β-胡萝卜素对人舌鳞癌Tca8113细胞增殖和端粒酶活性的影响

目的 观察β-胡萝卜素对体外培养的人舌鳞癌Tca8113细胞的细胞毒性、细胞周期、细胞凋亡以及端粒酶活性的影响.方法 四唑蓝法检测30、 50、 60和70 μmol/L 4种浓度β-胡萝卜素对Tca8113细胞增殖的抑制作用,流式细胞仪观察β-胡萝卜素处理后Tca8113细胞周期改变及细胞凋亡,端粒重复片段扩增银染法检测细胞端粒酶活性的变化.结果 从50 μmol/L开始,随β-胡萝卜素浓度的提高,对Tca8113细胞增殖抑制作用越来越强.70 μmol/L β-胡萝卜素作用Tca8113细胞4 d后,抑制率超过50%,细胞生长曲线的"S"形趋于平缓.70 μmol/L β-胡萝卜素作用Tca8113细胞3 d后,流式细胞仪检测未出现亚二倍体的"凋亡峰".与空白对照组相比,实验组S期细胞减少约50%(P<0.05),G0/G1期细胞同时显著增加(P<0.05),G2/M期细胞变化无统计学意义(P>0.05).β-胡萝卜素处理Tca8113细胞后,端粒酶活性显著下调.结论 β-胡萝卜素可以通过减少DNA合成,阻滞细胞于G0/G1期,抑制舌癌Tca8113细胞的增殖,抑制效果随浓度的增加而明显.作用机制可能与下调肿瘤细胞端粒酶活性、导致细胞死亡有关.     

水飞蓟宾对人舌癌细胞Tca的抑制作用

目的:研究水飞蓟宾在体外对舌癌细胞的作用,并进一步探讨其可能的作用机制.方法:MTT实验测定水飞蓟宾对舌癌细胞系(Tca)的半数抑制浓度(IC50);平板克隆形成实验检测加药前后细胞的克隆形成能力;流式细胞仪检测加药前后细胞周期的变化.结果:水飞蓟宾对舌癌细胞系有较强的抑制作用, 作用48 h时,IC50约为100 μmol/L;水飞蓟宾作用后,舌癌细胞的平板克隆形成能力降低;流式细胞仪检测结果显示细胞出现G1期阻滞.结论:水飞蓟宾在体外对舌癌细胞具有明显的抑制作用, 其作用机制可能是细胞出现G1期阻滞.     

丁酸钠对人口腔鳞状细胞癌Tca8113细胞增殖及p27Kip1蛋白表达的影响

目的 通过观察组蛋白去乙酰化酶抑制剂丁酸钠对人口腔鳞状细胞癌Tca8113细胞的增殖影响及p27Kip1蛋白表达改变,探讨丁酸钠调控人口腔癌细胞增殖的分子机制.方法 人口腔鳞状细胞癌Tca8113细胞经不同浓度丁酸钠[0 mmol/L(空白对照组),2、4、6、8 mmol/L(4个实验组)]作用后,甲基噻唑基四唑(MTT)法观察细胞增殖情况,流式细胞仪分析细胞周期分布,免疫组化检测p27Kip1蛋白的表达.结果 丁酸钠能够呈时间剂量依赖性抑制Tca8113细胞的增殖,丁酸钠处理后的Tca8113细胞出现了凋亡形态变化;细胞周期阻滞于G0～G1期,丁酸钠2 mmol/L组G0～G1期达(63.2±2.4)%,4 mmol/L组G0～G1期达(77.2±3.8)%,空白对照组G0～G1期达(48.1±2.4)%,P＜0.05;p27Kip1蛋白表达水平明显上调.结论 丁酸钠能够抑制人口腔癌细胞的增殖,该作用可能与p27KiP1蛋白表达上调及G0～G1期细胞周期阻滞有关.     

盐酸特比萘酚联合抗癌药物抑制口腔鳞癌细胞增殖的体外研究

目的 检测口腔癌常用的化疗药物5-FU、PTX、CDDP联合特比萘酚(terbinafine,TB)对癌细胞增殖的影响并初步探讨作用机制.方法 体外培养CAL27、HN4、HN6、HN30等4个口腔鳞癌细胞系,化疗药物在0～30μmol/L范围设置6个浓度,TB设置2个浓度:0μmol/L和10μmol/L.MTT法检测细胞增殖,免疫印迹方法检测SQLE蛋白表达水平.结果 与5-FU单独作用比较,联用TB时HN30和HN4细胞的增殖显著降低(P<0.05).与PTX单独使用比较,联用TB时CAL27细胞的IC50降低了49.3％;HN30细胞的IC50降低了43.7％.与CDDP单独使用比较,联用TB时HN6细胞的IC50降低了65.8％,细胞增殖降低18.2％～58.3％(P<0.05),SQLE蛋白表达明显上升.与CDDP联用时,20μmol/L TB的抑制效果明显高于10μmol/L.结论 TB联合5-FU、CDDP化疗药物可以提高对口腔鳞癌细胞增殖的抑制作用,其作用机制可能与TB抑制SQLE表达水平有关.     

NS-398诱导舌鳞癌细胞凋亡及其对E2F-1蛋白表达的影响

目的观察选择性环氧化酶-2(COX-2)抑制剂NS-398诱导舌鳞癌Tca8113细胞凋亡及其对细胞核转录因子E2F-1蛋白表达的影响。方法NS-398作用于舌癌Tca8113细胞,噻唑蓝法检测细胞生长抑制情况,流式细胞仪检测细胞周期及其凋亡率的改变,放射免疫法测定细胞上清液中前列腺素E2(PGE2)含量,Western blot法检测细胞中COX-2和E2F-1蛋白表达的变化。结果NS-398可以抑制Tca8113细胞的增殖,其抑制作用随着时间的延长和药物浓度的增加而增强。NS-398(50μmol/L)可引起Tca8113细胞G0/G1期细胞的逐渐增加,S期和G2/M期细胞比例数减少,并随着时间的延长细胞上清液中PGE2含量降低,细胞中COX-2和E2F-1蛋白表达下调。结论NS-398可以抑制Tca8113细胞的增殖,阻断细胞生长停滞于G0/G1期;该效应可能与其诱导细胞凋亡和降低细胞产生前列腺素E2有关;E2F-1蛋白可能参与了这些过程。     

白藜芦醇对人舌癌细胞Tca增殖及凋亡影响的研究

目的探讨白藜芦醇对人舌癌细胞Tca的增殖及凋亡影响。方法应用MTT比色法检测白藜芦醇对Tca细胞体外生长的抑制作用;平板克隆检测白藜芦醇处理前后Tca细胞的克隆形成能力;流式细胞仪测定细胞的周期和凋亡;Hoechst33258荧光染料染色,显示凋亡细胞形态。结果MTT实验观察,白藜芦醇对Tca细胞生长有抑制作用,随浓度升高和时间延长,作用增强,呈剂量-时间效应关系;白藜芦醇作用Tca细胞48 h和72 h后,半数细胞抑制作用所需浓度约为(IC50)100μmol/L和75μmol/L。白藜芦醇(75μmol/L)处理后,细胞的克隆形成能力明显下降。白藜芦醇(100μmol/L)作用48 h后使G1期细胞的比例增加;Hoechst33258染色,可见细胞呈明显的凋亡特征。结论白藜芦醇可抑制人舌癌细胞Tca增殖,使细胞周期呈G1阻滞并可诱导细胞发生凋亡。     

LY294002对唾液腺腺样囊性癌SACC-83细胞周期和细胞凋亡的影响

目的:研究LY294002对唾液腺腺样囊性癌SACC-83细胞周期和细胞凋亡的影响。方法:用LY294002处理体外培养的SACC-83细胞,采用透射电镜技术进行细胞形态观察,MTT法计算细胞存活率,流式细胞术测定各细胞周期细胞分布比例和细胞凋亡情况。实验数据采用SAS8.2软件包进行单因素方差分析和两因素方差分析。结果:SACC-83细胞经LY294002(50μmol/L)处理96h,细胞存活率显著降低(P<0.01);LY294002(50μmol/L)处理SACC-83细胞72h,G0/G1期细胞逐渐增加,而S期和G2/M期细胞逐渐减少,凋亡的细胞逐渐增加(P<0.01)。结论:LY294002能显著抑制体外培养的SACC-83细胞增殖,使SACC-83细胞周期阻滞于G0/G1期,并诱导SACC-83细胞凋亡。     

土曲霉酮对人舌鳞状细胞癌细胞系SCC9的作用

目的 观察土曲霉酮对人舌鳞状细胞癌细胞系SCC9的影响.方法 不同浓度的土曲霉酮处理SCC9细胞后,用四甲基偶氮唑盐法检测细胞活力,用Annexin V/PI试剂盒及碘化丙啶分别检测细胞凋亡及细胞周期情况.结果 土曲霉酮对SCC9细胞有抑制增殖的作用,抑制作用随着药物浓度的增大(P＜0.05)及给药时间的延长而增强(P＜0.05).此外,与不给药组相比,5 μmol/L和10 μmol/L土曲霉酮处理,可引起处于G2/M期的SCC9细胞比例由(24.8±0.6)％分别增加到(26.2±0.4)％和(34.2±1.2)％ (F=132.13,P＜0.01),但对SCC9细胞凋亡没有明显的影响.结论 土曲霉酮通过引起G2/M期阻滞来抑制人舌鳞状细胞癌细胞系SCC9细胞的增殖,提示土曲霉酮有望在舌鳞状细胞癌的治疗中发挥作用.     

NS-398对Tca8113细胞体外增殖的抑制作用

目的:探讨环氧合酶-2抑制剂NS-398对Tca8113细胞的生长抑制作用。方法:应用四甲基偶氮唑蓝(MTT)快速比色法,观察NS-398对Tca8113细胞生长的抑制作用,检测NS-398与Tca8113细胞间的时-效关系和量-效关系;通过流式细胞仪(FCM)研究NS-398对Tca8113细胞周期的影响和作用,采用SAS8.1统计软件包进行数据处理,均数比较采用t检验和重复测量方差分析。结果:含0、25、50、75、100、125、150、200μmol/LNS-398的Tca8113细胞培养液,在分别培养48、72、96、120h后,随着作用时间的延长和药物浓度的增加,抑制作用也增强。NS-398在浓度150μmol/L作用96h后,抑制作用明显(药物浓度:P<0.05;时间:P<0.001),NS-398可引起Tca8113细胞G0/G1期细胞的大量增加,S期和G2/M期细胞比例数减少,阻滞细胞生长于G0/G1期(P<0.001)。结论:NS-398可抑制Tca8113细胞的增殖,并具有时间和浓度依赖性;这种作用可能与阻止细胞周期进展有关,NS-398可能在口腔鳞癌治疗中发挥重要作用。     

Skp2在口腔鳞状细胞癌中的表达

口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)是最常见的口腔恶性肿瘤,它的发生与细胞周期调控机制的紊乱所导致细胞的失控性生长密切相关.细胞周期的运行受多种因素所调控.有两个主要调控点:一个处于G1/S转折点,是控制细胞进入S期的控制点(G1期检测点),另一个处于G2/M转折点,是控制细胞进入M期的控制点(G2期检测点).     

可利霉素对口腔鳞癌细胞生物学活性的影响

目的: 体外评价可利霉素对口腔鳞状细胞癌的抗肿瘤活性。方法: 梯度浓度可利霉素分别处理口腔鳞状细胞癌细胞（HN6/HB96细胞系）,采用CCK-8试剂盒检测细胞增殖,绘制生长曲线;采用划痕实验分析细胞迁移能力;采用平板克隆形成实验分析细胞克隆形成能力;采用流式细胞术分析细胞周期和细胞凋亡。采用 SPSS 18.0 软件包对数据进行统计学分析。结果: 可利霉素浓度依赖性抑制HN6/HB96的体外增殖,随着药物浓度增加,细胞迁移和克隆形成能力显著下降（P<0.05）;可利霉素使处理过的口腔鳞状细胞癌细胞G1期比例显著升高,S期和G2/M期比例显著下降,细胞周期阻滞于G1期（P<0.001）。可利霉素诱导口腔鳞状细胞癌细胞凋亡,随着药物浓度增加,细胞凋亡率显著上升（P<0.01）。结论: 可利霉素体外抑制口腔鳞状细胞癌细胞的生物学活性,为可利霉素用于口腔鳞状细胞癌的治疗提供了实验依据。     

Operculina turpethum extract inhibits growth and proliferation by inhibiting NF-κB,COX-2 and cyclin D1 and induces apoptosis by up regulating P53 in oral cancer cells

ObjectivesHerbal drugs are popularly emerging as complementary and alternative medicines in cancer patients because of their cost effectiveness and minimal side-effects. The extract of Operculina turpethum (OT) is known to have antipyretic, anti-inflammatory and purgative properties. Since it is popularly known have antiinflammatory activity, we investigated its anti-tumor activity on four oral squamous cell carcinoma cell lines (OSCC) namely, (SCC-4, KB, SCC-9 and SCC-25).DesignAntitumor activities of Operculina turpathum extract (OTE) was investigated by MTT and clonogenic assay, effect on cell cycle and apoptosis induction by Annexin-V/propidium iodide (PI) staining and flow cytometry and invasive potential of the tumor was determined by matrigel assay. The expression of various proteins involved in these mechanisms was analysed by western blotting.ResultsOTE specifically inhibited the growth and colony formation of OSCC cells in a dose-dependent manner via inhibiting NF-κB and its downstream target COX-2. It further arrested cell cycle at G0/G1 phase by inhibiting cyclin-D1 and induced early apoptosis by up-regulating P53 in OSCC cells. It also limits the invasion capacity of OSCC cells by up to 55–60%.ConclusionsOTE shows antitumor activities in OSCC cells by inhibiting NF-κB, COX-2 and cyclin D1 and upregulation of p53 expression. It may be developed as a safe and promising alternative chemopreventive/chemotherapeutic agent for oral cancer.     

Daxx and TCF4 interaction links to oral squamous cell carcinoma growth by promoting cell cycle progression via induction of cyclin D1 expression

Objectives

Death domain-associated protein (Daxx) has been recently implicated as a positive factor in ovarian cancer and prostate cancer, but the role of Daxx in oral squamous cell carcinoma (OSCC) has never been addressed. Herein, we investigate the expression and function of Daxx in OSCC.

Materials and methods

RT-quantitative PCR, Western blotting, and immunohistochemistry were used to evaluation of the expression of Daxx in human OSCC cell lines and clinical surgical specimens. Short hairpin RNA targeting Daxx was transduced by lentivirus infection to knockdown the expression of Daxx in SAS and SCC25 cell lines, and the influence of this knockdown was evaluated by analyzing the growth and the cell cycle in transduced cells. Immunoprecipitation and sequential chromatin immunoprecipitation-quantitative PCR were used to analyze the associations between Daxx, TCF4, and cyclin D1 promoter. Xenograft tumor model was used to evaluate the in vivo tumorigenicity of Daxx in OSCC.

Results

Daxx mRNA and protein expression are elevated in several OSCC cell lines and human OSCC samples in comparison to those in normal tissue. We further find that depletion of Daxx decreases OSCC cell growth activity through G1 cell cycle arrest. Daxx silencing reduces cyclin D1 expression via a Daxx-TCF4 interaction, whereas the Daxx depletion-mediated G1 arrest can be relieved by ectopic expression of cyclin D1. Moreover, we show that in OSCC clinical samples, the expression of Daxx is significantly correlated with that of cyclin D1.

Conclusion

Our data demonstrate the importance of Daxx in regulation of cyclin D1 expression and provide the first evidence that Daxx exhibits tumor-promoting activity in OSCC.

Clinical relevance

Daxx plays an important role in malignant transformation of OSCC and may serves as a target for cancer prevention and treatment.

     

转化生长因子β1对口腔鳞状细胞癌脑转移细胞系Tb细胞生长抑制作用的研究

目的 探讨转化生长因子(transforming growth factor,TGF)β1对口腔鳞状细胞癌脑转移细胞系Tb细胞的生长抑制作用及可能的机制.方法 采用细胞计数法检测TGF-β1对Tb细胞的生长抑制作用,流式细胞仪检测细胞周期的变化,微阵列分析法(superarray)筛查smad蛋白介导的TGF-B(TGF-β-Smads)信号通路中基因表达水平的变化,反转录聚合酶链反应(RT-PCR)验证差异表达基因.结果 TGF-β1对Tb细胞有明显的生长抑制作用(P<0.05).流式细胞仪检测显示,TGF-β1能够使Tb细胞周期阻滞于G1期(P<0.05).微阵列分析法筛查结果显示,TGF-β1作用后,活化素受体样激酶1(activin receptor-like kinase-1,ACVRL-1)、抗苗勒激素(anti-mullerian hirmine,AMH)、细胞周期蛋白依赖性激酶抑制因子2B(cyclim-dependent kinase inhibitor-2B,CDKN-2B)、TGF-相互作用因子(indnced factor,TGIF)基因表达增加,而畸形肿瘤衍生生长因子1(teratocarcinomaderived growth factor-1,TDGF-1)基因表达降低.RT-PCR验证结果表明,ACVR-1(0.67±0.08)、CDKN-2B基因表达(2.16±0.95)与微阵列分析法结果一致,差异有统计学意义(P<0.05),AMH(0.38±0.07)、TDGF-1(0.44±0.06)及TGIF(0.52±0.10)基因表达水平无统计学意义(P>0.05).结论 TGF±β1对口腔鳞状细胞癌脑转移细胞系Tb细胞有明显的生长抑制作用,其机制可能与细胞周期调控及调节TGF-β1-Smads信号通路中ACVRL-1、CDKN-2B基因表达有关.     

EMP1基因对口腔鳞癌细胞增殖、迁移和侵袭影响的体外研究

目的:体外观察EMP1基因对口腔鳞癌细胞增殖、迁移和侵袭的影响。方法:构建pEGFP-N1-EMP1表达载体,稳定转染口腔鳞癌细胞系Tb3.1,并以野生型Tb3.1和pEGFP-N1稳转的Tb3.1为对照,用MTT检测细胞增殖,绘制细胞生长曲线;流式细胞仪检测细胞周期变化;用Transwell小室检测细胞的迁移和侵袭能力;组间比较采用单因素方差分析。结果:相比野生型Tb3.1和Tb3.1-pEGFP-N1细胞,Tb3.1-pEGFP-N1-EMP1细胞生长受到抑制,同时其S+G2-M期细胞比例减少(P＜0.05),迁移和侵袭细胞数目减少(P＜0.05)。结论:EMP1在口腔鳞癌的发生、发展中作为抑癌基因可能对口腔鳞癌细胞的增殖、迁移和侵袭起抑制作用。     

The G1 cell cycle arrest of macrophages infected with Aggregatibacter actinomycetemcomitans

Oral Diseases (2010) 16 , 305–309 Objectives: Infection of murine macrophage cell line J774.1 with the periodontopathic bacterium Aggregatibacter actinomycetemcomitans induces apoptotic cell death. The infection induces cell cycle arrest in the G1 phase prior to the appearance of apoptotic cells. This study determined the involvement of various cell cycle‐related signal molecules in A. actinomycetemcomitans‐induced G1 cell cycle arrest. Materials and Methods: Cell cycle in J774.1 cells infected with A. actinomycetemcomitans was analyzed with a flow cytometer. Immunoblot analysis was also employed to determine the expression levels of intracellular signal molecules. Results: Flow cytometric analysis revealed that the percentage of cells in the G1 phase increased to 77.2% at 12 h after A. actinomycetemcomitans infection. Additionally, according to immunoblot analysis, expression levels of hyperphosphorylated forms of retinoblastoma protein (ppRb) declined in J774.1 cells following A. actinomycetemcomitans infection, whereas hypophosphorylated Rb (pRb) expression levels were elevated slightly. Expression levels of cyclin D1 and D2 in the cells decreased gradually postinfection; CDK2, CDK4, CDK6 and cyclin E levels were not changed. Furthermore, postinfection, p21^CIP1/WAF1 expression increased at 6 h, followed by a subsequent decrease. Conclusion: These findings suggest that cyclin D1 and D2 and p21^CIP1/WAF1 participate in G1 cell cycle arrest in A. actinomycetemcomitans‐infected J774.1 cells.     

沉默LASP1对口腔鳞癌细胞生物学行为及3种抗肿瘤药物IC50的影响

目的:评价LASP1 对口腔鳞癌细胞增殖、转移、侵袭和周期的影响,并对3种抗肿瘤药物顺铂、阿帕替尼和多西他赛的相关作用进行分析。方法:利用The human protein atlas数据分析LASP1与头颈部肿瘤生存率、预后的关系。RT-PCR 和Western免疫印迹检测LASP1在口腔鳞癌细胞系的mRNA和蛋白表达。慢病毒构建LASP1沉默的HN30稳转细胞株,CCK-8 法检测细胞增殖,平板克隆实验检测细胞克隆形成能力,Transwell法检测细胞转移和侵袭能力,流式细胞仪检测细胞周期变化。建立裸鼠口腔鳞癌肺部转移瘤。CCK-8法分析3种药物顺铂、阿帕替尼和多西他赛在细胞中的IC₅₀变化。采用SPSS 11.0 软件包对数据进行统计学分析。结果:LASP1与头颈部肿瘤生存率、预后密切相关。LASP1促进口腔鳞癌细胞系HN30增殖、克隆形成、转移和侵袭,促进细胞周期G2/M期过渡。沉默LASP1后,裸鼠肺部转移瘤显著减少,多西他赛IC₅₀显著下调,而顺铂和阿帕替尼IC₅₀无显著变化。结论:LASP1促进口腔鳞癌细胞增殖、平板克隆、转移和侵袭,细胞周期G2/M期过渡,促进裸鼠体内肺转移瘤形成和多西他赛耐药。     

Porphyromonas gingivalis gingipains cause G(1) arrest in osteoblastic/stromal cells

INTRODUCTION: The program for mammalian cell growth and division consists of four successive phases; G(1), S, G(2), and M. Porphyromonas gingivalis may manipulate the host cell cycle to benefit bacterial virulence expression, which likely causes the cell and tissue tropism observed in chronic periodontal infections. We examined P. gingivalis for its effects on cell-cycle modulation in mouse ST2 osteoblastic/stromal cells. METHODS: Synchronized ST2 cells were infected with P. gingivalis ATCC33277 (wild-type, WT), gingipain-mutants [KDP136 (DeltargpADeltargpBDeltakgp), KDP129 (DeltargpADeltargpB), and KDP133 (Deltakgp)], and a fimbria-deficient mutant (KDP150) for 24 h, then the cell cycle was evaluated using flow cytometry. Cell-cycle-related molecule expression was examined with a microarray, as well as with quantitative real-time polymerase chain reaction and Western blotting assays. RESULTS: Both the WT and KDP150 strains significantly inhibited cellular proliferation and arrested the cell cycle in the G(0)/G(1) phase, while the expression levels of the cell-cycle regulatory molecules cyclin D and cyclin E were also decreased. In contrast, KDP136 did not show any effects. G(1) arrest was also clearly induced by KDP129 and KDP133, with KDP129 being more effective. CONCLUSION: The present findings suggest that P. gingivalis gingipains reduce cyclin expression and cause early G(1) arrest, leading to the inhibition of cellular proliferation.     

重组人乳铁蛋白对口腔癌细胞周期及周期因子表达影响的实验研究

目的：探讨重组人乳铁蛋白（Recombinate human lactoferrin,rhLF）对口腔癌Tea8113细胞周期及周期调控因子cyclinD1、p19和p27基因表达的影响。方法：分别采用流式细胞仪和RT—PCR方法分析细胞周期及周期调控因子cyclinD1,p19和p27的mRNA水平。结果：50μg／mL、100gg／mLrhLF作用Tca8113细胞后,细胞增殖减慢,G0／G1期细胞明显增多,与对照组相比差异具有统计学意义（P〈0．05）;Tca8113细胞的cyclinDlmRNA表达下降显著,50μg／mL和25μg／mL rhLF实验组与对照组相比差异具有统计学意义（P〈0．05）;p19和p27mRNA表达水平的变化不明显。结论：rhLF通过下调口腔癌细胞周期蛋白cyclinD1的表达来调控周期阻滞,为乳铁蛋白用于肿瘤的预防及治疗提供实验基础。