- Research article
- Open Access
- Open Peer Review
Niclosamide induced cell apoptosis via upregulation of ATF3 and activation of PERK in Hepatocellular carcinoma cells
- Shunyan Weng†1, 2, 3,
- Liang Zhou†2,
- Qing Deng2,
- Jiaxian Wang1,
- Yan Yu2,
- Jianwei Zhu1 and
- Yunsheng Yuan1, 2Email author
© Weng et al. 2016
- Received: 9 October 2015
- Accepted: 18 February 2016
- Published: 25 February 2016
Hepatocellular carcinoma (HCC) is one of most common and aggressive human malignancies in the world, especially, in eastern Asia, and its mortality is very high at any phase. We want to investigate mechanism of niclosamide inducing cell apoptosis in HCC.
Two hepatoma cell lines were used to evaluate activity of niclosamide inducing cell apoptosis and study its mechanism. Quantitative real-time PCR and western blotting were used in analysis of genes expression or protein active regulated by niclosamide.
Niclosamide remarkably induced cell apoptosis in hepatoma cells. Furthermore, our study revealed that RNA-dependent protein kinase-like kinase (PERK) is activated and its expression is up-regulated in HCC cells which are exposed to niclosamide. niclosamide also significantly increase activating transcription factor 3 (ATF3), activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein-homologous protein (CHOP) expression in HCC cells. It’s suggested that the function of niclosamide was abrogated by PERK inhibitor or absent ATF3. Expression of PERK and CHOP is correlated with ATF3 level in the cells.
Taken together, our results indicate that ATF3 plays an integral role in ER stress activated and cell apoptosis induced by niclosamide in HCC cells. In this study, the new mechanism of niclosamide as anti-cancer we investigated, too.
- Activating transcription factor 3
- Endoplasmic reticulum stress
- Reactive oxygen species
- Liver cancer
Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies in the world . It is a major public health issue worldwide according to epidemiological data, and the incidence is high in East Asia.
HCC often develops caused by chronic tissue damage due to liver cirrhosis which could be induced by HBV, HCV, alcohol intake, hemochromatosis, nonalcoholic steatohepatits and so on .
Surgical resection is the primary treatment option for patients with early stage of HCC . After surgical treatment, patients should accept chemotherapy. However the rate of total 5-years survival is very low due to side effects of chemotherapeutic drugs and the chemo-resistance of tumor cells. Therefore it is very important to develop new drugs for HCC treatment . It is well known that Endoplasmic Reticulum (ER) stress could induce cell apoptosis or cell death in many tumor categories, including breast cancer , neuroectodermal tumor , HCC , etc. ER stress could activate unfolded protein response (UPR) whose signaling network consists of three stress sensors, namely protein kinase RNA like ER kinase (PERK) or RNA-dependent protein kinase-like kinase, inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6) . In cancer cells, UPR could be activated by exposure to hypoxia, oxidative stress and nutrient starvation. The tumor microenvironment usually features hypoxia and high content of reactive oxygen species (ROS) because of the highly active proliferation and metabolism status of cancer cells. Hypoxia and ROS could direct PERK activation, which in turn activates eukaryotic translation initiation factor 2α (eIF2α) which would induce the expression of activating transcription factor 4 (ATF4) for regulating redox homeostasis and metabolic homeostasis . However CCAAT/enhancer-binding protein-homologous protein (CHOP, also known as GADD153), a downstream element to ATF4 in the PERK pathway, induces apoptosis or cell death under intensive or prolonged ER stress conditions [9, 10]. Interestingly, activating transcription factor 3 (ATF3), a bZIP DNA-binding protein, is associated with CHOP and thus integrated into the PERK/eIF2α pathway under ER stress backgrounds . Expression of ATF3 could be induced by ER stress and involved in regulation of cell apoptosis . In the tumors, ATF3 might induce cell apoptosis or improve cell survival depending on tumor types. Currently, several studies have shown that ATF3 plays tumor suppressing roles in different cancer types, including colon cancer  and esophageal squamous cell carcinomas (ESCC) . It’s also reported that the overexpression of ATF3 suppresses growth of HeLa cells . Other data showed that niclosamide, an antihelminthic drug for treatment of tapeworm infections approved by FDA, has exhibited anticancer function in different tumor types, including leukemia, colon cancer, glioma, etc . We hypothesized that niclosamide also has effective function in anti-HCC. In this study, We demonstrated a new mechanism of niclosamide as anti-cancer with HCC cells.
Cell culture and drug treatment
HepG2 and QGY7701 cell lines were obtained from the Cell Bank of Shanghai Institute of Cell Biology (Chinese Academy of Sciences, Shanghai) and maintained in DMED (Hyclone, Logan, UT,) high glucose medium, supplemented with 10 % FBS (Gibco, NY). All cells were cultured in humidified 37 °C incubator supplied with 5 % CO2. Niclosamide (Sigma-Aldrich, St. Louis, MO) was dissolved in DMSO (Sigma-Aldrich, St. Louis, MO). To explore the regulation of niclosamide upon signal pathways, cells were seeded in 6-well plate and incubated for 24 h. Then cells were fed with fresh medium containing different concentrations of niclosamide or DMSO only as control. After 24 h of niclosamide treatment, cells was lysed with 1 % SDS lysis buffer (1%SDS,25 mM EDTA, 45 mM Tris-HCl, ph6.5) for western analysis, and total RNA was isolated directly with Trizol reagent (Life technologies, CA). In order to block ER stress, GSK2606414 (Selleck Chemicals, Houston, TX) was used to pretreat cells for 1 h before cells treated with niclosamide.
DNA constructs and lenti-virus packaging
Oligo of ATF3 shRNA was synthesized and inserted in pGV298 lentiviral vector (GeneChem, Shanghai). The ATF3 target sequence was 5’-GCAAAGTGCCGAAACAAGA-3’ according to as described in publication . The control GFP shRNA plasmid was purchased from GeneChem inc (GeneChem, Shanghai). The lentiviral vector is cotransfected with pVSV-G, pRev, pTat and pGag-pol (Gifts from Dr. Cheng, UMCM, Baltimore) in order to produce lentivirus particles in HEK293T cells with lipfectimin2000 (life technologies, CA) and supernatant were collected and were used to transduce HepG2 cells.
Stable ATF3 knockout HepG2 cell line
HepG2 cells were planted to 6-well plate and incubated overnight. Cells were fed with medium contained ATF3 shRNA lentivirus particles and 10 μg.ml−1 polybrene(Santa Cruz, CA) which could improve the efficiency of lentivirus transduction. The medium was changed after 24 h of lentivirus transduction. Positive cells were selected with 10 μM puromycin (Santa Cruz, CA) and efficiency of ATF3 knockout was analyzed with western blotting.
Cell viability assay
HepG2 and QGY7701 cells were seeded to 96-well plates and incubated overnight. Cells were fed with fresh complete medium containing different concentrations of niclosamide every 24 h and maintained for 3 days. Then cell viability was analyzed with CCK8 kit (Dojindo, Japan). Data were normalized with control group and presented as average ± SD. For the analysis of cell viability in ATF3 knock-down HepG2 and control cell lines, HepG2-ATF3KD and HepG2-control cells were planted in 96-well plates. Cells were treated with niclosamide or DMSO and cell number was counted every day for three days. Data was presented as average ± SD. Student t-test was used in statist analysis.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay
TUNEL assay was performed to analyze cells apoptosis induced by niclosamide. HepG2 and QGY7701 cells grown on polylysine-coated cover slides were fixed with 4 % Paraformalclehyde after 24 h of 10 μM niclosamide treatment. TUNEL APO-GREEN detect kit (biotool, Shanghai, China) was used in DNA labeling according to manufacturer’s instruction. Images were taken by NIKON fluorescence microscope (Nikon, Japan).
HepG2 and QGY7701 cells were treated with niclosamide, niclosamide/GSK2606414 or DMSO as control. Cells were harvested at 24 h of drug treatment and washed twice with cold PBS on ice. Cell pellets were resuspended with PBS and FITC-Annexin V Apoptosis Detection Kit (Wanlei Bio, Shenyang, China) was used to stain cells according to manufacturer’s manual book. Cells apoptosis were analyzed with flow cytometer (BD Bioscience, US).
RNA preparation and quantitative real-time PCR
Primers for qRT-PCR
Western immunoblotting was performed as previously described . The primary antibodies used in this study were anti-cleaved caspase 3 and anti- phospho-eIF2α (Ser51) (Cell signaling tech, Danvers), anti-GAPDH (Proteintech, Chicago), anti-ATF4 (Wanlei Bio, Shenyang, China), anti-GADD153/CHOP (Wanlei Bio, Shenyang,China), anti-ATF3, anti-eIF2α, anti-pPERK and anti-PERK (Santa Cruz biotech, Santa Cruz). HRP conjunct secondary Antibodies were purchased from Jackson immune Research-laboratories (Western Grove, PA). The PVDF membrane was purchased from Millipore (Millipore, Billerica) and ECL substrate was purchased from Thermo Fisher (Thermo Fisher, Waltham).
Immunofluorescence staining and confocal microscopy
HepG2 and QGY7701 cells were grown on polylysine-coated cover slides and were fixed with cold methanol after 24 h of 2 μM niclosamide treatment. The cells were incubated in block buffer (3%BSA in TBST) at room temperature (RT) for 30 min. Then, anti-ATF3 or anti-GADD153/CHOP antibodies was diluted to 1:100 in blotting buffer (1 % BSA in TBST with 0.3 % TritonX-100) and were used to blot cells overnight at 4 °C. After the cells were washed three times with TBST, Alex®488-goat anti-rabbit IgG (Life technologies, CA) were used to incubate cells at RT for 1 h. Cell nucleus was stained with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO). Confocal images were taken with Leica SP8 confocal microscope (Leica, Wetzlar, Germany).
All calculations and statistical analyses were performed with Excel software (Microsoft, WA). Student t test was used in comparing two groups in experiments. All data were presented as average ± SD. All tests were two-sided, and P values less than 0.05 were considered to be statistically significant.
Niclosamide suppressed cells growth by inducing ER-stress in HCC cells
Niclosamide increased nuclear accumulation of ATF3 and CHOP in HCC
Niclosamide induced apoptosis in the HCC suppressed by PERK inhibition
ATF3 upregulation during cell apoptosis induced by niclosamide
In this study, our data suggested that niclosamide remarkably induced cell apoptosis (Fig. 1b) and increase expression level of PERK and its down-stream genes in HCCs (Fig. 2). It’s known that sustaining ER stress could sequentially activate IRE1α, ATF6 and PERK pathways [4, 8]. PERK/ATF4/CHOP pathway is one of most important pathways to induce cancer cell apoptosis in the UPR . ATF3 is an important factor for stress response in cells and it interact with ATF4 and CHOP, these two transcription factors being PERK targeting genes under ER stress conditions [5, 9]. Current study demonstrated that ATF3 was involved in regulation of PERK function . We further tried to reveal whether their protein levels are related with dose of niclosamide or not. Data of western blotting indicated that niclosamide upregulates PERK, ATF4, ATF3 and CHOP depending on the niclosamide concentration (Fig. 3). The results also demonstrated that caspase-3 is activated under niclosamide treatment (Fig. 3). Many studies had shown that niclosamide blocked cancer cells proliferation in a number of tumors types by suppressing the activities of several oncogenic pathways, including NF-kB, STAT3, notch and Wnt [20-22]. It also increases the level of reactive oxygen species (ROS) in acute myelogenous leukemia cells and enhances sensitivity to ROS in lung cancer cells . Recently, it was identified that niclosamide could suppress HCC proliferation by blocking Wnt pathway . Interestingly, we found that PERK pathway should play a critical role in HCC apoptosis induced by niclosamide. The apoptosis-inducing role of niclosamide in HCC could be abrogated by PERK inhibitor, GSK2606414 (Fig. 5). Several studies have suggested PERK/ATF4/CHOP pathway being involved in regulation of cells apoptosis and death in HCC [6, 25, 26].
Our study reveals that niclosamide activates PERK and up-regulates both of ATF3 and CHOP expression in HCCs. The function of niclosamide could be abrogated by PERK inhibitor and suppression of ATF3 expression. Especially, ATF3 upregulates PERK and CHOP level in HCCs being exposed to niclosamide. Our data indicates that ATF3 plays a central role in the induction of cell apoptosis by niclosamide in HCC.
We would like to thank Weifeng Ma and Hao Xu from Shanghai Jiao Tong University(SJTU) help us to prepare some solutions and Evhy He from SJTU help us to review the manuscript. This work was supported by the National Natural and Science Foundation of China (Yunsheng Yuan, 81302825) and the Shanghai Key Laboratory of Veterinary Biotechnology (Yunsheng Yuan, klab201501).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Worns MA, Galle PR. HCC therapies--lessons learned. Nat Rev Gastroenterol Hepatol. 2014;11(7):447–52.View ArticlePubMedGoogle Scholar
- Tejeda-Maldonado J, Garcia-Juarez I, Aguirre-Valadez J, Gonzalez-Aguirre A, Vilatoba-Chapa M, Armengol-Alonso A, Escobar-Penagos F, Torre A, Sanchez-Avila JF, Carrillo-Perez DL. Diagnosis and treatment of hepatocellular carcinoma: an update. World J Hepatol. 2015;7(3):362–76.PubMed CentralView ArticlePubMedGoogle Scholar
- Greten TF, Wang XW, Korangy F. Current concepts of immune based treatments for patients with HCC: from basic science to novel treatment approaches. Gut. 2015;64(5):842–8.View ArticlePubMedGoogle Scholar
- Moenner M, Pluquet O, Bouchecareilh M, Chevet E. Integrated endoplasmic reticulum stress responses in cancer. Cancer Res. 2007;67(22):10631–4.View ArticlePubMedGoogle Scholar
- Armstrong JL, Flockhart R, Veal GJ, Lovat PE, Redfern CP. Regulation of endoplasmic reticulum stress-induced cell death by ATF4 in neuroectodermal tumor cells. J Biol Chem. 2009;285(9):6091–100.PubMed CentralView ArticlePubMedGoogle Scholar
- Koh IU, Lim JH, Joe MK, Kim WH, Jung MH, Yoon JB, Song J. AdipoR2 is transcriptionally regulated by ER stress-inducible ATF3 in HepG2 human hepatocyte cells. FEBS J. 2010;277(10):2304–17.View ArticlePubMedGoogle Scholar
- Wang WA, Groenendyk J, Michalak M. Endoplasmic reticulum stress associated responses in cancer. Biochim Biophys Acta. 2014;1843(10):2143–9.View ArticlePubMedGoogle Scholar
- Kato H, Nishitoh H. Stress responses from the endoplasmic reticulum in cancer. Front Oncol. 2015;5:93.PubMed CentralView ArticlePubMedGoogle Scholar
- Xu L, Su L, Liu X. PKCdelta regulates death receptor 5 expression induced by PS-341 through ATF4-ATF3/CHOP axis in human lung cancer cells. Mol Cancer Ther. 2012;11(10):2174–82.View ArticlePubMedGoogle Scholar
- Liao Y, Fung TS, Huang M, Fang SG, Zhong Y, Liu DX. Upregulation of CHOP/GADD153 during coronavirus infectious bronchitis virus infection modulates apoptosis by restricting activation of the extracellular signal-regulated kinase pathway. J Virol. 2013;87(14):8124–34.PubMed CentralView ArticlePubMedGoogle Scholar
- Park SH, Kim J, Do KH, Park J, Oh CG, Choi HJ, Song BG, Lee SJ, Kim YS, Moon Y. Activating transcription factor 3-mediated chemo-intervention with cancer chemokines in a noncanonical pathway under endoplasmic reticulum stress. J Biol Chem. 2014;289(39):27118–33.PubMed CentralView ArticlePubMedGoogle Scholar
- Spohn D, Rossler OG, Philipp SE, Raubuch M, Kitajima S, Griesemer D, Hoth M, Thiel G. Thapsigargin induces expression of activating transcription factor 3 in human keratinocytes involving Ca2+ ions and c-Jun N-terminal protein kinase. Mol Pharmacol. 2010;78(5):865–76.View ArticlePubMedGoogle Scholar
- Xie JJ, Xie YM, Chen B, Pan F, Guo JC, Zhao Q, Shen JH, Wu ZY, Wu JY, Xu LY, et al. ATF3 functions as a novel tumor suppressor with prognostic significance in esophageal squamous cell carcinoma. Oncotarget. 2014;5(18):8569–82.PubMed CentralView ArticlePubMedGoogle Scholar
- Lu D, Chen J, Hai T. The regulation of ATF3 gene expression by mitogen-activated protein kinases. Biochem J. 2007;401(2):559–67.PubMed CentralView ArticlePubMedGoogle Scholar
- Xiang D, Yuan Y, Chen L, Liu X, Belani C, Cheng H. Niclosamide, an anti-helminthic molecule, downregulates the retroviral oncoprotein Tax and pro-survival Bcl-2 proteins in HTLV-1-transformed T lymphocytes. Biochem Biophys Res Commun. 2015;464(1):221–8.View ArticlePubMedGoogle Scholar
- Wang H, Mo P, Ren S, Yan C. Activating transcription factor 3 activates p53 by preventing E6-associated protein from binding to E6. J Biol Chem. 2010;285(17):13201–10.PubMed CentralView ArticlePubMedGoogle Scholar
- Yuan Y, Wu X, Ou Q, Gao J, Tennant BC, Han W, Yu Y. Differential expression of the genes involved in amino acids and nitrogen metabolisms during liver regeneration of mice. Hepatol Res. 2009; 39(3):301–312.View ArticlePubMedGoogle Scholar
- Dicks N, Gutierrez K, Michalak M, Bordignon V, Agellon LB. Endoplasmic reticulum stress, genome damage, and cancer. Front Oncol. 2015;5:11.PubMed CentralView ArticlePubMedGoogle Scholar
- Edagawa M, Kawauchi J, Hirata M, Goshima H, Inoue M, Okamoto T, Murakami A, Maehara Y, Kitajima S. Role of activating transcription factor 3 (ATF3) in endoplasmic reticulum (ER) stress-induced sensitization of p53-deficient human colon cancer cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through up-regulation of death receptor 5 (DR5) by zerumbone and celecoxib. J Biol Chem. 2014;289(31):21544–61.Google Scholar
- Jin Y, Lu Z, Ding K, Li J, Du X, Chen C, Sun X, Wu Y, Zhou J, Pan J. Antineoplastic mechanisms of niclosamide in acute myelogenous leukemia stem cells: inactivation of the NF-kappaB pathway and generation of reactive oxygen species. Cancer Res. 2010;70(6):2516–27.View ArticlePubMedGoogle Scholar
- Li R, Hu Z, Sun SY, Chen ZG, Owonikoko TK, Sica GL, Ramalingam SS, Curran WJ, Khuri FR, Deng X. Niclosamide overcomes acquired resistance to erlotinib through suppression of STAT3 in non-small cell lung cancer. Mol Cancer Ther. 2013;12(10):2200–12.PubMed CentralView ArticlePubMedGoogle Scholar
- Osada T, Chen M, Yang XY, Spasojevic I, Vandeusen JB, Hsu D, Clary BM, Clay TM, Chen W, Morse MA, et al. Antihelminth compound niclosamide downregulates Wnt signaling and elicits antitumor responses in tumors with activating APC mutations. Cancer Res. 2011;71(12):4172–82.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee SL, Son AR, Ahn J, Song JY. Niclosamide enhances ROS-mediated cell death through c-Jun activation. Biomed Pharmacother. 2014;68(5):619–24.View ArticlePubMedGoogle Scholar
- Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S, Sueishi M, Yoshida T. Niclosamide suppresses Hepatoma cell proliferation via the Wnt pathway. Onco Targets Ther. 2013;6:1685–93.PubMed CentralView ArticlePubMedGoogle Scholar
- Shi YH, Ding ZB, Zhou J, Hui B, Shi GM, Ke AW, Wang XY, Dai Z, Peng YF, Gu CY, et al. Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis. Autophagy. 2011;7(10):1159–72.View ArticlePubMedGoogle Scholar
- Chen YJ, Su JH, Tsao CY, Hung CT, Chao HH, Lin JJ, Liao MH, Yang ZY, Huang HH, Tsai FJ, et al. Sinulariolide induced hepatocellular carcinoma apoptosis through activation of mitochondrial-related apoptotic and PERK/eIF2alpha/ATF4/CHOP pathway. Molecules. 2013;18(9):10146–61.View ArticlePubMedGoogle Scholar
- Jiang HY, Wek SA, McGrath BC, Lu D, Hai T, Harding HP, Wang X, Ron D, Cavener DR, Wek RC. Activating transcription factor 3 is integral to the eukaryotic initiation factor 2 kinase stress response. Mol Cell Biol. 2004;24(3):1365–77.PubMed CentralView ArticlePubMedGoogle Scholar
- Xiaoyan L, Shengbing Z, Yu Z, Lin Z, Chengjie L, Jingfeng L, Aimin H. Low expression of activating transcription factor 3 in human hepatocellular carcinoma and its clinicopathological significance. Pathol Res Pract. 2014;210(8):477–81.View ArticlePubMedGoogle Scholar