曲古抑菌素A对结肠癌细胞株SW480细胞周期影响的机制研究

为了研究组蛋白去乙酰化酶(HDACs)抑制剂曲古抑菌素A(TSA)对结肠癌细胞周期和凋亡的影响,初步探讨TSA作用细胞周期的可能机制,将人结肠癌细胞系SW480经TSA处理后,运用流式细胞术检测细胞周期、凋亡以及细胞周期素的变化,最后采用western-blot对细胞周期相关的基因进行检测.结果表明,TSA处理细胞后,TSA能够延缓细胞周期G1-S进程,阻滞细胞于G1期,并且影响细胞周期素cyclinE、cyclinA聚集,而对凋亡无明显的影响.Western-blot显示,TSA能够上调p21Waf1/Cip1、p27Kip1的表达,下调CDK2、cyclinE以及cycli-nA的表达.以上结果说明在结肠癌细胞中,TSA能够通过上调p21Waf1/Cip1、p27Kip1的表达以及下调CDK2、cy-clinE、cyclinA的表达,从而阻滞细胞周期于G1期,最终影响肿瘤细胞的生长,以上研究为HDAC抑制剂应用于结肠癌治疗提供了理论依据.     

Trichostatin A,a histone deacetylase inhibitor,suppresses proliferation and epithelial–mesenchymal transition in retinal pigment epithelium cells

The proliferation and epithelial–mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells are the major pathological changes in development of proliferative vitreoretinopathy (PVR), which leads to severe visual impairment. Histone deacetylases (HDACs)‐mediated epigenetic mechanisms play important roles in controlling various physiological and pathological events. However, whether HDACs are involved in the regulation of proliferation and EMT in PRE cells remains unidentified. In this study, we evaluated the expression profile of HDAC family (18 genes) and found that some of class I and class II HDACs were up‐regulated in transforming growth factor‐β2 (TGF‐β2)/TGF‐β1‐stimulated RPE cells. Tricostatin A (TSA), a class I and II HDAC inhibitor, suppressed the proliferation of RPE cells by G1 phase cell cycle arrest through inhibition of cyclin/CDK/p‐Rb and induction of p21 and p27. In the meantime, TSA strongly prevented TGF‐β2–induced morphological changes and the up‐regulation of α‐SMA, collagen type I, collagen type IV, fibronectin, Snail and Slug. We also demonstrated that TSA affected not only the canonical Smad signalling pathway but also the non‐canonical TGF‐β/Akt, MAPK and ERK1/2 pathways. Finally, we found that the underlying mechanism of TSA affects EMT in RPE cells also through down‐regulating the Jagged/Notch signalling pathway. Therefore, this study may provide a new insight into the pathogenesis of PVR, and suggests that epigenetic treatment with HDAC inhibitors may have therapeutic value in the prevention and treatment of PVR.     

Melanoma cell lines are susceptible to histone deacetylase inhibitor TSA provoked cell cycle arrest and apoptosis

Melanoma is the most aggressive of skin cancers because of its high resistance to currently available therapy. Although melanoma cells often retain wild-type p53 tumour suppressor protein and express it at high levels, the p53 mediated apoptosis pathway is suppressed. Histone deacetylase (HDAC) inhibitors are a promising group of compounds inducing differentiation, growth arrest and apoptosis in tumour cells in preclinical studies. We have studied the cellular effects of trichostatin A (TSA), a HDAC inhibitor, in a panel of melanoma cell lines and its mechanism of action in relation to p53. TSA stabilized wild-type p53, but p53 protein accumulation was overridden by simultaneous downregulation of p53 mRNA leading to a decrease in p53 protein. While growth arrest was induced in all cell lines studied and apoptosis in most (6/7), these cellular effects were independent of the p53 status of the cells. Inhibiting p53 function by a dominant negative p53 (p53(175His)) confirmed that the HDAC inhibitor induced apoptosis was independent of wild-type p53, even though TSA slightly activated p53 in a reporter assay. The results indicate that while the action of TSA is independent of p53, the activation of the apoptosis pathway by the HDAC inhibitors may provide therapeutic approaches for melanoma treatment.     

曲古抑菌素A（TSA）对骨肉瘤细胞株143B增殖和凋亡的作用及其机制

观察曲古抑菌素A（TSA）对骨肉瘤细胞株143B增殖及凋亡的作用,并探讨其机制。TSA与p38抑制剂（SB203580,3μmol／L）及JNK抑制剂（SP600125,0．5μmol／L）单独或同时处理143B细胞,分别以MTT、台盼蓝染色、流式细胞术和JC—1（测定线粒体跨膜电位）法检,TSA对143B细胞的增殖、存活、周期以及凋亡的影响。应用RT-PCR、Westernblot检测Bax、Bcl．2、p38／JNK表达。结果显示,TsA能够以时间和剂量依赖方式抑制143B细胞增殖,使细胞周期阻滞于GdG。与G2／M期,并能诱导143B细胞凋亡,引起线粒体膜电位降低,促凋亡蛋白Bax表达上调,抑凋亡蛋白Bcl．2表达下调,同时使p38／ⅢK活化增加。p38／JNK4检测剂则能逆转TSA对Bax／Bcl．2的上调及抑制作用。研究结果揭示,TSA可以时间剂量依赖方式抑制143BN胞增殖,阻滞细胞周期,诱导细胞凋亡;其诱导细胞凋亡的机制可能与活化MAPK通路中p38和JNK的活性从而激发线粒体凋亡通路有关。     

The epigenetic modifier trichostatin A,a histone deacetylase inhibitor,suppresses proliferation and epithelial–mesenchymal transition of lens epithelial cells

Proliferation and epithelial–mesenchymal transition (EMT) of lens epithelium cells (LECs) may contribute to anterior subcapsular cataract (ASC) and posterior capsule opacification (PCO), which are important causes of visual impairment. Histone deacetylases (HDACs)-mediated epigenetic mechanism has a central role in controlling cell cycle regulation, cell proliferation and differentiation in a variety of cells and the pathogenesis of some diseases. However, whether HDACs are involved in the regulation of proliferation and EMT in LECs remain unknown. In this study, we evaluated the expression profile of HDAC family (18 genes) and found that class I and II HDACs were upregulated in transforming growth factor β2 (TGFβ2)-induced EMT in human LEC lines SRA01/04 and HLEB3. Tricostatin A (TSA), a class I and II HDAC inhibitor, suppressed the proliferation of LECs by G1 phase cell cycle arrest not only through inhibition of cyclin/CDK complexes and induction of p21 and p27, but also inactivation of the phosphatidylinositol-3-kinase/Akt, p38MAPK and ERK1/2 pathways. Meanwhile, TSA strongly prevented TGFβ2-induced upregulation of fibronectin, collagen type I, collagen type IV, N-cadherin, Snail and Slug. We also demonstrated that the underlying mechanism of TSA affects EMT in LECs through inhibiting the canonical TGFβ/Smad2 and the Jagged/Notch signaling pathways. Finally, we found that TSA completely prevented TGFβ2-induced ASC in the whole lens culture semi-in vivo model. Therefore, this study may provide a new insight into the pathogenesis of ASC and PCO, and suggests that epigenetic treatment with HDAC inhibitors may be a novel therapeutic approach for the prevention and treatment of ASC, PCO and other fibrotic diseases.     

The enhanced antiproliferative response to combined treatment of trichostatin A with raloxifene in MCF-7 breast cancer cells and its relevance to estrogen receptor β expression

Antiestrogen is one type of the endocrine therapeutic agents for estrogen receptor α (ERα)-positive breast cancer. Unfortunately, this treatment alone is insufficient. Here we reported a novel potential anticancer strategy by using histone deacetylase (HDAC) inhibitor to enhance the action of endocrine therapy in ERα-positive breast cancer cell. The well-described HDAC inhibitor, trichostatin A (TSA), and antiestrogen raloxifene were found to, respectively, inhibit E2-induced proliferation of MCF-7 breast cancer cell in a dose-responsive and time-dependent manner. TSA and raloxifene enhanced the antiproliferative activity of each other by promoting cell death via apoptosis and cell cycle arrest. Thus, they displayed better antiproliferative effects in combined treatment than that with either agent alone. The expression level of estrogen receptor β (ERβ) showed a marked increase after TSA or/and raloxifene treatment. Treatments with TSA or/and raloxifene resulting in the up-regulation of ERβ are in accordance with the antiproliferative effects of the two agents. Furthermore, the over-expression of ERβ by adenovirus delivery could inhibit the proliferation of MCF-7 tumor cells and drastically enhanced the antiproliferative effects of TSA and raloxifene. These results demonstrated that the interference of ERβ on the antiproliferative effects of HDAC inhibitor and antiestrogen constitutes a promising approach for breast cancer treatment.     

Activation of the growth-differentiation factor 11 gene by the histone deacetylase (HDAC) inhibitor trichostatin A and repression by HDAC3

Histone deacetylase (HDAC) inhibitors inhibit the proliferation of transformed cells in vitro, restrain tumor growth in animals, and are currently being actively exploited as potential anticancer agents. To identify gene targets of the HDAC inhibitor trichostatin A (TSA), we compared the gene expression profiles of BALB/c-3T3 cells treated with or without TSA. Our results show that TSA up-regulates the expression of the gene encoding growth-differentiation factor 11 (Gdf11), a transforming growth factor beta family member that inhibits cell proliferation. Detailed analyses indicated that TSA activates the gdf11 promoter through a conserved CCAAT box element. A comprehensive survey of human HDACs revealed that HDAC3 is necessary and sufficient for the repression of gdf11 promoter activity. Chromatin immunoprecipitation assays showed that treatment of cells with TSA or silencing of HDAC3 expression by small interfering RNA causes the hyperacetylation of Lys-9 in histone H3 on the gdf11 promoter. Together, our results provide a new model in which HDAC inhibitors reverse abnormal cell growth by inactivation of HDAC3, which in turn leads to the derepression of gdf11 expression.     

Distinct HDACs regulate the transcriptional response of human cyclin-dependent kinase inhibitor genes to Trichostatin A and 1alpha,25-dihydroxyvitamin D3

The anti-proliferative effects of histone deacetylase (HDAC) inhibitors and 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] converge via the interaction of un-liganded vitamin D receptor (VDR) with co-repressors recruiting multiprotein complexes containing HDACs and via the induction of cyclin-dependent kinase inhibitor (CDKI) genes of the INK4 and Cip/Kip family. We investigated the effects of the HDAC inhibitor Trichostatin A (TSA) and 1alpha,25(OH)2D3 on the proliferation and CDKI gene expression in malignant and non-malignant mammary epithelial cell lines. TSA induced the INK4-family genes p18 and p19, whereas the Cip/Kip family gene p21 was stimulated by 1alpha,25(OH)2D3. Chromatin immunoprecipitation and RNA inhibition assays showed that the co-repressor NCoR1 and some HDAC family members complexed un-liganded VDR and repressed the basal level of CDKI genes, but their role in regulating CDKI gene expression by TSA and 1alpha,25(OH)2D3 were contrary. HDAC3 and HDAC7 attenuated 1alpha,25(OH)2D3-dependent induction of the p21 gene, for which NCoR1 is essential. In contrast, TSA-mediated induction of the p18 gene was dependent on HDAC3 and HDAC4, but was opposed by NCoR1 and un-liganded VDR. This suggests that the attenuation of the response to TSA by NCoR1 or that to 1alpha,25(OH)2D3 by HDACs can be overcome by their combined application achieving maximal induction of anti-proliferative target genes.     

TSA Suppresses miR-106b-93-25 Cluster Expression through Downregulation of MYC and Inhibits Proliferation and Induces Apoptosis in Human EMC

Histone deacetylase (HDAC) inhibitors are emerging as a novel class of anti-tumor agents and have manifested the ability to decrease proliferation and increase apoptosis in different cancer cells. A significant number of genes have been identified as potential effectors responsible for the anti-tumor function of HDAC inhibitor. However, the molecular mechanisms of these HDAC inhibitors in this process remain largely undefined. In the current study, we searched for microRNAs (miRs) that were affected by HDAC inhibitor trichostatin (TSA) and investigated their effects in endometrial cancer (EMC) cells. Our data showed that TSA significantly inhibited the growth of EMC cells and induced their apoptosis. Among the miRNAs that altered in the presence of TSA, the miR-106b-93-25 cluster, together with its host gene MCM7, were obviously down-regulated in EMC cells. p21 and BIM, which were identified as target genes of miR-106b-93-25 cluster, increased in TSA treated tumor cells and were responsible for cell cycle arrest and apoptosis. We further identified MYC as a regulator of miR-106b-93-25 cluster and demonstrated its down-regulation in the presence of TSA resulted in the reduction of miR-106b-93-25 cluster and up-regulation of p21 and BIM. More important, we found miR-106b-93-25 cluster was up-regulated in clinical EMC samples in association with the overexpression of MCM7 and MYC and the down-regulation of p21 and BIM. Thus our studies strongly indicated TSA inhibited EMC cell growth and induced cell apoptosis and cell cycle arrest at least partially through the down-regulation of the miR-106b-93-25 cluster and up-regulation of it''s target genes p21 and BIM via MYC.