trichostatin-a and Burkitt-Lymphoma

Histone deacetylase inhibitors (HDACi) manifest great potential for treatment of Burkitt's lymphoma (BL), an aggressive B-cell lymphoma. Epidermal growth factor receptor pathway substrate 8 (EPS8) is confirmed overexpressed and associated with poor prognosis in solid tumors and leukemia. However, EPS8 expression and the relationship between EPS8 and HDACi on BL remains obscure. Here, we hypothesized that trichostatin A (TSA), a pan-HDACi, could inhibit BL cells by downregulating EPS8. We demonstrated that TSA reduced cell viability, induced apoptosis and cell arrest at G0/G1. Mechanismly, TSA attenuated EPS8 and downstream Phospho-Erk1/2 pathway. Knockdown of EPS8 resulted in a significant reduction in cellular proliferation and suppressed Phospho-Erk1/2 pathway activity, particularly when combined with TSA. Conversely, overexpression of EPS8 rescued this phenomenon. Then we showed that the combination of TSA and Epirubicin had a more significant effect when compared with TSA or Epirubicin alone. Finally, knockdown of EPS8 and TSA had a synergistic suppression effect on BALB/c nude mice. In conclusion, this study reveals that TSA affects BL cells by suppressing Phospho-Erk1/2 pathway through downregulating EPS8.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Burkitt Lymphoma; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Epirubicin; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; MAP Kinase Signaling System; Mice; Mice, Nude

We examined the consequences of 3-deazaneplanocin A (DZNep) on HACE1 expression in human Burkitt- Lymphoma-derived cells to investigate fundamental molecular mechanisms that control its expression. We treated the human Burkitt- Lymphoma-derived cells lines Ramos and Raji with DZNep and examined HACE1 mRNA expression by RT-PCR. We also studied the effect of DZNep on the methylation of lysine 9 and 27 of histone 3 (H3K27me3 and H3K9me2) associated with the CpG88 and CpG177 islands of the HACE1 promoters by chromatin immunoprecipitation and quantitative PCR. CpG88 (hypomethylated) of the HACE1 promoter was enriched for histone marks H3K27me3 and H3K9me2 whereas CpG177 (hypermethylated) was only enriched for H3K9me2. DZNep treatment increased HACE1 gene expression which was further increased by the addition of trichostatine A (TSA), a promising therapeutic compound for the treatment of human B-Lymphoma. Histone methylation (both H3K9me2 and H3K27me3) of the HACE1 promoter concomitantly decreased. Our experiments suggest that HACE1 can be downregulated by methylation of its promoter region chromatin (H3K27me3 and H3K9me2), making HACE1 a potential target for DZNep combined with TSA. These results highlight the heterogeneity of HACE1 regulation in B-lymphoma and suggest that successful drug-induced restoration of epigenetically silenced tumor suppressor genes will require accurate characterization of cell type- and locus-specific gene silencing mechanisms.

Topics: Adenosine; Burkitt Lymphoma; Cell Line, Tumor; CpG Islands; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histones; Humans; Hydroxamic Acids; Promoter Regions, Genetic; RNA, Messenger; Ubiquitin-Protein Ligases

Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in two human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9-Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.

Topics: Burkitt Lymphoma; Cell Line, Tumor; Cell Proliferation; Curcumin; Epigenesis, Genetic; Gene Expression; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Tetraspanin 29; Transfection

In Epstein-Barr virus (EBV)-associated malignancies, the virus is harbored in every tumor cell and persists in tightly latent forms expressing a very limited number of viral latent proteins. Induction of EBV lytic cycle leads to expression of a much larger number of viral proteins, which may serve as potential therapeutic targets. We found that 4 histone deacetylase inhibitors, trichostatin A (TSA), sodium butyrate (SB), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), all significantly induced EBV lytic cycle in EBV-positive gastric carcinoma cells (AGS/BX1, latency II) but only weakly induced in Burkitt lymphoma cells (AK2003, latency I) and did not induce in lymphoblastoid cells (LCLs, latency III). Interestingly, SAHA potently induced viral lytic cycle in AGS/BX1 cells at micromolar concentrations (evidenced by 8-fold increase in viral DNA replication, strong expression of viral lytic proteins and production of infectious virus particles) and mediated enhanced cell death of EBV-positive AGS/BX1 cells when compared with that of EBV-negative AGS cells, possibly related to cell cycle arrest at G2/M phase. Furthermore, SAHA effected strong induction of EBV lytic cycle in nasopharyngeal carcinoma but not in NK lymphoma cells (both expressing EBV latency II pattern), indicating preferential viral lytic induction in epithelial rather than lymphoid malignancies. In conclusion, SAHA is found to be a potent EBV lytic cycle inducing agent, which warrants further investigation into its potential application as a novel virus-targeted drug for treatment of EBV-associated epithelial malignancies.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Burkitt Lymphoma; Butyrates; Carcinoma; Cell Cycle; Cell Line, Tumor; Cell Survival; Fluorescent Antibody Technique; Herpesvirus 4, Human; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia; Polymerase Chain Reaction; Stomach Neoplasms; Valproic Acid; Vorinostat

Recently, it has been found that inappropriate expression of microRNAs (miRNAs) is strongly associated with carcinogenesis. In this study, we demonstrated that the expression of miRNAs (miRs) -143 and -145, the levels of which were previously shown to be reduced in colon cancers and various kinds of established cancer cell lines, was also decreased in most of the B-cell malignancies examined, including chronic lymphocytic leukemias (CLL), B-cell lymphomas, Epstein-Barr virus (EBV)-transformed B-cell lines, and Burkitt lymphoma cell lines. All samples from 13 CLL patients and eight of nine B-cell lymphoma ones tested exhibited an extremely low expression of miRs-143 and -145. The expression levels of miRs-143 and -145 were consistently low in human Burkitt lymphoma cell lines and were inversely associated with the cell proliferation observed in the EBV-transformed B-cell lines. Moreover, the introduction of either precursor or mature miR-143 and -145 into Raji cells resulted in a significant growth inhibition that occurred in a dose-dependent manner and the target gene of miRNA-143 was determined to be ERK5, as previously reported in human colon cancer DLD-1 cells. Taken together, these findings suggest that miRs-143 and -145 may be useful as biomarkers that differentiate B-cell malignant cells from normal cells and contribute to carcinogenesis in B-cell malignancies by a newly defined mechanism.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Burkitt Lymphoma; Cell Line, Tumor; Cell Survival; Decitabine; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Leukemia, Lymphocytic, Chronic, B-Cell; Lymph Nodes; Lymphoma, B-Cell; MicroRNAs; RNA, Neoplasm; Tumor Cells, Cultured

Epstein-Barr virus (EBV) infects more than 90% of the human population and has a potential oncogenic nature. Trichostatin A (TSA) has potent antitumor activity, but its exact mechanism on EBV-infected cells is unclear. This study examined the effects of TSA on proliferation and apoptosis of the Burkitt's lymphoma cell line, Akata. TSA treatment inhibited cell growth and induced cytotoxicity in both the EBV-negative and -positive Akata cells. TSA sensitively induced apoptosis in both cells, as demonstrated by the increased number of positively stained cells in the TUNEL assay, the migration of many cells to sub-G1 phase by flow cytometric analysis, and the formation of DNA ladders. This suggests that EBV has no effect on the sensitivity to TSA. Western blot analysis showed that the cleavage of PARP and Bid and the activation of caspases are closely related to the TSA-induced apoptosis of the cells. The reduction in mitochondrial transition potential and the release of apoptosis-inducing factor from mitochondria to cytosol was also observed after the TSA treatment, but was suppressed by treating the cells with a cathepsin B inhibitor. Overall, these findings suggest that besides the caspase-dependent pathway, mitochondrial events are also associated with the TSA-induced apoptosis of Akata cells.

Topics: Apoptosis; BH3 Interacting Domain Death Agonist Protein; Burkitt Lymphoma; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; DNA Damage; Enzyme Activation; Enzyme Inhibitors; Herpesvirus 4, Human; Humans; Hydroxamic Acids; In Situ Nick-End Labeling; Mitochondria; Poly(ADP-ribose) Polymerases

The expression of human general control of amino acid synthesis protein 5 (hGCN5) in human Burkitt's lymphoma Daudi cells in vitro, effects of Trichostatin A (TSA) on cell proliferation and apoptosis and the molecular mechanism of TSA inhibiting proliferation of Daudi cells were investigated. The effects of TSA on the growth of Daudi cells were studied by 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. The effect of TSA on the cell cycle of Daudi cells was assayed by a propidium iodide method. Immunochemistry and Western blot were used to detect the expression of hGCN5. The proliferation of Daudi cells was decreased in TSA-treated group with a 24 h IC50 value of 415.3979 microg/L. TSA induced apoptosis of Daudi cells in a time- and dose-dependent manner. Treatment with TSA (200 and 400 microg/L) for 24 h, the apoptosis rates of Daudi cells were (14.74+/-2.04) % and (17.63+/-1.25) %, respectively. The cell cycle was arrested in G0/G1 phase (50, 100 microg/L) and in G2/M phase (200 microg/L) by treatment with TSA for 24 h. The expression of hGCN5 protein in Daudi cells was increased in 24 h TSA-treated group by immunochemistry and Western blot (P<0.05). It was suggested that TSA as HDACIs could increase the expression of hGCN5 in Daudi cells, and might play an important role in regulating the proliferation and apoptosis of B-NHL cell line Daudi cells.

Topics: Apoptosis; Burkitt Lymphoma; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; p300-CBP Transcription Factors

The frequent rearrangement of chromosome band 1q12 constitutive heterochromatin in hematologic malignancies suggests that this rearrangement plays an important pathogenetic role in these diseases. The oncogenic mechanisms linked to 1q12 heterochromatin are unknown. Constitutive heterochromatin can epigenetically regulate gene function through the formation of transcriptional-silencing compartments. Thus, as a first step toward understanding whether 1q12 rearrangements might compromise such activity in tumor cells, we investigated the 3-D organization of the 1q12 heterochromatin domain (1q12HcD) in normal and tumor B lymphocytes. Strikingly, in normal B cells, we showed that the 1q12HcD dynamically organizes to the nuclear periphery in response to B-cell receptor engagement. Specifically, we observed an almost twofold increase in 1q12Hc domains at the extreme nuclear periphery in activated versus resting B lymphocytes. Remarkably, 1q12Hc organization was noticeably altered in tumor cells that showed structural alterations of 1q12; the 1q12Hc domains were significantly displaced from the extreme nuclear periphery compared to normal activated B lymphocytes (P > 0.0001), although overall peripheral localization was maintained. In a case in which there was a translocation of IGL enhancer to 1q, the altered nuclear positioning of the 1q12HcD was even more pronounced (5% of the 1q12Hc domains at the nuclear periphery compared to 20% in other lymphoma lines), and we were able to mimic this effect in two additional B-cell tumor lines by treatment with trichostatin A, a histone deacetylase (HDAC) inhibitor. Taken together, these results point to the 1q12HcD having a specific, nonrandom, and regulated peripheral organization in B lymphocytes. This organization is significantly disrupted in lymphoma cells harboring 1q rearrangements.

Topics: B-Lymphocytes; Burkitt Lymphoma; Cell Line, Tumor; Cell Nucleus; Chromosome Aberrations; Chromosomes, Human, Pair 1; Heterochromatin; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; In Situ Hybridization, Fluorescence; Lymphocyte Activation; Lymphoma, Follicular; Translocation, Genetic

Chromatin structure and thereby transcription is controlled by the level of acetylation of histones, which is determined by the balance between histone acetyl transferase (HAT) activity and histone deacetylase (HDAC) activity. HDAC inhibitors are a class of compounds able to regulate gene expression by modulating chromatin structure. There are two major classes of HDAC inhibitors: the hydroxamic acid derivatives such as trichostatin A (TSA) or SAHA, and the butyrates such as phenyl-butyrate. HDAC inhibitors interfere with differentiation, proliferation and apoptosis in tumor cells. Here, we investigated the activity of a new hydroxamic acid derivative, LAQ824, on lymphoblastic cells.. Four different pre-B lymphoblastic cell lines: Sup-B15 and TMD-5, both t(9;22) positive, SEM, t(4;11) positive, and NALM-6 cells were exposed to the hydroxamic acid derivatives, LAQ824 and TSA. Histone hyperacetylation, apoptosis, cell cycle and related pathways were assessed by flow cytometry and Western blotting.. LAQ824 significantly inhibited the proliferation of leukemic lymphoblastic cell lines. The effect of LAQ824 was due to increased apoptosis accompanied by activation of caspase-3 and caspase-9, cleavage of poly(ADP-ribose)-polymerase (PARP) as well as by down-regulation of Bcl-2 and disruption of the mitochondrial membrane potential. Surprisingly, LAQ824-induced apoptosis was at least partially independent of caspase activation as indicated by the fact that LAQ824-induced apoptosis was inhibited only partially in both t(9;22) positive Sup-B15 and TMD-5 cells, whereas no inhibition was observed in t(4;11) positive SEM cells upon exposure to the polycaspase inhibitor zVAD-fmk.. Our study establishes that LAQ824 is a promising agent for the therapy of acute lymphoblastic leukemia.

Topics: Apoptosis; Burkitt Lymphoma; Cell Line, Tumor; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Translocation, Genetic