was shown (A, top), and the signal intensity of DMPO-OH was represented (A, bottom). ROS levels of the cadmium-transformed BEAS-2B cells and their parent BEAS-2B cells also were measured by using flow cytometry (B), fluorescence microscopy (C), and fluorescence microplate reader (D) after staining with CM-H2DCFDA (5 ␮M) for 30 min. In addition, the basal cellular levels of catalase, SOD1, and SOD2 were measured (E). The effects of cadmium (0 –10 ␮M) on the antioxidant enzymes of the transformed cells or normal cells also were analyzed by Western blot (F). For the inhibition assay, both cells were incubated with cadmium (10 ␮M) for 24 h in the presence or absence of 3AT (10 mM), LCS-1 (10 ␮M), and 2ME (1 ␮M), and then cell viability was performed by an MTT assay (G). The ESR spectrometer settings were as follows: frequency, 9.8 GHz; power, 39.91 milliwatts; modulation frequency, 100 kHz; receiver gain, 5.02 ⫻ 105; time constant, 40.96 ms; modulation amplitude, 1.00 G; scan time, 60 s; magnetic field, 3,451 ⫾ 100 G. All spectra shown are an accumulation of five scans. The results are shown as the mean ⫾ S.E. (error bars) of three separate experiments. **, p ⬍ 0.01; ***, p ⬍ 0.001 versus the normal BEAS-2B cells. #, p ⬍ 0.05; ###, p ⬍ 0.001 versus the cadmium-treated control cells (ANOVA, Scheffe’s test). GAPDH was used as a loading control. CdT, cadmium-induced transformed BEAS-2B cells; 3AT, catalase inhibitor; LCS-1, SOD1 inhibitor; 2ME, SOD2 inhibitor.


FIGURE 5. Nrf2 plays a critical role for cell survival in the cadmium-transformed BEAS-2B cells. The basal expression level of Nrf2 was measured in the cadmium-transformed BEAS-2B cells and non-transformed BEAS-2B cells by Western blot analysis (A). The normal and transformed BEAS-2B cells were exposed to various concentrations of cadmium (0 –10 ␮M) for 24 h (B) or various times (0 –24 h) with 10 ␮M cadmium (C); then the levels of Nrf2 were detected. To diminish Nrf2 levels, cells were transfected with siRNA specific to Nrf2. After overnight transfection, cells were exposed to 10 ␮M cadmium for an additional 24 h. The expression level of Nrf2 was evaluated by Western blot (D), and an apoptosis assay was performed after staining with annexin V/PI and is represented in dot blots (E) and a graph (F). Finally, the total number of cell deaths was calculated by the early (annexin V⫹/PI⫺) and late apoptotic (annexin V⫹/PI⫹) cells and necrotic (annexin V⫺/PI⫹) cells (G). The results represent the mean ⫾ S.E. (error bars) of three independent experiments. **, p ⬍ 0.01, significant difference between the experiments (ANOVA, Scheffe’s test). GAPDH was used as loading control.

ROS in the transformed cells, we measured ROS levels after depletion of either Nrf2 or p62. The low basal levels of ROS were obviously up-regulated in the siRNA p62 and in the siRNA Nrf2-transfected transformed cells, as shown in fluorescence


microscopy (Fig. 7A), fluorimetric (Fig. 7B), and ESR (Fig. 7, C and D) analysis. These results strongly suggest that high expression of p62 and Nrf2 is involved in a low level of ROS in the transformed cells. VOLUME 289 • NUMBER 41 • OCTOBER 10, 2014


FIGURE 4. Cadmium-transformed BEAS-2B cells highly expressed the antiapoptotic proteins Bcl-2 and Bcl-xL. Cellular proteins were prepared from the normal BEAS-2B cells and cadmium-transformed BEAS-2B cells, and the cellular levels of Bcl-2 and Bcl-xL were detected by Western blot analysis (A). Cadmiumtransformed BEAS-2B cells and non-transformed BEAS-2B were exposed to various concentrations of cadmium (0 –10 ␮M) for 24 h (B); the cells were exposed to 10 ␮M cadmium for various times (0 –24 h) (C), and then the levels of Bcl-2 and Bcl-xL were analyzed by Western blot. For the inhibition assay, cells were incubated with cadmium (10 ␮M) for 24 h in the presence or absence of ABT-263 (10 ␮M). Thereafter, the expression levels of Bcl-2 and Bcl-xL were detected by Western blot analysis (D), and the cell viability was determined by an MTT assay (E). ***, p ⬍ 0.001, significant difference between the experiments (ANOVA, Scheffe’s test). ABT-263 is a Bcl-2 family inhibitor. Error bars, S.E.


FIGURE 7. Nrf2 and p62 regulate the intracellular ROS levels in the cadmium-transformed cells. Cadmium-transformed BEAS-2B cells were transfected with siRNA specific to p62 or Nrf2. After overnight transfection, ROS levels were measured by using fluorescence microscopy (A) and a fluorescence microplate reader (B) after staining with CM-H2DCFDA (5 ␮M) for 30 min. In addition, the generation of a 1:2:2:1 quartet ESR signal of the ROS level was shown (C), and the signal intensity of DMPO-OH was represented on a graph (D). The results are shown as the mean ⫾ S.E. (error bars) of three separate experiments. **, p ⬍ 0.01; ***, p ⬍ 0.001 versus the vehicle control (ANOVA, Scheffe’s test).

Nrf2 and p62 Cross-talk and Cadmium Increases Binding of Nrf2 to ARE Regions of the p62 Promoter in Cadmium-transformed BEAS-2B Cells—Nrf2 regulates p62 expression by increasing the ARE binding of the p62 promoter (7), and p62 in turn increases Nrf2 activity through inactivation of Keap1 (6), suggesting that Nrf2 and p62 have a positive feedback loop. We investigated the correlation between Nrf2 and p62 in our system. When we knocked down Nrf2 with siRNA transfection, the basal level and increased level of the Nrf2 by cadmium were abolished along with the p62 level (Fig. 8A). Further, depletion of p62 by siRNA transfection reduced the basal level and OCTOBER 10, 2014 • VOLUME 289 • NUMBER 41

increased levels of both p62 and Nrf2 by cadmium in the normal or transformed cells (Fig. 8A). This result suggests that Nrf2 and p62 cross-talk with each other in cadmium-exposed cells. To observe the role of Nrf2 and p62 in detail, we performed ChIP assays. Nucleotide sequence analysis of the 2.9-kb p62 promoter revealed the presence of two consensus and five putative AREs in the forward strand (Fig. 8B). We also found three AREs in the reverse strand of the p62 promoter (Fig. 8B). ChIP assay results demonstrated Nrf2 binding to the ARE F1 (⫺2664 to ⫺2653), F4 (⫺963 to ⫺952), F5 (⫺493 to ⫺481), and R3 (⫺353 to ⫺343) in the p62 gene promoter (Fig. 8C). InterestJOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 6. p62 is important for cell death resistance in the cadmium-transformed BEAS-2B cells. The basal expression level of p62 was measured in the cadmium-transformed BEAS-2B cells and the normal BEAS-2B cells by Western blot analysis (A). The non-transformed cells or transformed cells were exposed to various concentrations of cadmium (0 –10 ␮M) for 24 h (B) and various times (0 –24 h) with 10 ␮M cadmium (C); then the levels of p62 were measured. To deplete p62 levels, cells were transfected with siRNA specific to p62. After overnight transfection, the cells were exposed to 10 ␮M cadmium for an additional 24 h. The expression levels of p62 were examined by Western blot (D), and an apoptosis assay (E and F) was performed. The total cell death was analyzed from the annexin V/PI assay and is represented in G. The results represent the mean ⫾ S.E. (error bars) of three independent experiments. **, p ⬍ 0.01; ***, p ⬍ 0.001, significant difference between the experiments (ANOVA, Scheffe’s test). GAPDH was used as a loading control.


ingly, the Nrf2 binding to the AREs decreased in response to cadmium treatment in the normal BEAS-2B cells, whereas the binding was dramatically enhanced in the transformed BEAS-2B cells (Fig. 8C). When chromatin was immunoprecipitated with the control mouse IgG, the AREs of p62 gene were not amplified in the same experimental conditions (Fig. 8C). Furthermore, the Nrf2 binding activities to the ARE F1, F4, F5, and R3 of the p62 promoter by cadmium also greatly increased in quantitative real-time PCR (Fig. 8D). These results indicate that a specific interaction between Nrf2 and p62 contributes to cell survival mechanisms in the transformed cells. Nrf2 Regulates Bcl-2/Bcl-xL through Binding of the ARE Regions of the Bcl-2/Bcl-xL Promoter in Cadmium-transformed BEAS-2B Cells—Niture and Jaiswal (8, 9) recently reported that Nrf2 regulates the antiapoptotic protein Bcl-2/Bcl-xL and enhances cell death resistance. We performed an siRNA transfection assay to investigate whether Nrf2 or p62 regulates Bcl2/Bcl-xL expression. The decrease of Bcl-2/Bcl-xL expression levels by cadmium and basal levels of Bcl-2/Bcl-xL was further accelerated by siRNA Nrf2 transfection in the normal or transformed cells (Fig. 9A). This result was similar to siRNA p62 transfection (Fig. 9A). These results further confirmed the positive feedback loop between Nrf2 and p62 and confirmed that Nrf2 regulates Bcl-2/Bcl-xL expression. Next, we determined


whether Nrf2 regulates Bcl-2/Bcl-xL through the ARE binding of promoter regions. We found one consensus and six putative AREs on the 3.6-kb Bcl-xL promoter (Fig. 9B) and two putative AREs on the 8-kb Bcl-2 promoter (Fig. 9E). ChIP analysis revealed that Nrf2 binds to the ARE R1 (⫺3386 to ⫺3378), R2 (⫺3239 to ⫺3231), F1 (⫺2992 to ⫺2984), F2 (⫺2183 to ⫺2175), and R3 (⫺2046 to ⫺2038) in the Bcl-xL gene promoter (Fig. 9C). Similar to the p62 promoter assay, Nrf2 binding activities to AREs decreased in response to cadmium in non-transformed BEAS-2B cells, whereas the binding activities were enhanced in the CdT cells (Fig. 9C). We used mouse IgG as control for the chromatin immunoprecipitation experiment. ChIP assay and quantitative real-time PCR revealed that the binding of Nrf2 to the Bcl-xL AREs (R1, R2, F1, F2, and R3) was increased by cadmium in the transformed cells (Fig. 9D). The binding activity on the ARE region of the Bcl-2 promoter (F1, ⫺2991 to ⫺2980) was similar to that of Bcl-xL (Fig. 9, E, F, and G). These results suggest that Nrf2 regulates Bcl-2/Bcl-xL expression by regulating transcription of these genes. Cadmium-transformed BEAS-2B Cells Have a Property of Autophagy Deficiency—It has been shown that a high expression level of p62 is associated with an autophagy defect in the cells (3, 6). We investigated whether the transformed cells have an autophagy defect and the defect’s relationship with cell VOLUME 289 • NUMBER 41 • OCTOBER 10, 2014


FIGURE 8. Nrf2 and p62 have a positive feedback loop, and the binding activities of Nrf2 to the ARE regions of the p62 promoter were increased in the cadmium-exposed transformed BEAS-2B cells. Normal or cadmium-transformed BEAS-2B cells were transfected with siRNA specific to p62 or Nrf2. After overnight transfection, the cells were treated with or without 10 ␮M cadmium for an additional 24 h. The expression levels of p62, Nrf2, and NQO1 were analyzed by Western blot (A). For the ChIP assay, consensus or putative, the ARE regions of the p62 promoter were explored (B). The cadmium-transformed cells and normal BEAS-2B cells were treated with cadmium (10 ␮M) for 6 h, fixed with formaldehyde, and cross-linked, and the chromatin was isolated. The chromatin was immunoprecipitated with an anti-Nrf2 antibody or control mouse IgG. The Nrf2 binding to the p62 promoter was analyzed by normal real-time PCR (C) or quantitative real-time PCR (D) with the specific primers for each ARE region of the promoter. The presented data follow the percentage input method and are normalized for each control. The results are expressed as the mean ⫾ S.E. relative to the control of triplicate experiments. *, p ⬍ 0.05; ***, p ⬍ 0.001 versus the vehicle control (ANOVA, Scheffe’s test). ␤-Actin was used as a loading control. ND, not detectable.


death resistance. Cadmium treatment dramatically increased the LC3-II level, a hallmark of autophagy, in the normal BEAS-2B cells in a dose- and time-dependent manner (Fig. 10, A and B). LC3-II accumulated at 12 h after cadmium treatment (10 ␮M) and attenuated at 24 h. However, LC3-II did not accumulate significantly in the CdT cells in dose and time experiments (Fig. 10, A and B). Importantly, the autophagy flux rate was increased by cadmium only in the non-transformed BEAS-2B cells. When cells were treated with bafilomycin A1, an inhibitor of autophagosome and lysosome fusion, increased LC3-II levels were observed. LC3-II levels further increased when cells were treated with a combination of cadmium and OCTOBER 10, 2014 • VOLUME 289 • NUMBER 41

bafilomycin A1, suggesting that cadmium increased autophagy flux rather than blocking the fusion of autophagosomes with lysosomes (Fig. 10C, top). However, no increase in autophagy flux was observed in the transformed cells (Fig. 10C, bottom). When we treated cells with cadmium in the presence or absence of wortmannin, an inhibitor of autophagosome initiation, the cadmium-induced up-regulation of LC3-II was attenuated in the non-transformed cells (Fig. 10C, top). To further confirm autophagy flux in the normal or transformed cells, we used tandem fluorescence-tagged LC3. When the transfected cells were exposed to cadmium, both yellow (autophagosome) and red (autolysome) puncta were increased in normal BEAS-2B JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 9. Nrf2/p62 up-regulates the expression of Bcl-2/Bcl-xL, and cadmium increases binding activities of Nrf2 to the ARE regions of the Bcl-2/ Bcl-xL promoter in the cadmium-transformed BEAS-2B cells. Normal or transformed BEAS-2B cells were transfected with siRNA specific to p62 or Nrf2. After overnight transfection, the cells were treated with or without 10 ␮M cadmium for an additional 24 h. The expression levels of Bcl-2/Bcl-xL or p62/Nrf2 were analyzed by Western blot (A). For the ChIP assay, consensus or putative ARE regions of the Bcl-xL (B) and Bcl-2 promoter (E) were discovered, respectively. Cadmium-transformed cells and non-transformed cells were treated with cadmium (10 ␮M) for 6 h. After isolating chromatin, it was immunoprecipitated with an anti-Nrf2 antibody or control mouse IgG. The Nrf2 binding to the Bcl-xL or Bcl-2 promoter was analyzed by a normal real-time PCR (C and F) or quantitative real-time PCR (D and G) with the specific primers for each ARE region of the promoter. The presented data follow the percentage input method and are normalized to each control. The results were expressed as the mean ⫾ S.E. (error bars) relative to the control of triplicate experiments. *, p ⬍ 0.05; ***, p ⬍ 0.001 versus the vehicle control (ANOVA, Scheffe’s test). ␤-Actin was used as a loading control. ND, not detectable.



FIGURE 10. Cadmium-transformed BEAS-2B cells characterize an autophagy deficiency. The cadmium-transformed BEAS-2B cells and normal BEAS-2B cells were exposed to various concentrations of cadmium (0 –10 ␮M) for 24 h (A), or the cells were exposed with 10 ␮M cadmium for various times (0 –24 h) (B), and then the levels of LC3-I and LC3-II were detected by Western blot. To analyze autophagy flux, the cells were preincubated with bafilomycin A1 (100 nM) or wortmannin (100 nM) for 1 h before treatment with cadmium (10 ␮M). After a 24-h incubation, the levels of LC3-I and LC3-II were detected by Western blot (C). The expression level of LC3-II was quantified and represented in D. In addition, the cells were transfected with the mCherry-EGFP-LC3 or GFP-LC3 plasmid and treated with cadmium (10 ␮M). The yellow puncta (mCherry⫹/GFP⫹) and red puncta (mCherry⫹/GFP⫺) were visualized using fluorescence microscopy (E), and quantification of each punctum is represented in F. The GFP-LC3 punctal cells are also visualized in G, and the number of GFP-LC3 puncta-positive cells was counted and is presented in H. For the inhibition assay, the cells were incubated with cadmium (10 ␮M) for 24 h in the presence and absence of wortmannin (100 nM). Thereafter, the total numbers of dead cells (I) and GFP-LC3 punctal cells (J) were determined by the trypan blue assay and fluorescence microscopy, respectively. The shRNA Beclin-1-transfected stable BEAS-2B cells were used for the study of autophagy function. After treatment with cadmium (0 –10 ␮M) for 24 h, the trypan blue-positive staining cells (K) and fluorescent puncta-positive cells (L) were determined. The results are shown as the mean ⫾ S.E. (error bars) in triplicate or three independent experiments. *, p ⬍ 0.05; **, p ⬍ 0.01; ***, p ⬍ 0.001, significant difference between the experiments (ANOVA, Scheffe’s test). GAPDH or control shRNA was used as an internal control.




cells; however, only yellow puncta was increased in CdT cells (Fig. 10, E and F). When compared with cadmium-exposed normal and transformed cells, there were lower levels of both mCherry⫹/GFP⫹ and mCherry⫹/GFP⫺ puncta in the transformed cells (Fig. 10, E and F). These results suggest that autophagic flux is increased in the normal cells and is less induced or blocked in the transformed cells. Consistently, when we used normal GFP-LC3 plasmid, the fluorescence in punctal cells increased in the non-transformed BEAS-2B cells (Fig. 10G, top), but fluorescence was not seen in the CdT cells (Fig. 10G, bottom). The total number of GFP-LC3 puncta-positive cells also dramatically increased in the non-transformed cells, whereas far fewer cells were observed in the transformed cells (Fig. 10H). To verify whether autophagy is involved in cell survival or cell death in our system, we performed a trypan blue assay. Cadmium-induced death cells and autophagy were attenuated by treatment with wortmannin in both cells (Fig. 10, I and J). Moreover, cadmium-induced cell death was significantly attenuated in autophagy-defective Beclin1 knockdown cells when compared with the vehicle control (Fig. 10K). This result corresponded to the GFP-LC3 punctal cell population rate (Fig. 10L). Taken together, these results suggest that autophagy is involved in cell death rather than survival mechaOCTOBER 10, 2014 • VOLUME 289 • NUMBER 41

nisms and that autophagy deficiency contributes to cell death resistance in the transformed cells. Nrf2/p62 Is an Important Regulator for Growth and Survival of Transformed Cells—Previously, we reported that chronic exposure to cadmium increases cell transformation (21). To investigate whether Nrf2/p62 signaling is involved in the cadmium carcinogenesis, the expression levels of Nrf2 and p62 were analyzed in chronic medium long or long exposure periods (Fig. 11, A and B). The expression level of p62 did not change within 4 weeks, but this protein level was dramatically increased after 1-month cadmium exposure periods when compared with the non-treated cells or vehicle control cells cultured for 5 months (Fig. 11, A and B). The expression level of Nrf2 was increased at the earliest time point (2 weeks), and this level was sustained through the entire experiment period (Fig. 11, A and B). Apparently, the antiapoptotic proteins Bcl-2/ Bcl-xL and antioxidant enzymes (CAT, SOD1, and SOD2) also increased after 1- or 2-month cadmium exposure periods (Fig. 11B). In addition, the increasing of LC3-II was started at the first week of cadmium exposure, increased in an accelerated fashion until 2 months, and then sharply decreased (Fig. 11, A and B). Moreover, p62, Nrf2, antiapoptotic protein, and antioxidant enzymes were overexpressed in the tumor tissues from JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 11. Nrf2 and p62 play a critical role in cadmium-induced carcinogenesis. BEAS-2B cells were exposed to cadmium (0.5 ␮M) for either 4 weeks or 5 months. The cells were harvested each week or month, and the expression levels of p62, Nrf2, Bcl-2, Bcl-xL, catalase, SOD, and LC3 were measured by Western blot (A and B). BEAS-2B cells exposed to cadmium for 2 months were injected (1 ⫻ 106 cells/site) subcutaneously into 6-week-old male athymic nude mice. After 4 months, each tumor was dissected, and the tumor tissues were lysed for Western blot analysis (C). In addition, cadmium-transformed BEAS-2B cells were transfected with siRNA specific for either Nrf2 or p62, and then a soft agar assay (D) and clonal assay (E) were performed. The results are shown as the mean ⫾ S.E. (error bars) in three independent experiments. **, p ⬍ 0.01; ***, p ⬍ 0.001 versus the vehicle control cells (ANOVA, Scheffe’s test). Actin was used as an internal control.

Nrf2/p62 Signaling in Cadmium Carcinogenesis mouse xenograft models, where chronic cadmium-exposed cells were injected into nude mice (Fig. 11C). More importantly, soft agar and clonal assays showed that the colony growth was attenuated significantly when transformed cells knocked down Nrf2 or p62 expression with siRNA transfection (Fig. 11, D and E). Collectively, the Nrf2 and p62 play a pivotal role in cadmium-induced carcinogenesis, and Bcl-2/Bcl-xL and the antioxidant enzymes contribute to these processes.




DISCUSSION Cadmium is a group I human carcinogen that is an important environmental and industrial pollutant (30). Cadmium is used in industrial materials such as batteries, pigments, metal coatings, and plastics (30). Cadmium is also used for smelting and refining during the production of zinc, lead, and copper (31). Recently, cadmium use in nanotechnology has increased, primarily to construct particles known as quantum dots (32). Cigarette smoke and cadmium-contaminated food are nonoccupational sources of human exposure to cadmium (33). Furthermore, cadmium ranks seventh on the 2013 Priority List of Hazardous Substances (34). Recent epidemiologic studies have reported that cadmium levels of ⬍1 ␮g/liter in blood are a health risk for humans (33). The concentrations of cadmium (0.125–10 ␮M) that we used in chronic or acute exposure were higher than the blood level of cadmium (1 ␮g/liter). However, cadmium has a long biological half-life (20 years) and occupationally or environmentally accumulates to tissues and organs for a long time. Thus, the concentration used in the present study is highly relevant to human exposure. Our previous study has shown that chronic exposure of human bronchial epithelial cells to cadmium generates ROS and that these species are responsible for cadmium-induced transformation of these cells (21). In the present study, however, we have shown that the cadmium-transformed cells have low ROS and highly express antiapoptotic proteins (Bcl-2 and Bcl-xL) and antioxidant enzymes (catalase, SOD1, and SOD2) as well as Nrf2/p62. We have also shown that cadmium-transformed cells have the properties of apoptosis resistance and autophagy dysfunction. Apoptosis is best described as a programmed cell death and well defined process of cellular dismantling that leads to cell corpses that are engulfed by phagocytosis without inflammation. Evading apoptosis is a critical cellular event in acquiring the capabilities of cancer (35). Further, most types of cancer cells show an apoptosis resistance and have a high proliferative potential (35). The present study shows that CdT cells exhibit apoptosis resistance. The cell death resistance of cadmiumtransformed cells might be due to high expression of Bcl-2 and Bcl-xL. Because Bcl-2 and Bcl-xL are well established antiapoptotic proteins, the high expression of these proteins is responsible for apoptosis resistance. The basal levels of Bcl-2 and Bcl-xL were higher in CdT cells than in non-transformed cells (Fig. 4A) and were found attenuated in both transformed and normal cells when the cells were further exposed to cadmium. However, the CdT cells showed high expression of Bcl-2/Bcl-xL even at the highest concentration of cadmium (10 ␮M) exposure. The transformed cells carry a low ROS level that makes the cells less sensitive to cadmium toxicity (Fig. 3, A–D). Further, higher expression of antioxidant enzymes, such as cata-

lase, SOD1, and SOD2, helps keep a low ROS concentration and contributes to cell death resistance in the transformed BEAS-2B cells (Fig. 3, E–G). Consistent with this, cadmiuminduced cell death was enhanced when antioxidant enzymes were blocked by the addition of each antioxidant enzyme inhibitor (Fig. 3G). Taken together, our data suggest that intracellular ROS levels are an important factor in deciding cell fate when cells are exposed to toxic substances. Nrf2 is an important transcription factor that regulates antioxidant enzymes and several apoptosis-regulatory proteins (8, 9, 29). Above, we demonstrated that antioxidant enzymes are highly expressed and that Bcl-2/Bcl-xL is up-regulated in transformed cells. Nrf2 also acts as a regulator of antioxidant and antiapoptotic proteins in cadmium-transformed cells. Nrf2 contributes to keeping a low level of ROS in the cadmiumtransformed cells. Knockdown of Nrf2 expression by siRNA enhanced ROS generation in the transformed cells (Fig. 7). This suggests that Nrf2 down-regulates intracellular ROS generation through up-regulation of antioxidant proteins (36, 37). High expression levels of Nrf2 in the cadmium-transformed cells are more likely to be responsible for cell death resistance (Fig. 5). This is because the cell death (or apoptosis) rate is correlated with Nrf2 levels when the cells are exposed to cadmium (Figs. 1, 2, and 5). The cell defense mechanism of Nrf2 against metals or oxidative stress was also investigated in various cell types (27, 29, 38). It might be that constitutive activation of Nrf2 increases the expression of antioxidant enzymes, which deplete the ROS levels that help transformed cells resist oxidative stress and apoptosis. In addition, Nrf2 also activates the antiapoptotic proteins Bcl-2 and Bcl-xL in cadmium-exposed transformed cells. The Nrf2 binding to the ARE regions of Bcl-2 or Bcl-xL promoter was up-regulated by cadmium in the transformed cells (Fig. 9). The up-regulation of antiapoptotic proteins by Nrf2 might represent apoptosis resistance in the cadmium-exposed transformed cells. Our findings suggest that Nrf2 plays a critical cytoprotective role in the transformed cells. Autophagy is a catabolic process that enables cells to break down intracellular organelles, such as ribosomes and mitochondria, and allows them to recycle for biosynthesis and energy metabolism (11). This supports cell survival mechanisms against a stress environment (16). However, autophagy is a type II programmed cell death, which can mediate cellular demise through excessive self-digestion and degradation of essential cellular constituents (11). Thus, the cytotoxic effect of autophagy is well recognized in type II cell deaths (i.e. autophagy cell death). Further, autophagy triggers proapoptotic signals, depending on specific circumstances (39), and cadmium-induced autophagy triggers cell death mechanisms in mouse skin epidermal cells (24). In our experimental setting using BEAS-2B cells, the autophagy involves a cell death pathway and not a survival function. This is because cell death is attenuated by the addition of pharmacological inhibitors or the genetic depletion of autophagy in the cadmium-exposed normal BEAS-2B cells (Fig. 10, I and K). Notably, it seems that defective autophagy in the transformed cells has a beneficial effect for survival mechanisms (Fig. 10).


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ious tumor cells (40, 41). Moreover, p62 accumulation is frequently observed in a variety of solid tumors due to impairment of the autophagy pathway (2, 42– 44). Nrf2/p62 signaling is also involved in cadmium-induced carcinogenesis. The induction of Nrf2 or p62 was much higher in the chronic cadmium-exposed conditions, which is a malignant transformation setting, than the vehicle controls (21). For example, the induction of p62 dramatically increased and the expression of Nrf2 obviously increased during long term chronic cadmium exposure periods (Fig. 11B). The antiapoptotic proteins Bcl-2 and Bcl-xL and antioxidant enzymes (catalase, SOD1, and SOD2) also increased in long term chronic cadmium-exposed conditions (Fig. 11B). However, the p62 levels did not change in chronic medium long cadmium exposure periods (within 4 weeks), but the Nrf2 levels were increased significantly after 2 weeks of cadmium exposure (Fig. 11A). This result suggests that Nrf2 is a primary regulator of p62, antiapoptotic protein, and antioxidant enzymes in cadmium-induced carcinogenesis. Notably, the induction of Nrf2 by cadmium is very specific to lung tissue in chronic exposure conditions. The Nrf2 did not induce in other cell lines, such as skin epidermal cells and embryonic fibroblast cells in chronic low dose cadmium exposure (data not shown). It seems that autophagy was induced well until 2 months in chronic cadmium exposure periods. The LC3-II levels were increased until 2 months of cadmium exposure, and after that, the levels were dramatically reduced (Fig. 11, A and B). These data imply that cell transformation occurred mainly after 2 months of cadmium exposure, and those transformed cells might have acquired autophagy deficiency. This time point also correlated with high expression of antiapoptotic proteins, which is related with apoptosis resistance (Fig. 11B). More importantly, tumor tissues from the mouse xenograft model overexpressed Nrf2/p62 and highly expressed antiapoptotic proteins and antioxidant enzymes (Fig. 11C). Furthermore, due to depletion of Nrf2 and p62 by siRNA transfection, the colony growth of transformed cells was attenuated in the soft agar and the clonal assay (Fig. 11, D and E). These results further verified the critical role of Nrf2 and p62 in cadmiuminduced carcinogenesis. Overall, this study demonstrates the integration of Nrf2/p62, ROS, autophagy, apoptosis, and cell survival in cadmium-induced carcinogenesis (Fig. 12). Cadmium-induced ROS have shown oncogenic properties in the premalignant stage; exposure of human lung bronchial epithelial cells to cadmium generates ROS, which are responsible for malignant transformation of these cells (21). After transformation (e.g. the postmalignant stage), ROS plays an antioncogenic role. A low level of ROS is one of the main reasons for increasing survival of transformed cells and tumorigenesis. High expression of p62 due to defective autophagy leads to constitutive Nrf2 activation. This activation was followed by the induction of p62, antiapoptotic proteins (Bcl-2/Bcl-xL), and antioxidant enzymes (catalase, SOD1, and SOD2). The decreased ROS and increased Bcl2/Bcl-xL levels due to constitutive Nrf2/p62 expression are responsible for the development of apoptosis resistance. This process promotes cell survival, proliferation, and carcinogenesis of transformed cells. Based on the results presented in this study and our previous one (21), the following approach is proJOURNAL OF BIOLOGICAL CHEMISTRY

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It has been reported that dysfunction of autophagy or defective autophagy causes up-regulation of p62 (3, 5). As expected, cadmium-transformed cells showed autophagy deficiency (Fig. 10). The high expression of p62 in transformed cells may occur due to autophagy deficiency (Figs. 6A and 10). Notably, up-regulation of Bcl-2/Bcl-xL may further contribute to dysregulation of autophagy in the transformed cells because Bcl-2/Bcl-xL inhibits the autophagy function through the binding of the BH-3 domain of Beclin-1, which is necessary for the induction of autophagy (11). Accumulation of p62 in transformed cells may contribute to apoptosis resistance and tumorigenesis (Figs. 6 and 11) through down-regulation of ROS generation (Fig. 7). These results are consistent with a previous report that p62 has a survival function through the regulation of ROS levels in the cells (2). Indeed, ROS levels were elevated, and the cell death was accelerated in the p62 knockdown transformed cells (Figs. 6 (D–G) and 7). Recently, several novel findings were reported regarding the relationship between p62 and Nrf2. p62 activates an Nrf2 function through direct interaction with the Nrf2binding site on Keap1 (5, 6). A previous study found the binding site of p62 on Keap1 (5). Thus, overexpression of p62 due to dysfunction of autophagy or defective autophagy competes with the Nrf2-Keap1 interaction, leading to inhibition of Keap1-mediated Nrf2 ubiquitination and its subsequent degradation by the proteasome (5). This leads to the stabilization of Nrf2 and transcriptional activation of Nrf2 target genes. Actually, depletion of p62 by siRNA transfection resulted in a decrease of cadmium-induced Nrf2 expression in normal or transformed BEAS-2B cells (Fig. 8A). More interestingly, knockdown of Nrf2 by siRNA transfection also attenuated cadmium-induced p62 expression in the cells (Fig. 8A). These results create a possible positive feedback loop between Nrf2 and p62 in cadmium-exposed cells. Furthermore, it has been reported that Nrf2 is an activator with specificity for the binding on the p62 promoter (7). We found two ARE consensus and five ARE putative sequences on the forward strand and three ARE putative sequences on the reverse strand of the p62 promoter region (⫺2901 to ⫺51) (Fig. 8B). ChIP analysis revealed that four ARE regions of the p62 promoter have an Nrf2 binding activity (Fig. 8, C and D). The binding activity decreased when the normal cells were exposed to cadmium, whereas the binding activity apparently was enhanced in the cadmium-exposed transformed cells (Fig. 8, C and D). This might be the main reason for sustenance of high levels of p62 during cadmium exposure periods in the transformed cells (Fig. 6, B and C). The p62 level significantly decreased when the normal cells were exposed to cadmium (10 ␮M) for 24 h, but the high expression level of p62 was sustained during periods of exposure to cadmium in the transformed cells. Collectively, these results suggest that Nrf2 up-regulates p62 expression in response to toxic stimuli. It is notable that the basal high level of p62 in the transformed cells is not a direct cause of Nrf2 activation. This is because Nrf2 binding activation on the ARE regions of the p62 promoter is not higher in the non-stimulated transformed cells than the normal cells. High expression of p62 seems mainly due to autophagy deficiency in the transformed cells. Constitutive activation of Nrf2 contributes to a malignant transformation and high expression of Nrf2, as observed in var-


posed to prevent metal-induced carcinogenesis. In the first stage of metal carcinogenesis or before cell transformation, upregulation of Nrf2 may reduce metal-induced ROS generation and prevent metal-induced cell transformation. In the second state of metal carcinogenesis or after cell transformation, down-regulation of constitutive Nrf2 expression may reduce antioxidant protein levels and increase ROS generation. Both increased ROS generation and reduced Bcl-2 and Bcl-xL expression will increase apoptosis sensitivity. These steps would result in decreased proliferation and survival of transformed cells as well as a decrease in carcinogenesis. In short, our findings provide a novel insight into the oncogenic and antioncogenic role of ROS and its regulatory proteins (Nrf2/p62) in cadmium-induced carcinogenesis, which can be used as an effective strategy for chemoprevention and chemotherapy. REFERENCES 1. Nezis, I. P., and Stenmark, H. (2012) p62 at the interface of autophagy, oxidative stress signaling, and cancer. Antioxid. Redox Signal. 17, 786 –793 2. Duran, A., Linares, J. F., Galvez, A. S., Wikenheiser, K., Flores, J. M., DiazMeco, M. T., and Moscat, J. (2008) The signaling adaptor p62 is an important NF-␬B mediator in tumorigenesis. Cancer Cell 13, 343–354 3. Mathew, R., Karp, C. M., Beaudoin, B., Vuong, N., Chen, G., Chen, H. Y., Bray, K., Reddy, A., Bhanot, G., Gelinas, C., Dipaola, R. S., Karantza-Wadsworth, V., and White, E. (2009) Autophagy suppresses tumorigenesis through elimination of p62. Cell 137, 1062–1075 4. Lau, A., Zheng, Y., Tao, S., Wang, H., Whitman, S. A., White, E., and Zhang, D. D. (2013) Arsenic inhibits autophagic flux, activating the Nrf2-Keap1 pathway in a p62-dependent manner. Mol. Cell. Biol. 33, 2436 –2446




FIGURE 12. Proposed model of cadmium-induced cell transformation and carcinogenesis. Chronic exposure of BEAS-2B cells to cadmium increases ROS, which induces cell transformation. After transformation, the transformed cells exhibit a property of autophagy dysfunction. Hyperactivation of p62 due to autophagy dysfunction activates positive feedback loop of Nrf2 and p62, which leads to up-regulation of antiapoptotic proteins (Bcl-2/Bcl-xL) and antioxidant enzymes (catalase, SOD1, and SOD2). Up-regulation of the antiapoptotic protein leads to apoptosis resistance. A low level of ROS due to up-regulation of the antioxidant enzymes causes less sensitivity to an apoptosis. Those factors contribute to cell proliferation and survival and carcinogenesis.

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Homeostatic imbalance in epithelial ducts and its role in carcinogenesis.

p62 in Cancer: Signaling Adaptor Beyond Autophagy.

Hypoxia-inducible factor 1 and its role in viral carcinogenesis.

CD95L signaling pathway: a role in carcinogenesis.

H2S and its role in redox signaling.

IL-1 Receptor-Associated Kinase Signaling and Its Role in Inflammation, Cancer Progression, and Therapy Resistance.

Apoptosis resistance-related ABCB5 and DNaseX (Apo10) expression in oral carcinogenesis.

SQSTM1 levels predicts radiotherapy resistance in hypopharyngeal carcinomas.

The role of Wnt signaling pathway in carcinogenesis and implications for anticancer therapeutics.

Chrysin and its emerging role in cancer drug resistance.

Cell-mediated immunity and its role in resistance to infection.

Role of mitochondria in carcinogenesis.

Aldehyde dehydrogenases and their role in carcinogenesis.

Role of Akt signaling in resistance to DNA-targeted therapy.

Role of carcinogenesis related mechanisms in cataractogenesis and its implications for ionizing radiation cataractogenesis.

The hepatic role in carcinogenesis and its early detection--the vinyl chloride model.

The role of the androgen receptor in ovarian cancer carcinogenesis and its clinical implications.

Hypoxia inducible factor-1α: Its role in colorectal carcinogenesis and metastasis.

The CK1 Family: Contribution to Cellular Stress Response and Its Role in Carcinogenesis.

Glutathione S-transferase in chemotherapy resistance and in carcinogenesis.

SOCS3 signaling pathway playing a regulatory role in ulcerative colitis carcinogenesis.

Sequestosome 1 as a signaling hub.

β-Catenin Signaling Pathway in Skin Carcinogenesis and Therapy.

Dysregulation of SOCS-Mediated Negative Feedback of Cytokine Signaling in Carcinogenesis and Its Significance in Cancer Treatment.