沉默DEPDC1抑制鼻咽癌细胞HNE-1和CNE-1侵袭迁移

DEPDC1(DEP domain containing 1)是一个新的肿瘤相关基因,在多种恶性肿瘤的发生发展进程中起着重要作用。我们前期工作中在鼻咽癌细胞内沉默了DEPDC1的表达,发现抑制细胞增殖并诱发细胞凋亡。本研究旨在探讨沉默DEPDC1表达后,对鼻咽癌细胞HNE-1和CNE-1侵袭迁移能力的影响及其分子机制。结果显示,siRNA介导DEPDC1表达沉默后,细胞侧向运动能力、侵袭及迁移能力显著降低。qRT-PCR及Western印迹检测发现DEPDC1沉默导致EMT上游关键转录因子Twist1及间质细胞标志分子Vimentin表达显著下调。这些研究表明,鼻咽癌细胞中DEPDC1通过调节Twist1等EMT关键分子的表达在细胞侵袭转移过程中起关键作用。推测DEPDC1在鼻咽癌中高表达可能对于促进其侵袭转移具有重要作用,进而促进肿瘤发生发展,但具体分子机制仍有待更深入研究。     

JNK相互作用蛋白通过JNK途径影响鼻咽癌的增殖和凋亡

EB病毒编码的瘤蛋白潜伏膜蛋白（LMP1）所介导的活化蛋白（AP-1）信号转导途径在细胞增殖、分化、转化与凋亡方面发挥着重要作用.越来越多的证据表明,AP-1信号转导通路中上游激酶JNK在鼻咽癌的发生发展过程中起着重要作用.最近克隆出来的JNK相互作用蛋白(JIP-1)是一种能抑制JNK核移位的胞浆锚蛋白.为探讨JIP在LMP1调控AP-1信号通路中的作用机制,采用间接免疫荧光法和报告基因法,发现JIP通过有效地抑制磷酸化的JNK从胞浆移位入核,从而抑制LMP1上调的AP-1活性.同时,JIP导入鼻咽癌细胞中,MTT法发现JIP能够明显抑制鼻咽癌细胞的生长.进一步发现转染JIP后细胞的集落形成率与对照组相比大约降低了53.6%,也抑制了细胞. 提示JIP可明显抑制细胞的增殖作用.进一步采用流式细胞术分析,结果发现JIP引起细胞G1/S期细胞阻滞,说明JIP是抑制细胞增殖的重要调节子.进一步采用流式细胞术定量发现,转染JIP后细胞的24 h凋亡百分率由1.25%上升到8.25%,上升约6.6倍,48 h由1.04%上升到31.45%,上升约30倍. 采用激光共聚焦显微镜发现,转染JIP后细胞核发生显著变化,核质由均匀状态固缩成高凝集状态,形成了典型的胞膜体.提示JIP可有效地促进细胞凋亡.结果表明,JIP可通过抑制活化的JNK核移位,降低LMP1所介导的AP-1信号通路.并进一步发现JIP可有效地抑制细胞增殖和细胞凋亡,从而提示JIP可作为新的治疗肿瘤潜在靶分子.     

Bub1基因对人肝癌MHCC97-H细胞增殖、周期和凋亡的影响

目的:研究Bub1基因在肝癌中的表达以及对肝癌细胞系MHCC97-H增殖、周期和凋亡的影响。方法:利用RNA干扰技术下调肝癌细胞系MHCC97-H中Bub1的表达;qRT-PCR和Western Blot分别检测Bub1在mRNA和蛋白水平表达的变化;CCK-8实验检测肿瘤细胞增殖能力的改变;流式细胞术检测细胞周期和凋亡的变化。结果:qRT-PCR和Western Blot结果显示si-Bub1能够成功下调Bub1的表达;下调Bub1后肝癌MHCC97-H细胞的增殖能力下降(P0.05),细胞的凋亡比例升高(P0.05),细胞发生S期阻滞。结论:Bub1基因在肝癌中高表达,下调Bub1的表达后能够降低肝癌细胞的增殖能力,促进细胞凋亡,诱导细胞发生S期阻滞。     

槲皮素对鼻咽癌CNE2细胞的增殖抑制和诱导凋亡作用

本研究以人鼻咽癌细胞CNE2为研究对象,探讨了槲皮素在鼻咽癌细胞CNE2中抑制细胞增殖、诱导细胞凋亡的效果和分子机制。本研究采用细胞增殖检测试剂盒-8(CCK-8)实验测定了槲皮素在CNE2细胞中的半数抑制浓度(IC50);并采用Ca依赖性磷脂结合蛋白Annexin V和荧光染料碘化丙啶(PI)双染的方法,检测了槲皮素诱导CNE2细胞凋亡的情况;然后采用平板克隆实验,评价了槲皮素对CNE2细胞克隆形成能力的影响;最后采用免疫印迹法,检测了槲皮素对CNE2细胞中凋亡标志分子、Wnt通路及其下游靶标分子的作用。结果表明,槲皮素不仅呈浓度依赖性诱导CNE2细胞凋亡,也呈浓度依赖性阻遏Wnt信号通路、下调其靶标蛋白c-Myc和Survivin的表达,进而抑制CNE2细胞的恶性增殖。综上所述,本研究发现了槲皮素抑制鼻咽癌细胞增殖、诱导鼻咽癌细胞凋亡的效果,并阐明了其分子机制,提供了槲皮素作为鼻咽癌临床治疗候选药物的实验数据。     

无花果果浆对肿瘤细胞增殖抑制和诱导凋亡作用

为了探讨无花果果浆(Fig fruit latex,FFL)对人肿瘤细胞的生长抑制作用及其机制,用无花果果浆处理体外培养的人肿瘤细胞,细胞增殖试验(MTT法)、克隆形成试验研究FFL对人肿瘤细胞的增殖抑制作用,Brdu掺人试验、吖啶橙/溴乙啶(AO/EB)染色、流式细胞术检测FFL对肿瘤细胞DNA合成、凋亡和细胞周期的影响.结果,用FFL处理后,肿瘤细胞增殖活性降低(P＜0.05),克隆形成下降(P＜0.05),Brdu标记指数降低(P＜0.05),吖啶橙染色凋亡细胞增多(P＜0.05);细胞周期分布改变,凋亡指数升高(P＜0.01),G0/G1期细胞数增加(P＜0.01),S期细胞数减少(P＜0.01),在一定剂量内对正常细胞无明显影响.试验结果提示,FFL对所试肿瘤细胞的增殖有显著地抑制作用,其作用机理可能与抑制肿瘤细胞DNA合成,诱导肿瘤细胞凋亡及细胞周期阻滞有关.     

选择性COX-2抑制剂celecoxib对K562细胞的增殖抑制、凋亡诱导作用和分子机制

环氧化酶(cyclooxygenase, COX)家系被显示与恶性肿瘤的增殖和凋亡耐受有关,COX-2可作为恶性肿瘤治疗和预防的重要分子靶标.应用COX-2特异抑制剂——celecoxib,观察了药物对人慢性粒细胞白血病急变细胞株——K562细胞的增殖抑制和凋亡诱导效应.结果证明,celecoxib能够有效地抑制K562细胞增殖(台盼蓝染色,MTT试验及集落形成抑制试验证实),并呈一定的剂量依赖性.Celecoxib抑制K562细胞增殖的IC₅₀为46 μmol/L.通过DNA ladder胶电泳和流式细胞仪检测,凋亡细胞的AO/EB染色等方法证明celecoxib能够诱导K562细胞凋亡,这一效应与Caspase-3蛋白表达上调和裂解激活有关,当阻断Caspase-3的活性,celecoxib诱导的K562细胞凋亡明显受抑.利用RT-PCR分析技术及蛋白质印迹,证明K562细胞存在COX-2 mRNA和COX-2蛋白表达;而且,K562细胞COX-2蛋白表达可被IL-1β诱导性刺激,从而确认K562细胞为COX-2表达阳性细胞;celecoxib在较高浓度(80～160μmol/L)既可抑制K562细胞COX-2 mRNA表达,也可下调COX-2蛋白质表达,提示celecoxib抗K562白血病细胞活性与COX-2的抑制相关,其抗白血病的分子机制部分涉及到COX-2依赖性途径.     

槐米中天然产物槲皮素抗人鼻咽癌CNE1细胞活性研究

本研究以槐米为原材料提纯天然产物槲皮素,以人鼻咽癌细胞系CNE1为试验对象,探索天然产物槲皮素对人鼻咽癌细胞的增殖抑制效应及凋亡诱导效应。研究方法采用超声醇提法从槐米中提取芸香苷,再经酸水解和重结晶制备槲皮素精制品;采用槲皮素标准曲线比色法检测槲皮素精制品中槲皮素的纯度;采用MTT比色法进行细胞毒性试验,检测槲皮素精制品对人鼻咽癌细胞CNE1增殖的抑制效应及半数抑制浓度(IC50);采用流式细胞术结合AnnexinV、PI双染色法进行细胞凋亡试验,检测槲皮素精制品对CNE1细胞凋亡的诱导效应。结果表明,从槐米中提纯的天然产物槲皮素精制品,能呈剂量依赖性地抑制人鼻咽癌细胞CNE1增殖并诱导其凋亡。综上所述,应用超声醇提法,从槐米中制备的天然产物槲皮素精制品,不仅纯度高,且有着较优的抗癌活性,进一步明确了槐米等中药材开发天然产物有效成分,服务于人类健康的潜在应用价值和社会与经济效益。     

虎杖提取物对人胰腺癌细胞系Panc-1增殖与凋亡的影响

目的:通过虎杖提取物干预人胰腺癌细胞系Panc-1,探讨虎杖提取物对人胰腺癌细胞增殖凋亡表型的影响。方法:制备不同浓度(0、10、50、100、150、200μg/m L)的虎杖提取物,将各个浓度的虎杖提取物分别加入待处理的人胰腺癌Panc-1细胞系中持续培养24 h后,利用CCK-8(cell counting kit-8)法检测细胞株Panc-1的增殖活性;将100μg/m L虎杖提取物处理人胰腺癌细胞系Panc-124 h后,利用流式细胞术(FCM)检测其细胞周期及凋亡分布;100μg/m L虎杖提取物处理人胰腺癌细胞株Panc-124 h后,提取细胞总RNA及总蛋白,后续利用实时荧光定量PCR及Western blot分别检测人胰腺癌细胞株Panc-1增殖标志基因PCNA、CDK2及凋亡标志基因BAD、BAX的转录和翻译水平。结果:CCK-8结果表明虎杖提取物对人胰腺癌细胞系Panc-1细胞增殖的抑制率随浓度增加;流式细胞术结果显示虎杖提取物抑制人胰腺癌细胞增殖促进其凋亡;荧光定量PCR和Western blot结果显示虎杖提取物能使人胰腺癌细胞增殖标志基因PCNA,CDK2表达量下降,凋亡标志基因BAD,BAX表达量上升。结论:虎杖提取物能够抑制人胰腺癌细胞系Panc-1细胞增殖并促进其凋亡。     

RNA干扰ABCE1基因后可抑制肺癌95-D/NCI-H446细胞的增殖和诱导细胞凋亡

ABCE1作为RNase L抑制剂首先是在脊椎动物中被发现的．前期研究结果显示ABCE1与肺腺癌的发生率及临床分期显著相关．为了进一步研究ABCE1的新功能,构建了ABCE1基因的siRNA表达质粒(RNAi-Ready pSIREN-DNR-DsRed-　Express vector),培养肺癌细胞(95-D和 NCI-H446),用FuGENE 6作为转染试剂转染后,使用荧光显微镜观察转染效果,RT-PCR分析ABCE1基因表达,Western blot 分析ABCE1蛋白的表达,MTT法检测细胞的活性,流式细胞仪分析细胞周期,ELISA法检测细胞凋亡．结果显示：质粒的转染效果较满意,阳性率约为42.70%;在实验组,细胞活性和生长指数明显受到抑制,细胞凋亡明显增加,与对照组比较差异显著(P < 0.05)．上述结果显示,RNA干扰ABCE1基因可显著抑制肺癌细胞(95-D/NCI-H446) RNA的转录、蛋白质的表达,并增加细胞凋亡,为进一步研究ABCE1基因提供必要的基础．     

人参皂苷单体Rh2对人鼻咽癌CNE-2S细胞增殖及凋亡的影响

目的:探讨人参皂苷单体Rh2对人鼻咽癌CNE-2S细胞增殖及凋亡的影响。方法:将生长在对数期的人鼻咽癌CNE-2S细胞分为空白对照组、阴性对照组和实验组。对照组常规培养,阴性对照组采用含有DMSO的培养液培养,实验组在对照组细胞的基础上加入不同浓度人参皂苷单体Rh2处理。采用MTT法测定细胞增殖,PI单染流式细胞术分析各时期细胞所占百分比,Annexin V-PI双染流式细胞仪检测细胞的凋亡情况。结果:与阴性对照组相比,实验组各浓度下的Rh2对CNE-2S细胞均具有显著的增殖抑制作用(P0.05),且随着Rh2浓度的增加而呈现增强的趋势,其中浓度为12.5 mg·L-1 Rh2增值抑制率最低,浓度为100 mg·L-1Rh2增值抑制率最高。不同浓度人参皂苷单体Rh2 G0/G1期细胞分布显著高于阴性对照组(P0.001),且G2/M、S期细胞比例显著低于阴性对照组(P0.01),且随着人参皂苷单体Rh2浓度的增加作用呈现增强的趋势(P0.05);不同浓度的Rh2单体作用24h,CNE-2S细胞早期、晚期凋亡率及总凋亡率均较阴性对照组明显增高(P0.001),并且在Rh2单体浓度为100 mg·L-1时,凋亡率最高。结论:人参皂苷单体Rh2对人鼻咽癌CNE-2S细胞增殖及凋亡具有显著的影响,并且可能对单体Rh2的浓度存在依懒性。     

Transient overexpression of TGFBR3 induces apoptosis in human nasopharyngeal carcinoma CNE-2Z cells

NPC (nasopharyngeal carcinoma) is a common malignancy in southern China without defined aetiology. Recent studies have shown that TGFBR3 (transforming growth factor type III receptor, also known as betaglycan), exhibits anticancer activities. This study was to investigate the effects of TGFBR3 on NPC growth and the mechanisms for its actions. Effects of TGFBR3 overexpression on cell viability and apoptosis were measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], AO/EB (acridine orange/ethidium bromide) staining and electron microscopy in human NPC CNE-2Z cells. The expression of apoptosis-related proteins, p-Bad, Bad, XIAP (X-linked inhibitor of apoptosis), AIF (apoptosis-inducing factor), Bax and Bcl-2, was determined by Western blot or immunofluorescence analysis. Caspase 3 activity was measured by caspase 3 activity kit and [Ca²⁺]_i (intracellular Ca²⁺ concentration) was detected by confocal microscopy. Transfection of TGFBR3 containing plasmid DNA at concentrations of 0.5 and 1 μg/ml reduced viability and induced apoptosis in CNE-2Z in concentration- and time-dependent manners. Forced expression of TGFBR3 up-regulated pro-apoptotic Bad and Bax protein, and down-regulated anti-apoptotic p-Bad, Bcl-2 and XIAP protein. Furthermore, transient overexpression of TGFBR3 also enhanced caspase 3 activity, increased [Ca²⁺]_i and facilitated AIF redistribution from the mitochondria to the nucleus in CNE-2Z cells, which is independent of the caspase 3 pathway. These events were associated with TGFBR3-regulated multiple targets involved in CNE-2Z proliferation. Therefore transient overexpression of TGFBR3 may be a novel strategy for NPC prevention and therapy.     

RPA1低表达对辐射抵抗人鼻咽癌CNE-2R细胞侵袭迁移及细胞周期的影响*

目的: 探讨复制蛋白A1(RPA1)沉默对人鼻咽癌CNE-2R细胞侵袭、迁移及细胞周期的影响。方法: 采用shRNA技术构建RPA1低表达的CNE-2R细胞模型并通过RT-PCR和Western blot实验验证。选用空白对照组(CNE-2R)、阴性对照组(NC-shRNA)、RPA1低表达组(RPA1-shRNA)3组细胞完成后续实验,通过CCK8和克隆形成实验检测细胞增殖能力、Transwell实验检测侵袭能力、划痕实验检测迁移能力,流式细胞术检测细胞周期;Western blot实验检测Chk2、p-Chk2、Cdc25c和p-cdc25c蛋白的表达。结果: 与CNE-2R和NC-shRNA组比较,RPA1-shRNA组细胞的RPA1mRNA和蛋白质均显著降低(P＜0.01和＜0.05);RPA1-shRNA组组细胞的增殖、侵袭、迁移能力显著下降(P均＜ 0.05),细胞周期被阻滞在G2/M期(P＜0.01);RPA1-shRNA组细胞Chk2、Cdc25c的表达低于CNE-2R和NC-shRNA组细胞(P＜0.05), 而p-Chk2、p-cdc25c的表达高于其它两组(P ＜0.05)。结论: RPA1低表达抑制辐射抵抗人鼻咽癌CNE-2R细胞的增殖、迁移以及使细胞周期阻滞于G2/M期。     

MicroRNA-1 induces apoptosis by targeting prothymosin alpha in nasopharyngeal carcinoma cells

Background  

MiR-1 (microRNA-1) has been used as a positive control in some microRNA experiments. We found that miR-1 transfection of nasopharyngeal carcinoma cells reveals a typical apoptotic process as shown by time-lapse microscopy so we investigated the mechanisms of miR-1 inducing apoptosis.     

microRNA-136对人鼻咽癌细胞5-8f的抑制作用研究

CpG岛的高甲基化是肿瘤中作为抑癌基因microRNA失活的重要表观遗传机制之一。利用UCSC预测hsa-miR-24、hsa-miR-126、hsa-miR-132、以及hsa-miR-136定位于CpG岛内部或附近,采用甲基化酶抑制剂5-Aza-CdR处理鼻咽癌细胞5-8f,经RT-PCR与MSP检测,结果表明hsa-miR-136在5-8f中存在高甲基化,5-Aza-CdR能逆转hsa-miR-136甲基化,恢复hsa-miR-136的表达。外源过表达hsa-miR-136能明显抑制鼻咽癌细胞5-8f的增殖;同时流式细胞技术检测显示,hsa-miR-136能诱导5-8f细胞发生晚期凋亡,迁移实验也显示hsa-miR-136能明显抑制5-8f细胞的迁移。综上所述,hsa-miR-136可作为治疗鼻咽癌潜在的靶标。     

Inhibition of cell proliferation and induction of apoptosis by genistein in experimental hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the leading cause of cancer related deaths in the world, with increasing incidence in many developed countries. Epidemiological data suggest that consumption of soy products may be associated with a decreased risk of cancer. We investigate the effects of genistein on cell proliferation, apoptosis and caspase-3 in DEN induced (200 mg/kg body weight; by single intraperitoneal injection) and Phenobarbital promoted (0.05% through drinking water for 14 successive weeks) cancer-bearing rats. Immunohistochemistry was employed to detect cell proliferating markers proliferating cell nuclear antigen (PCNA), DNA fragmentation was determined by agarose gel electrophoresis and terminal deoxynucleatide transferase dUTP nick labeling (TUNEL) staining and caspase by enzyme-linked immunosorbent assay. We found inhibition of cell proliferation, induction of apoptosis and activation of caspase-3 in genistein treated animals. From these results, we conclude that genistein inhibit cell proliferation, induced apoptosis. This activation of caspsase-3 in genistein treated liver cancer bearing animals correlated well with its apoptosis inducing effect.     

Treatment with vector-expressed small hairpin RNAs against Ki67 RNA-induced cell growth inhibition and apoptosis in human renal carcinoma cells

Short hairpin RNAs (shRNAs) transcribed by.RNA polymerase Ⅲ promoters can triggersequence-selective gene silencing in mammalian cells.By virtue of their excellent function in knocking downexpression of cancer-associated genes,shRNAs could be used as new therapeutic agents for cancer.Asoverexpression of Ki67 in renal cancer has been correlated to a more aggressive tumor phenotype,inhibitionof Ki67 protein expression by means of shRNAs seems to be a promising approach for the therapy of renalcancer.In this study,we constructed an expression plasmid encoding shRNAs against the Ki67 gene,namedpSilencerKi67,and transfected it into human renal carcinoma cells.The pSilencerKi67 was shown to signifi-cantly knock down the expression of the Ki67 gene in human renal carcinoma cells,resulting in inhibitingproliferation and inducing apoptotic cell death that can be maintained for at least 6d.These findings offer thepromise of using vector-based shRNAs against Ki67 in renal cancer gene therapy.     

Calcium-activated nucleotidase 1 silencing inhibits proliferation,migration, and invasion in human clear cell renal cell carcinoma

Calcium-activated nucleotidase 1 (CANT1, belongs to the apyrase family, is widely expressed in various organs. However, the biological function of CANT1 remains poorly explored. In this study, we aimed to investigate the expression profile and functions of CANT1 in clear cell renal cell carcinoma (ccRCC). Our data show that the protein level of CANT1 was significantly higher in tumor tissues than in adjacent normal tissues. CANT1 silencing suppressed cell proliferation, migration, and invasion obviously in 769-P and 786-O cells, arrested cell cycle in S phase and promoted apoptosis in 769-P cells. In conclusion, the present study shows the different expression mode of CANT1 in human ccRCC tumor tissue and adjacent normal tissue, denotes the function of CANT1 in ccRCC cells and provides potential molecular mechanisms and pathways of CANT1 antitumor function in ccRCC.