9-顺式维甲酸诱导胃癌MGC803细胞周期阻滞和凋亡的作用机制

背景与目的:9-顺式维甲酸(9-cisretinoicacid,9-cisRA)对胃癌的抗癌活性及机制尚不清楚,本研究以MGC803为靶细胞观察9-cisRA对胃癌的作用。方法:采用RT-PCR法检测维甲类X受体α(recinoidXreceptor!,RXRα)、细胞周期蛋白CyclinD1和细胞周期蛋白依赖性激酶CDK4mRNA表达,流式细胞术检测细胞周期,MTT实验检测9-cisRA作用MGC803细胞后生长抑制情况,Hoechst33342/PI双荧光染色和琼脂糖凝胶电泳检测凋亡,免疫细胞化学检测凋亡相关基因Bcl-2蛋白表达。结果:0.1～10μmol/L9-cisRA作用MGC803细胞96h,能显著抑制细胞增殖。10μmol/L9-cisRA分别作用48、72和96h,G1期细胞随着作用时间的延长而增加,呈明显的G1期阻滞。作用72h后,细胞出现核染色质凝集、DNA片段化等凋亡特征;从作用48h开始,Bcl-2表达即显著下降。在MGC803细胞中RXRα表达较弱,经10μmol/L9-cisRA作用48h其表达水平显著增加(P<0.01);作用96h,细胞中CyclinD1和CDK4表达显著降低(P<0.01)。结论:9-cisRA能明显诱导MGC803细胞周期G1期阻滞和凋亡,该作用可能与其下调细胞周期因子CyclinD1和CDK4表达有关。     

9-顺式维甲酸抑制甲状腺鳞癌细胞SW579增殖的作用机制

  目的  观察9-顺式维甲酸(9-cis-retinoic acid, 9-cisRA)对甲状腺鳞癌细胞株SW579细胞RARβ、p21、CDK4、CyclinDl表达的影响, 进而探讨9-cisRA的抗癌作用机制。  方法  分别以终浓度为1×10-7、1×10-6、5×10-6、1×10-5mol/L 9-cisRA作用SW579细胞, 各组细胞均在培养24 h后加药, 继续培养48 h。用RT-PCR方法检测SW579细胞RARβ、p21、CDK4、CyclinD1的mRNA表达; 用Western Blot检测SW579细胞RARβ、p21、CDK4、CvclinD1的蛋白表达。  结果  9-cisRA作用后, RT-PCR检测结果表明, 经不同浓度9-cisRA处理的细胞RARB、p21的mRNA表达水平均显著上调; CDK4的mRNA表达水平无明显变化; CyclinD1的表达水平明显下调; Western Blot检测结果表明经不同浓度9-cisRA处理的细胞RARβ、p21蛋白表达水平均显著上调; CDK4蛋白表达水平无明显变化; CyclinD1蛋白表达水平明显下调。  结论  9-cisRA在一定浓度范围内可能通过上调RARβ、p21的表达进而下调细胞周期蛋白CvclinD1的表达, 从而抑制细胞增殖。      

香加皮杠柳苷对人食管癌细胞TE-13生长抑制作用

目的：分析中药香加皮醇提物杠柳苷（periplocin from cortex periplocae，CPP）对食管癌细胞株TE-13的生长抑制作用，探讨其诱导细胞周期阻滞的机制。方法：应用MTr法检测CPP对TE-13细胞的抑制作用；Gimsa染色法分析TE-13细胞的形态学变化；FCM法检测细胞周期分布和凋亡率；Western印迹法检测TE-13细胞经药物处理前后细胞周期蛋白依赖性激酶CDK4、CDK2蛋白表达的变化。结果：CPP对TE-13细胞增殖具有明显的抑制作用（P〈0．01），并呈时间和浓度依赖性，药物浓度越大，作用时间越长，抑制效应越强，CPP作用48h时，对TE-13细胞的半数抑制浓度（IC50）为0．61μg／mL。经2μg／mL的CPP作用48h后，TE-13细胞发生明显的凋亡形态学变化；G0／G1期细胞明显增多（P〈0．01），S期细胞明显减少（P〈0．01），G2／M期细胞没有明显变化（P〉0．05）。不同浓度CPP作用48h后能降低TE-13细胞中CDK4蛋白的表达（P〈0．01），对CDK2蛋白的表达则没有明显影响（P〉0．05）。结论：CPP能显著抑制，TE-13细胞的增殖，其作用机制可能与CPP诱导细胞周期阻滞和细胞凋亡有关。     

过氧化物酶体激活物活化受体γ活化对人胃癌细胞周期的影响

目的:探讨过氧化物酶体激活物活化受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)活化在诱导人胃癌MGC803细胞周期停滞中的作用。方法:MTT法检测吡格列酮(pioglitazone,PGZ)对MGC803细胞增殖的抑制作用;流式细胞术检测肿瘤细胞周期的改变;RT-PCR方法检测PPARγ、细胞周期蛋白Cyclin D1和细胞周期蛋白依赖性激酶CDK4的表达。结果:0.1~10μmol/L PGZ作用MGC803细胞96 h后能显著抑制细胞增殖;10μmol/L PGZ分别作用48、72和96 h,G1期细胞随着作用时间的延长而增加,呈明显的G1期阻滞;在MGC803细胞中PPARγ表达较弱,经10μmol/L PGZ作用48 h表达水平显著增加;作用96 h,细胞中细胞周期调节因子CDK4表达显著降低(P<0.01),Cyclin D1轻微下调。结论:PPARγ活化能诱导MGC803细胞周期G1期阻滞,该作用可能与其下调细胞周期因子CDK4和Cyclin D1的表达有关。     

PPARγ的配体15d-PGJ2对人类胃癌细胞生长的影响

目的：探讨过氧化物酶体增殖物激活受体γ（peroxisome proliferator activated receptor γ，PPARγ）的配体15-脱氧前列腺素J2（15-deoxy-Δ^12,14-prostaglandin J2，15d—PGj2）对人类胃癌MGC803细胞生长的影响及其机制。方法：RT—PCR检测PPARγ和survivin mRNA，Western blot检测PPARγ、survivin、S期激酶相关蛋白2（S-phase kinase-assoeiated protein2，Skp2）和p27蛋白;MTT法及流式细胞术检测细胞增殖、凋亡及细胞周期分布；Hoechst33342染色观察细胞凋亡的形态学变化。结果：PPARγmRNA和蛋自在MGC803细胞均表达。15d-PGJ2作用MGC803细胞24h后，5、10、20、30、40μmol／L组的增殖抑制率和凋亡率均高于0μmol／L组（P〈0．001），且随浓度的增加而升高。30μmol／L组的MGC803细胞增殖抑制率和凋亡率，随时间的延长而升高。G0／G1期细胞比例，30μmol／L组明显高于0μmol／L组（P〈0．001）;S和G2／M期细胞比例，30μmol／L组均明显低于0μmol／L组（分别为P〈0．001，P〈0．01）。随15d0PGJ2作用MGC803细胞浓度的增加，survivin mRNA和蛋白及Skp2蛋白的表达均降低，p27蛋白表达升高。结论：15d-PGJz抑制人胃癌MGC803细胞的增殖、诱导其凋亡并将其阻滞在G0／G1期，survivin和Skp2表达下调及p27表达上调在这个过程中起重要作用，提示15d—PGJ2可能对治疗胃癌有效。     

西达本胺对结肠癌LoVo细胞增殖抑制的体外研究

[目的]研究去乙酰化酶抑制剂（histone deacetylase inhibitor,HDACi）西达本胺（Chi-damide,实验室编号：CS055）对结肠癌细胞株LoVo体外生物学特性的影响、诱导细胞凋亡的分子机制。[方法]MTT法观察CS055（2、4、8、16、32、64μmol/L）对LoVo增殖抑制情况,流式细胞仪检测细胞周期阻滞,Westernblot检测p21、CDK4、caspase-3蛋白表达情况。[结果]CS055体外能明显抑制LoVo细胞的增殖,呈剂量、时间依赖性（P〈0.05）。CS055（16μmol/L、48h）诱导后,流式细胞仪检测示细胞被阻滞于G0/G1期,并且出现典型的亚二倍体（sub-G1）峰。CS055可明显提高p21、caspase-3蛋白的表达水平,下调CDK4蛋白的表达水平。[结论]CS055可通过诱导细胞周期阻滞及细胞凋亡而发挥体外抗结肠癌LoVo细胞增殖作用,其作用机制可能涉及对p21、CDK4、caspase-3蛋白表达的调控。     

丙戊酸钠对Kasumi-1细胞周期蛋白及周期蛋白依赖性激酶抑制剂的影响

 目的　探讨组蛋白脱乙酰化酶（HDAC）抑制剂丙戊酸钠（VPA）对t(8;21)／AML细胞株Kasumi-1细胞周期蛋白及其周期蛋白依赖性激酶抑制剂基因的影响。方法　将Kasumi-1细胞经不同浓度VPA处理不同时间,应用反转录聚合酶链反应（RT-PCR）技术检测细胞周期调节因子mRNA水平的变化。结果　VPA下调cyclinD1、cyclinE1及cyclinB1 mRNA水平,上调p21WAF1／CIP1 mRNA表达,对p27KIP1 mRNA表达无明显影响。结论　VPA可通过对Kasumi-1细胞周期蛋白及周期蛋白依赖性激酶抑制剂的影响调节细胞周期,使细胞阻滞于G0／G1期。     

蟾毒灵对人肝癌细胞HepG2细胞周期的影响及其机制研究

目的：探讨蟾毒灵对人肝癌细胞HepG2细胞周期的影响及其相关机制。方法：MTT方法检测蟾毒灵单体对肝癌细胞HepG2的生长抑制作用;FCM法检测蟾毒灵单体诱导HepG2细胞周期阻滞的作用;WestelTlblot方法观察蟾毒灵单体对细胞周期相关蛋白的作用。结果：蟾毒灵能够抑制HepG2细胞的生长,该抑制作用呈时间和剂量依赖性。蟾毒灵能够诱导HepG2细胞周期阻滞于G2／M期。蟾毒灵对HepG2细胞周期的阻滞作用可能与cyclinB1、CDK1的表达下调有关。结论：蟾毒灵能够通过下调细胞周期相关蛋白的表达,诱导HepG2细胞阻滞于G2／M期。     

下调TPX2抑制喉癌细胞生长的体外实验研究

目的:研究TPX2对喉癌细胞增殖、克隆形成能力及细胞周期的影响。方法:Hep-2细胞中转染TPX2 siRNA、siRNA control记为TPX2 siRNA、siRNA -NC组，以不做转染的细胞为Con组。荧光定量PCR和Western blot分别测定细胞中TPX2 mRNA和蛋白水平，MTT测定各组细胞增殖，平板克隆实验测定各组细胞克隆形成能力，流式细胞术测定各组细胞周期情况，Western blot测定各组细胞中增殖细胞核抗原（PCNA）、细胞核增殖抗原(Ki-67)、细胞周期依赖性蛋白激酶4(CDK4)、细胞周期蛋白D1（Cyclin D1）蛋白水平。结果:siRNA control中TPX2 mRNA和蛋白水平、细胞存活率、克隆形成数目、细胞周期以及细胞中PCNA、Ki-67、CDK4、Cyclin D1蛋白水平与Con相比均没有明显变化（P＞0.05）。TPX2 siRNA细胞中TPX2 mRNA和蛋白水平均明显低于Con（P＜0.05）。TPX2 siRNA细胞存活率、克隆形成数目均明显低于Con，细胞G0/G1期比例明显高于Con，细胞中PCNA、Ki-67、CDK4、Cyclin D1蛋白水平明显低于Con（P＜0.05）。结论:TPX2敲低可以降低喉癌细胞增殖、克隆形成能力，将细胞周期阻滞在G0/G1期，降低细胞中PCNA、Ki-67、CDK4、Cyclin D1蛋白表达。     

丙戊酸钠对白血病细胞Jurkat的增殖抑制作用

 目的　探讨丙戊酸钠（VPA）对Jurkat细胞增殖抑制作用及对组蛋白乙酰化修饰调控的影响。方法　采用CCK-8法检测VPA对Jurkat细胞增殖的抑制作用;流式细胞术检测VPA作用前后Jurkat细胞周期的变化情况;半定量RT-PCR检测VPA作用前后Jurkat细胞中组蛋白去乙酰化酶1（HDAC1）mRNA的表达变化;Western blotting检测VPA作用前后HDAC1及组蛋白H3、H4乙酰化蛋白水平的变化。结果　VPA对Jurkat细胞的增殖抑制作用呈时间-浓度依赖性;不同浓度的VPA处理细胞48 h后,细胞周期检测显示随浓度增加,G0／G1期比例增高,S期比例下降,细胞被阻滞在G0／G1期（P＜0.05）;RT-PCR证实VPA能够抑制HDAC1 mRNA水平的表达;Western blotting方法分析证实,不同浓度VPA作用细胞48 h后降低HDAC1蛋白水平,提高组蛋白H3、H4乙酰化表达水平。结论　VPA能抑制Jurkat细胞增殖,阻滞细胞周期于G0／G1期,这可能与VPA抑制HDAC1表达,上调组蛋白H3、H4乙酰化水平有关。     

肿瘤的细胞周期治疗

肿瘤细胞周期治疗时代已经来临。直接调节细胞周期的蛋白如细胞周期素依赖性激酶（CDKs）、检查点激酶（CHK）、Aurora激酶、Polo样激酶和WEE1激酶是细胞周期治疗药物的主要靶标。除了阻滞细胞周期从G1期进入S期外,细胞周期治疗还能够激活抗肿瘤免疫、控制代谢功能和调节转录水平。细胞周期治疗与激素药物、磷脂酰肌醇-3激酶（PI3K）抑制剂、表皮生长因子（EGFR）抑制剂和自噬抑制剂的联合应用提高了肿瘤内科治疗的效果。在整合基因组学时代,细胞周期治疗为肿瘤精准治疗增加了新内容。本文对肿瘤细胞周期治疗的主要靶标和作用机制进行综述。      

Cytotoxic effects of cell cycle phase specific agents: result of cell cycle perturbation

Although agents which act in a cell cycle phase specific manner are commonly used in the clinic and in basic research, it is as yet unclear why these agents are cytotoxic. In this paper, we examine the cellular events associated with the cytotoxicity of aphidicolin and vincristine in CHO strain AA8 cells. Cell killing resulting from aphidicolin treatment was found to require a period of inhibition-free growth following removal of the drug and was associated with characteristic aberrant mitotic processes. The cytotoxic effects of aphidicolin could be antagonized by the concomitant inhibition of protein synthesis with cycloheximide in the period of DNA synthesis inhibition. Cell killing resulting from treatment with vincristine was associated with the aberrant segregation of nuclear material and the formation of multiple partial nuclei. Vincristine cytotoxicity was found to be antagonized by concomitant administration of cycloheximide or cytochalasin D. These data support a hypothesis that the cytotoxic effects of cell cycle phase specific agents do not derive directly from their biochemical actions per se. We propose that cell death results from processes that are evoked by dissociation of normally integrated cell cycle events, and that dissociation involves replicative/mitotic events in the case of aphidicolin and karyokinetic/nuclear reformation events in the case of vincristine.     

Control of the cell cycle

Cell biology has made major progress in identifying the molecules that drive the cell cycle. The evidence accumulating from these studies indicates that derangements in the cell cycle machinery contribute to the uncontrolled cell growth of tumours. The cell cycle machinery has been found to be substantially altered in tumour cells and also may be crucial for carcinogenesis. In this context, various aspects of tumour cell growth have been studied in an effort to understand 1) why tumour cells display uncontrolled growth, 2) why radiation selectively affects growing cells, and 3) whether aspects of the cell cycle and tumour cell growth may be used in tumour diagnosis and prognosis.     

肿瘤细胞自噬的诱导及其细胞周期分析

背景与目的:自噬作为Ⅱ型程序性死亡——自噬性死亡过程中的主要现象,与细胞的自噬性死亡有着密切的关系。研究者们对凋亡与细胞周期的关系进行了深入而细致的研究,但对自噬性细胞死亡与细胞周期的关系却知之甚少。本研究的目的是探讨不同方法诱导的细胞自噬与细胞周期之间的关系。方法:用Hanks’液替代培养基的饥饿诱导和用长春新碱诱导两种方法分别处理对数生长期的HeLa细胞、SW480细胞以及经过和未经过植物血凝素(phytohemagglutinin,PHA)刺激的健康人外周血淋巴细胞;应用激光共聚焦显微镜和透射电镜检测细胞自噬的发生,兔抗人微管相关蛋白1轻链3Ⅱ(microtubule-associatedprotein1lightchain3,MAP1-LC3-Ⅱ)/DNA双参数流式细胞术分析自噬细胞的细胞周期。结果:HeLa细胞和SW480细胞用饥饿和长春新碱两种方法诱导的细胞自噬在G1、S、G2/M期均可以发生,且自噬发生率随诱导时间的延长逐渐增加。处于静止期(未经PHA刺激)的外周血淋巴细胞没有自噬的发生,48h时LC3-Ⅱ表达率<2.62%(HanksL液饥饿诱导)或<6.16%(长春新碱诱导);经PHA刺激48h进入细胞周期的外周血淋巴细胞,2h时已有明显的自噬发生。结论:MAP1-LC3-Ⅱ/DNA双参数流式细胞术是对细胞自噬与细胞周期进行同步分析的一种新的简便可靠的方法;细胞自噬只发生在细胞进入周期后,而静止期细胞对自噬诱导因素不敏感。     

Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines

Flow cytometric analysis of fibroblasts, normal breast epithelial cells and breast or other cancer cell lines identified variation in the abilities of cell lines to undergo cell cycle arrest as a response to hypoxia. Human mammary epithelial cells (HMEC), normal fibroblasts (Hs68 and WI38), HeLa cervical carcinoma and HTB-30 breast carcinoma cells arrest in G(1)/S in response to severe hypoxia. Hep3B hepatocellular carcinoma cells did not exhibit orderly G(1)/S arrest in response to severe hypoxia. We found a general decrease in p16(INK4a) (p16) mRNA levels, with an associated decrease in p16 protein levels in both normal cells and in cancer cells, regardless of their cell cycle response to hypoxia. p27 protein levels did not correlate with the cell line's ability to enter a hypoxic G(1)/S arrest. Furthermore, cell lines that underwent G(1)/S arrest showed decreased expression of hypoxia inducible factor 1 (HIF-1alpha) and at least one member of INK4 or Sdi cell cycle kinase inhibitors families after 12-24 h of hypoxia. Conversely, Hep3B, which did not exhibit orderly hypoxia-associated G(1)/S arrest, also did not show decreased HIF-1alpha, INK4 or Sdi protein levels in hypoxia. Furthermore, Hep3B showed constitutive activating phosphorylation of Akt and inhibitory phosphorylation of GSK3beta, which was the opposite pattern to that exhibited by the cell lines showing the G(1)/S arrest phenotype. Inhibition of GSK3beta by lithium chloride treatment of HeLa cells converted the HIF-1alpha, p16 and p27 loss to levels unchanged by hypoxic exposure. Our results suggest that regulation of the cell cycle during hypoxia in either normal or cancer cells is not simply due to up-regulation of cell cycle kinase inhibitors. Furthermore, decreased protein expression of HIF-1alpha, p16 and p27 was associated with both a hypoxia-induced G(1)/S arrest phenotype and increased GSK3beta activity.     

Retinoic acid-induced cell cycle arrest of human myeloid cell lines

Retinoic acid-induced terminal differentiation of myeloid cells involves the sequential regulation of cell cycle regulatory genes, coordinating the process of differentiation with arrest in the G0/G1 phase of the cell cycle. In this review we have summarized changes in expression and activity of cell cycle regulatory proteins associated with retinoic acid induced-growth arrest in human myeloid cell lines. These changes involve: (i) an early down-regulation of c-Myc; (ii) up-regulation of p21CIP1 and p27KIP1 and, in some cases, p15INK4b or p18INK4c; (iii) down-regulation of cyclin E and cyclin D1/D3, and, at later stages, cyclin A and cyclin B; and (iv) decreased CDK activity and dephosphorylation of pRb.     

Evaluation of novel cell cycle inhibitors in mantle cell lymphoma

Signature abnormalities in the cell cycle and apoptotic pathway have been identified in mantle cell lymphoma (MCL), affording the opportunity to develop targeted therapies. In this study, we tested a novel class of kinase inhibitors, styryl sulfones, which differ from prior cell cycle inhibitors in that they are not related to purines or pyrimidines. We observed that two closely related compounds, ON013100 and ON01370, altered the growth and cell cycle status of MCL lines and potently inhibited the expression of several important molecules, including cyclin-dependent kinase 4, p53, mouse double minute 2 (MDM2), and cyclin D as well as increased cyclin B expression. Using both terminal deoxy transferase uridine triphosphate nick end-labelling and poly ADP-ribose polymerase assays, we found that these compounds caused apoptosis in MCL cells. In addition, using molecular analyses, we observed the modulation of caspase-3 activity but not the expression of B-cell lymphoma family molecules. Next, we investigated the cytotoxicity of the MCL lines upon treatment with styryl sulfone compounds in combination with other currently used chemotherapeutic agents, such as doxorubicin (DOX) or vincristine (VCR). We found that the combination of DOX plus styryl sulfone or VCR plus styryl sulfone increased cytotoxicity by one log scale, compared with the single styryl sulfone compound. Thus, styryl sulfones alone, or in combination with chemotherapeutic agents, present attractive opportunities for new drug development in MCL.