Abstract
Aim:
C/EBP homologous protein (CHOP) is a transcription factor that is activated at multiple levels during ER stress and plays an important role in ER stress-induced apoptosis. In this study we identified a novel CHOP activator, and further investigated its potential to be a therapeutic agent for human lung cancer.
Methods:
HEK293-CHOP-luc reporter cells were used in high-throughput screening (HTS) to identify CHOP activators. The cytotoxicity against cancer cells in vitro was measured with MTT assay. The anticancer effects were further examined in A549 human non-small cell lung cancer xenograft mice. The mechanisms underlying CHOP activation were analyzed using luciferase assays, and the anticancer mechanisms were elucidated in A549 cells.
Results:
From chemical libraries of 50 000 compounds, LGH00168 was identified as a CHOP activator, which showed cytotoxic activities against a panel of 9 cancer cell lines with an average IC50 value of 3.26 μmol/L. Moreover, administration of LGH00168 significantly suppressed tumor growth in A549 xenograft bearing mice. LGH00168 activated CHOP promoter via AARE1 and AP1 elements, increased DR5 expression, decreased Bcl-2 expression, and inhibited the NF-κB pathway. Treatment of A549 cells with LGH00168 (10 μmol/L) did not induce apoptosis, but lead to RIP1-dependent necroptosis, accompanied by cell swelling, plasma membrane rupture, lysosomal membrane permeabilization, MMP collapse and caspase 8 inhibition. Furthermore, LGH00168 (10 and 20 μmol/L) dose-dependently induced mito-ROS production in A549 cells, which was reversed by the ROS scavenger N-acetyl-L-cysteine (NAC, 10 mmol/L). Moreover, NAC significantly diminished LGH00168-induced CHOP activation, NF-κB inhibition and necroptosis in A549 cells.
Conclusion:
LGH00168 is a CHOP activator that inhibits A549 cell growth in vitro and lung tumor growth in vivo.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Stewart BW, Wild PC . World Cancer Report 2014. p 632.
Wang FW, Wang SQ, Zhao BX, Miao JY . Discovery of 2′-hydroxychalcones as autophagy inducer in A549 lung cancer cells. Org Biomol Chem 2014; 12: 3062–70.
Keith RL, Miller YE . Lung cancer chemoprevention: current status and future prospects. Nat Rev Clin Oncol 2013; 10: 334–43.
Pirker R . Novel drugs against non-small-cell lung cancer. Curr Opin Oncol 2014; 26: 145–51.
Goffin J, Lacchetti C, Ellis PM, Ung YC, Evans WK . First-line systemic chemotherapy in the treatment of advanced non-small cell lung cancer: a systematic review. J Thorac Oncol 2010; 5: 260–74.
Minguet J, Smith KH, Bramlage P . Targeted therapies for treatment of non-small cell lung cancer — recent advances and future perspectives. Int J Cancer 2016; 138: 2549–61.
Malhotra JD, Kaufman RJ . Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword? Antioxid Redox Signal 2007; 9: 2277–93.
Saveljeva S, Mc Laughlin SL, Vandenabeele P, Samali A, Bertrand MJ . Endoplasmic reticulum stress induces ligand-independent TNFR1-mediated necroptosis in L929 cells. Cell Death Dis 2015; 6: e1587.
Kitsis RN, Molkentin JD . Apoptotic cell death “Nixed” by an ER-mitochondrial necrotic pathway. Proc Natl Acad Sci U S A 2010; 107: 9031–2.
Janssen K, Horn S, Niemann MT, Daniel PT, Schulze-Osthoff K, Fischer U . Inhibition of the ER Ca2+ pump forces multidrug-resistant cells deficient in Bak and Bax into necrosis. J Cell Sci 2009; 122: 4481–91.
Logue SE, Cleary P, Saveljeva S, Samali A . New directions in ER stress-induced cell death. Apoptosis 2013; 18: 537–46.
van Schadewijk A, van't Wout EF, Stolk J, Hiemstra PS . A quantitative method for detection of spliced X-box binding protein-1 (XBP1) mRNA as a measure of endoplasmic reticulum (ER) stress. Cell Stress Chaperones 2012; 17: 275–9.
Lee AS . The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 2005; 35: 373–81.
Yang J, Zong YN, Zhou L, Liu QL . Close and allosteric opening of the polypeptide-binding site in a human Hsp70 chaperone BiP. Structure 2015; 23: 2191–203.
Dalton LE, Clarke HJ, Knight J, Lawson MH, Wason J, Lomas DA, et al. The endoplasmic reticulum stress marker CHOP predicts survival in malignant mesothelioma. Br J Cancer 2013; 108: 1340–7.
Nishitoh H . CHOP is a multifunctional transcription factor in the ER stress response. J Biochem 2012; 151: 217–9.
Kim I, Xu W, Reed JC . Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov 2008; 7: 1013–30.
Oyadomari S, Mori M . Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 2004; 11: 381–9.
Yamaguchi H, Wang HG . CHOP is involved in endoplasmic reticulum stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells. J Biol Chem 2004; 279: 45495–502.
Degterev A, Yuan J . Expansion and evolution of cell death programmes. Nat Rev Mol Cell Biol 2008; 9: 378–90.
Hengartner MO . The biochemistry of apoptosis. Nature 2000; 407: 770–6.
Majno G, Joris I . Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 1995; 146: 3–15.
Christofferson DE, Yuan J . Necroptosis as an alternative form of programmed cell death. Curr Opin Cell Biol 2010; 22: 263–8.
Thapa RJ, Nogusa S, Chen P, Maki JL, Lerro A, Andrake M, et al. Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases. Proc Natl Acad Sci U S A 2013; 110: E3109–18.
Guicciardi ME, Gores GJ . Life and death by death receptors. FASEB J 2009; 23: 1625–37.
Walczak H . Death receptor-ligand systems in cancer, cell death, and inflammation. Cold Spring Harb Perspect Biol 2013; 5: a008698.
Giampietri C, Starace D, Petrungaro S, Filippini A, Ziparo E . Necroptosis: molecular signalling and translational implications. Int J Cell Biol 2014; 2014: 490275.
Marshall KD, Baines CP . Necroptosis: is there a role for mitochondria? Front Physiol 2014; 5: 323.
Dillon CP, Weinlich R, Rodriguez DA, Cripps JG, Quarato G, Gurung P, et al. RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3. Cell 2014; 157: 1189–202.
Green DR, Oberst A, Dillon CP, Weinlich R, Salvesen GS . RIPK-dependent necrosis and its regulation by caspases: a mystery in five acts. Mol Cell 2011; 44: 9–16.
Degterev A, Hitomi J, Germscheid M, Chen IL, Korkina O, Teng X, et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol 2008; 4: 313–21.
Lin Y, Choksi S, Shen HM, Yang QF, Hur GM, Kim YS, et al. Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation. J Biol Chem 2004; 279: 10822–8.
Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 2005; 1: 112–9.
Jaramillo ML, Banville M, Collins C, Paul-Roc B, Bourget L, O'Connor-McCourt M . Differential sensitivity of A549 non-small lung carcinoma cell responses to epidermal growth factor receptor pathway inhibitors. Cancer Biol Ther 2008; 7: 557–68.
Zang Y, Yu LF, Pang T, Fang LP, Feng X, Wen TQ, et al. AICAR induces astroglial differentiation of neural stem cells via activating the JAK/STAT3 pathway independently of AMP-activated protein kinase. J Biol Chem 2008; 283: 6201–8.
McKeague AL, Wilson DJ, Nelson J . Staurosporine-induced apoptosis and hydrogen peroxide-induced necrosis in two human breast cell lines. Br J Cancer 2003; 88: 125–31.
Mpoke SS, Wolfe J . Differential staining of apoptotic nuclei in living cells: application to macronuclear elimination in Tetrahymena. J Histochem Cytochem 1997; 45: 675–83.
Qiu BY, Turner N, Li YY, Gu M, Huang MW, Wu F, et al. High-throughput assay for modulators of mitochondrial membrane potential identifies a novel compound with beneficial effects on db/db mice. Diabetes 2010; 59: 256–65.
Dive C, Gregory CD, Phipps DJ, Evans DL, Milner AE, Wyllie AH . Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry. Biochim Biophys Acta 1992; 1133: 275–85.
Kondo Y, Kanzawa T, Sawaya R, Kondo S . The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 2005; 5: 726–34.
Janku F, McConkey DJ, Hong DS, Kurzrock R . Autophagy as a target for anticancer therapy. Nat Rev Clin Oncol 2011; 8: 528–39.
Mansilla S, Bataller M, Portugal J . Mitotic catastrophe as a consequence of chemotherapy. Anticancer Agents Med Chem 2006; 6: 589–602.
Amaravadi RK, Thompson CB . The roles of therapy-induced autophagy and necrosis in cancer treatment. Clin Cancer Res 2007; 13: 7271–9.
Caffrey PB, Frenkel GD . Selenite cytotoxicity in drug resistant and nonresistant human ovarian tumor cells. Cancer Res 1992; 52: 4812–6.
Shilo S, Tirosh O . Selenite activates caspase-independent necrotic cell death in Jurkat T cells and J774.2 macrophages by affecting mitochondrial oxidant generation. Antioxid Redox Signal 2003; 5: 273–9.
Duanmu J, Cheng J, Xu J, Booth CJ, Hu Z . Effective treatment of chemoresistant breast cancer in vitro and in vivo by a factor VII-targeted photodynamic therapy. Br J Cancer 2011; 104: 1401–9.
Festjens N, Vanden Berghe T, Vandenabeele P . Necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response. Biochim Biophys Acta 2006; 1757: 1371–87.
Henriquez M, Armisen R, Stutzin A, Quest AF . Cell death by necrosis, a regulated way to go. Curr Mol Med 2008; 8: 187–206.
Murphy MP . How mitochondria produce reactive oxygen species. Biochem J 2009; 417: 1–13.
Zong WX, Thompson CB . Necrotic death as a cell fate. Genes Dev 2006; 20: 1–15.
Li L, Han W, Gu Y, Qiu S, Lu Q, Jin J, et al. Honokiol induces a necrotic cell death through the mitochondrial permeability transition pore. Cancer Res 2007; 67: 4894–903.
Miyoshi N, Watanabe E, Osawa T, Okuhira M, Murata Y, Ohshima H, et al. ATP depletion alters the mode of cell death induced by benzyl isothiocyanate. Biochim Biophys Acta 2008; 1782: 566–73.
Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (No 81072667, 81473244, and 91029716) and the State Key Laboratory of Natural and Biomimetic Drugs (K20130202).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ma, Ym., Peng, Ym., Zhu, Qh. et al. Novel CHOP activator LGH00168 induces necroptosis in A549 human lung cancer cells via ROS-mediated ER stress and NF-κB inhibition. Acta Pharmacol Sin 37, 1381–1390 (2016). https://doi.org/10.1038/aps.2016.61
Received:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/aps.2016.61
Keywords
This article is cited by
-
Normobaric hyperoxia re-sensitizes paclitaxel-resistant lung cancer cells
Molecular & Cellular Toxicology (2022)
-
USP18 alleviates neurotoxicity induced by sevoflurane via AKT and NF-κB pathways
Molecular & Cellular Toxicology (2022)
-
Shikonin induces glioma cell necroptosis in vitro by ROS overproduction and promoting RIP1/RIP3 necrosome formation
Acta Pharmacologica Sinica (2017)
-
2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-glucoside protects murine hearts against ischemia/reperfusion injury by activating Notch1/Hes1 signaling and attenuating endoplasmic reticulum stress
Acta Pharmacologica Sinica (2017)
-
Necrostatin-1 Mitigates Endoplasmic Reticulum Stress After Spinal Cord Injury
Neurochemical Research (2017)
