Cell growth inhibition and gene expression induced by the histone deacetylase inhibitor, trichostatin A, on human hepatoma cells

OBJECTIVE: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation in tumor cells. The effect of the HDAC inhibitor, trichostatin A (TSA), on hepatoma cells, however, has not been well studied. In this study, we examined cell viability and gene expression profile in hepatoma cell lines treated with TSA. METHODS: To study cell growth inhibition and induction of apoptosis by TSA on human hepatoma cell lines including HuH7, Hep3B, HepG2, and PLC/PRF/5, cells were treated with TSA at various concentrations and analyzed by the 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively. Changes in gene expression profile after exposure to TSA were assessed using a cDNA microarray consisting of 557 distinct cDNA of cancer-related genes. The levels of acetylated histones were examined by the chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 or H4 antibody. RESULTS: The MTT assay demonstrated that TSA showed cell growth inhibition not only in a concentration-dependent but also a time-dependent manner on all cell lines studied. The TUNEL assay also revealed the potential of TSA to induce apoptosis. The microarray analysis revealed that 8 genes including collagen type 1, alpha2 (COL1A2), insulin-like growth factor binding protein 2 (IGFBP2), integrin, alpha7 (ITGA7), basigin (BSG), quiescin Q6 (QSCN6), superoxide dismutase 3, extracellular (SOD3), nerve growth factor receptor (NGFR), and p53-induced protein (PIG11) exhibited substantial induction (ratio >2.0) after TSA treatment in multiple cell lines. ChIP assay, in general, showed a good correlation between the expression level of mRNA and levels of acetylated histones in these upregulated genes. CONCLUSIONS: This study showed cell growth inhibition and the gene expression profile in hepatoma cell lines exposed to TSA. The alteration in levels of acetylated histones was closely associated with expression of specific cancer-related genes in hepatoma cells.     

Doxorubicin-induced apoptosis and chemosensitivity in hepatoma cell lines

PURPOSE: Doxorubicin (DOX) is a commonly used anticancer drug which causes DNA damage and kills cancer cells mainly by apoptosis. However, the process leading to killing of cancer cells and the molecular basis of resistance to DOX are not well understood. To evaluate the role of p53 and the cellular effects of DOX on hepatoma cell lines, we examined three hepatoma cell lines with different p53 status--Huh-7 (mutated p53), HepG2 (wild-type p53) and Hep3B (deleted p53). METHODS: The chemosensitivity of the three hepatoma cell lines was assessed using the MTT assay, and cell cycle distribution was evaluated by flow cytometry. Western blotting and immunostaining were employed to examine the protein alterations in response to DOX treatment, and a DNA fragmentation assay was performed to detect apoptosis. RESULTS: Of the three cell lines, HepG2 was found to be most resistant to DOX, followed by Hep3B, and Huh-7 was most sensitive to DOX treatment. HepG2 showed G1 arrest 24 h after drug administration and upregulation of p53 protein level in a time-dependent manner. In Hep3B cells (deleted p53), G2/M phase arrest was observed soon after drug administration, accompanied by induced apoptosis that was p53-independent. In Huh-7 cells (mutated p53), which were most sensitive to DOX, there was neither G1 nor G2 arrest, and the level of p53 mutated protein was downregulated after DOX treatment. MDM2 and p27 proteins were downregulated in all cell lines independently of p53 status. p21 was upregulated following p53 activation at low doses of DOX in HepG2 cells, but at higher doses, p21 was downregulated in Huh-7 and HepG2 cells. CONCLUSION: DOX confers different chemosensitivity on different hepatoma cell lines with different p53 status. The contrasting relationships between chemosensitivity and p53 status and expression suggest that DOX-induced apoptosis and cell death involve pathways that are independent of p53.     

Notch信号通路对肝癌细胞迁移的调控机制研究

目的：探讨Notch信号通路对肝癌细胞迁移的调控机制。方法体外培养HepG2、MHCC97H、Huh-7肝癌细胞株和HL-7702正常肝细胞株,Transwell小室检测细胞迁移能力,蛋白免疫印迹法检测各细胞系Snail、E-cadherin蛋白表达水平,实时定量RT-PCR检测Snail、E-cadherin mRNA表达水平;采用DAPT阻断Notch信号通路,比较其对细胞迁移能力和Snail、E-cadherin蛋白及mRNA表达水平的影响。结果 HepG2、MHCC97H、Huh-7细胞株迁移能力高于HL-7702细胞株（P﹤0.05）,HepG2、MHCC97H、Huh-7肝癌细胞株迁移能力比较差异无统计学意义（P﹥0.05）。HepG2、MHCC97H、Huh-7细胞Snail蛋白及mRNA高于HL-7702细胞,E-cadherin蛋白及mRNA表达低于HL-7702细胞（P﹤0.05）,HepG2、MHCC97H、Huh-7细胞间Snail、E-cadherin蛋白及mRNA表达差异无统计学意义（P﹥0.05）。HepG2、MHCC97H、Huh-7细胞经DAPT处理后迁移能力低于未经DAPT处理的细胞株（P﹤0.05）,HL-7702细胞DAPT处理与未经DAPT处理迁移能力比较差异无统计学意义（P﹥0.05）。DAPT处理后,HepG2、MHCC97H、Huh-7细胞株Snail蛋白及mRNA表达水平降低,E-cadherin蛋白及mRNA表达增加,与未经DAPT处理的细胞株比较差异具有统计学意义（P﹤0.05）,HepG2、MHCC97H、Huh-7细胞间Snail、E-cadherin蛋白及mRNA表达差异无统计学意义（P﹥0.05）。结论 Notch信号通路可能是通过调控Snail、E-cad-herin通路调控肝癌细胞的迁移过程。     

TGF-β1对人肝癌细胞系凋亡的调控作用研究

目的肝细胞对TGF-β1敏感性的丧失据认为是肝癌重要的致病因素之一。本研究旨在明确人肝肿瘤细胞系中TGF-β1的作用及其与凋亡的关系。方法选用三种含不同p53基因状态的人肝肿瘤细胞系,应用脱氧核糖核苷酸末瑞转移酶介导的dUTP缺口末端标记技术(TUNEL)对TGF-β1诱导的肝肿瘤细胞的凋亡进行定量检测。结果在应用TUNEL检测三个细胞系中,TGF-β1仅能诱导Hep3B细胞(缺失p53)则凋亡较少。这提示凋亡p53基因的表达具有明确的联系。结论HepG2细胞系比Huh-7和Hep3B系细胞更易发生TGF-β1诱导的凋亡,TGF-β1通过p53依赖性途径诱导肝癌细胞系发生凋亡。     

Antitumor activity of histone deacetylase inhibitor trichostatin A in osteosarcoma cells

Background: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest,apoptosis and differentiation of tumor cells. The present study aimed to examine the effects of trichostatin A(TSA), one such inhibitor, on the cell cycle, apoptosis and invasiveness of osteosarcoma cells. Methods: MG-63 cells were treated with TSA at various concentrations. Then, cell growth and apoptosis were determinedby 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively; cell cyclingwas assessed by flow cytometry; invasion assays were performed with the transwell Boyden Chamber system.Results: MTT assays revealed that TSA significantly inhibited the growth of MG-63 cells in a concentrationand time dependent manner. TSA treated cells demonstrated morphological changes indicative of apoptosisand TUNEL assays revealed increased apoptosis of MG-63 cells after TSA treatment. Flow cytometry showedthat TSA arrested the cell cycle in G1/G2 phase and annexin V positive apoptotic cells increased markedly. Inaddition, the invasiveness of MG-63 cells was inhibited by TSA in a concentration dependent manner. Conclusion:Our findings demonstrate that TSA inhibits the proliferation, induces apoptosis and inhibits invasiveness ofosteosarcoma cells in vitro. HDAC inhibitors may thus have promise to become new therapeutic agents againstosteosarcoma.     

Histone deacetylase inhibitors such as sodium butyrate and trichostatin A induce apoptosis through an increase of the bcl-2-related protein Bad

The effects of sodium butyrate (SB) and trichostatin A (TSA) on cell proliferation and apoptosis against human glioma T98G, U251MG, and U87MG cells were investigated. Upon exposure to either SB or TSA, cell proliferation was reduced, and apoptosis detected by DNA fragmentation analysis and the cleavage of CPP32 was induced. Previously, we reported that SB increased the expression levels of p21 (WAF-1) and inhibited G1-S transition of the cell cycle. In this study, we showed that TSA also increased p21 expression, suggesting that histone deacetylase (HDAC) inhibitors may up-regulate p21 protein in common and thus arrest proliferation in the G1 phase of the cell cycle. To further determine the underlying molecular mechanisms of apoptosis with either SB or TSA treatment, we studied the expression levels of apoptosisrelated proteins in human glioma cells. SB increased the expression of the Bad protein, although the expression of Bcl-2, Bcl-xL, Bax, and Fas was not changed by the addition of SB. TSA treatment also up-regulated the expression of Bad protein. The results suggest that HDAC inhibitors such as SB and TSA induce apoptosis through an increase in Bad protein in human glioma cells in vitro.     

Bevacizumab and CCR2 Inhibitor Nanoparticles Induce Cytotoxicity-Mediated Apoptosis in Doxorubicin-Treated Hepatic and Non-Small Lung Cancer Cells

Non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC) are very common in certain populationaround the world. Despite the recent advances in their diagnosis and therapy, their prognosis remains poor due to thedevelopment resistance to drug. Although doxorubicin (DOX) is considered to be one of the most anti-solid tumordrugs, developed resistance is contributing to unsuccessful outcome. The rationale of the current study is to explorethe sensitizing capability of the DOX-treated cancer cells using the anticancer agents; bevacizumab (avastin; AV) andCCR2 inhibitor (CR) in their free- and nano-formulations. Here, the average size, polydispersity index (PDI), zetapotential, and entrpment effeciency (EE%) of the synthesized nanoparticles were measured. We investigated the effectof these platforms on the proliferation, apoptosis, necrosis, nitric oxide (NO), malondialdehyde (MDA), and zinc levelsof human HCC (HepG2 and Huh-7) and NSCLC (A549) cancer cell lines. Glucose consumption rates using Huh-7and A549 cancer cells were tested upon treatments. We demonstrated that AV and CR nano-treatments significantlysuppressed A549 cell viability and activated apoptosis by NO level elevation. We concluded that AVCR NP plusDOX significantly induces A549 cytotoxicity-mediated apoptosis more than Huh-7 and HepG2 cells. This drug-drugnano-combination induced Huh-7 cytotoxicity-mediated apoptosis more than HepG2 cells. In conclusion, AVCR NPsensitized DOX-treated A549 and Huh-7 cells through reactive oxygen species (ROS)-stimulated apoptosis. Takentogether, our data suggested that the CR plus AV nano-platforms would be a potential personalized medicine-basedstrategy for treating CCR2-positive NSCLC and HCC patients in the near future.     

Trichostatin A is a histone deacetylase inhibitor with potent antitumor activity against breast cancer in vivo.

PURPOSE: Trichostatin A (TSA), an antifungal antibiotic with cytostatic and differentiating properties in mammalian cell culture, is a potent and specific inhibitor of histone deacetylase (HDAC) activity. The purpose of this study was to evaluate the antiproliferative and HDAC inhibitory activity of TSA in vitro in human breast cancer cell lines and to assess its antitumor efficacy and toxicity in vivo in a carcinogen-induced rat mammary cancer model. EXPERIMENTAL DESIGN AND RESULTS: TSA inhibited proliferation of eight breast carcinoma cell lines with mean +/- SD IC(50) of 124.4 +/- 120.4 nM (range, 26.4-308.1 nM). HDAC inhibitory activity of TSA was similar in all cell lines with mean +/- SD IC(50) of 2.4 +/- 0.5 nM (range, 1.5-2.9 nM), and TSA treatment resulted in pronounced histone H4 hyperacetylation. In randomized controlled efficacy studies using the N-methyl-N-nitrosourea carcinogen-induced rat mammary carcinoma model, TSA had pronounced antitumor activity in vivo when administered to 16 animals at a dose of 500 microg/kg by s.c. injection daily for 4 weeks compared with 14 control animals. Furthermore, TSA did not cause any measurable toxicity in doses of up to 5 mg/kg by s.c. injection. Forty-one tumors from 26 animals were examined by histology. Six tumors from 3 rats treated with TSA and 14 tumors from 9 control animals were adenocarcinomas. In contrast, 19 tumors from 12 TSA-treated rats had a benign phenotype, either fibroadenoma or tubular adenoma, suggesting that the antitumor activity of TSA may be attributable to induction of differentiation. Two control rats each had tumors with benign histology. CONCLUSIONS: The present studies confirm the potent dose-dependent antitumor activity of TSA against breast cancer in vitro and in vivo, strongly supporting HDAC as a molecular target for anticancer therapy in breast cancer.     

艾迪注射液对肝癌HepG2细胞放疗增敏的研究

目的:研究艾迪注射液对人肝癌HepG2细胞放疗增敏作用及相关机制。方法:流式细胞仪检测不同浓度艾迪注射液对HepG2细胞失活率、细胞周期及凋亡的影响;克隆形成法测定细胞存活分数,拟合细胞存活曲线,计算放疗增敏比。结果:艾迪注射液使HepG2细胞失活率明显升高,表现出一定的细胞毒作用,可以诱导其早期凋亡及引起细胞周期的重新分布,增加G1期细胞的比例,减少S期细胞的比例,这些作用与时间和浓度有一定的相关。10mg/ml浓度的艾迪注射液对HepG2细胞有放疗增敏作用,放疗增敏比达1.62。结论:艾迪注射液对HepG2细胞有放疗增敏作用,其机制可能与诱导细胞凋亡、直接的细胞毒作用有关。     

Cell cycle arrest and lytic induction of EBV-transformed B lymphoblastoid cells by a histone deacetylase inhibitor, Trichostatin A

Latent infection of the Epstein-Barr virus (EBV) is strongly associated with the pathogenesis of several human tumor types. The restricted expression of the latent EBV antigens is critical for EBV-associated tumors to escape from immune surveillance. EBV lytic replication can be triggered by various treatments and the induced lytic genes cause strong cytotoxic T lymphocyte (CTL) responses. Histone acetylation or deacetylation is associated with chromatin remodeling and regulates gene expression. Histone deacetylase (HDAC) inhibitors affect cell cycle progression as well as gene expression in a wide variety of transformed cells. We examined whether an HDAC inhibitor, TSA, can affect cell cycle progression and induce EBV lytic replication in EBV-transformed lymphoblastoid cell lines (LCLs). TSA caused cell cycle arrest at low concentrations and induced apoptosis at higher (>300 nM) concentrations in the LCLs and EBV negative BJAB cells. To clarify the underlying mechanism of TSA-induced cell cycle arrest, expression of cell cycle regulatory factors was examined by RNase protection assay and Western blot analysis. Following TSA treatment, a reduced expression of cyclin D2 and an induction of p21 may have played an essential role for G1 arrest in LCLs, while p21 induction might have arrested BJAB cells in G1 phase. A Cdk inhibitor, p57, was increased by 300 nM TSA in both LCLs and BJAB cells, indicating its role in apoptosis. Moreover, immunofluorescene assay and Western blotting showed that TSA induced EBV lytic replication in LCL cells. These results suggest that TSA may exert an enhanced anti-tumor effect for EBV-associated tumors not only by inducing a cell cycle arrest and apoptosis, but also by triggering an EBV lytic cycle.     

艾迪注射液对肝癌HepG2细胞放疗增敏的研究

目的：研究艾迪注射液对人肝癌HepG2细胞放疗增敏作用及相关机制。方法：流式细胞仪检测不同浓度艾迪注射液对HepG2细胞失活率、细胞周期及凋亡的影响;克隆形成法测定细胞存活分数,拟合细胞存活曲线,计算放疗增敏比。结果：艾迪注射液使HepG2细胞失活率明显升高,表现出一定的细胞毒作用,可以诱导其早期凋亡及引起细胞周期的重新分布,增加G1期细胞的比例,减少S期细胞的比例,这些作用与时间和浓度有一定的相关。10mg/ml浓度的艾迪注射液对HepG2细胞有放疗增敏作用,放疗增敏比达1.62。结论：艾迪注射液对HepG2细胞有放疗增敏作用,其机制可能与诱导细胞凋亡、直接的细胞毒作用有关。     

曲古霉素A对肺腺癌细胞株NCI-H1299增殖的抑制作用及其机制

背景与目的：曲古霉素A（trichostatin A,TSA）是具有组蛋白去乙酰化酶（histone deacetylases,HDAC@强效非竞争性抑制剂,对血液系统肿瘤和实质性肿瘤均有较强的生长抑制作用。本文观察HDACs抑制剂TSA对体外培养的肺腺癌NCI-H1299细胞株的增殖、凋亡和周期以及相关基因表达的影响,并探讨其可能的作用机制。方法：MTT法检测不同浓度（0.1、0.2、0.4、2.0μmol/L@的TSA对人肺腺癌NCI-H1299细胞株体外增殖的影响,流式细胞术检测药物处理后细胞周期及凋亡率的变化;Western blot法检测细胞内组蛋白H4乙酰化水平的变化;Real-time PCR检测NCI-H1299细胞内p21、CyclinB1、Bcl-2和Bax的基因表达。结果：TSA能明显抑制NCI-H1299细胞的体外生长,其抑制作用呈明显的剂量和时间依赖性。TSA诱导后,流式细胞术检测结果显示细胞阻滞于G2/M期,细胞凋亡增加。TSA可明显提高NCI-H1299细胞内组蛋白H4的乙酰化水平,诱导p21和Bax的mRNA表达增加,同时抑制Bcl-2和CyclinB1表达。结论：TSA可通过诱导细胞凋亡及阻滞细胞周期而发挥体外抗肺腺癌细胞生长的作用,其机制可能与组蛋白乙酰化水平的提高以及调控相关基因p21、Bax、Bcl-2和CyclinB1的表达变化有关。     

FR901228, a novel histone deacetylase inhibitor, induces cell cycle arrest and subsequent apoptosis in refractory human pancreatic cancer cells

Histone deacetylase (HDAC) inhibitors have antiproliferative activity against human cancer cells via cell cycle arrest, differentiation, and apoptosis. However, no report has focused on the apoptotic potential of HDAC inhibitors in refractory human pancreatic cancer. This study was designed to examine the apoptotic potential of FR901228, a novel HDAC inhibitor, in five human pancreatic cancer cell lines: Capan-1, BxPC-3, HPAF, Panc-1, and MIAPaCa-2. FR901228 markedly inhibited the proliferation of all five cell lines (IC50: 1-500 nM), with the greatest effect in MIAPaCa-2 cells. Treatment of each cell line with FR901228 (10-100 nM) caused cell cycle arrest at the G1 or G2/M phase and subsequent apoptosis. FR901228 induced expression of hyperacetylated histone H3 after 3 h of treatment and overexpression of p21Waf-1 after 6 h. In addition, FR901228 induced apoptosis by activating caspase-3, which led to cleavage of p21Waf-1 into a 15-kDa breakdown product and drove cancer cells from cell cycle arrest into apoptosis. FR901228 also decreased the protein level of survivin dramatically. Our results show that FR901228 markedly inhibits the growth of pancreatic cancer cells, not only through cell cycle arrest, but also through subsequent apoptosis; this was accompanied by caspase-3 activation, survivin degradation, and p21Waf-1 cleavage. FR901228 may prove clinically useful as an agent for refractory pancreatic cancers.     

Hybrid polar histone deacetylase inhibitor induces apoptosis and CD95/CD95 ligand expression in human neuroblastoma.

Inhibitors of histone deacetylase (HDAC) have been shown to have both apoptotic and differentiating effects on various tumor cells. M-carboxycinnamic acid bishydroxamide (CBHA) is a recently developed hybrid polar compound structurally related to hexamethylene bisacetamide. CBHA is a potent inhibitor of HDAC activity. CBHA induces cellular growth arrest and differentiation in model tumor systems. We undertook an investigation of the effects of CBHA on human neuroblastoma cell lines in vitro. When added to cultures of a panel of neuroblastoma cell lines, CBHA induced the accumulation of acetylated histones H3 and H4, consistent with the inhibition of HDAC. Concentrations of CBHA between 0.5 microM and 4 microM led to apoptosis in nine of nine neuroblastoma cell lines. Apoptosis was assessed by DNA fragmentation analysis and the appearance of a sub-G1 (<2N ploidy) population by flow cytometric analysis. The addition of a caspase inhibitor (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) completely abrogated CBHA-induced apoptosis in three of three cell lines. The addition of cycloheximide greatly reduced CBHA-induced apoptosis, suggesting that apoptotic induction was dependent on de novo protein synthesis. In addition, CBHA induced the expression of both CD95 (APO-1/Fas) and CD95 ligand within 12 h. The effect of CBHA on human neuroblastoma cells suggests that this agent and structurally related synthetic hybrid polar compounds have therapeutic potential for the treatment of this malignancy.