Elsevier

Oral Oncology

Volume 47, Issue 11, November 2011, Pages 1032-1038
Oral Oncology

Apicidin, a histone deaceylase inhibitor, induces both apoptosis and autophagy in human oral squamous carcinoma cells

https://doi.org/10.1016/j.oraloncology.2011.07.027Get rights and content

Summary

Apicidin acts as a potent histone deacetylases (HDAC) inhibitor and the precise mechanism for its anti-tumor activity in human oral squamous cell carcinoma (OSCC) cells has not been examined. The aim of this study was to evaluate the anti-tumor efficacy of apicidin through apoptosis and autophagy in OSCC cells. Cells were treated with apicidin and cell death was quantified. Cell cycle and apoptosis were measured using flow cytometry assay, immunoblot. Autophagy was characterized by the increase of LC3B-II and the formation of acidic vesicular organelles (AVOs). Apicidin significantly inhibited the proliferation of OSCC cells in a dose-dependent manner. Apicidin markedly up-regulated p21WAF1 led to G2/M phase arrest. Apicidin significantly increased the number of apoptotic cells compared to untreated control. Apicidin induced not only apoptosis but also autophagy in OSCC cells. Apicidin dramatically increased the levels of LC3 type II expression, ATG5 protein expression and the accumulation of AVOs. Inhibition of autophagy enhanced apicidin-mediated cytotoxicity through an increase in apoptosis. These results suggest that apicidin exerts anti-tumor effects by inducing apoptosis and autophagy and provide novel evidence of apicidin-induced autophagy and autophagy inhibition enhances apicidin-mediated apoptosis in OSCC cells.

Introduction

Oral squamous cell carcinoma (OSCC) is the main malignant neoplasm of the oral cavity and the fifth most common cancer worldwide, with variations in its global incidence.[1], [2], [3] Surgical removal of the tumor is the primary therapy. However it causes a lack of quality of life in patients by facial distortion. Radiation and/or chemotherapy are alternative treatment regimens against OSCC.[4], [5] In spite of recent improvements and advances in surgery as well as medicine, the 5-year survival rate for oral cancer patients has remained at 50% over the past 5 decades.[6], [7] Therefore, it is necessary to develop new therapeutic strategies of improved efficacy against OSCC.

Histone deacetylases (HDACs) play a key role in the epigenetic regulation of genes by catalyzing the removal of acetyl groups, stimulating chromatin condensation and promoting transcriptional repression.8 The inhibition of HDAC can reverse epigenetic silencing that is commonly observed in cancer, and various HDAC inhibitors have been developed for cancer therapy.9 Despite many HDAC inhibitors have entered pre-clinical or clinical trials for various types of human cancers, there are few reports on the anti-tumor effects of HDAC inhibitor on OSCC cells.[10], [11], [12], [13], [14] Apicidin isolated from Fusarium sp. was first reported to be a reversible inhibitor of the in vitro development of apicomplexan parasites. Apicidin acts by inhibiting the HDAC enzyme of the parasite, and it was later shown to have anti-proliferative and cyto-differentiation activity on mammalian cells.[15], [16] Apicidin has been shown to exhibit anti-tumor activities in several human cancer cells, including leukemia, cervical cancer, gastric and breast cancer cells.[17], [18], [19] However, there was no report has examined the apicidin-induced cell death in human OSCC cells.

Autophagy is a self-catabolic process that maintains intracellular homeostasis and prolongs cell survival under stress via lysosomal degradation.20 Autophagy can ultimately prevent genome damage by clearing away damaged proteins and organelles that drives tumorigenesis. On the other hand, autophagy enables tumor cells to tolerate stress and can prolong their survival.21

The aim of this study was to evaluate the effects of apicidin on the modulation of cell death, cell cycle arrest, apoptosis and autophagy in OSCC cells. The results showed that apicidin has significant anti-proliferative effect which is mediated through G2/M phase cell cycle arrest and apoptotic pathway. Apicidin also induced the autophagy in OSCC cells and inhibition of autophagy enhanced the apicidin-mediated cytotoxicity through an increase in apoptosis.

Section snippets

Chemicals and cell culture

Apicidin [cyclo(N-O-methyl-l-tryptophanyl-l-isoleucinyl-d-pipecolinyl-l-2-amino-8-oxodecanoyl)] and all chemicals were purchased from Sigma (St. Louis, MO, USA). Apicidin was dissolved in sterile dimethyl sulfoxide (DMSO) to generate a 5 mM stock solution, and stored at −80 °C. Subsequent dilutions were made in RPMI-1640 (Gibco, Rockville, MD, USA).

The YD-8 and YD-10B human OSCC cells were purchased from Korea Cell Line Bank (KCLB, Seoul, Korea).22 The cells were maintained as monolayers at 37 °C

Apicidin inhibits the cell proliferation and induces histone H3 and H4 acetylation

The YD-8 and YD-10B cells were treated with various concentrations of apicidin for 48 h, and the cell viability was assessed to determine the anti-proliferative effects of apicidin. As shown in Fig. 1A, apicidin significantly inhibited the proliferation of OSCC cells in a dose-dependent manner. The 50% inhibitory concentration (IC50) of apicidin in this culture system was approximately 1.0 μM.

The effect of apicidin on the level of histone acetylation was next examined by Western blotting. The

Discussion

Apicidin is a novel cyclic tetrapeptide with a broad spectrum of anti-proliferative activity against a variety of cancer cell lines.[17], [18], [19] In this study, the anti-tumor efficacy of apicidin was examined against YD-8 and YD-10B human OSCC cells. We first have examined the effects of apicidin on the inhibition of cell proliferation and apoptosis. Our data showed that apicidin dramatically induced cell cycle arrest at G2/M phases, which is mediated by inducing the levels of p21WAF1 and

Conflict of interest statement

None declared.

Acknowledgment

This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. R13-2008-010-00000-0).

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