Molecular Mechanisms Involved in the Synergistic Interaction of the EZH2 Inhibitor 3-Deazaneplanocin A with Gemcitabine in Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDAC) is characterized by overexpression of enhancer of Zeste homolog-2 (EZH2), which plays a pivotal role in cancer stem cell (CSC) self-renewal through methylation of histone H3 lysine-27 (H3K27me3). Against this background, EZH2 was identified as an attractive target, and we investigated the interaction of the EZH2 inhibitor DZNeP with gemcitabine. EZH2 expression was detected by quantitative PCR in 15 PDAC cells, including seven primary cell cultures, showing that expression values correlated with their originator tumors (Spearman R(2) = 0.89, P = 0.01). EZH2 expression in cancer cells was significantly higher than in normal ductal pancreatic cells and fibroblasts. The 3-deazaneplanocin A (DZNeP; 5 μmol/L, 72-hour exposure) modulated EZH2 and H3K27me3 protein expression and synergistically enhanced the antiproliferative activity of gemcitabine, with combination index values of 0.2 (PANC-1), 0.3 (MIA-PaCa-2), and 0.7 (LPC006). The drug combination reduced the percentages of cells in G(2)-M phase (e.g., from 27% to 19% in PANC-1, P < 0.05) and significantly increased apoptosis compared with gemcitabine alone. Moreover, DZNeP enhanced the mRNA and protein expression of the nucleoside transporters hENT1/hCNT1, possibly because of the significant reduction of deoxynucleotide content (e.g., 25% reduction of deoxycytidine nucleotides in PANC-1), as detected by liquid chromatography/tandem mass spectrometry. DZNeP decreased cell migration, which was additionally reduced by DZNeP/gemcitabine combination (-20% in LPC006, after 8-hour exposure, P < 0.05) and associated with increased E-cadherin mRNA and protein expression. Furthermore, DZNeP and DZNeP/gemcitabine combination significantly reduced the volume of PDAC spheroids growing in CSC-selective medium and decreased the proportion of CD133+ cells. All these molecular mechanisms underlying the synergism of DZNeP/gemcitabine combination support further studies on this novel therapeutic approach for treatment of PDACs. Mol Cancer Ther; 11(8); 1735-46. ?2012 AACR.     

A rare case of adult diffuse midline glioma with H3 K27M mutant in the prepontine cistern

Diffuse midline glioma with the H3.3 histone A (H3F3A) or H3 clustered histone 2/3 (HIST1H3B/C) K27M mutation occurs primarily in children and less frequently in adults involving the midline structures of the central nervous system. This case report describes an adult patient with a diffuse midline glioma H3 K27M mutant in the prepontine cistern, which is an unusual site in clinical practice. The clinical, radiographic and histopathological data from the case are presented. Magnetic resonance imaging showed a progressively enlarged and enhanced nodule in the right prepontine cistern, with diffuse involvement of the meninges and communicating hydrocephalus. Analysis of the cerebrospinal fluid occasionally found suspiciously atypical cells with hyperchromatic nuclei and multiple nucleoli, as well as a severely elevated opening pressure and protein level, slightly elevated white cell count and decreased chloride level. Empirical antituberculosis treatment was administered but eventually proved to be ineffective. The definite diagnosis was made by histopathological analysis of the lesion based on the features of positive H3 K27M mutant protein and diffusely infiltrating growth. A diffuse midline glioma with the H3 K27M mutation may rarely present in an unusual site. A biopsy is recommended at an early stage for suspected cases to facilitate a definite diagnosis.     

西达本胺对人B淋巴瘤细胞株的作用及其机制研究

本研究旨在探讨组蛋白去乙酰化酶抑制剂(HDACI)西达本胺对3种B淋巴瘤细胞株Raji(Burkitt淋巴瘤)、Maver及Z-138(套细胞淋巴瘤)的抑制增殖和诱导凋亡的作用及其机制。以不同浓度的西达本胺及不同时间作用于体外培养的3种B淋巴瘤细胞,采用CCK-8法检测细胞的增殖;流式细胞术检测细胞的凋亡及线粒体膜电位;Western blot方法检测细胞内组蛋白H3、H4乙酰化水平及caspase-3蛋白的表达。结果表明,西达本胺可抑制这3种B淋巴瘤细胞的增殖,其抑制作用具有一定的时间和浓度依赖性,尤其能较早较快地抑制Z-138细胞的增殖;另外,西达本胺还可诱导3种B淋巴瘤细胞发生凋亡并引起细胞线粒体膜电位下降,Maver及Z-138细胞对西达本胺较Raji细胞更为敏感;西达本胺可以提高细胞内组蛋白H3、H4乙酰化水平及Maver和Z-138细胞的caspase-3活性。结论:西达本胺能够抑制B淋巴瘤细胞株的增殖,诱导细胞凋亡,其机制可能与西达本胺上调组蛋白H3、H4乙酰化水平,触发线粒体凋亡途径及活化caspase-3有关。     

Germinal center dysregulation by histone methyltransferase EZH2 promotes lymphomagenesis

Protection against deadly pathogens requires the production of high-affinity antibodies by B cells, which are generated in germinal centers (GCs). Alteration of the GC developmental program is common in many B cell malignancies. Identification of regulators of the GC response is crucial to develop targeted therapies for GC B cell dysfunctions, including lymphomas. The histone H3 lysine 27 methyltransferase enhancer of zeste homolog 2 (EZH2) is highly expressed in GC B cells and is often constitutively activated in GC-derived non-Hodgkin lymphomas (NHLs). The function of EZH2 in GC B cells remains largely unknown. Herein, we show that Ezh2 inactivation in mouse GC B cells caused profound impairment of GC responses, memory B cell formation, and humoral immunity. EZH2 protected GC B cells against activation-induced cytidine deaminase (AID) mutagenesis, facilitated cell cycle progression, and silenced plasma cell determinant and tumor suppressor B-lymphocyte–induced maturation protein 1 (BLIMP1). EZH2 inhibition in NHL cells induced BLIMP1, which impaired tumor growth. In conclusion, EZH2 sustains AID function and prevents terminal differentiation of GC B cells, which allows antibody diversification and affinity maturation. Dysregulation of the GC reaction by constitutively active EZH2 facilitates lymphomagenesis and identifies EZH2 as a possible therapeutic target in NHL and other GC-derived B cell diseases.     

异硫氰酸苯己酯对Molt-4细胞组蛋白甲基化、乙酰化调控的实验研究

目的 研究异硫氰酸苯己酯（PHI）在体外对淋巴细胞白血病Molt-4细胞系的作用，观察PHI对Molt-4细胞组蛋白甲基化、乙酰化调控的影响。方法 采用MTT法、克隆抑制实验观察PHI对Molt-4细胞增殖的影响；采用流式细胞术检测PHI诱导细胞凋亡和对细胞周期的影响；用Western blot法观察PHI作用后细胞的组蛋白乙酰化酶、组蛋白甲基化及乙酰化状态的变化。结果 PHI可上调Molt-4细胞组蛋白乙酰化酶P300／CBP水平，显著提高组蛋白H3、H4乙酰化及H3K4甲基化水平，抑制组蛋白甲基化H3K9表达，阻滞细胞于G0／G1期，并诱导细胞凋亡。结论 PHI可能是一种组蛋白去乙酰化酶抑制剂，同时能调控组蛋白甲基化，影响其表观遗传学，可能作为新的抗白血病治疗药物。     

DNA methyltransferases inhibitors effectively induce gene expression changes suggestive of cardiomyogenic differentiation of human amniotic fluid‐derived mesenchymal stem cells via chromatin remodeling

Human amniotic fluid‐derived mesenchymal stem cells (AF‐MSCs) are a new potential stem cell source for cell therapy and regenerative medicine. These are fetal mesenchymal stem cells with multilineage differentiation potential found in amniotic fluid. The aim of the present study was to evaluate in vitro differentiation initiation of AF‐MSCs into cardiac progenitors upon application of inhibitors of DNA methyltransferases (DNMT), such as Decitabine (DEC; 5‐aza‐2′‐deoxycytidine) and Zebularine (ZEB). We assessed epigenetic changes and explored patterns of genes, enriched in association with hyperacetylated H4 after induced differentiation. Upregulation of cardiomyogenesis‐related genes (TNNT2, MYH6, ACTN2, and DES) and cardiac ion channels genes, downregulation of pluripotency genes markers as well as increase in Connexin43 expression indicated cardiomyogenic commitment. Evaluation of global epigenetic changes showed that levels of chromatin modifying enzymes, such as Polycomb repressive complex 2 proteins (EZH2, SUZ12), DNMT1, histone deacetylases 1 and 2 were reduced to the similar extent by both differentiation agents. Levels of specific histone marks keeping active state of chromatin (H3K4me3, H3K9Ac, and H4hyperAc) increased and marks of repressed chromatin state (H3K27me3 and H3K9me3) decreased after DEC or ZEB treatment. Chip‐Seq analysis after chromatin immunoprecipitation with H4hyperAc demonstrated enrichment of around 100 functionally annotated genes, related to chromatin reorganization and cardiomyogenesis and confirmed relation between H4 hyperacetylation and gene expression. Our results demonstrate that both DEC and ZEB can be potentially used as cardiomyogenic differentiation inducers in AF‐MSCs, and they cause various genetic and epigenetic changes resulting in global chromatin remodeling.     

PHI对Burkitt淋巴瘤Daudi细胞株组蛋白甲基化和乙酰化调控的实验研究

本研究观察异硫氰酸苯己酯(PHI)在体外对Burkitt淋巴瘤Daudi细胞株的作用及对淋巴瘤细胞表观遗传学调控的影响,初步探讨其可能的机制。用流式细胞术检测PHI处理后的细胞凋亡率,蛋白免疫印迹法检测PHI处理后的细胞的组蛋白H3、H4乙酰化和H3K4、H3K9甲基化状态改变。结果表明,PHI诱导细胞凋亡并提高组蛋白H3、H4乙酰化水平,组蛋白H3K4甲基化增强,而H3K9甲基化减弱。结论:PHI能上调与转录激活相关的组蛋白H3乙酰化水平及甲基化H3K4,下调转录抑制相关的组蛋白甲基化H3K9,促进肿瘤细胞凋亡,PHI可作为淋巴瘤的靶向治疗药物。     

Histone modifications patterns in tissues and tumours from acute promyelocytic leukemia xenograft model in response to combined epigenetic therapy

Xenograft models are suitable for in vivo study of leukemia’s pathogenesis and the preclinical development of anti-leukemia agents but understanding of epigenetic regulatory mechanisms linking to adult cell functions in pathological conditions during different in vivo treatments is yet unknown. In this study, for the first time epigenetic chromatin modifications were characterized in tissues and tumours from murine xenograft model generated using the human acute promyelocytic leukemia (APL) NB4 cells engrafted in immunodeficient NOG mice. Xenografts were subjected to combined epigenetic treatment by histone deacetylase inhibitor Belinostat, histone methyltransferase inhibitor 3-DZNeaplanocin A and all-trans-retinoic acid based on in vitro model, where such combination inhibited NB4 cell growth and enhanced retinoic acid-induced differentiation to granulocytes. Xenotransplantation was assessed by peripheral blood cells counts, the analysis of cell surface markers (CD15, CD33, CD45) and the expression of certain genes (PML-RAR alpha, CSF3, G-CSFR, WT1). The combined treatment prolonged APL xenograft mice survival and prevented tumour formation. The analysis of the expression of histone marks such as acetylation of H4, trimethylation of H3K4, H3K9 and H3K27 in APL xenograft mice tumours and tissues demonstrated tissue-specific changes in the level of histone modifications and the APL prognostic mark, WT1 protein. In summary, the effects of epigenetic agents used in this study were positive for leukemia prevention and linked to a modulation of the chromatin epigenetic environment in adult tissues of malignant organism.     

Long non-coding RNA PVT1 facilitates cell proliferation by epigenetically regulating FOXF1 in breast cancer

Background: plasmacytoma variant translocation 1 (PVT1) has been identified as an oncogenic long non-coding RNA (lncRNA) in multiple cancers including breast cancer. However, its molecular basis has not been exhaustively elucidated. Methods: RT-qPCR assay was used to detect PVT1 expression in tissues and cells. The effect of PVT1 and FOXF1 on breast cancer cell proliferation was assessed by MTT, colony formation and cell cycle assays. Cell apoptotic rate was measured by flow cytometry via double-staining of Annexin V-FITC and PI. The protein expression patterns of forkhead box f1 (FOXF1) and enhancer of zeste homolog 2 (EZH2) were detected using western blot assays. The subcellular location of PVT1 was analyzed using subcellular fractionation assays. The interaction between PVT1 and EZH2 were demonstrated by RNA-protein pull down and RIP assays. ChIP assay was used to explore whether PVT1 affected FOXF1 expression by recruiting EZH2. In vivo assays were performed to further investigate the roles of PVT1 in breast cancer tumorigenesis. Results: PVT1 expression was elevated in breast cancer tissues and cells. Moreover, higher PVT1 level was positively associated with aggressive pathological status and poor prognosis of breast cancer. PVT1 knockdown suppressed proliferation and induced apoptosis in breast cancer cells. PVT1 silenced FOXF1 expression by recruiting EZH2 to the promoter region of FOXF1, resulting in the increase of H3K27me3 level. EZH2 inhibitor EPZ005687 counteracted PVT1-mediated enrichment effect on H3K27me3 and EZH2 to FOXF1 promoter region. FOXF1 overexpression hampered proliferation and facilitated apoptosis in breast cancer cells. Furthermore, down-regulation of FOXF1 partly abrogated PVT1-knockdown-mediated anti-proliferation and pro-apoptosis effect in breast cancer cells. Finally, PVT1 deficiency suppressed tumor growth by promoting FOXF1 expression in vivo. Conclusion: PVT1 promoted cell proliferation and suppressed apoptosis by epigenetically silencing FOXF1 expression through EZH2 in breast cancer.

Plasmacytoma variant translocation 1 (PVT1) expression was elevated in breast cancer tissues and correlated to breast cancer progression and prognosis.     

Non-toxic dose chidamide synergistically enhances platinum-induced DNA damage responses and apoptosis in Non-Small-Cell lung cancer cells

Combination of low doses of histone deacetylases inhibitors and chemotherapy drugs is considered as one of the most promising strategies to increase the anticancer efficacy. Chidamide is a novel benzamide chemical class of HDAC inhibitor that selectively inhibited HDAC1, 2, 3 and 10. We sought to determine whether chidamide may enhance platinum-induced cytotoxicity in NSCLC cells. In this study, the combination of chidamide with carboplatin showed a good synergism on growth inhibition with the mean combination index value as 0.712 and 0.639 in A549 and NCI-H157 cells, respectively. The used concentration of chidamide was non-toxic on cells by itself as low as 0.3 μM. All of our experiments were comparisons between combination regimen and single carboplatin regimen in A549 and NCI-H157 cell lines. Phosphorylated histone H2A.X (γH2A.X), a hall marker of DNA damage response, was dramatically increased by the combination treatment. Cell cycle analysis by flow cytometry and phosphorylation level analysis of histone H3 (Ser10) by western blotting showed that combination treatment significantly increased the percentage of G2/M phase of cells. Mitochondrial membrane potential and cleaved-PARP1 level analysis indicate that chidamide synergistically enhances carboplatin-induced apoptosis. Additionally, synergistic effects of chidamide were found when it was combined with two other platinum drugs (cisplatin and oxaliplatin). The results suggest that Chidamide in combination with platinum drugs may be a novel therapeutic option for NSCLC.     

Methyltransferase inhibitors restore SATB1 protective activity against cutaneous T cell lymphoma in mice

Cutaneous T cell lymphoma (CTCL) has a poorly understood etiology and no known cure. Using conditional knockout mice, we found that ablation of the genomic organizer special AT-rich sequence–binding protein 1 (Satb1) caused malignant transformation of mature, skin-homing, Notch-activated CD4⁺ and CD8⁺ T cells into progressively fatal lymphoma. Mechanistically, Satb1 restrained Stat5 phosphorylation and the expression of skin-homing chemokine receptors in mature T cells. Notably, methyltransferase-dependent epigenetic repression of SATB1 was universally found in human Sézary syndrome, but not in other peripheral T cell malignancies. H3K27 and H3K9 trimethylation occluded the SATB1 promoter in Sézary cells, while inhibition of SUV39H1/2 methyltransferases (unlike EZH2 inhibition) restored protective SATB1 expression and selectively abrogated the growth of primary Sézary cells more effectively than romidepsin. Therefore, inhibition of methyltransferases that silence SATB1 could address an unmet need for patients with mycosis fungoides/Sézary syndrome, a set of incurable diseases.     

三丁酸甘油酯诱导白血病细胞分化及凋亡机制的研究

为了探讨组蛋白去乙酰化酶抑制剂三丁酸甘油酯（tributyrin,TB）诱导NB4、K562白血病细胞分化和（或）凋亡的作用机制,利用Western blot方法及逆转录聚合酶链反应（RT-PCR）检测TB作用前后NB4、K562细胞组蛋白H3乙酰化水平以及p21^WAF1表达量的改变.结果表明：NB4（或K562）细胞经TB0.1 mmol/L（或TB 0.5 mmol/L）作用16小时后可见组蛋白H3乙酰化水平明显上升,并有剂量依赖效应.NB4、K562细胞经TB 0.1 mmol/L作用后可见p21^WAF1 mRNA表达上调,且具有剂量依赖效应.p21^WAF1 mRNA的这种上调开始于TB作用2小时后,16小时达高峰,可维持48小时.结论：TB诱导NB4、K562细胞发生分化和（或）凋亡的机制与TB引起细胞组蛋白高乙酰化和继而上调p21^WAF1表达有关.     

Novel genotyping and quantitative analysis of neuraminidase inhibitor resistance-associated mutations in influenza a viruses by single-nucleotide polymorphism analysis

Neuraminidase (NA) inhibitors are among the first line of defense against influenza virus infection. With the increased worldwide use of the drugs, antiviral susceptibility surveillance is increasingly important for effective clinical management and for public health epidemiology. Effective monitoring requires effective resistance detection methods. We have developed and validated a novel genotyping method for rapid detection of established NA inhibitor resistance markers in influenza viruses by single nucleotide polymorphism (SNP) analysis. The multi- or monoplex SNP analysis based on single nucleotide extension assays was developed to detect NA mutations H275Y and I223R/V in pandemic H1N1 viruses, H275Y in seasonal H1N1 viruses, E119V and R292K in seasonal H3N2 viruses, and H275Y and N295S in H5N1 viruses. The SNP analysis demonstrated high sensitivity for low-content NA amplicons (0.1 to 1 ng/μl) and showed 100% accordant results against a panel of defined clinical isolates. The monoplex assays for the H275Y NA mutation allowed precise and accurate quantification of the proportions of wild-type and mutant genotypes in virus mixtures (5% to 10% discrimination), with results comparable to those of pyrosequencing. The SNP analysis revealed the lower growth fitness of an H275Y mutant compared to the wild-type pandemic H1N1 virus by quantitatively genotyping progeny viruses grown in normal human bronchial epithelial cells. This novel method offers high-throughput screening capacity, relatively low costs, and the wide availability of the necessary equipment, and thus it could provide a much-needed approach for genotypic screening of NA inhibitor resistance in influenza viruses.