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Depsipeptide induces cell death in Hodgkin lymphoma-derived cell lines
Leukemia Research, 7, 33, pages 929 - 936
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.
Keywords: Depsipeptide, HDAC, Apoptosis, Hodgkin Lymphoma.
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  . So far, no uniform genetic mechanism has been identified that explains malignant transformation sufficiently in most cases  . As it was shown that NFkB is necessary for survival of H-RS cell lines in vitro  , 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  and , NOTCH  , Jak/Stat  and , and receptor tyrosine kinases  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  . 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  . 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  . 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  .
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  and  might be also promising in HL patients.
2. Material and methods
2.1. Cell lines
cHL derived cell lines L1236, L428, Km-H2, and L540Cy were used for in vitro studies and cultured using standard conditions  .
2.2. 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 Aqueous One Solution Reagent following the manufacturer's instructions (Promega, Mannheim, Germany). Where indicated, cells were preincubated with 50 μM of the Z-Val-Ala-Asp(OCH3)-Fluoromethylketone (z-VAD-fmk) (Biomol, Hamburg, Germany) before addition of depsipeptide. Calculation of EC50 values was done using Prism software (version 4.0a).
2.3. RNA expression profiling
L428 and KmH2 cell lines were incubated with depsipeptide or DMSO for 20 h. Total RNA was extracted using RNeasy (Qiagen, Hilden, Germany). The RNA concentration was determined photometrically (Nanodrop 1000, Thermo Scientific, Schwerte, Germany) and the quality of the RNA was assessed with the Agilent BioAnalyzer 2100 (Agilent, Waldbronn, Germany). 1 μg of total RNA was used for transcription into Biotin-labeled cRNA (One-Cycle cDNA Synthesis Kit, Affymetrix, Santa Clara, USA) which was subjected to the Affymetrix U133 Plus2.0 GeneChips (Santa Clara, USA) according to the recommendations of the supplier. Two independent experiments were used for data analysis. Gene expression profiles were analyzed using dCHIP software  , BioRag (Bioresource for array genes at www.biorag.org ) and GeneTRAIL  database.
2.4. FACS analysis
5 × 10e4 cells were seeded in 96 well plates in 100 μL medium and incubated with cell line specific EC50 concentrations of depsipeptide, or DMSO and/or 50 μM z-VAD-fmk. At 24 h, cells were analyzed for Annexin V expression using an Annexin V-FITC Apoptosis Detection Kit (BioVision, Wertheim, Germany) and a FACS Calibur (Becton Dickinson, Heidelberg, Germany) with Cell Quest software.
2.5. Cell cycle analysis
1 × 10e5 cells were seeded in 96 well plates in 200 μl medium, incubated with cell line specific EC50 concentrations of depsipeptide for 20 h and prepared using a Cell Cycle Test Plus Kit (BD Biosciences, Heidelberg, Germany). Briefly, after trypsin and RNase digestion nuclei were stained with propidium iodide and analyzed on a FACS Calibur, aggregates and fragments were excluded using appropriate gating strategies measuring FL2-A versus FL2-W intensities.
2.6. Western blot
2 × 10e6 cells were seeded in 6 well plates in 2 ml medium and incubated with EC50 concentrations of depsipeptide. Proteins (40 μg/lane) were extracted in RIPA buffer and loaded on a 10% SDS-PAGE, blotted on nitrocellulose membranes and incubated with a mouse anti-PARP monoclonal antibody (BD #611038, dilution 1:5000). Membranes were developed using ECL Western Blotting Detection Reagents and Hyperfilm ECL (both Amersham, Munich, Germany).
2.7. NFkB (p65) binding assay
2 × 10e5 cells were seeded in 96 well plates in 200 μl medium and incubated with EC50 concentrations of depsipeptide or DMSO for 24 h. Nuclear extracts were prepared using the Nuclear Cytosol Extraction Kit (BioVision). NFkB p65 binding activity of 5 μg nuclear extract was measured quantitatively with the NFkB p65 TransAM ELISA (Active Motif, Vinci, Italy).
3.1. 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 follows: 0.07 μM for L1236, 0.43 μM for L428, 0.58 μM for Km-H2, and 0.16 μM for L540Cy.
3.2. Depsipeptide induced gene expression profiles
To determine changes of gene expression patterns, cHL cell lines L428 and Km-H2 were treated for 20 h with previously determined EC50 concentrations of depsipeptide. Gene expression data obtained from 2 independent hybridizations were analyzed. Genes showing a fold-change greater than 2, a minimal absolute expression value difference of 100, a significant present call >75% in at least one group, and a p-value < 0.05 were filtered as differentially expressed (treated versus non-treated cells). Overall, expression pattern of 1779 transcripts was changed by depsipeptide. Half of those (49.6%) were transcriptionally upregulated in both cell lines while the other half (50.4%) was repressed. None of the filtered transcripts were discordantly regulated in opposite directions in the two cell lines studied. This is also reflected by the fact that in an unsupervised analysis L428 and Km-H2 cell lines are clustered together depending on the function of their treatment and independent of their cellular origin. The most prominent changes for cell cycle, apoptosis, and NFkB-related genes regulated by depsipeptide are summarized in supervised cluster analysis shown in Fig 2, Fig 3, Fig 4, and Fig 5.
Depsipeptide directly affected the expression of several cyclins (e.g. cyclins D2, D3, G2), cyclin-dependent kinases and their inhibitors (CDK4, CDK6, CDKN1a, and CDKN2b). It also down-regulated genes involved in DNA replication initiation (MCM2, MCM3, MCM5, and MCM5). The transcription factor E2F3 as well as its co-factor Dp-1 that both control progression from G1 to S phase are down-regulated. In addition, depsipeptide induced gene transcription of TGFbeta downstream targets ID2 (inhibitor of differentiation/DNA binding gene) and GADD45beta, both of which can induce lymphocyte growth arrest in G2/M and apoptosis  .
Genes encoding members of the tumor necrosis factor receptor/ligand superfamily were activated (e.g. TNFSF4, encoding for OX40L; and TNFSF9, encoding for 4-1BBL/CD137L) or repressed (e.g. TNFRSF6, encoding for Fas/CD95; and TNFRSF8 encoding for CD30). Downregulation of Fas/CD95 is associated with downregulation of genes encoding for the death-inducing signaling complex (DISC) (e.g. FADD and caspase 8). This indicates that the CD95 pathway does not mediate depsipeptide induced cytotoxicity in H-RS cells. With regard to micromilieu interaction of depsipeptide treated H-RS cells, a downregulation of the immunosuppressive transmembrane protein CD200 was seen indicating a disruption of CD200-mediated induction of regulatory T-cells and T-cell anergy  .
Target genes of NFkB show a mixed signature of activation or repression. For example, genes such as BCL2L1 (encoding for BCL-XL/S) and Birc4 (encoding for XIAP) are downregulated while Birc3 (encoding for CIAP2) is upregulated. Caspase 6 which is a downstream effector of caspase 3 is also induced by depsipeptide. Interestingly, transcription of NFKB1A (encoding for IKBA) is upregulated. Genes of the toll-like receptor pathway such as IRAK1 and IRAK2 are repressed or activated, respectively, whereas genes of the phosphatidylinositol 3-kinase pathway (PIK3CA encoding for the catalytic subunit of PI3K and its downstream target PDK1) are repressed.
3.3. Depsipeptide induces apoptosis in cHL cell lines
Gene expression changes were validated using several apoptotic tests. In MTS assays, cytotoxicity of depsipeptide was blocked significantly in 3 out of 4 cHL cell lines if cells were preincubated with the pan-caspase inhibitor z-VAD-fmk at EC50 concentrations ( Fig. 6 ). This supports the concept that depsipeptide induced cell death is at least in part caspase-mediated.
Depsipeptide induced exposure of phosphatidlyserines as measured by Annexin V-FITC staining by FACS analyses. Early and late apoptotic phases were detected in all cHL cell lines ( Fig. 7 ). Exposure of phosphatidylserines was blocked significantly by the addition of z-VAD-fmk.
Evidence for apoptosis was also obtained at the protein level as cleavage of poly-ADP-ribose polymerase (PARP) was clearly demonstrated in L428 and L1236 cells and to a weaker extent in the L540Cy and Km-H2 cells ( Fig. 8 ).
3.4. G2/M arrest in cHL cell lines induced by depsipeptide
As RNA expression experiments indicated a profound disturbance of cell cycle associated genes, we performed a cell cycle analysis of L428 and Km-H2 cells that were previously analyzed with Affymetrix gene expression arrays. We showed, that L428 and Km-H2 cells were arrested in G2/M phase after incubation with depsipeptide at EC50 concentrations ( Fig. 9 ). Additional analyses at later time points were hampered by cytotoxic effects of the compound.
3.5. RelA/p65 DNA binding activity is enhanced by depsipeptide
Constitutive NFkB activity is known to be one key mechanism of apoptosis resistance in H-RS cells. We thus analyzed the DNA binding activity of RelA/p65 in nuclear extracts of depsipeptide treated cell lines L428 and Km-H2 using a quantitative plate based immunoassay. Our results show marked differences of basal NFkB activity in cHL cell lines. L428 showed low and Km-H2 showed strong basal NFkB binding activity. RelA/p65 binding activity was increased in both cHL cell lines at least two-fold after incubation with depsipeptide for 24 h ( Fig. 10 ).
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  . 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 histone acetylase transferases p300 and CBP. As a consequence, RelA/p65 is acetylated at lysine 310, resulting in an increased DNA binding activity of NFkB  . The possible relevance of this finding was underlined by a recent study, where HDAC inhibitor mediated toxicity was potentiated by concomitant pharmacological inhibition of NFkB  .
In depsipeptide treated HRS cells, NFkB activity might even enhance cell cycle arrest cells as it induces transcription of growth arrest and DNA damage-induced gene GADD45beta  and . High levels of GADD45 are known to cause both apoptosis and lymphocyte growth arrest in G2/M in cooperation with p21 (WAF1/CIP1)  . It could thus be speculated that NFkB mediated upregulation of GADD45beta represents a mechanism of depsipeptide induced toxicity in HRS cells. Cell cycle arrest of H-RS cells in G2/M might be also explained by upregulation of the cyclin-dependent kinase (CDK) inhibitor p21 that is known to be induced by acetylation of p21 promoter associated histones  . At higher concentrations, p21 induces both G1 and G2/M arrest by inhibition of CDK1 function. At lower concentrations it causes predominantly G1 arrest by interference with the function of CDK4/6 and CDK2  . As we used higher concentrations of depsipeptide in our in vitro model, we found a G2/M arrest of H-RS cells.
It is known, that inhibition of HDAC activity alters both the intrinsic and the extrinsic apoptosis pathways: levels of antiapoptotic proteins are decreased while levels of proapoptotic proteins are increased , , and . In our study, depsipeptide induced cell death was associated with PARP cleavage and exposure of phosphatidylserines, both effects being suppressed by pan-caspase inhibition. At a transcriptional level, the main effect of depsipeptide consisted in the decreased transcription of antiapoptotic proteins, e.g. of the inhibitor of apoptosis (IAP) genes XIAP and BCL-XL. As published recently, H-RS cells express almost exclusively the BCL-XL but not the BCL-XS splice variant  . In contrast, many other BCL2 family encoding genes were not affected. We found that transcripts of IRAK1 were also downregulated. As IRAK1 was linked to the pathogenesis of cHL, its depsipeptide mediated downregulation might be of functional importance  .
H-RS cells are known to be part of a complex cytokine and chemokine network  . At a molecular level this is reflected by an activated JAK/STAT pathway. This is partly a direct consequence of cytokine stimulation, partly it is caused by somatic mutations of the gene encoding for the suppressors of cytokine signaling (SOCS)  . We observed that depsipeptide reduced the number of STAT6 transcripts in H-RS cells. STAT6 mainly mediates signals from Interleukin IL-4 and IL-13 receptors  that are involved in the expression of Th2 chemokines. Although molecular mechanisms responsible for STAT6 downregulation are not known, it might be intriguing to speculate that deacetylase activity is required for STAT6 transactivation  . Basal expression of 4-1BBL/CD137L was found in our study as described recently in HL cell lines  and might contribute to the shaping of a H-RS cell specific micorenvironment in HL involved tissue.
Induction of terminal cell differentiation was discussed to be another characteristic of HDAC treated cells. In our study, histone acetylation did not re-induce a B-cell phenotype of cultured H-RS cells but it drastically reduced the number of CD30 transcripts which is one of the pathognomonic histopathological markers of H-RS cells in cHL. A recent report focused on the importance of epigenomic events such as promoter DNA methylation and histone acetylation for silencing of B-cell associated genes  .
Taken together, we here demonstrate that the HDAC inhibitor depsipeptide can efficiently induce cell death in H-RS cells despite induction of NFkB binding activity. Combination of HDAC inhibitors with NFkB inhibitors might be a promising approach to further increase the efficacy of HDAC inhibition as a new and attractive therapeutic option for patients with cHL.
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.
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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a Department of Internal Medicine II, University Hospital of Wuerzburg, 97080 Wuerzburg, Germany
b Department of Internal Medicine I, University Hospital of Cologne, 50931 Cologne, Germany
c Institute of Pathology, Campus Benjamin Franklin, 12200 Berlin, Germany
d Centre Hospitalier Antibes-Juan les Pins, Route Nationale 7, 06606 Antibes, Cedex, France
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