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

DOI: 10.1007/s12253-014-9794-6

Cite this article as:
Pereira, D.L., dos Santos Ferreira, A.C., de Faria, G.P. et al. Pathol. Oncol. Res. (2015) 21: 103. doi:10.1007/s12253-014-9794-6


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 cancerTrisenoxL-buthionine sulfoximineAutophagyApoptosis



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)

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