组蛋白去乙酰化转移酶抑制剂MS-275对胃癌细胞增殖的影响

目的：探讨组蛋白去乙酰化转移酶抑制剂MS-275对胃癌细胞（MKN-45）和人正常胃黏膜细胞（GES-1）及张氏肝细胞（changliver）生长及凋亡的影响。方法：用不同浓度的MS-275分别处理体外培养的胃癌细胞和人正常胃黏膜细胞及张氏肝细胞,用水溶性四唑盐（WST-1）法检测对细胞的增殖抑制作用;流式细胞仪和TUNEL法检测细胞凋亡情况。结果：MS-275可明显抑制胃癌细胞的生长且呈时间和浓度依赖性,细胞凋亡率明显增加,但对正常细胞没有促凋亡作用。低浓度的MS-275能诱导MKN-45细胞发生G0/G1期阻滞,随着药物处理时间的延长,可以明显检测到G0/G1期比例增大。结论：去乙酰化转移酶抑制剂MS-275对胃癌细胞MKN-45具有选择性细胞毒作用,为其临床应用提供了有价值的实验依据,有望成为新的抗胃癌药物。     

人MUC4启动子驱动的HSV-TK重组腺病毒靶向杀伤SGC-7901胃癌细胞的实验研究

目的:构建人粘蛋白( MUC4)启动子驱动下的单纯疱疹胸苷激酶基因(HSV-TK)重组腺病毒,研究其对SGC-7901胃癌细胞的靶向杀伤作用.方法:克隆MUC4启动子区625 bp活性序列,构建重组荧光素酶报告基因载体pGL3-MUC4,检测其在SGC-7901胃癌细胞及NIH3T3成纤维细胞中的转录活性.以AdEasyTM腺病毒系统为载体,构建MUC4启动子驱动下的HSV-TK重组腺病毒rAdeno-MUC4-TK,感染SGC-7901及NIH3T3,经更昔洛韦(GCV)处理,MTT法检测细胞活力,TUNEL法检测细胞凋亡.结果:成功扩增出大小为625 bp的MUC4启动子序列.pGL3-MUC4在SGC-7901细胞中具有强转录活性,转录活性高于强启动子SV40 6.6倍,而NIH3T3成纤维细胞系中几乎无转录活性.构建重组腺病毒rAdeno-MUC4-TK,MTT法和TUNEL检测结果显示,其与GCV联合能够诱导SGC-7901细胞凋亡,产生靶向细胞毒作用.结论:人MUC4启动子驱动下的HSV-TK重组腺病毒联合GCV对SGC-7901胃癌细胞具有靶向杀伤作用,MUC4启动子可以作为胃癌靶向基因治疗的工具.     

MS-275通过上调ROS释放选择性杀伤胃癌细胞

目的：研究组蛋白去乙酰化酶抑制剂MS-275通过线粒体凋亡途径,选择性杀伤胃癌细胞的具体作用机制。方法：MS-275分别处理GES-1、MKN-45细胞,通过WST-1法检测细胞存活率,分析MS-275细胞毒性及选择性杀伤作用;通过流式细胞术检测线粒体膜电位变化,及ROS抑制剂对MS-275凋亡诱导作用的影响;Western blot、PCR分别检测处理后胃癌细胞中p21、p57、cyclin D1、cyclin E1、和TBP-2的mRNA及蛋白水平表达情况。结果：MS -275对正常胃黏膜上皮细胞GES -1存活率无显著性影响,但对胃癌细胞MKN-45影响显著（P＜0．05）。MS-275对胃癌细胞MKN-45线粒体膜电位影响不显著,但能够诱导ROS显著升高,ROS抑制剂显著降低由MS-275诱导的ROS释放。MS-275显著激活肿瘤抑制基因p21、p57、TBP-2以及细胞周期蛋白相关基因cyclin D1、cyclin E1的表达。结论：MS-275通过促进细胞周期阻滞因子、凋亡诱导基因、肿瘤抑制基因的表达,诱导ROS生成,选择性杀伤胃癌细胞。     

人MUC4启动子驱动HSV-TK腺病毒靶向杀伤胃癌细胞的研究

目的：构建人MUC4启动子驱动下的HSV-TK重组腺病毒,研究其对胃癌细胞的靶向杀伤作用。方法：免疫荧光法检测MUC4在胃癌细胞系中的表达。克隆MUC4启动子区625bp活性序列,利用重组荧光素酶检测系统检测其在SGC-7901胃腺癌细胞及NUGC4胃印戒细胞癌细胞中的转录活性。以AdEasyTM腺病毒系统为载体,构建MUC4启动子驱动下的HSV-TK重组腺病毒,与前体药物GCV联合,检测其对上述两种胃癌细胞的细胞毒作用。结果：MUC4蛋白在两种胃癌细胞的胞浆和胞膜均有表达,而在成纤维细胞中无表达。克隆的MUC4启动子片段在SGC-7901和NUGC4细胞中具有强转录活性,转录活性高于强启动子SV40,而NIH3T3成纤维细胞系中几乎无转录活性。MUC4启动子驱动下的HSV-TK重组腺病毒与GCV联合能够诱导SGC-7901和NUGC4胃癌细胞凋亡,产生特异性靶向细胞毒作用。结论：人MUC4启动子驱动下的HSV-TK重组腺病毒联合GCV对SGC-7901和NUGC4胃癌细胞具有靶向杀伤作用,MUC4启动子可以作为胃癌靶向基因治疗的工具。     

HIF-1α反义寡核苷酸增强胃癌细胞对顺铂的敏感性

目的研究缺氧诱导因子-1α(hypoxia inducible factor1α,HIF-1α)反义寡核苷酸(antisense ol-igodexynucleotide,ASODN)对人胃癌细胞凋亡及化疗药物敏感性的影响。方法人工合成HIF-1αASODN经阳离子脂质体包裹后瞬时转染人胃癌SGC-7901细胞系。采用RT-PCR和免疫细胞化学检测转染后HIF-1α基因表达情况,MTT法观察化疗药物敏感性的变化,AO/EB染色及TUNEL检测SGC-7901细胞转染后顺铂诱导的凋亡。结果经HIF-1αASODN处理的SGC-7901细胞HIF-1α基因表达明显下调,HIF-1αASODN处理的SGC-7901细胞加顺铂作用后与对照组比较,细胞凋亡率明显增加,化疗药物敏感性增强。结论阳离子脂质体转染HIF-1αASODN具有促进化疗药物诱导胃癌SGC-7901细胞凋亡及增强化疗药物敏感性作用。     

Ad-PTEN体外对SGC-7901胃癌细胞生长的抑制作用

目的:探讨腺病毒介导的PTEN基因表达体外对SGC-7901胃癌细胞生长抑制作用及其分子机制。方法:将携有PTEN基因的复制缺陷型腺病毒载体(Ad-PTEN)感染SGC-7901胃癌细胞,用RT-PCR法检测Ad-PTEN在细胞中的表达,光学显微镜及荧光显微镜下观察Ad-PTEN感染细胞前后形态的变化,MTT法检测Ad-PTEN对SGC-7901胃癌细胞生长的抑制作用,用流式细胞术(FCM)检测SGC-7901胃癌细胞凋亡率。RT-PCR分析Bax、Bcl-2、p53、Survivin细胞凋亡相关基因的表达。结果:Ad-PTEN基因组感染SGC-7901胃癌细胞后,RT-PCR结果显示PTEN目的基因能在SGC-7901胃癌细胞中转录,其表达可明显抑制该胃癌细胞的生长,并诱导细胞凋亡。其凋亡机制可能与Bax/Bcl-2比值、p53基因表达上调、Survivin下调有关。结论:重组腺病毒Ad-PTEN具有抑制SGC-7901胃癌细胞生长和诱导细胞凋亡的作用。     

Ad-PTEN体外对SGC-7901胃癌细胞生长的抑制作用

目的：探讨腺病毒介导的PTEN基因表达体外对SGC-7901胃癌细胞生长抑制作用及其分子机制。方法：将携有PTEN基因的复制缺陷型腺病毒载体（Ad-PTEN）感染SGC-7901胃癌细胞,用RT-PCR法检测Ad-PTEN在细胞中的表达,光学显微镜及荧光显微镜下观察Ad-PTEN感染细胞前后形态的变化,MTT法检测Ad-PTEN对SGC-7901胃癌细胞生长的抑制作用,用流式细胞术（FCM）检测SGC-7901胃癌细胞凋亡率。RT-PCR分析Bax、Bcl-2、p53、Survivin细胞凋亡相关基因的表达。结果：Ad-PTEN基因组感染SGC-7901胃癌细胞后,RT-PCR结果显示PTEN目的基因能在SGC-7901胃癌细胞中转录,其表达可明显抑制该胃癌细胞的生长,并诱导细胞凋亡。其凋亡机制可能与Bax/Bcl-2比值、p53基因表达上调、Survivin下调有关。结论：重组腺病毒Ad-PTEN具有抑制SGC-7901胃癌细胞生长和诱导细胞凋亡的作用。     

氧化苦参碱对人胃癌SGC-7901细胞株的杀伤作用

目的探讨氧化苦参碱（OM）对于人胃癌SGC-7901细胞株的杀伤作用。方法采用四甲基偶氮唑蓝（MTT）比色法检测OM对人胃癌SGC-7901细胞的杀伤作用。结果证实OM在浓度〉2gm/mL时对人胃癌SGC-7901细胞具有杀伤作用，而当浓度〈1mg/mL则无明显的细胞毒性作用。结论OM对人胃癌SGC-7901细胞株具有剂量依赖性杀伤作用。     

人MUC4启动子驱动的HSV—TK重组腺病毒靶向杀伤SGC-7901胃癌细胞的实验研究

目的：构建人粘蛋白（MUC4）启动子驱动下的单纯疱疹胸苷激酶基因（HSV-TK）重组腺病毒,研究其对SGC一7901胃癌细胞的靶向杀伤作用。方法：克隆MUC4启动子区625bp活性序列,构建重组荧光素酶报告基因载体pGL3MUC4,检测其在sGD790l胃癌细胞及NIH3T3成纤维细胞中的转录活性。以AdEasy。”腺病毒系统为载体,构建Muc4启动子驱动下的HsV—TK重组腺病毒rAdeno—MUC4一TK,感染SGC7901及NIH3T3,经更昔洛韦（GCV）处理,MTT法检测细胞活力,TUNEL法检测细胞凋亡。结果：成功扩增出大小为625bp的MUC4启动子序列。pGL3一MUC4在SGC一790l细胞中具有强转录活性,转录活性高于强启动子SV406．6倍,而NIH3T3成纤维细胞系中几乎无转录活性。构建重组腺病毒rAdeno—MUC4一TK,MTT法和TUNEI。检测结果显示,其与GCV联合能够诱导SGC-7901细胞凋亡,产生靶向细胞毒作用。结论：人MUC4启动子驱动下的HSV—TK重组腺病毒联合GCV对SGC一7901胃癌细胞具有靶向杀伤作用,MUC4启动子可以作为胃癌靶向基因治疗的工具。     

pGL3-hTERT-tk/GCV对胃癌细胞的促凋亡作用

目的：探讨重组质粒pGL3-hTERT—tk／GCV对胃癌细胞的促调亡作用。方法：以基因工程方法构建重组质粒pGL3-hTERT-tk和相应的荧光报告质粒pGL3-hTERT-tk-Luc＋；脂质体LipofectamineTM2000瞬时转染胃癌细胞系SGC-7901并用GCV干预，荧光显微镜观察细胞形态变化和转染效率，TUNEL标记和流式细胞术观察转染后胃癌细胞的凋亡；以上实验均以正常肝细胞L-02为对照。结果：经鉴定，重组质粒pGL3-hTERT-tk中tk片段的长度为1100bp。荧光素酶标记的阳性、阴性对照及治疗报告质粒pGL3-hTERT-tk-Luc。均能有效转染高表达端粒酶活性的胃癌细胞SGC-7901，转染效率为（8．2±1．14）％。重组质粒转染胃癌细胞后与GCV共育4d，细胞的凋亡率为（60．0±1．56）％；被pGL3-hTERT—tk转染的肿瘤细胞细胞周期发生了变化，处于细胞周期早期的细胞大量凋亡，早期凋亡率为（47．1±1．35）％。结论：pGL3-hTERT-tk／GCV对胃癌细胞有强烈的杀伤作用，但不影响正常细胞的生长，有潜在临床应用前景。     

Histone deacetylase inhibitor-mediated sensitization to TRAIL-induced apoptosis in childhood malignancies is not associated with upregulation of TRAIL receptor expression, but with potentiated caspase-8 activation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has great potential for the treatment of cancer because it targets tumor cells while sparing normal cells. Several cancers, however, fail to respond to TRAIL's antineoplastic effects. These resistant tumors require cotreatment with sensitizing agents in order for TRAIL to exert anticancer activity. Histone deacetylase inhibitors (HDACi) have been recognized as potent TRAIL sensitizers. In searching for the determinants of TRAIL responsiveness, HDACi-mediated TRAIL sensitization has been predominantly attributed to TRAIL receptor upregulation. This explanation, however, has been challenged by a few studies. The aim of the present study was to explore the relevance of TRAIL receptor expression for HDACi-mediated TRAIL sensitization in childhood tumors, i.e., in medulloblastoma, Ewing's sarcoma and osteosarcoma. In previous studies, we had shown that TRAIL and HDACi were synergistic in inducing apoptosis in medulloblastoma and Ewing's sarcoma. In the present study, we demonstrate that HDACi cooperated with TRAIL in eliciting cell death in osteosarcoma. However, HDACi treatment did not alter or even reduced cell surface expression of TRAIL receptors in the three childhood tumors. In gaining insight into the apoptotic pathway involved in TRAIL sensitization, HDACi were found to potentiate TRAIL-induced caspase-8 activation. Taken together, our findings suggest that HDACi-mediated TRAIL sensitization is not the result of TRAIL receptor upregulation, but the result of a receptor-proximal event in childhood tumor cells.     

The histone deacetylase inhibitors depsipeptide and MS-275, enhance TRAIL gene therapy of LNCaP prostate cancer cells without adverse effects in normal prostate epithelial cells

Gene therapy of cancer using adenovirus as a single treatment modality has met limited success and efforts to enhance therapeutic outcomes have included combination of gene therapy with chemotherapy. The goal of this study was to investigate which chemotherapeutic agents may be suitable for combination with gene therapy of prostate cancer. Using an adenovirus expressing green fluorescent protein (GFP), we determined the effect of cisplatin, gemcitabine, doxorubicin, depsipeptide and MS-275 on adenoviral infectivity and transgene expression in LNCaP cells. We found that the two histone deacetylase inhibitors (HDACi), depsipeptide and MS-275, and to a lesser extent doxorubicin, increased infectivity and transgene expression. However, only the HDACi selectively increased infectivity in LNCaP cells while doxorubicin increased infectivity to a greater extent in normal prostate epithelial cells (PrEC). The increase in infectivity but not transgene expression correlated to increased surface expression of coxsackie and adenovirus receptor (CAR). Increased transgene expression following infection with an adenovirus expressing tumor necrosis factor-related apoptosis inducing ligand (TRAIL) was observed only in LNCaP cells treated with depsipeptide or MS-275. Combination of TRAIL gene therapy with HDACi but not doxorubicin resulted in increased induction of apoptosis in LNCaP cells. In contrast, apoptosis was not enhanced by HDACi in normal PrEC. These results suggest that combination of HDACi with adenoviral TRAIL gene therapy may be a new therapeutic approach for the treatment of prostate cancer that warrants further investigation.     

Alteration of cancer stem cell‐like phenotype by histone deacetylase inhibitors in squamous cell carcinoma of the head and neck

Recent progression in the understanding of stem cell biology has greatly facilitated the identification and characterization of cancer stem cells (CSCs). Moreover, evidence has accumulated indicating that conventional cancer treatments are potentially ineffective against CSCs. Histone deacetylase inhibitors (HDACi) have multiple biologic effects consequent to alterations in the patterns of acetylation of histones and are a promising new group of anticancer agents. In this study, we investigated the effects of two HDACi, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), on two CD44+ cancer stem‐like cell lines from squamous cell carcinoma of the head and neck (SCCHN) cultured in serum‐free medium containing epidermal growth factor and basic fibroblast growth factor. Histone deacetylase inhibitors inhibited the growth of SCCHN cell lines in a dose‐dependent manner as measured by MTS assays. Moreover, HDACi induced cell cycle arrest and apoptosis in these SCCHN cell lines. Interestingly, the expression of cancer stem cell markers, CD44 and ABCG2, on SCCHN cell lines was decreased by HDACi treatment. In addition, HDACi decreased mRNA expression levels of stemness‐related genes and suppressed the epithelial‐mesencymal transition phenotype of CSCs. As expected, the combination of HDACi and chemotherapeutic agents, including cisplatin and docetaxel, had a synergistic effect on SCCHN cell lines. Taken together, our data indicate that HDACi not only inhibit the growth of SCCHN cell lines by inducing apoptosis and cell cycle arrest, but also alter the cancer stem cell phenotype in SCCHN, raising the possibility that HDACi may have therapeutic potential for cancer stem cells of SCCHN.     

New histone deacetylase inhibitors improve cisplatin antitumor properties against thoracic cancer cells

Histone deacetylase inhibitors (HDACi) have shown promising antitumor effects on numerous cancer cells including malignant pleural mesothelioma (MPM) and lung adenocarcinoma (ADCA) cells. However, clinical trials using these compounds alone have shown limited efficacy against solid tumors. Therefore, new molecules are being developed and combinations with classical chemotherapeutic drugs are being tested.Here, we have evaluated on three MPM and three lung ADCA cell lines the antitumor potential of four new HDACi compounds, either alone or in combination with cisplatin. These effects were compared with those of vorinostat, an HDACi approved for cancer treatments.First, we characterized the HDAC mRNA expression profiles of tumor cells and showed an increase of the classI/classII HDAC ratio. We then treated cancer cells with these new HDACi and observed a cell-death induction and an increase of HDACi target genes and proteins expression. This was particularly evident for NODH compound (pan-HDACi) which had similar effects at nanomolar concentrations as micromolar concentrations of vorinostat. Interestingly, we observed that the HDACi/cisplatin combination strongly increased cell-death and limited resistance-phenotype emergence as compared with results obtained when the drugs were used alone.These results could be exploited to develop MPM and lung ADCA treatments combining chemotherapeutic approaches.     

The DNA damage mark pH2AX differentiates the cytotoxic effects of small molecule HDAC inhibitors in ovarian cancer cells

High grade epithelial ovarian cancers are relatively sensitive to DNA damaging platinum-based chemotherapy, suggesting that the dependencies of ovarian tumors on DNA damage response pathways can be harnessed for therapeutic purposes. Our goal was to determine if the DNA damage mark gamma-H2AX phosphorylation (pH2AX) could be used to identify suitable cytotoxic histone deacetylase inhibitors (HDACi) for ovarian cancer treatment. Nineteen chemically diverse HDACi compounds were tested in 7 ovarian cancer cell lines. Fluorescent, biochemical and cell-based assays were performed to assess DNA damage by induction of pH2AX and to measure cell viability and apoptosis. The relationships between pH2AX and the cellular effects of cell viability and apoptosis were calculated. Selected HDACi were tested in combination with cisplatin and other DNA damaging agents to determine if the HDACi improved upon the effects of the DNA damaging agents. The HDACi compounds induced differing levels of pH2AX expression. High levels of pH2AX in HDACi-treated ovarian cancer cells were tightly associated with decreased cell viability and increased apoptosis. Consequently, a ketone-based HDACi was chosen and found to enhance the effects of cisplatin, even in ovarian cancer cells with extreme resistance to DNA damaging drugs. In conclusion, a fluorescent-based assay for pH2AX can be used to determine cellular responses to HDACi in vitro and may be a useful tool to identify potentially more effective HDACi for the treatment of ovarian cancer. In addition, these results lend support to the inclusion of ketone-derived HDACi compounds for future development.     

Histone Deacetylase Inhibitors Resensitize EGFR/EGFRvIII-Overexpressing,Erlotinib-Resistant Glioblastoma Cells to Tyrosine Kinase Inhibition

Although the epidermal growth factor receptor (EGFR) is overexpressed and/or amplified in more than 50 % of all glioblastomas (GBM), therapeutic targeting of the EGFR has not yet been successful. Since histone deacetylases (HDAC) have been described as controlling EGFR expression, we combined the EGFR tyrosine kinase inhibitor erlotinib with different HDAC inhibitors (HDACi) and investigated the benefit of combinatorial therapy for glioblastoma cells. Using representative models of EGFR-amplified, erlotinib-sensitive and -resistant GBM with or without EGFRvIII expression, we determined proliferation, migration, and EGFR-dependent signaling in response to erlotinib and HDACi alone or in combination. HDACi significantly inhibited proliferation of erlotinib-resistant GBM cells, partially restored their sensitivity to erlotinib, and also significantly reduced proliferation of all treatment-naïve cell lines tested. In combination with erlotinib, the development of resistance was prevented. The multitargeted EGFR/HDAC-inhibitor CUDC-101 exhibited similar effects. However, inhibition of cell migration was only achieved by targeting EGFR, and HDACi exhibited no additive effect. Mechanistically, we identified an HDACi-dependent decrease of EGFR/EGFRvIII protein expression underlying the anti-proliferative effects of HDACi. In conclusion, HDACi in combination with erlotinib might serve as a treatment option for newly diagnosed, treatment-naïve tumors irrespective of their EGFR status, as well as for treatment-refractory, EGFR-overexpressing GBM.     

Long-term antiepileptic treatment with histone deacetylase inhibitors may reduce the risk of prostate cancer

Various antiepileptic drugs such as valproic acid, carbamazepine, oxcarbazepine, lamotrigine and levetiracetam are known to exert histone deacetylase inhibitory (HDACi) properties, which can modify aberrantly silenced gene expression by an epigenetic mechanism. This study was initiated to examine a potential beneficial effect of these drugs on prostate cancer (PC) development. The prostate-specific antigen (PSA) levels of 106 patients under long-term treatment with antiepileptic drugs and known HDACi properties were examined. PSA represents a hallmark in the early detection of PC, and its levels may predict an invasive disease in subsequent years. For in-vitro experiments, the PC cell line LNCaP was treated with HDACi drugs; subsequently, PSA and further PC markers were assessed. When men over 50 years of age were treated with HDACi drugs they had lower age-corrected PSA levels compared with control groups, according to the following ranking: valproic acid>levetiracetam>carbamazepine/oxcarbazepine>lamotrigine. Furthermore, there was a correlation between PSA reduction and the number of HDACi drugs within the medication, lending credence to the idea that a synergistic effect might be possible. Moreover, in vitro, HDACi drugs decrease PSA on mRNA and protein levels and exhibit further oncoprotective properties.The fact that HDACi drugs exert antiproliferative effects on neoplastic cells in vitro and in vivo, which are paralleled by expression alterations of aberrantly regulated genes, underlines the potential therapeutic value of HDACi drugs. These data suggest that long-term HDACi treatment can positively influence the characteristically slow transformation of tumour precursor cells in the prostate and may thus reduce a patient's risk of developing PC.     

HDAC inhibitors effectively induce cell type-specific differentiation in human glioblastoma cell lines of different origin

The anti-neoplastic effects of histone deacetylase inhibitors (HDACi), Trichostatin A (TSA) and 4-phenylbutyrate (4-PB) on the human glioblastoma cell lines GBM-29, U-343 MG and U-343 MGa Cl. 2:6 were investigated. TSA and 4-PB induced apoptosis in the three cell lines in a dose- and time-dependent manner. Whereas caspase-3 activation was detected in all three cell lines, U-343 MG cells were more sensitive to the apoptotic effect of HDACi compared with U-343 MGa Cl. 2:6. TSA and 4-PB induced differentiation in the three cell lines, each cell line developing unique phenotypic characteristics. During long-term treatment with a low dose of HDACi U-343 MGa Cl. 2:6 cells developed an astrocytic morphology with expression of glial fibrillary acidic protein (GFAP). GFAP-negative U-343 MG cells changed their morphology in response to HDACi and down-regulated their expression of vimentin. The nestin and vimentin positive GBM-29 cells also showed a morphological differentiation, while the expression of the two malignancy markers decreased. In summary, our results showed that these three glioblastoma cell lines display unique phenotypes and differentiation patterns in response to HDACi.