Histone deacetylase regulation of immune gene expression in tumor cells

Epigenetic modifications of chromatin, such as histone acetylation, are involved in repression of tumor antigens and multiple immune genes that are thought to facilitate tumor escape. The status of acetylation in a cell is determined by the balance of the activities of histone acetyltransferases and histone deacetylases. Inhibitors of histone deacetylase (HDACi) can enhance the expression of immunologically important molecules in tumor cells and HDACi treated tumor cells are able to induce immune responses in vitro and in vivo. Systemic HDACi are in clinical trails in cancer and also being used in several autoimmune disease models. To date, 18 HDACs have been reported in human cells and more than thirty HDACi identified, although only a few immune targets of these inhibitors have been identified. Here, we discuss the molecular pathways employed by HDACi and their potential role in inducing immune responses against tumors. We review data suggesting that selection of target specific HDACi and combinations with other agents and modalities, including those that activate stress pathways, may further enhance the efficacy of epigenetic therapies.     

As_2O_3对胃癌细胞中肿瘤干性相关因子表达的抑制作用

目的观察As_2O_3对胃癌干性相关因子CD44、EpCAM表达的影响,并探讨其可能的作用机制。方法体外实验:以MGC803细胞株为实验对象,给予4μmol/L的As_2O_3处理48 h后,Western blot及RT-PCR法检测CD44、EpCAM的表达。体内实验:建立人胃癌MGC803细胞裸鼠移植瘤模型,分为对照组和As_2O_3(5 mg/kg/天)组,观察裸鼠体内肿瘤的形成及瘤体组织的体积和重量;给药10天后处死裸鼠,分别用Western blot和RT-PCR法检测两组肿瘤中CD44、EpCAM的表达。结果体外:细胞在用As_2O_3处理后,与对照组相比,As_2O_3组中CD44、Ep CAM的蛋白和mRNA表达均减少(P0.05)。体内:与对照组相比,As_2O_3组中裸鼠的移植瘤体积和重量明显降低;Western blot和RT-PCR检测结果均表明在As_2O_3组中移植瘤的CD44、EpCAM的蛋白和mRNA表达均降低,差异有统计学意义(P0.05)。结论 As_2O_3在体内和体外水平均抑制了CD44、EpCAM的表达,因此As_2O_3对胃癌中的肿瘤干细胞种群具有明显的抑制作用。     

Increased expression of histone deacetylase 2 is found in human gastric cancer

Accumulated evidence has established that aberrant regulation of histone deacetylases (HDACs) is one of the major causes of the development of human malignancies. Among different iso-enzymes of HDAC and sirtuins grouped as the HDAC super family, little is known as to how histone deacetylase 2 (HDAC2) causes carcinogenesis in solid tumors. Here, in order to investigate the possible role of HDAC2 in gastric carcinogenesis, we analyzed the expression of HDAC2 in 71 gastric adenocarcinomas by immunohistochemistry. Moderate to strong expression of HDAC2 was found in 44 (62%) out of a total of 71 tumors. The majority of positive tumors, which were detected in the nucleus but not in normal gastric epithelium, did not express HDAC2 or showed only weak positive staining. Interestingly, we also noted that HDAC2 expression appeared to be associated with tumor aggressiveness as HDAC2 expression was observed to be statistically significant in advanced gastric cancer (P=0.0023, Chi-square test) and in positive lymph node metastasis (P=0.0713, Chi-square test). Taken together, these results suggest that HDAC2 may play an important role in the aggressiveness of gastric cancer.     

PUMA增强依托泊苷诱导宫颈癌细胞凋亡

目的探讨PUMA蛋白在依托泊苷诱导的宫颈癌细胞凋亡中的作用。方法采用MTT方法检测依托泊苷对宫颈癌细胞系SiHa细胞活力的影响。通过Annexin-v／PI双染检测和光学显微镜形态观察检测依托泊苷对宫颈癌SiHa细胞凋亡的影响。通过转染siRNA干扰PUMA的表达,检测低表达PUMA后,宫颈癌细胞对依托泊苷的敏感性。通过免疫印迹方法检测PUMA干扰后PUMA的表达水平。结果依托泊苷显著抑制宫颈癌细胞的活力和诱导其凋亡,且随着浓度和时间的增加而细胞活力依次减少而凋亡率依次升高,且光学显微镜下有明显的细胞皱缩等凋亡形态。当转染PUMA的siRNA后．依托泊苷诱导的细胞凋亡显著减少。结论PUMA在依托泊苷诱导宫颈癌细胞凋亡中起了重要作用,它能够增强宫颈癌细胞对依托泊苷的敏感性。     

5,7,2,5-tetrahydroxy-8,6-dimethoxyflavone up-regulates miR-145 expression and inhibits proliferation of gastric cancer cells

IntroductionGastric cancer is a frequently detected malignancy, and its incidence has increased over the past decades in East Asia. The present study investigated the effect of 5,7,2, 5-tetrahydroxy-8,6-dimethoxyflavone (THDMF) on gastric cancer cells and explored the underlying mechanism. The study analysed cell viability changes, apoptotic features, and metastasis potential of treatment with THDMF.Material and methodsMTT colorimetric assay was used for measurement of MKN28, MKN45, and GES-1 cell proliferation and flow cytometry for the detection of apoptosis. Transwell and wound healing assays were used to observe the invasion and migration abilities of MKN28 cells. The expression of p21, MMP2/-9, PI3K, and c-Myc proteins was detected by western blotting.ResultsThe THDMF treatment significantly (p < 0.05) reduced MKN28 and MKN45 cell proliferation without changing GES-1 cell viability. A significant increase in apoptotic cell population on treatment with THDMF was observed. Treatment of MKN28 cells with THDMF increased the percentage of cells in the G1 phase. Exposure of MKN28 cells to THDMF caused a marked decrease in invasion and migration potential in comparison to control cells. The expression of miR-145 was markedly increased in MKN28 cells on treatment with THDMF. In MKN28 cells expression of c-Myc, PI3K, p-AKT, MMP-2, and MMP-9 was suppressed markedly on exposure to THDMF. The expression of p21 protein in MKN28 cells was markedly promoted on exposure to THDMF.ConclusionsTHDMF exhibits anti-cancer effect on gastric cancer cells in vitro by activation of cell apoptosis and arrest of cell cycle. In addition, THDMF promoted miR-145 expression and down-regulation of PI3K/AKT signalling pathway in MKN28 cells. Therefore, THDMF may be utilised as a potential novel therapeutic agent for the treatment of gastric cancer.     

Histone deacetylase inhibitors increase neuronal differentiation in adult forebrain precursor cells

Chromatin modification plays a key role in fate decision of neural stem cells. Here, we explored the impact of epigenetic remodelling onto neuronal fate determination using specific inhibitors of histone deacetylases (iHDAC). Adult subventricular zone (SVZ) precursor cells were expanded as neurospheres and treated in vitro with second generation iHDAC MS-275, M344 and suberoylanilide hydroxamic acid (SAHA). All tested compounds revealed a significant increase of βIII-tubulin positive neurons (ranging from 258 to 431%) in a concentration-dependent manner. The number of oligodendrocytes was decreased by almost 50%, accompanied by a reduction of Olig2 mRNA expression. In contrast, astrocyte quantity remained unaffected after iHDAC treatment. Both control and iHDAC treated cells expressed markers of mature GABAergic and dopaminergic neurons. Increased expression levels of NeuroD, Cyclin D2 and B-lymphocyte translocation gene 3 (Btg3) point to a shift towards neuronal fate determination targeted by HDAC inhibitors.     

Decreased expression of histone deacetylase 10 predicts poor prognosis of gastric cancer patients

Aberrant expression of histone deacetylase (HDACs) was associated with carcinogenesis and progression of various tumors. However, the association of HDAC10 with clinical outcomes in gastric cancer patients is unclear. Thus, the objective of the current study was to evaluate the association of expression level of HDAC10 with clinicopathologic factors and prognosis of patients with gastric cancer. The expression level of HDAC10 in 179 paraffin-embedded gastric cancer tissue specimens was examined by immunohistochemistry (IHC). As a result, we found that expression of HDAC10 in gastric cancer was significantly decreased in gastric cancer tissues as compared with adjacent tissues (51.4% vs. 87.3%, P < 0.001). HDAC10 expression was significantly correlated with gender (P = 0.023), tumor size (P = 0.015), histological grade (P = 0.009), tumor invasion (P = 0.033), lymph node metastatic status (P = 0.019) and tumor stage (P = 0.004), but not correlated with age and lauren classification (all P > 0.05). Kaplan-Meier survival curves showed that the overall survival rate was significantly lower in the patients with low expression of HDAC10 compared with those patients with high HDAC10 (P < 0.001). Moreover, multivariate analysis revealed that HDAC10 expression was an independent prognostic factor for gastric cancer patients (P = 0.001). These results suggest that HDAC10 expression could see as a prognosis marker for gastric cancer patients.     

组蛋白去乙酰化酶3抑制剂通过减轻氧化应激降低缺氧/复氧引起的PC12细胞凋亡

目的 探讨组蛋白去乙酰化酶3（HDAC3）抑制剂（HDAC3I）RGFP966在PC12细胞缺氧/复氧（H/R)损伤中的作用。 方法 采用PC12细胞缺氧4 h复氧24 h培养建立H/R细胞损伤模型。H/R+抑制剂组采用RGFP966预处理1 h后进行H/R处理。实验分为3组,对照组、H/R组和H/R+抑制剂组,每组重复3次。采用MTT法测定细胞活性,比色法检测细胞乳酸脱氢酶(LDH),流式细胞术分别检测细胞凋亡率和胞内活性氧簇（ROS）,黄嘌呤氧化酶法测定超氧化物歧化酶（SOD）活性,硫代巴比妥酸法测定丙二醛（MDA）含量,Western blotting法检测Bcl-2促凋亡基因(Bax)、B淋巴细胞瘤-2基因（Bcl-2）、剪切型Caspase-3（cleaved-Caspase-3）和HDAC3蛋白表达。 结果 与对照组相比,H/R组与H/R+抑制剂组细胞活力均显著降低（P<0.05）,细胞LDH（P<0.05）和凋亡率（P<0.05）均显著升高。H/R+抑制剂组与H/R组相比,H/R+抑制剂组细胞活力显著高于H/R组（P<0.05）,而H/R+抑制剂组细胞LDH（P<0.05）和凋亡率显著低于H/R组（P<0.05）。此外,与对照组相比,H/R组与H/R+抑制剂组ROS和MDA（P<0.05）均显著增加,SOD显著降低（P<0.05）;H/R+抑制剂组与H/R组相比,H/R+抑制剂组ROS和MDA（P<0.05）显著低于H/R组,而SOD水平高于H/R组（P<0.05）;Western blotting结果表明,与对照组相比,H/R组与H/R+抑制剂组Bax、cleaved-Caspase-3均显著升高,Bcl-2显著降低（P<0.05）,H/R+抑制剂组Bax、cleaved-Caspase-3均显著低于H/R组,Bcl-2显著高于H/R组（P<0.05）;与对照组相比,H/R组HDAC3蛋白表达显著升高（P<0.05）,而H/R+抑制剂组HDAC3蛋白显著降低（P<0.05）。 结论 HDAC3I通过减轻氧化应激降低缺氧/复氧引起的PC12细胞凋亡。     

组蛋白去乙酰化酶抑制剂促大鼠胚胎干细胞向神经元分化

目的探讨组蛋白去乙酰化酶（HDAC）抑制剂丁酸钠（NaB）对大鼠胚胎干细胞（ESC）向神经元分化的影响。方法利用含bFGF、EGF的DMEM／F12培养基培养原代ESC;应用DAPI染色,荧光显微镜观察不同药物浓度NaB（0．2、1、2μmol／L）作用48h后对细胞凋亡的影响;免疫荧光检测NaB（1μmol／L）作用72h后和对照组（PBS）ESC双肾上皮质激素（DCX）和DAPI,计算DCX／DAPI的比值;免疫印迹检测不同浓度NaB（0．2、1、2μmol／L）作用48h后和对照组（PBS）ESC组蛋白H3、H4乙酰化水平。结果在1、2／μmol／LNaB组ESC出现明显凋亡,细胞形状不规则,核固缩,核内可见致密的颗粒荧光,视野下细胞碎片较多,且凋亡细胞百分比明显高于0．2μmol／LNaB组和对照组[（7．85±0．73）％、（18．42±2．04）％比（3．48±0．35）％、（2．16±0．32）％,均P〈0．05]。1μmol／LNaB组ESCDCX／DAPI比值高于对照组（38．51±4．33比14．81±1．77,P〈0．05）。随NaB药物浓度的增加,ESC蛋白H3、H4乙酰化程度较对照组增强,0．2±mol／L组处理后乙酰化组蛋白H3和H4表达变化不明显,1、2μmol／L时组蛋白H3和H4乙酰化程度明显增高（均P〈0．05）。结论HDAC抑制剂NaB可明显促使ESC向神经元分化。     

纺锤菌素抑制胃癌细胞的侵袭和转移

目的探讨纺锤菌素(netropsin)对胃癌细胞侵袭和转移能力的影响及其分子机制。方法用Transwell检测胃癌细胞侵袭和转移能力,用Western blot检测EMT相关标志物E-cadherin和vimentin表达,用免疫荧光检测β-catenin的细胞定位,验证netropsin作用前后Wnt/β-catenin信号通路活性。结果 Netropsin浓度25μmol/L时对MKN28细胞增殖有抑制作用,netropsin可以降低上皮标志物E-cadherin的表达,上调间质标志物vimentin的表达;netropsin可以通过抑制胃癌细胞的EMT,降低胃癌细胞侵袭和转移能力(P0.05),同时可阻止β-catenin进入细胞核。结论 Netropsin可以通过与HMGA2竞争结合转录因子结合位点抑制Wnt/β-catenin信号通路,降低胃癌细胞EMT的发生,从而抑制胃癌细胞侵袭能力。     

Histone deacetylase inhibitors promote apoptosis and senescence in human mesenchymal stem cells

Histone deacetylase inhibitors (HDACi) have received a great amount of attention for their antitumoral properties. Suberoyl anilide hydroxamic acid (SAHA) and MS-275 are among the more promising HDACi for cancer treatments. Although these HDACi compounds exert low toxicity on normal cells, the therapies based on these molecules can cause side effects that can greatly impair the functions of the bone marrow microenvironment. This is a complex system that contains several types of stem cells, such as mesenchymal stem cells (MSCs). We conducted comparative studies on the effects of SAHA and MS-275 on human MSCs in order to ascertain if these compounds can impair the physiology of MSCs. Both SAHA and MS-275 induced an arrest in the cell cycle along with the induction of apoptotic pathways as evidenced by flow cytometry, annexin assay, detection of activated caspase 9, and molecular analysis of Bax/Bcl-2 expression. The MS-275 treatment induced an increase of senescent cells, whereas in cells treated with SAHA, we detected a reduction of senescent cells compared to the control. We hypothesize that SAHA preferentially transactivates apoptotic genes, thereby inducing a great majority of the damaged cells to die by programmed cell death rather than senescence. Following the HDACi treatment, we observed a decrease in the expression of some genes that are involved in the regulation of stem cell properties. This suggests that SAHA and MS-275 could also be involved in the impairment of the stemness characteristics of MSCs. The phenomena that were induced by HDACi treatment were associated with an upregulation of several cyclin kinase inhibitors. By contrast, the p53-p21 pathway is apparently not involved in these processes.     

黄芩素抑制胃癌MGC-803细胞增殖和迁移

目的:探讨黄芩素(BAI)对胃癌MGC-803细胞增殖和迁移的作用及机制。方法:MGC-803细胞用不同浓度BAI处理后,采用MTT法检测细胞的存活率;平板集落形成实验检测细胞的集落形成能力;划痕愈合实验和Transwell小室迁移实验检测细胞的迁移能力;ELISA检测12-羟基二十碳四烯酸(12-HETE)的浓度;Western blot实验检测血小板型12-脂氧合酶(p12-LOX)、血管内皮生长因子(VEGF)、p-ezrin和上皮-间充质转化(EMT)标志物蛋白的表达。结果:BAI可显著抑制MGC-803细胞的增殖、平板集落形成及迁移(P0.05或P0.01),显著下调p12-LOX代谢产物12-HETE的浓度(P0.05或P0.01),并显著下调p12-LOX、VEGF、p-ezrin、vimentin和Snail蛋白的表达水平(P0.05或P0.01),上调E-cadherin蛋白的表达水平(P0.01)。结论:BAI可有效抑制胃癌MGC-803细胞的增殖和迁移,其机制与BAI调控p12-LOX、VEGF、p-ezrin及EMT相关蛋白的表达变化有关。     

A novel long non-coding RNA NFIA-AS1 is down-regulated in gastric cancer and inhibits proliferation of gastric cancer cells

Gastric cancer is one of the most common malignant gastrointestinal tumors whose morbidity and mortality account for the second and third place respectively in malignant tumors in China. As an important participant in tumor biology, the abnormal expression of long non-coding RNA (lncRNAs) in cancer cells is closely related to the occurrence and development of tumors and plays the role of oncogenes or tumor suppressor genes. In this study, we identified a novel lncRNA NFIA antisense RNA 1 (NFIA-AS1) and explored its role and clinical significance in gastric cancer. Real-time quantitative PCR was performed to detect the expression of NFIA-AS1 in tumor tissues and corresponding normal tissues from 42 pairs of gastric cancer samples. The lower expression of NFIA-AS1 was significantly associated with larger tumor size, lower histological grade, and advanced TNM stage. Kaplan-meier analysis showed that NFIA-AS1 expression could be used as an independent predictor of overall survival. We also demonstrated that overexpression of NFIA-AS1 significantly inhibited the proliferation of gastric cancer cells through affecting p16 levels. In conclusion, our results suggest that the lncRNA NFIA-AS1 may play the role of tumor suppressor gene, and serve as a biomarker for prognosis or progression of gastric cancer.     

Histone deacetylase inhibitor valproic acid affects plasmacytoid dendritic cells phenotype and function

Objective

Plasmacytoid dendritic cells (PDC) represent a rare subset of dendritic cells specialized in the production of type I IFN in response to microbial pathogens. Recent data suggested that histone deacetylase (HDAC) inhibitors possess potent immunomodulatory properties both in vitro and in vivo. In this study, we assayed the ability of the HDAC inhibitor, valproic acid (VPA), to influence the phenotype and functional properties of human PDC isolated from peripheral blood.

Methods and results

We showed that VPA inhibited the production of IFN-α and the proinflammatory cytokines TNF-α and IL-6 by CpG-activated PDC. VPA also affected the phenotype of PDC by reducing the expression of costimulatory molecules induced by CpG activation. Moreover, VPA reduced the capacity of CpG-stimulated PDC to promote CD4⁺ T cell proliferation and IFN-γ production, while enhancing the proportion of IL-10 positive T cells.

Conclusion

These results suggest that HDAC inhibition by VPA alters essential human PDC functions, highlighting the need for monitoring immune functions in cancer patients receiving HDAC inhibitors, but also making these drugs attractive therapies in inflammatory, and autoimmune diseases implicating PDC.