Elsevier

Leukemia Research

Volume 33, Issue 7, July 2009, Pages 929-936
Leukemia Research

Depsipeptide induces cell death in Hodgkin lymphoma-derived cell lines

https://doi.org/10.1016/j.leukres.2008.12.013Get rights and content

Abstract

A variety of genetic and epigenetic abnormalities were characterized over the last years in Hodgkin and Reed-Sternberg (H-RS) cells of classic Hodgkin Lymphoma (cHL). It was speculated that simultaneous inhibition of multiple signalling pathways might be a promising strategy to target this tumor entity. In the present study we tested the effect of histone deacetylase (HDAC) inhibition using depsipeptide (also known as romidepsin, FK228, FR901228 or NSC-630176) in cHL cell lines in vitro. Molecular mechanisms of toxicity were analyzed using RNA expression analysis and functional assays. It is shown that depsipeptide is effective at submicromolar concentrations and acts mainly by apoptosis induction, upregulation of p21 and cell cycle inhibition in G2/M. Of special note, HDAC mediated toxicity in H-RS cells does not require RelA/p65 downregulation, which was previously shown to drive the malignant phenotype of H-RS cells. In summary, depsipeptide induced protein acetylation results in transcriptional changes of a large number of pathogenetically relevant genes and increased RelA/p65 binding activity in cHL cell lines. Our preclinical data suggest that HDAC inhibition using depsipeptide might be a promising approach for the treatment of cHL patients.

Introduction

Hodgkin and Reed-Sternberg (H-RS) cells are pathognomonic for classic Hodgkin Lymphoma (cHL). Although germinal center B-cell derived in most instances, they are characterized by a downregulated B-cell program and apoptosis resistance [1]. So far, no uniform genetic mechanism has been identified that explains malignant transformation sufficiently in most cases [2]. As it was shown that NFkB is necessary for survival of H-RS cell lines in vitro [3], it was speculated, that targeting NFkB might represent a valuable strategy to sensitize H-RS cells to cell death. In addition, gene expression and functional studies of H-RS cells showed that pathways of PI3K/AKT [4], [5], NOTCH [6], Jak/Stat [7], [8], and receptor tyrosine kinases [9] contribute to the malignant phenotype of those cells.

More recently, it became evident that epigenetic mechanisms such as promoter methylation are also involved in the deregulation of transcriptional programs of H-RS cells [10]. In contrast, remodeling of histone and non-histone chromatin proteins by histone deacetylases (HDACs) and histone acetyltransferases (HATs) has not been well analyzed in H-RS cells so far.

Eighteen human HDACs enzymes belonging to 3 different classes of molecules are known to date and seem to exert non-redundant functions in cellular processes [11]. Especially class I HDACs, namely HDAC1 and HDAC2, are discussed to regulate proliferation of cancer cells in cooperation with different transcription factors or corepressors. There are a number of structurally distinct classes of compounds that inhibit HDAC including aliphatic acids, hydroxamates, cyclic tetrapeptides and benzamides. The main functional effect of HDAC inhibitors consists in transcriptional activation of differentiation, arrest of cell cycle in G1 and/or G2, and induction of apoptosis. Transcription-independent effects include disruption of the spindle assembly checkpoint causing a segregation defect [12]. The cyclic peptide depsipeptide shows some preference to class I over class II HDACs and is known to be active in the submicromolar range in several tumor models [13].

In this study we describe molecular mechanisms of action of depsipeptide in H-RS cell lines. Gene expression profiles identified a large number of genes that are regulated at an epigenetic level. Apoptosis induction and cell cycle arrest in G2/M despite induction of RelA/p65 binding activity represent the main effects of depsipeptide in cHL. Our result indicates that clinical trials with HDAC inhibitors [14], [15] might be also promising in HL patients.

Section snippets

Cell lines

cHL derived cell lines L1236, L428, Km-H2, and L540Cy were used for in vitro studies and cultured using standard conditions [16].

MTS assay

5 × 10e4 cells were seeded in 96 well plates in 100 μL RPMI medium and incubated with depsipeptide (Gloucester Pharmaceuticals, USA) or DMSO in triplicates at the indicated concentrations. OD490 was determined after 48 h of incubation using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt], using CellTiter96

Depsipeptide induced cytotoxiciy in H-RS cell lines

Four cHL cell lines (L1236, L428, KM-H2, and L540Cy) were cultured in the presence of depsipeptide at different concentrations for 24 and 48 h. Depsipeptide induced cytotoxicity in all H-RS cell lines in a time- and dose-dependent manner (Fig. 1). In contrast, viability of Daudi and Reh cells used as control cell lines was not affected by depsipeptide (10 μM at 48 h; data not shown). The effective concentrations of depsipeptide to inhibit viability of H-RS cells by 50% (EC50) were determined as

Discussion

Gene expression and functional data presented in our study show that the HDAC inhibitor depsipeptide induces apoptosis and cell cycle arrest in H-RS cells at submicromolar concentrations. Results from gene expression analysis are in line with earlier studies showing that HDAC inhibition changes RNA transcription of 10–20% of genes [21]. Interestingly, our functional data indicate that depsipeptide increases DNA binding capacity of RelA/p65 in H-RS cells. This can be explained by activation of

Contributions

All authors have made substantial contributions and approved the final version submitted. IH, CP, GW, and AK were responsible for data acquisition, data analysis and data interpretation. MH was responsible for the analysis and interpretation of data. DR was responsible for the conception and design of the study, analysis and interpretation of data, and drafting the article.

Conflict of interest

There is no conflict of interest.

Acknowledgments

We would like to thank Hedwig Lammert for excellent technical assistance. This work was supported by the German Cancer Aid, Weiskam Ruranski Foundation and Cologne Fortune.

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