Cell Stress and Chaperones

, Volume 18, Issue 6, pp 733–743 | Cite as

Comparative cytoprotective effects of carbocysteine and fluticasone propionate in cigarette smoke extract-stimulated bronchial epithelial cells

  • Elisabetta PaceEmail author
  • Maria Ferraro
  • Serena Di Vincenzo
  • Chiara Cipollina
  • Stefania Gerbino
  • Diego Cigna
  • Valentina Caputo
  • Rossella Balsamo
  • Luigi Lanata
  • Mark Gjomarkaj
Original Paper


Cigarette smoke extracts (CSE) induce oxidative stress, an important feature in chronic obstructive pulmonary disease (COPD), and oxidative stress contributes to the poor clinical efficacy of corticosteroids in COPD patients. Carbocysteine, an antioxidant and mucolytic agent, is effective in reducing the severity and the rate of exacerbations in COPD patients. The effects of carbocysteine on CSE-induced oxidative stress in bronchial epithelial cells as well as the comparison of these antioxidant effects of carbocysteine with those of fluticasone propionate are unknown. The present study was aimed to assess the effects of carbocysteine (10−4 M) in cell survival and intracellular reactive oxygen species (ROS) production (by flow cytometry) as well as total glutathione (GSH), heme oxygenase-1 (HO-1), nuclear-related factor 2 (Nrf2) expression and histone deacetylase 2 (HDAC-2) expression/activation in CSE-stimulated bronchial epithelial cells (16-HBE) and to compare these effects with those of fluticasone propionate (10−8 M). CSE, carbocysteine or fluticasone propionate did not induce cell necrosis (propidium positive cells) or cell apoptosis (annexin V-positive/propidium-negative cells) in 16-HBE. CSE increased ROS production, nuclear Nrf2 and HO-1 in 16-HBE. Fluticasone propionate did not modify intracellular ROS production, GSH and HDCA-2 but reduced Nrf2 and HO-1 in CSE-stimulated 16-HBE. Carbocysteine reduced ROS production and increased GSH, HO-1, Nrf2 and HDAC-2 nuclear expression/activity in CSE-stimulated cells and was more effective than fluticasone propionate in modulating the CSE-mediated effects. In conclusion, the present study provides compelling evidences that the use of carbocysteine may be considered a promising strategy in diseases associated with corticosteroid resistance.


Cigarette smoke Airway epithelial cells Reactive oxygen species 



Cigarette smoke extracts




Fluticasone propionate


Reactive oxygen species




Heme oxygenase-1


Nuclear-related factor 2


Histone deacetylase 2



This work was mainly supported by the Italian National Research Council and by Dompè, Italy. Elisabetta Pace declares that she has had access to and takes responsibility for the integrity of the data and the accuracy of the data analysis.


  1. Adcock IM, Marwick J, Casolari P, Contoli M, Chung KF, Kirkham P, Papi A, Caramori G (2010) Mechanisms of corticosteroid resistance in severe asthma and chronic obstructive pulmonary disease (COPD). Curr Pharm Des 16:3554–3573PubMedCrossRefGoogle Scholar
  2. Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL (2009) Glutathione dysregulation and the etiology and progression of human diseases. Biol Chem 390:191–214PubMedCrossRefGoogle Scholar
  3. Barnes PJ (2011) Glucocorticosteroids: current and future directions. Br J Pharmacol 163:29–43PubMedCrossRefGoogle Scholar
  4. Barnes PJ, Adcock IM (2009) Glucocorticoid resistance in inflammatory diseases. Lancet 373:1905–1917PubMedCrossRefGoogle Scholar
  5. Benhar M, Forrester MT, Stamler JS (2009) Protein denitrosylation: enzymatic mechanisms and cellular functions. Nat Rev Mol Cell Biol 10:721–732PubMedGoogle Scholar
  6. Biswas SK, Rahman I (2009) Environmental toxicity, redox signaling and lung inflammation: the role of glutathione. Mol Aspects Med 30:60–76PubMedCrossRefGoogle Scholar
  7. Bruno A et al (2011) Apigenin affects leptin/leptin receptor pathway and induces cell apoptosis in lung adenocarcinoma cell line. Eur J Cancer 47:2042–2051PubMedCrossRefGoogle Scholar
  8. Brusselle GG, Joos GF, Bracke KR (2011) New insights into the immunology of chronic obstructive pulmonary disease. Lancet 378:1015–1026PubMedCrossRefGoogle Scholar
  9. Cosio BG, Tsaprouni L, Ito K, Jazrawi E, Adcock IM, Barnes PJ (2004) Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages. J Exp Med 200:689–695PubMedCrossRefGoogle Scholar
  10. Cozens AL, Yezzi MJ, Yamaya M et al (1992) A transformed human epithelial cell line that retains tight junctions post crisis. In Vitro Cell Dev Biol 28A:735–744PubMedCrossRefGoogle Scholar
  11. Dolinay T, Choi AM, Ryter SW (2012) Heme oxygenase-1/CO as protective mediators in cigarette smoke-induced lung cell injury and chronic obstructive pulmonary disease. Curr Pharm Biotechnol 13:769–776PubMedCrossRefGoogle Scholar
  12. Drost EM, Skwarski KM, Sauleda J, Soler N, Roca J, Agusti A, MacNee W (2005) Oxidative stress and airway inflammation in severe exacerbations of COPD. Thorax 60:293–300PubMedCrossRefGoogle Scholar
  13. Faux SP, Tai T, Thorne D, Xu Y, Breheny D, Gaca M (2009) The role of oxidative stress in the biological responses of lung epithelial cells to cigarette smoke. Biomarkers 14:90–96PubMedCrossRefGoogle Scholar
  14. Garavaglia ML, Bononi E, Dossena S, Mondini A, Bazzini C, Lanata L, Balsamo R, Bagnasco M, Conese M, Bottà G, Paulmichl M, Meyer G (2008) S-CMC-Lys protective effects on human respiratory cells during oxidative stress. Cell Physiol Biochem 22:455–464PubMedCrossRefGoogle Scholar
  15. Ghezzi P (2011) Role of glutathione in immunity and inflammation in the lung. Int J Gen Med 4:105–113PubMedCrossRefGoogle Scholar
  16. Goven D, Boutten A, Leçon-Malas V, Boczkowski J, Bonay M (2009) Prolonged cigarette smoke exposure decreases heme oxygenase-1 and alters NRF2 and Bach1 expression in human macrophages: roles of the MAP kinases ERK(1/2) and JNK. FEBS Lett 583:3508–3518PubMedCrossRefGoogle Scholar
  17. Hanaoka M, Droma Y, Chen Y, Agatsuma T, Kitaguchi Y, Voelkel NF, Kubo K (2011) Carbocisteine protects against emphysema induced by cigarette smoke extract in rats. Chest 139:1101–1108PubMedCrossRefGoogle Scholar
  18. Ito K, Yamamura S, Essilfie-Quaye S, Cosio B, Ito M, Barnes PJ, Adcock IM (2006) Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NF-kappaB suppression. J Exp Med 203:7–13PubMedCrossRefGoogle Scholar
  19. Kosmider B, Messier EM, Chu HW, Mason RJ (2011) Human alveolar epithelial cell injury induced by cigarette smoke. PLoS One 6:e26059PubMedCrossRefGoogle Scholar
  20. Kratschmar DV, Calabrese D, Walsh J, Lister A, Birk J, Appenzeller-Herzog C, Moulin P, Goldring CE, Odermatt A (2012) Suppression of the Nrf2-dependent antioxidant response by glucocorticoids and 11β-HSD1-mediated glucocorticoid activation in hepatic cells. PLoS One 7:e36774PubMedCrossRefGoogle Scholar
  21. Luppi F, Aarbiou J, van Wetering S, Rahman I, de Boer WI, Rabe KF, Hiemstra PS (2005) Effects of cigarette smoke condensate on proliferation and wound closure of bronchial epithelial cells in vitro: role of glutathione. Respir Res 6:140PubMedCrossRefGoogle Scholar
  22. Maestrelli P, Páska C, Saetta M, Turato G, Nowicki Y, Monti S, Formichi B, Miniati M, Fabbri LM (2003) Decreased haem oxygenase-1 and increased inducible nitric oxide synthase in the lung of severe COPD patients. Eur Respir J 21:971–976PubMedCrossRefGoogle Scholar
  23. Malhotra D, Thimmulappa RK, Mercado N, Ito K, Kombairaju P, Kumar S, Ma J, Feller-Kopman D, Wise R, Barnes P, Biswal S (2011) Denitrosylation of HDAC2 by targeting Nrf2 restores glucocorticosteroid sensitivity in macrophages from COPD patients. J Clin Invest 121:4289–4302PubMedCrossRefGoogle Scholar
  24. Mizuno S, Yasuo M, Bogaard HJ, Kraskauskas D, Natarajan R, Voelkel NF (2011) Inhibition of histone deacetylase causes emphysema. Am J Physiol Lung Cell Mol Physiol 300:L402–L413PubMedCrossRefGoogle Scholar
  25. Moodie FM, Marwick JA, Anderson CS, Szulakowski P, Biswas SK, Bauter MR, Kilty I, Rahman I (2004) Oxidative stress and cigarette smoke alter chromatin remodeling but differentially regulate NF-kappaB activation and proinflammatory cytokine release in alveolar epithelial cells. FASEB J 18:1897–1899PubMedGoogle Scholar
  26. Morse D, Choi AM (2002) Heme oxygenase-1: the “emerging molecule” has arrived. Am J Respir Cell Mol Biol 27:8–16PubMedCrossRefGoogle Scholar
  27. Nogawa H et al (2009) Carbocisteine can scavenge reactive oxygen species in vitro. Respirology 14:53–59PubMedCrossRefGoogle Scholar
  28. Pace E, Bruno TF, Berenger B, Mody CH, Melis M, Ferraro M, Tipa A, Bruno A, Profita M, Bonsignore G, Gjomarkaj M (2007) Elevated expression of prostaglandin receptor and increased release of prostaglandin E2 maintain the survival of CD45RO+ T cells in the inflamed human pleural space. Immunology 121:427–436PubMedCrossRefGoogle Scholar
  29. Pace E, Ferraro M, Siena L, Melis M, Montalbano A, Johnson M, Bonsignore MR, Bonsignore G, Gjomarkaj M (2008a) Cigarette smoke increases TLR4 and modifies LPS mediated responses in airway epithelial cells. Immunology 124:401–411PubMedCrossRefGoogle Scholar
  30. Pace E, Ferraro M, Mody CH, Melis M, Bonanno A, Profita M, Giarratano A, Gjomarkaj M (2008b) Pleural mesothelial cells express both BLT2 and PPARa and mount an integrated response to pleural LTB4. J Immunol 181:7292–7299PubMedGoogle Scholar
  31. Pace E, Scafidi V, Di Bona D, Siena L, Chiappara G, Ferraro M, La Grutta S, Gallina S, Speciale R, Ballacchino A, Bachert C, Bousquet J, Gjomarkaj M (2012a) Increased expression of IL-19 in the epithelium of patients with chronic rhinosinusitis and nasal polyps. Allergy 67:878–886CrossRefGoogle Scholar
  32. Pace E, Ferraro M, Minervini MI, Vitulo P, Pipitone L, Chiappara G, Siena L, Montalbano AM, Johnson M, Gjomarkaj M (2012b) Beta defensin-2 is reduced in central but not in distal airways of smoker COPD patients. PLoS One 7:e33601CrossRefGoogle Scholar
  33. Puchelle E, Zahm JM, Tournier JM, Coraux C (2006) Airway epithelial repair, regeneration, and remodeling after injury in chronic obstructive pulmonary disease. Proc Am Thorac Soc 3:726–733PubMedCrossRefGoogle Scholar
  34. Rahman I, Macnee W (2012) Antioxidant pharmacological therapies for COPD. Curr Opin Pharmacol 12:256–265PubMedCrossRefGoogle Scholar
  35. Rahman I, Kode A, Biswas SK (2006) Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc 1:3159–3165PubMedCrossRefGoogle Scholar
  36. Raval CM, Lee PJ (2010) Heme oxygenase-1 in lung disease. Curr Drug Targets 11:1532–1540PubMedCrossRefGoogle Scholar
  37. Shakespear MR, Halili MA, Irvine KM, Fairlie DP, Sweet MJ (2011) Histone deacetylases as regulators of inflammation and immunity. Trends Immunol 32:335–343PubMedCrossRefGoogle Scholar
  38. Su Y, Han W, Giraldo C, De Li Y, Block ER (1998) Effect of cigarette smoke extract on nitric oxide synthase in pulmonary artery endothelial cells. Am J Respir Cell Mol Biol 19:819–825PubMedCrossRefGoogle Scholar
  39. Yang SR et al (2006) Cigarette smoke induces proinflammatory cytokine release by activation of NF-kappaB and posttranslational modifications of histone deacetylase in macrophages. Am J Physiol Lung Cell Mol Physiol 291:L46–L57PubMedCrossRefGoogle Scholar
  40. Yasuda H, Yamaya M, Sasaki T, Inoue D, Nakayama K, Yamada M, Asada M, Yoshida M, Suzuki T, Nishimura H, Sasaki H (2006) Carbocisteine inhibits rhinovirus infection in human tracheal epithelial cells. Eur Respir J 28:51–58PubMedCrossRefGoogle Scholar
  41. Zhang H, Forman HJ (2008) Acrolein induces heme oxygenase-1 through PKC-delta and PI3K in human bronchial epithelial cells. Am J Respir Cell Mol Biol 38:483–490PubMedCrossRefGoogle Scholar
  42. Zheng JP, Kang J, Huang SG, Chen P, Yao WZ, Yang L, Bai CX, Wang CZ, Wang C, Chen BY, Shi Y, Liu CT, Chen P, Li Q, Wang ZS, Huang YJ, Luo ZY, Chen FP, Yuan JZ, Yuan BT, Qian HP, Zhi RC, Zhong NS (2008) Effect of carbocisteine on acute exacerbation of chronic obstructive pulmonary disease (PEACE study): a randomised placebo-controlled study. Lancet 371:2013–2018PubMedCrossRefGoogle Scholar

Copyright information

© Cell Stress Society International 2013

Authors and Affiliations

  • Elisabetta Pace
    • 1
    • 6
    Email author
  • Maria Ferraro
    • 1
  • Serena Di Vincenzo
    • 1
    • 2
  • Chiara Cipollina
    • 1
    • 3
  • Stefania Gerbino
    • 1
  • Diego Cigna
    • 1
  • Valentina Caputo
    • 4
  • Rossella Balsamo
    • 5
  • Luigi Lanata
    • 5
  • Mark Gjomarkaj
    • 1
  1. 1.Institute of Biomedicine and Molecular ImmunologyNational Research Council—PalermoPalermoItaly
  2. 2.Scienze e Biotecnologie Mediche e Sperimentali-Pneumologia Sperimentale e ClinicaUniversità degli Studi—PalermoPalermoItaly
  3. 3.Fondazione Ri.MedPalermoItaly
  4. 4.Department of DermatologyUniversity of PalermoPalermoItaly
  5. 5.Medical AffairDompé SPA MilanMilanItaly
  6. 6.Istituto di Biomedicina e Immunologia MolecolareConsiglio Nazionale delle RicerchePalermoItaly

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