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

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Pathology & Oncology Research

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.

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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

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Acknowledgments

This work was supported by grants from Fundação do Câncer (2301415 and 2301402) and Ministério da Saúde (MS).

Competing Interests

The authors have declared that no competing interest exists.

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Correspondence to Jolie Kiemlian Kwee.

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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)

(TIFF 193 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)

(TIFF 167 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)

(TIFF 244 kb)

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Pereira, D.L., dos Santos Ferreira, A.C., de Faria, G.P. et al. Autophagy Interplays with Apoptosis and Cell Cycle Regulation in the Growth Inhibiting Effect of Trisenox in HEP-2, a Laryngeal Squamous Cancer. Pathol. Oncol. Res. 21, 103–111 (2015). https://doi.org/10.1007/s12253-014-9794-6

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  • DOI: https://doi.org/10.1007/s12253-014-9794-6

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