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

Authors

  • Débora Lima Pereira
    • Departamento de EstomatologiaHospital AC Camargo
  • Ana Carolina dos Santos Ferreira
    • Programa de Pós-Graduação em Oncologia do Instituto Nacional de Câncer (PPGO-INCA) Instituto Nacional de Câncer José Alencar Gomes da Silva (INCA)
  • Giselle Pinto de Faria
    • Departamento de Biorregulação, Instituto de Ciências da Saúde (ICS)Universidade Federal da Bahia (UFBA)
    • Coordenação de PesquisaINCA
Research

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

Abstract

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.

Keywords

Laryngeal squamous cancerTrisenoxL-buthionine sulfoximineAutophagyApoptosis

Abbreviations

HNSCC

Head and neck squamous cell carcinomas

LSCC

Laryngeal squamous cell carcinoma

CDDP

Cisplatin

As2O3

Arsenic Trioxide

BSO

L-buthionine sulfoximine

MTT

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

DCFH-DA

2′,7′-dichlorodihydrofluorescein diacetate

PI

Propidium iodide

DMSO

Dimethylsulfoxide

3-MA

3-methyladenine

AO

Acridine orange

H2O2

Hydrogen peroxide

ROS

Reactive oxygen species

AVO

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 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_MOESM2_ESM.tif (168 kb)
(TIFF 167 kb)
12253_2014_9794_Fig9_ESM.gif (178 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_MOESM3_ESM.tif (245 kb)
(TIFF 244 kb)

Copyright information

© Arányi Lajos Foundation 2014