Supplementary MaterialsSupplementary information 41598_2019_52714_MOESM1_ESM
Supplementary MaterialsSupplementary information 41598_2019_52714_MOESM1_ESM. suggest that the combination of HDACi and BRD4i should be pursued in further pre-clinical testing. expression could be a potential target for therapy in lymphomas. Indeed, BCL6 inhibition using specific inhibitors was able to produce apoptosis and cell cycle arrest of these cells10, 11 suggesting that BCL6 may be a promising therapeutic target in lymphoma12,13. We and others, have recently shown that epigenetic mechanisms are involved in regulation14C16. Histone deacetylase inhibitors (HDACi) are a novel class of antitumor agents that have shown very promising results for the treatment of a number of hematologic malignancies17,18. Regulation of the reversible acetylation status of an increasing number of non-histone proteins, many of them being proto-oncogenes, allows to modulate a number of essential cellular processes such as protein interactions, protein stability, apoptosis, cell proliferation and cell survival19. Particularly, HDAC inhibitors have been shown to inhibit BCL6 function by inducing its acetylation, which leads to de-repression of its target genes20. Romidepsin is an HDACi with high inhibitory activity for class I histone deacetylases that is approved by the FDA for the treatment of cutaneous T-cell lymphoma or refractory/relapsed peripheral T-cell lymphoma21,22. HDACi synergize with other agents including hypomethylating agents in pre-clinical models of DLBCL23. MYC translocations occur in 10C15% of DLBCL1. High expression of MYC, independent of the presence of chromosomal translocations involving MYC, is associated with Simvastatin poor clinical outcome in B-cell lymphoma24,25. There is interest in the bromodomain and extra-terminal (BET) family member BRD4, which recognizes acetylated histones and plays an essential role in the regulation of expression26. BRD4 (bromodomain-containing protein-4) inhibitors27 such as JQ1 are able to cause oncogene downregulation in a variety of human cancers, including leukemia and lymphoma28. BET inhibitors are currently being used in clinical trials29. Promising Simvastatin data on combining HDACi with BRD4 inhibitors has been reported18. This combination has a specific rationale in DLBCL and BL as it potentially targets MYC in poor prognosis disease. Thus, the aim of this study was to investigate the effects of romidepsin alone or in combination with the BRD4 inhibitor, JQ1, in the treatment of aggressive lymphomas, and to identify the molecular mechanisms involved in its effects. Results Romidepsin promotes apoptosis in cells from agressive lymphomas As a first approach, we measured cell proliferation (based on metabolic activity) upon romidepsin treatment to establish a dose-response assessment and to analyze the effect of the HDACi on proliferation at different time points MGC102762 (Fig.?1a). Romidepsin was tested in different types of aggressive B-cell lymphoma cell lines: three Burkitt lymphoma cell lines (Raji, DG75 and Ramos), one GC-DLBCL (Toledo) and one ABC-DLBCL (Ly03) (see Supplementary Table?S1). Open in a separate window Figure 1 Romidepsin effect on B-cell lymphoma cells proliferation and apoptosis. (a) The indicated cell lines were treated with different concentrations of romidepsin and Simvastatin metabolic activity was determined using WST-1 method at the designated times. Untreated cells represented 100% of metabolic activity. The data show the means??s.e.m. of four measurements in two independent experiments. (b) Annexin V staining to assess early apoptosis in B-cell lymphoma cells untreated (control) or cells treated with 5?nM romidepsin for 48?h. One representative experiment is shown for each cell line. The graphs on Simvastatin the right represent percentages of Annexin V positive cells. The data show the means??s.e.m. of two or three independent experiments; significance difference (*p? ?0.05) from the control untreated cells. (c) Western blot showing PARP1 and cleaved-PARP1 (indicated with an asterisk) in B-cell lymphoma cells treated Simvastatin with romidepsin at the indicated times and concentrations. Actin was used as loading control. The blots were cropped for improved clarity and the full-length blots were included in the Supplementary Information file. At 48?h, Raji and DG75 cells showed little (10C20%) reduction of metabolic activity (Fig.?1a), even with the highest doses tested (10?nM). Ramos cells were the most sensitive, showing a metabolic reduction 50% after treatment with romidepsin (5?nM) while both Toledo and Ly03, showed intermediate sensitivity. Very high doses of romidepsin inhibit almost completely the proliferation of all the lymphoma cell lines studied (not shown). Given that with 1?nM concentration did not show any significant effect on the studied cell lines and 10?nM treatment resulted in cell death for the most sensitive cell lines, we chose 2?nM and 5?nM as optimal concentrations for further experiments. To evaluate the effects of romidepsin on apoptosis, Annexin V binding was determined (Fig.?1b). No significant cell death was observed for the metabolically less-sensitive cell lines Raji and DG75, while the sensitive cell.