组蛋白去乙酰化酶抑制剂MS-275在体外抑制胃癌细胞的实验研究

背景与目的:组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitors,HDACis)对于很多肿瘤细胞具有选择性的诱导凋亡及分化作用,而对正常细胞的细胞毒性作用很低,这一选择性杀伤作用使HDACis成为目前抗肿瘤分子靶向药物的研究热点.本文研究了HDACis MS-275对人胃癌细胞SGC-7901、人胃黏膜正常细胞GES-1和张氏肝细胞(CHANG-LIVER)的选择性作用及机制,为HDACis的临床应用提供了理论依据.方法:不同终浓度MS-275处理SGC-7901细胞、GES-1和张氏肝细胞,用WST-1方法检测药物对细胞的生长抑制作用;AnnexinV、PI双标流式细胞术检测MS-275对肿瘤细胞SGC-7901和正常细胞的选择性杀伤作用;TUNEL染色观察肿瘤细胞核内凋亡情况.结果:MS-275对胃癌细胞SGC-7901具有明显的生长抑制作用,并且这种作用具有时间及剂量依赖性;MS-275的杀伤作用具有选择性,对正常细胞GES-1和张氏肝细胞并不表现出促凋亡作用;TUNEL染色证明MS-275能诱导SGC-7901细胞核内发生DNA断裂,是细胞发生凋亡的重要指标.结论:MS-275具有体外选择性杀伤胃癌细胞的作用,对正常细胞没有明显的杀伤作用,有希望成为新一代的胃癌临床治疗的肿瘤分子靶向药物.     

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。     

藤梨根提取物对人胃癌MKN－45细胞增殖及凋亡的影响

目的：探讨太白山藤梨根提取物（Radix Actinidiae extractive,RAE）在体外对胃癌MKN-45细胞增殖与凋亡的影响。方法：用0．01—100μg／ml浓度范围内的RAE和胃癌MKN-45细胞共培养24、48、72、96小时,采用MTF法检测RAE对MKN-45细胞增殖的影响;用流式细胞仪检测其对MKN-45细胞凋亡及细胞周期分布的影响;DNA琼脂糖凝胶电泳检测细胞凋亡。结果：RAE在体外能够显著抑制胃癌MKN-45细胞的增殖,且呈现浓度和时间依赖性;RAE对胃癌细胞的作用表现为周期特异性,使细胞阻滞于Go／G1期,进一步诱导细胞凋亡。对照组凋亡率为0．53％,1μg／ml、10μg／ml、100μg／ml浓度的RAE干预胃癌细胞48小时后均出现凋亡峰,细胞凋亡率分别为3．27％、8．97％、12．6％。DNA琼脂糖凝胶电泳检测细胞凋亡,不同浓度RAE处理的细胞样品中均可看到梯形凋亡条带,并呈现浓度依赖性。结论：RAE在体外对人胃癌MKN-45细胞有显著的抑制细胞增殖及诱导细胞凋亡作用,其作用机制可能是使胃癌细胞周期阻滞于G0／G1期。     

组蛋白去乙酰化酶抑制剂MS-275诱导骨髓瘤细胞株U266凋亡过程中Survivin的表达

背景与目的：组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitors,HDACi）是-类具有抗肿瘤活性的新型药物,主要通过诱导细胞凋亡发挥作用。本实验研究HDACi（MS-275）对人骨髓瘤细胞系U266细胞增殖和凋亡的影响及其与Survivin表达的关系。方法：将不同浓度的MS-275作用于U266细胞不同时间后,用台盼蓝拒染法观察药物对细胞活力的影响：通过瑞氏-姬姆萨染色观察药物作用后细胞形态学的变化;用流式细胞仪分析细胞周期;用Westernblot检测Survivin、P21和Cdk4等的蛋白表达,以及凋亡信号通路中Caspase-3活化及蛋白聚ADP核糖聚合酶[poly（ADP—ribose）polymerase,PARP]的裂解情况。结果：MS-275呈时间和剂量依赖性抑制U266细胞增殖,阻断细胞周期于G0／G1期。MS-275作用U266细胞48h时的Ic50为1．39μmol／L：2μmol／L的MS-275作用U266细胞24h后,G／G,期细胞占66．39％．36h后G0／G1期细胞占89．80％。瑞氏-姬姆萨染色显示细胞形态发生明显变化。Western blot检测结果显示。MS-275作用U266细胞后,Survivin和Cdk4表达下降,P21表达增加,Caspase3被裂解活化,其底物蛋白PARP发生剪切。结论：MS-275抑制人多发性骨髓瘤细胞系U266增殖并诱导细胞凋亡,可能与下调Survivin蛋白的表达有关。     

TSA对人肝癌细胞SMMC-7721的抑制作用及其机制

目的：研究去乙酰化转移酶抑制剂TSA对肝癌细胞SMMC-7721的作用及其机理。方法：利用细胞计数,流式细胞仪分析细胞凋亡及细胞周期,Tunel试验研究TSA对肝癌细胞SMMC-7721的作用;利用western研究TSA对肝癌细胞蛋白表达的影响。结果：TSA可明显抑制肝癌细胞SMMC-7721的生长,并可诱导细胞凋亡。可阻滞肝癌细胞SMMC-7721细胞周期于G0/G1期。可增加p53,p21,bax等基因的表达,降低BCL-2的表达。结论：去乙酰化转移酶抑制剂TSA可明显抑制肝癌细胞SMMC-7721的生长并诱导其凋亡,其主要通过调控一些肿瘤相关基因的表达起作用。     

藤梨根提取物对人胃癌MKN-45细胞增殖及凋亡的影响

目的:探讨太白山藤梨根提取物(Radix Actinidiae extractive,RAE)在体外对胃癌MKN-45细胞增殖与凋亡的影响。方法:用0.01-100μg/ml浓度范围内的RAE和胃癌MKN-45细胞共培养24、48、72、96小时,采用MTT法检测RAE对MKN-45细胞增殖的影响;用流式细胞仪检测其对MKN-45细胞凋亡及细胞周期分布的影响;DNA琼脂糖凝胶电泳检测细胞凋亡。结果:RAE在体外能够显著抑制胃癌MKN-45细胞的增殖,且呈现浓度和时间依赖性;RAE对胃癌细胞的作用表现为周期特异性,使细胞阻滞于G0/G1期,进一步诱导细胞凋亡。对照组凋亡率为0.53%,1μg/ml、10μg/ml、100μg/ml浓度的RAE干预胃癌细胞48小时后均出现凋亡峰,细胞凋亡率分别为3.27%、8.97%、12.6%。DNA琼脂糖凝胶电泳检测细胞凋亡,不同浓度RAE处理的细胞样品中均可看到梯形凋亡条带,并呈现浓度依赖性。结论:RAE在体外对人胃癌MKN-45细胞有显著的抑制细胞增殖及诱导细胞凋亡作用,其作用机制可能是使胃癌细胞周期阻滞于G0/G1期。     

组蛋白去乙酰化酶抑制剂MS-275对人宫颈癌SiHa细胞增殖及凋亡的影响

目的： 观察组蛋白去乙酰化酶抑制剂MS-275对人宫颈癌SiHa细胞增殖和凋亡的影响,并探讨其可能的机制。 方法： 以MS-275（0、5、10、20 μmol/L）作用于人宫颈癌SiHa细胞,MTT法检测细胞的增殖,流式细胞术检测细胞的凋亡,Western blotting法检测细胞内乙酰化组蛋白H4（acetylhistone4,Ac-H4）水平,RT-PCR检测p21和p53 mRNA的表达。 结果： MS-275作用后,人宫颈癌SiHa细胞增殖受到抑制、细胞凋亡增加,20 μmol/L MS-275作用下,细胞增殖率仅为（1395±891）%,凋亡率高达（3838±178）%,显著高于其他各组（P< 001）。MS-275作用后,细胞内Ac-H4水平增加（P< 001）,p21和p53 mRNA表达增加（均P< 001）。 结论： MS-275可抑制人宫颈癌SiHa细胞的增殖,诱导SiHa细胞凋亡;上调p21和p53表达、提高组蛋白乙酰化水平可能是其作用机制之一。     

组蛋白去乙酰化酶抑制剂MS-275对人骨髓瘤U266细胞生长的影响及其可能机制

目的 探讨组蛋白去乙酰化酶抑制剂(HDACi)MS-275对人骨髓瘤细胞U266的凋亡抑制基因生存素(survivin)和核因子-κB(NF-κB)表达的影响.方法 台盼蓝拒染法观察MS-275对细胞活力的影响;瑞氏-姬姆萨染色观察细胞形态学变化;流式细胞仪分析细胞周期;Western blot检测survivin、p21、细胞周期依赖激酶4(CDK4)和NF-κB的抑制蛋白(IKB-α)等的表达,以及凋亡信号通路中caspase-3活化及蛋白聚ADP核糖聚合酶(PARP)裂解情况.结果 MS-275抑制U266细胞增殖,阻断细胞周期于G0/G1期,呈时间-剂量依赖性.MS-275作用U266细胞48 h的IC50为1.39μmol/L.2 μmol/L MS-275作用U266细胞24 h后,G0/G1期细胞所占比例为(64.57±4.09)%;作用36 h后,G0/G1期细胞所占比例为(87.20±2.83)%;瑞氏-姬姆萨染色显示,U266细胞形态发生明显变化.Western blot检测表明,MS-275作用U266细胞后,survivin和CDK4表达下降,p21表达增加,IκB-α磷酸化水平受到明显抑制,caspase-3被裂解活化,其底物蛋白PARP发生剪切,细胞发生凋亡.结论 MS-275可诱导人骨髓瘤细胞系U266凋亡,NF-κB信号通路阻断是凋亡发生的机制之一.     

TSA对人肝癌细胞SMMC-7721的抑制作用及其机制

目的:研究去乙酰化转移酶抑制剂TSA对肝癌细胞SMMC-7721的作用及其机理。方法:利用细胞计数,流式细胞仪分析细胞凋亡及细胞周期,Tunel试验研究TSA对肝癌细胞SMMC-7721的作用;利用western研究TSA对肝癌细胞蛋白表达的影响。结果:TSA可明显抑制肝癌细胞SMMC-7721的生长,并可诱导细胞凋亡。可阻滞肝癌细胞SMMC-7721细胞周期于G0/G1期。可增加p53,p21,bax等基因的表达,降低BCL-2的表达。结论:去乙酰化转移酶抑制剂TSA可明显抑制肝癌细胞SMMC-7721的生长并诱导其凋亡,其主要通过调控一些肿瘤相关基因的表达起作用。     

miR-133在胃癌组织中的表达及对胃癌细胞恶性行为的影响

目的 探讨microRNA-133（miR-133）在胃癌组织及细胞系中的表达情况,并探讨其对胃癌细胞凋亡和侵袭的影响。方法 采用实时定量PCR（qPCR）检测64例胃癌组织及对应癌旁组织中的miR-133水平,分析miR-133表达与临床病理参数（性别、年龄、肿瘤位置、临床分期、分化程度、浸润深度及淋巴结转移）的关系,并检测其在胃癌细胞株AGS、SGC-7901、MKN-1、MKN-45、MGC-803和BGC-823及正常胃黏膜细胞GES-1细胞的表达情况,同时选取miR-133水平最低的胃癌细胞并转染过表达miR-133的真核重组质粒pCDNA3.1+miR-133,转染后分别采用流式细胞仪PI/Annexin V双染法及Transwell法检测miR-133对细胞凋亡和侵袭能力的影响。结果 胃癌组织的miR-133相对表达量为0.347±0.024,低于癌旁组织（P＜0.05）,且其表达与临床分期、分化程度、浸润深度及淋巴结转移有关均有关（P＜0.05）;与GES-1细胞相比（其miR-133表达水平设为1.00）,胃癌细胞的miR-133水平均较低（P＜0.05）,且MKN-45的表达水平最低;与对照组和空转染组相比,过表达组转染48、96 h后的细胞凋亡水平均升高,且穿膜细胞数均降低（P＜0.05）。结论miR-133在胃癌组织和细胞中均为低表达,上调miR-133水平可抑制胃癌细胞的侵袭并诱导凋亡。     

miR-26a/b调控p53/MDM2通路对胃癌细胞凋亡的影响

目的 探讨miR-26a/b调控p53/MDM2通路对胃癌细胞凋亡的影响。方法 实时荧光定量PCR检测胃癌细胞系MGC803、MKN-45和MKN-28中miR-26a/b的表达水平,荧光素酶报告系统分析miR-26a/b与MDM2 3’非翻译区（3’UTR）的结合情况;将化学合成miR-26a/b前体分子mimics和无关序列转染胃癌细胞MKN-45,化学合成miR-26a/b inhibitor转染胃上皮细胞GES-1后,Western blotting检测MDM2、p53及其下游分子p21和Bcl-2的表达,MTT法检测转染24、48、72 和96 h的细胞增殖情况;通过Annexin Ⅴ/PI双染检测miR-26a/b对MKN-45细胞凋亡情况。结果 与永生化胃上皮细胞GES-1相比,miR-26a/b在肿瘤细胞系MGC803、MKN-45和MKN-28中的表达均下调,在MKN-45中表达水平最低;荧光素酶活性检测显示,miR-26a/b过表达抑制MDM2 3’UTR报告载体（Wild）的荧光素酶活性,而对3’UTR突变型报告载体（Mutation）荧光素酶活性无明显影响。miR-26a/b抑制MKN-45细胞中MDM2表达并增强p53及下游分子表达,而在GES-1细胞中抑制miR-26a/b可以增强MDM2表达,降低p53及下游分子表达。MTT结果显示,miR-26a/b抑制MKN-45细胞的增殖,miR-26a/b inhibitor则明显促进GES-1细胞的增殖。通过AnnexinⅤ/PI检测细胞凋亡发现,miR-26a/b过表达的MKN-45细胞的凋亡率高于对照细胞（P＜0.01）。结论 miR-26a/b 能与MDM2 3’UTR 特异结合,调控p53/MDM2通路影响胃癌细胞的增殖及凋亡。     

长链非编码RNA NR2F2-AS1对胃癌细胞增殖、凋亡的影响

目的:研究lncRNA NR2F2-AS1对胃癌细胞增殖、凋亡的影响,并探讨其机制。方法:运用qRT-PCR法检测细胞中 NR2F2-AS1、miR-425-5p的mRNA表达情况;将pcDNA组（转染pcDNA）、pcDNA-NR2F2-AS1组（转染pcDNA-NR2F2-AS1）、sh-NR2F2-AS1组(转染sh-NR2F2-AS1)、sh-NC组(转染sh-NC)、anti-miR-NC组（转染anti-miR-NC）、anti-miR-425-5p组（转染anti-miR-425-5p）、pcDNA-NR2F2-AS1+miR-NC组（共转染pcDNA-NR2F2-AS1和miR-NC）、pcDNA-NR2F2-AS1+miR-425-5p组（共转染pcDNA-NR2F2-AS1和miR-425-5p mimics）转染至MGC-803、MKN-45细胞;MTT法检测细胞的增殖;流式细胞术检测细胞的凋亡;双荧光素酶报告基因检测实验检测细胞的荧光活性。结果:与正常胃黏膜上皮细胞GES-1相比,胃癌细胞MGC-803、MKN-45中NR2F2-AS1的表达显著降低,miR-425-5p的表达显著升高;过表达NR2F2-AS1、抑制miR-425-5p均可抑制胃癌细胞增殖,促进细胞凋亡;miR-425-5p可抑制野生型NR2F2-AS1的MGC-803、MKN-45细胞的荧光活性;过表达miR-425-5p可逆转过表达NR2F2-AS1对MGC-803、MKN-45细胞增殖和凋亡的作用。结论:lncRNA NR2F2-AS1可抑制胃癌细胞的增殖,促进细胞凋亡,其机制可能与靶向miR-425-5p有关,将可为胃癌的治疗提供新靶点。     

Reactivation of death receptor 4 (DR4) expression sensitizes medulloblastoma cell lines to TRAIL

Object Apoptosis, a key cellular response to therapeutic agents is often inactivated in tumor cells. In this study, we evaluated the expression of the tumor necrosis family of death receptors, DR4 and DR5, in medulloblastoma tumor samples and cell lines to determine if epigenetic modulation of gene expression could sensitize tumor cell lines to TRAIL-mediated apoptosis. Methods Human medulloblastoma samples and cell lines were analyzed for DR4 and DR5 expression by quantitative PCR and immunofluorescence assays. Cell lines with downregulated expression of one or both genes were treated with the histone deacetylase inhibitor, MS-275, and the expression of DR4 and DR5 measured by quantitative PCR, Western blotting, flow cytometry and chromatin immunoprecipitation assays. Induction of apoptosis in the presence of MS-275 was evaluated by TUNEL assay and its ability to augment TRAIL-mediated cytotoxicity was determined by MTT assays, Western blotting and flow cytometry. Results Compared to normal cerebellum, DR4, but not DR5 expression was consistently downregulated in medulloblastoma tumor samples and in Daoy and D283 cell lines. Interestingly, MS-275 decreased cell growth and induced apoptosis in Daoy and D283 cells. In Daoy cells, this coincided with increased histone H3 and H4 acetylation at the DR4 promoter and enhanced DR4 gene and protein expression as well as elevated Caspase-8 activity. The involvement of DR4 in the cellular response to MS-275 was further confirmed by the observation that knockdown of DR4 and FADD abrogated apoptosis. Further, addition of TRAIL to MS-275 treated cells resulted in an enhancement of apoptosis, suggesting that the upregulated death receptors were functional. Conclusion Our study provides an understanding of the role of DR4 in apoptosis of medulloblastoma cell lines and suggests a potential contribution of aberrant histone deacetylation to the resistance of medulloblastoma cells to therapeutic death. This work was supported in part by Award P50CA127001 from the National Cancer Institutes and Grants from the National Brain Tumor Foundation and the American Cancer Society-IRG to VG.     

miR-325-3p靶向CLDN1基因调控胃癌上皮间质转化和侵袭转移

目的 探讨miR-325-3p靶向CLDN1基因对胃癌上皮间质转化和侵袭转移的影响。方法 选取人胃黏膜上皮细胞株GES-1以及人胃癌细胞株HGC27、SGC-7901、MKN-45和MGC-803,并检测细胞中miR-325-3p和CLDN1的表达。双荧光素酶报告实验验证miR-325-3p和CLDN1的靶向关系,干预胃癌细胞中miR-325-3p和CLDN1的表达,qRT-PCR和Western blot检测细胞中N-cadherin、Vimentin和MMP2的表达,CCK-8检测细胞增殖活力,Transwell和流式细胞仪分别检测细胞侵袭和凋亡能力。结果 相对于GES-1细胞,MGC-803细胞中miR-325-3p表达降低而CLDN1表达增高（均P<0.05）,双荧光素酶报告实验证实CLDN1为miR-325-3p的靶基因。过表达miR-325-3p能够抑制胃癌细胞的增殖、侵袭和上皮间质转化,促进胃癌细胞凋亡,抑制miR-325-3p则能够促进胃癌细胞的增殖、侵袭和上皮间质转化,抑制胃癌细胞凋亡（均P<0.05）。而过表达CLDN1则能够逆转miR-325-3p过表达对胃癌细胞生物学行为的影响。结论 miR-325-3p能够靶向抑制CLDN1进而抑制胃癌细胞的侵袭转移和上皮间质转化,促进胃癌细胞凋亡,miR-325-3p有望成为治疗胃癌的新靶点。     

Effect of trichostatin A on cell growth and expression of cell cycle- and apoptosis-related molecules in human gastric and oral carcinoma cell lines

The effect of trichostatin A (TSA), histone deacetylase inhibitor, on cell growth and the mechanism of growth modulation was examined in 8 gastric and 3 oral carcinoma cell lines which included 9-cis-retinoic acid resistant (MKN-7 and Ho-1-N-1) and IFN-beta resistant cell lines (MKN-7, -28 and -45). TSA inhibited growth in all cell lines examined. Apoptotic cell death was confirmed by apoptotic ladder formation and induction of a cleaved form (85 kDa) of poly (ADP-ribose) polymerase (PARP) induction. TSA enhanced the protein expression of p21(WAF1), CREB-binding protein, cyclinE, cyclin A, Bak and Bax, while it reduced the expression of E2F-1, E2F-4, HDAC1, p53 and hyperphosphorylated form of Rb. Furthermore, TSA induced morphological changes, such as elongation of cytoplasm and cell-to-cell detachment, in gastric and oral carcinoma cell lines. These results suggest that TSA may inhibit cell growth and induce apoptosis of gastric and oral carcinoma cells through modulation of the expression of cell cycle regulators and apoptosis-regulating proteins.     

Enhanced radiation-induced cell killing and prolongation of gammaH2AX foci expression by the histone deacetylase inhibitor MS-275

Histone deacetylase (HDAC) inhibitors are undergoing clinical evaluation for cancer therapy. Because HDAC modulates chromatin structure and gene expression, parameters considered to influence radioresponse, we have investigated the effects of the HDAC inhibitor MS-275 on the radiosensitivity of two human tumor cell lines (DU145 prostate carcinoma and U251 glioma). Acetylation status of histones H3 and H4 was determined as a function of time after MS-275 addition to and removal from culture medium. Histone acetylation increased by 6 h after MS-275 addition, reaching a maximum between 24 and 48 h of exposure; providing fresh drug-free medium then resulted in a decrease in histone acetylation that began by 6 h and approached untreated levels by 16 h. Treatment of cells with MS-275 for 48 h followed by irradiation had little or no effect on radiation-induced cell death. However, exposure to MS-275 before and after irradiation resulted in an increase in radiosensitivity with dose enhancement factors of 1.9 and 1.3 for DU145 and U251 cells, respectively. This MS-275 treatment protocol did not result in a redistribution of the cells into a more radiosensitive phase of the cell cycle or in an increase in apoptosis. However, MS-275 did modify the time course of gammaH2AX expression in irradiated cells. Whereas there was no significant difference in radiation-induced gammaH2AX foci at 6 h, the number of cells expressing gammaH2AX foci was significantly greater in the MS-275-treated cells at 24 h after irradiation. These results indicate that MS-275 can enhance radiosensitivity and suggest that this effect may involve an inhibition of DNA repair.