Pathology & Oncology Research

, Volume 21, Issue 1, pp 103–111

Autophagy Interplays with Apoptosis and Cell Cycle Regulation in the Growth Inhibiting Effect of Trisenox in HEP-2, a Laryngeal Squamous Cancer

  • Débora Lima Pereira
  • Ana Carolina dos Santos Ferreira
  • Giselle Pinto de Faria
  • Jolie Kiemlian Kwee


Laryngeal squamous cell carcinoma (LSCC) is the most common among several types of head and neck cancers. Current treatments have a poor effect on early and advanced cases, and further investigations for novel agents against LSCCs are desirable. In this study, we elucidate the cytotoxic enhancing effect of arsenic trioxide (As2O3) combined with L-buthionine sulfoximine (BSO) in LSCC. The effect of BSO with As2O3 or Cisplatin (CDDP) on cell viability was examined using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The reactive oxygen species (ROS) levels, cell cycle, and apoptosis were measured by flow cytometry using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), propidium iodide (PI) and annexin V/PI. The acidic vacuolar organelles were visualized by fluorescence microscope and quantified using flow cytometry. Neither CDDP nor As2O3 when used alone reduced the cell viability. BSO was found to enhance only As2O3 sensitivity, leading to G2/M arrest and autophagy with no correlation of ROS induction. This result suggests that modulation of glutathione enhances autophagy, which interplays with apoptosis. In this study, we obtained initial preclinical evidence for the potential efficacy of these drugs in a combined therapy protocol.


Laryngeal squamous cancer Trisenox L-buthionine sulfoximine Autophagy Apoptosis 



Head and neck squamous cell carcinomas


Laryngeal squamous cell carcinoma




Arsenic Trioxide


L-buthionine sulfoximine


3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide


2′,7′-dichlorodihydrofluorescein diacetate


Propidium iodide






Acridine orange


Hydrogen peroxide


Reactive oxygen species


Acidic vacuolar organelle

Supplementary material

12253_2014_9794_Fig7_ESM.gif (154 kb)
Supplementary Figure 1

ROS production in HEp-2 treated cells. HEp-2 cells were pre-incubated with or without BSO and treated with As2O3 (1.0 and 4.0 μM) for 72 h. Positive control comprised cells that were separately incubated with H2O2. ROS production was determined by DCFH-DA fluorescence (FITC channel) probe. Dead cells were excluded by morphology [R8 gate] SS x FS (side scatter x forward scatter) and PI staining [R26] PE channel. Representative figure of three independent experiments using flow cytometry. (GIF 153 kb)

12253_2014_9794_MOESM1_ESM.tif (193 kb)
(TIFF 193 kb)
12253_2014_9794_Fig8_ESM.gif (124 kb)
Supplementary Figure 2

Cell cycle arrests by BSO/As2O3 treatment. HEp-2 cells were pre-incubated with or without BSO and treated with As2O3 (1.0 and 4.0 μM) for 48 h. Cells were gated by morphology [R29 gate] SS x FS (side scatter x forward scatter) and PI fluorescence [R28] (PE channel) to exclude debris and doublets. The cell cycle phases [R34-R59] were determined by using PI fluorescence (PE channel). Representative figure of five independent experiments using flow cytometry. (GIF 124 kb)

12253_2014_9794_MOESM2_ESM.tif (168 kb)
(TIFF 167 kb)
12253_2014_9794_Fig9_ESM.gif (178 kb)
Supplementary Figure 3

Effect of BSO on As2O3-induced apoptosis. HEp-2 cells were pre-incubated with or without BSO and treated with As2O3 (1.0 and 4.0 μM) for 72 h by flow cytometry. Cells were gated by FITC and PI fluorescence (FITC and PE channels respectively). This assay distinguishes viable cells (FITC-annexin V−/PI−) [R3, R7, R11, R15] from cells in early apoptosis (FITC-annexin V+/PI−) [R4, R8, R12, R16], late apoptosis/secondary necrosis (FITC-annexin V+/PI+) [R2, R6, R10, R14] or those undergoing necrosis (FITC-annexin V−/PI+) [R1, R5, R9, R3]. Representative figure of five independent experiments. (GIF 178 kb)

12253_2014_9794_MOESM3_ESM.tif (245 kb)
(TIFF 244 kb)


  1. 1.
    Khariwala SS, Hatsukami D, Hecht SS (2012) Tobacco carcinogen metabolites and DNA adducts as biomarkers in head and neck cancer: Potential screening tools and prognostic indicators. Head and Neck 34:441–447PubMedCrossRefGoogle Scholar
  2. 2.
    Marur S, Forastiere AA (2010) Challenges of integrating chemotherapy and targeted therapy with radiation in locally advanced head and neck squamous cell cancer. Curr Opin Oncol 22:206–211PubMedCrossRefGoogle Scholar
  3. 3.
    Cancela MC, Voti L, Guerra-Yi M, Chapuis F, Mazuir M, Curado MP (2010) Oral cavity cancer in developed and in developing countries: Population-Based Incidence. Head and Neck 32:357–367Google Scholar
  4. 4.
    Schmitz S, Machiels JP (2010) Molecular biology of squamous cell carcinoma of the head and neck: relevance and therapeutic implications. Expert Rev. Anticancer Ther 10:1471–1484CrossRefGoogle Scholar
  5. 5.
    Genden EM, Ferlito A, Rinaldo A, Silver CE, Fagan JJ, Suárez C et al (2008) Recent changes in the treatment of patients with advanced laryngeal cancer. Head and Neck 30:103–110PubMedCrossRefGoogle Scholar
  6. 6.
    Ma X, Herbert Y (2007) Global Burden of Cancer. Yale J Biol Med 79:85–94PubMedCentralGoogle Scholar
  7. 7.
    Holsinger FC, Lin HY, Bassot V, Laccourreye O (2009) Platin-based exclusive chemotherapy for selected patients with squamous cell carcinoma of the larynx and pharynx. Cancer 115:3909–3918PubMedCrossRefGoogle Scholar
  8. 8.
    Hoffman HT, Porter K, Karnell LH, Cooper JS, Weber RS, Langer CJ et al (2006) Laryngeal cancer in the United States: Changes in demographics, patterns of care, and survival. Laryngoscope 116:1–13PubMedCrossRefGoogle Scholar
  9. 9.
    Shi N, Han X, Yu W, Wang L, Lu A (2012) Adoption in China of clinical practice guidelines for hypertension using traditional Chinese medical approaches: a literature review based on clinical studies. J Altern Complement Med 18:1–8CrossRefGoogle Scholar
  10. 10.
    Chen SJ, Zhou GB, Zhang XW, Mao JH, de Thé H, Chen Z (2011) From an old remedy to a magic bullet: molecular mechanisms underlying the therapeutic effects of arsenic in fighting leukemia. Blood 117:6425–6437PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Soignet SL, Maslak P, Wang ZG, Jhanwar S, Calleja E, Dardashti LJ et al (1998) Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N Engl J Med 339:1341–1348PubMedCrossRefGoogle Scholar
  12. 12.
    Zhang TD, Chen GQ, Wang ZG, Wang ZY, Chen SJ, Chen Z (2001) Arsenic trioxide, a therapeutic agent for APL. Oncogene 20:7146–7153PubMedCrossRefGoogle Scholar
  13. 13.
    Sahu GR, Jena RK (2005) Significance of intracellular arsenic trioxide for therapeutic response in acute promyelocytic leukemia. Am J Hematol 78:113–116PubMedCrossRefGoogle Scholar
  14. 14.
    Konig H, Härtel N, Schultheis B, Schatz M, Lorentz C, Melo JV et al (2007) Enhanced Bcr-Abl-specific antileukemic activity of arsenic trioxide through glutathione-depletion in imatinib-resistant cells. Haematologica 92:838–841PubMedCrossRefGoogle Scholar
  15. 15.
    Chiu HW, Ho YS, Wang YJ (2011) Arsenic trioxide induces autophagy and apoptosis in human glioma cells in vitro and in vivo through downregulation of survivin. J Mol Med 89:927–941PubMedCrossRefGoogle Scholar
  16. 16.
    Dizaji MZ, Malehmir M, Ghavamzadeh A, Alimoghaddam K, Ghaffari SH (2012) Synergistic effects of arsenic trioxide and silibinin on apoptosis and invasion in human glioblastoma U87MG cell line. Neurochem Res 37:370–380PubMedCrossRefGoogle Scholar
  17. 17.
    Smith D, Patel S, Raffoul F, Haller E, Mills GB, Nanjundan M (2010) Arsenic trioxide induces a beclin-1 independent autophagic pathway via modulation of snon/skil expression in ovarian carcinoma cells. Cell Death Differ 17:1867–1881PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Liu N, Tai S, Ding B, Thor RK, Bhuta S, Sun Y et al (2012) Arsenic trioxide synergizes with everolimus (Rad001) to induce cytotoxicity of ovarian cancer cells through increased autophagy and apoptosis. Endocrine-Related Cancer 19:711–723PubMedCrossRefGoogle Scholar
  19. 19.
    Kumar P, Gao Q, Ning Y, Wang Z, Krebsbach PH, Polverini PJ (2008) Arsenic trioxide enhances the therapeutic efficacy of radiation treatment of oral squamous carcinoma while protecting bone. Mol Cancer Ther 7:2060–2069PubMedCrossRefGoogle Scholar
  20. 20.
    Zhang X, Su Y, Zhang M, Sun Z (2012) Opposite effects of arsenic trioxide on the Nrf2 pathway in oral squamous cell carcinoma in vitro and in vivo. Cancer Lett 318:93–98PubMedCrossRefGoogle Scholar
  21. 21.
    Diepart C, Karroum O, Magat J, Feron O, Verrax J, Calderon PB et al (2012) Arsenic trioxide treatment decreases the oxygen consumption rate of tumor cells and radiosensitizes solid tumors. Cancer Res 72:482–490PubMedCrossRefGoogle Scholar
  22. 22.
    Chen G, Wang K, Yang BY, Tang B, Chen JX, Hua ZC (2012) Synergistic antitumor activity of oridonin and arsenic trioxide on hepatocellular carcinoma cells. Int J Oncol 40:139–147PubMedGoogle Scholar
  23. 23.
    Li H, Zhu X, Zhang Y, Chen H (2009) Arsenic trioxide exerts synergistic effects with cisplatin on non-small cell lung cancer cells via apoptosis induction. J Exp Clin Cancer Res 28:110PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Brown M, Bellon M, Nicot C (2007) Emodin and DHA potently increase arsenic trioxide interferon-α-induced cell death of HTLV-I- transformed cells by generation of reactive oxygen species and inhibition of AKT and AP-1. Blood 109:1653–1659PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    You BR, Park WH (2012) Arsenic trioxide induces human pulmonary fibroblast cell death via increasing ROS levels and GSH depletion. Oncol Rep 28:749–757PubMedGoogle Scholar
  26. 26.
    Sun RC, Board PG, Blackburn AC (2011) Targeting metabolism with arsenic trioxide and dichloroacetate in breast cancer cells. Mol Cancer 10:142PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Xie LX, Lin XH, Li DR, Chen JY, Hong CQ, Du CW (2007) Synergistic therapeutic effect of arsenic trioxide and radiotherapy in BALB/C nude mice bearing nasopharyngeal carcinoma xenografts. Exp Oncol 29:45–48PubMedGoogle Scholar
  28. 28.
    Sides MD, Sosulski ML, Luo F, Lin Z, Flemington EK, Lasky JA (2013) Co-treatment with arsenic trioxide and ganciclovir reduces tumor volume in murine xenograft model of nasopharyngeal carcinoma. Virol J 10:1–7CrossRefGoogle Scholar
  29. 29.
    Kotowski U, Heiduschka G, Brunner M, Erovic BM, Martinek H, Thurnher D (2012) Arsenic trioxide enhances the cytotoxic effect of cisplatin in head and neck squamous cell carcinoma cell lines. Oncol Lett 3:1326–1330PubMedCentralPubMedGoogle Scholar
  30. 30.
    Brozovic A, Majhen D, Roje V, Mikac N, Jakopec S, Fritz G et al (2008) αvβ3 Integrin-mediated drug resistance in human laryngeal carcinoma cells is caused by glutathione-dependent elimination of drug-induced reactive oxidative species. Mol Pharmacol 74:298–306PubMedCrossRefGoogle Scholar
  31. 31.
    Yang X (2012) Proteosome inhibitor bortezomi-induced the apoptosis of laryngeal squamous cell carcinoma Hep-2 cell line disrupting redox equilibrium. Biomed Pharmacother 66:607–611PubMedCrossRefGoogle Scholar
  32. 32.
    Sobhakumari A, Love-Homan L, Fletcher EVM, Martin SM, Parsons AD, Spitz DR (2012) Susceptibility of human head and neck cancer cells to combined inhibition of glutathione and thioredoxin metabolism. PLoS ONE 7:e48175PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and citotoxicity assays. J Immunol Methods 65(1–2):55–63PubMedCrossRefGoogle Scholar
  34. 34.
    Griffith OW, Meister A (1979) Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J Biol Chem 254(16):7558–7560PubMedGoogle Scholar
  35. 35.
    Fischel J-L, Formento P, Milano G (2005) Epidermal growth factor receptor double targeting by a tyrosine kinase inhibitor (Iressa) and a monoclonal antibody (Cetuximab). Impact on cell growth and molecular factors. Br J Cancer 92(6):1063–1068PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Chou T, Talalay P (1984) Quantitative analysis of dose-effects relationships: the combined effects of multiple drugs or enzyme inhibtors. Adv Enzym Regul 22:27–55CrossRefGoogle Scholar
  37. 37.
    Kwee JK, Luque DG, Ferreira ACS, Vasconcelos FC, Silva KL, Klumb CE, Maia RC (2008) Modulation of reactive oxygen species by antioxidants in chronic myeloid leukemia cells enhances imatinib sensitivity through surviving downregulation. Anti-Cancer Drugs 19:975–981PubMedCrossRefGoogle Scholar
  38. 38.
    Dos Santos Ferreira AC, Fernandes RA, Kwee JK, Klumb CE (2012) Histone deacetylase inhibitor potentiates chemotherapy-induced apoptosis through Bim upregulation in Burkitt’s lymphoma cells. J Cancer Res Clin Oncol 138:317–325PubMedCrossRefGoogle Scholar
  39. 39.
    Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K et al (2012) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 8:445–544PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Jiang H, White EJ, Conrad C, Gomez-Manzano C, Fueyo J (2009) Autophagy pathways in glioblastoma. Methods Enzymol 453:273–286PubMedCrossRefGoogle Scholar
  41. 41.
    Burtness B, Goldwasser MA, Flood W, Mattar B, Forastiere AA (2005) Phase III randomized trial of cisplatin plus placebo compared with cisplatin plus cetuximab in metastatic/recurrent head and neck cancer: an Eastern Cooperative Oncology Group study. J Clin Oncol 23:8646–8654PubMedCrossRefGoogle Scholar
  42. 42.
    Razak AR, Siu LL, Le Tourneau C (2010) Molecular targeted therapies in all histologies of head and neck cancers: an update. Curr Opin Oncol 22:212–220PubMedCrossRefGoogle Scholar
  43. 43.
    Specenier P, Vermorken JB (2013) Cetuximab: its unique place in head and neck cancer treatment. Biologics 7:77–90PubMedCentralPubMedGoogle Scholar
  44. 44.
    Bailey HH, Mulcahy RT, Tutsch KD, Arzoomanian RZ, Alberti D, Tombes MB et al (1994) Phase 1 clinical trial of intravenous L-buthionine sulfoximine and melphalan: an attempt at modulation of glutathione. J Clin Oncol 12:194–205PubMedGoogle Scholar
  45. 45.
    O’Dwyer PJ, Hamilton TC, LaCreta FP, Gallo JM, Kilpatrick D, Halbherr T et al (1996) Phase I trial of buthionine sulfoximine in combination with melphalan in patients with cancer. J Clin Oncol 14:249–256PubMedGoogle Scholar
  46. 46.
    Bailey HH, Ripple G, Tutsch KD, Arzoomanian RZ, Alberti D, Feierabend C et al (1997) Phase I study of continuous-infusion L-S,R-buthionine sulfoximine with intravenous melphalan. J Natl Cancer Inst 89:1789–1796PubMedCrossRefGoogle Scholar
  47. 47.
    Bailey HH (1998) L-S, R-Buthionine sulfoximine: historical development and clinical issues. Chem Biol Interact 111–112:239–254PubMedCrossRefGoogle Scholar
  48. 48.
    Wu JH, Batist G (2013) Glutathione and glutathione analogues; therapeutic potentials. Biochim Biophys Acta 1830:3350–3353PubMedCrossRefGoogle Scholar
  49. 49.
    Ramanathan B, Jan KY, Chen CH, Hour TC, Yu HJ, Pu YS (2005) Resistance to paclitaxel is proportional to cellular total antioxidant capacity. Cancer Res 65:8455–8460PubMedCrossRefGoogle Scholar
  50. 50.
    Ong PS, Chan SY, Ho PC (2011) Differential augmentative effects of buthionine sulfoximine and ascorbic acid in As2O3-induced ovarian cancer cell death: oxidative stress-independent and –dependent cytotoxic potentiation. Int J Oncol 38:1731–1739PubMedGoogle Scholar
  51. 51.
    Giansanti V, Torriglia A, Scovassi AI (2011) Conversation between apoptosis and autophagy: “Is it your turn or mine?”. Apoptosis 16:321–333PubMedCrossRefGoogle Scholar
  52. 52.
    Huang L, Wang S, Li SS, Yang XM (2013) Prognostic significance of beclin-1 expression in laryngeal squamous cell carcinoma. Pathol Oncol Res 2013 in pressGoogle Scholar
  53. 53.
    Church DN, Talbot DC (2012) Survivin in solid tumors: rationale for development of inhibitors. Curr Oncol Rep 14:120–128PubMedCrossRefGoogle Scholar
  54. 54.
    Thomas DJ (2010) Arsenolysis and thiol-dependent arsenate reduction. Toxicol Sci 117:249–252PubMedCrossRefGoogle Scholar

Copyright information

© Arányi Lajos Foundation 2014

Authors and Affiliations

  • Débora Lima Pereira
    • 1
  • Ana Carolina dos Santos Ferreira
    • 2
  • Giselle Pinto de Faria
    • 3
  • Jolie Kiemlian Kwee
    • 4
  1. 1.Departamento de EstomatologiaHospital AC CamargoSão PauloBrazil
  2. 2.Programa de Pós-Graduação em Oncologia do Instituto Nacional de Câncer (PPGO-INCA) Instituto Nacional de Câncer José Alencar Gomes da Silva (INCA)Rio de JaneiroBrazil
  3. 3.Departamento de Biorregulação, Instituto de Ciências da Saúde (ICS)Universidade Federal da Bahia (UFBA)BahiaBrazil
  4. 4.Coordenação de PesquisaINCARio de JaneiroBrazil

Personalised recommendations