Effects of Meloxicam on Vascular Endothelial Growth Factor and Angiopoietin-2 Expression in Colon Carcinoma Cell Line HT-29

To investigate the effect of meloxicam, a selected NSAIDs, on cell growth, expression of VEGF and angiopointin-2 （Ang-2） protein in HT-29 cell line, cultured HT-29 cells were treated with meloxicam of various concentrations for various lengths of time. The proliferation of HT-29 was detected by cell counting kit-8 （CCK-8）, the cell cycle was determined by flow cytometer and the levels of VEGF and Ang-2 protein in supernatants were examined by enzyme linked immunosorbent assay （ELISA）. The mRNA expressions of VEGF and Ang-2 in cultured HT-29 were determined by real-time quantitative reverse-transcription polymerase chain reaction. Our results showed that treatment of meloxicam of different concentrations and for various lengths of time had a cytotoxicic effect on the cell proliferation of HT-29 cells in a concentration-dependant and time-dependant manner. Cell cycle analysis showed that the cells were mainly blocked in G0/G1 phase. The VEGF and Ang-2 protein levels in supernatants of the culture medium were decreased gradually in a concentration-dependent or time-dependent fashion. The mRNA expression of cox-2, VEGF and Ang-2 showed a gradual and concentration-dependent reduction. It is concluded that meloxicam can reduce the expression of VEGF and Ang-2 at the protein and mRNA level in colon carcinoma cell line.

STUDY ON THE EXPRESSION OF CYCLOOXYGENASE-2 IN HEPATOCELLULAR CARCINOMA CELL LINES AND ON THE GROWTH INHIBITION EFFECT OF NS-398

Objective： To investigate the expression of cyclooxygenase -2 （COX-2） in hepatocellular carcinoma cell lines and to explore the effect of NS-398, a selective inhibitor for COX-2, on HepG-2 cell line. Methods： lmmunohistochemistry and RT-PCR were used to investigate COX-2 expression in 6 HCC cell lines. MTT and Flowcytometry were used to evaluate the effect of the selective inhibitor of COX-2, NS-398, on HepG-2 cell lines. Results： All six HCC cell lines showed COX-2 expression at protein level. Five out of 6 cell lines showed COX-2 expression at mRNA level. NS-398 could suppress the growth of HepG-2 cell line, in a time and dose dependant manner. Conclusion： NS-398, a selective inhibitor of COX-2, showed inhibition effect on HepG-2 HCC cell line. The efficacy of inhibition was time and dose dependent, providing a new evidence for chemoprovention of hepatocellular carcinorma with COX-2 selective inhibitors.

Effect of 5-Aza-2'-deoxycytidine on the expression of p16 in hepatocellular carcinoma cells in vitro

Objective: To study the effect of 5-Aza-2’ -deoxycytidine (5-Aza-cdR) on tumour suppressor gene p16 expres- sion in hepatocellular carcinoma cells. Method: Expression of pl6 mRNA and protein in hepatocellular carcinoma cell lines SMMC-7721 and HePG2 before and after treatment with 5-Aza-cdR were analyzed via reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistrty Results: The expression levels of p16 mRNA and protein were increased dramatically after treatment with 5-Aza-cdR. Conclusion: Our data show that, 5-Aza-2’ -deoxycytidine can increase the expression of pl6 gene both at transcription and translation. The findings suggested that 5-Aza-cdR may reactivate the pl6 gene by demethylation.

白细胞介素24对鼻咽癌CNE-2Z细胞株增殖的影响

目的探讨白细胞介素24(IL-24)对鼻咽癌CNE-2Z细胞株增殖的影响。方法取鼻咽癌CNE-2Z细胞株,进行细胞培养;用细胞计数、四甲基偶氮唑蓝(MTT)抑制试验,以IL-240 pg/L为对照组,检测不同浓度、不同时间的IL-24对鼻咽癌CNE-2Z细胞株增殖的影响。结果细胞计数及MTT比色法检测得出:体外培养的鼻咽癌CNE-2Z细胞株在培养的48 h增殖状况最差,并且随着IL-24浓度的增加,其活细胞数也随着减少,当IL-24为50 pg/L时,其增殖状况最差;当IL-24为100 pg/L时,其增殖状况反而好转。结论 IL-24可呈时间和剂量依赖性地抑制鼻咽癌CNE-2Z细胞株增殖。

吡咯烷二硫代氨基甲盐酸对人鼻咽癌CNE-2Z细胞增殖及其细胞核抗原表达的影响

目的研究核转录因子-κB(NF-κB)特异性抑制剂吡咯烷二硫代氨基甲盐酸(PDTC)对人鼻咽癌CNE-2Z细胞生长增殖及其细胞核抗原(PCNA)的影响。方法不同浓度的PDTC(25、50、100μmol/L)作用CNE-2Z细胞不同时间后(24、48、72 h),四甲基偶氮唑蓝(MTT)比色法检测PDTC对CNE-2Z细胞增殖的抑制效应;不同浓度的PDTC(25、50、100μmol/L)作用于CNE-2Z细胞48 h后,流式细胞术(FCM)分析细胞周期,免疫组化(SP)法检测不同强度PDTC作用下CNE-2Z细胞增殖细胞核抗原(PCNA)的表达情况。结果 25、50、100μmol/L PDTC处理的CNE-2Z细胞增殖活性明显受到抑制(P<0.05),并呈时间和剂量依赖;流式细胞术结果表明S期细胞数减少,细胞受阻于G0/G1期。与对照(无PDTC处理)比较,PDTC作用后CNE-2Z细胞PCNA表达降低(P<0.05),并呈明显的剂量依赖。结论 PDTC具有显著抑制人鼻咽癌CNE-2Z细胞生长的作用,是一种有潜力的抑制鼻咽癌细胞增殖的药物。

Relation between the Expression of K-ras in Hep-2 Cells and Development of Laryngeal Carcinoma~*

Objective： To investigate the expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and its significance for establishing a solid foundation for further study of the relationship between human laryngeal squamous cell carcinoma and K-ras gene point mutations. Methods： The expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and human pancreatic carcinoma cell lines （MIAPaCa-2） was detected by using RT-PCR. Results： The expression of K-ras mRNA in Hep-2 and MIAPaCa-2 was strong and positive. Conclusion： The expression of K-ras mRNA in human laryngeal squamous cell carcinoma cell lines （Hep-2） is positive. Development of laryngeal carcinoma might be related to the activation of K-ras gene point mutation.

检测细胞周期分布的改变以探讨温和性高温对鼻咽癌细胞系CNE-2Z放射增敏作用的机制

目的 探讨温和性高温对鼻咽癌细胞系CNE-2Z放射增敏作用的机制.方法 实验分4个组,对照组、单纯加热组、单纯照射组和联合处理组.采用流式细胞仪分析G2/M期分布比例及凋亡百分比,Western-Blotting检测CyclinB1的表达,改良的荧光染色法观察核碎片形态.结果 联合处理组G2/M期分布比例较高,CvclinB1表达较高,并产生更多的核碎片,放射诱导的凋亡百分比并不增加.结论 G2/M 期分布比例增高,CyclinB1表达增加是温和性高温对鼻咽癌细胞系CNE-2Z放射增敏作用的机制.

Celecoxib Inhibits Proliferation and Induces Apoptosis via Cyclooxygenase-2 Pathway in Human Pancreatic Carcinoma Cells

In order to evaluate the effects and mechanisms of celecoxib in inhibiting proliferation and inducing apoptosis on human pancreatic carcinoma cells, the anti-proliferative effect was measured by using methabenzthiazuron (MTT) assay. Cell cycle and apoptosis were analyzed by using flow cytometry (FCM), and the PGE 2 levels in the supernatant of cultured pancreatic carcinoma cells were quantitated by enzyme-linked immunoabsordent assay (ELISA). Our results showed that celecoxib suppressed the production of PGE 2 and inhibited the growth of JF-305 cells, and the anti-proliferative effect of celecoxib could be abolished by addition of PGE 2. FCM revealed that celecoxib could inhibit proliferation and induce apoptosis by G 1-S cell cycle arrest. It was concluded that cyclooxygenase-2 specific inhibitor celecoxib could inhibit proliferation and induced apoptosis of human pancreatic carcinoma cells via suppression of PGE 2 production in vitro.

温和性高温对鼻咽癌细胞系CNE-2Z的放射增敏作用

目的:探讨温和性高温是否对鼻咽癌细胞系CNE-2Z具有放射增敏作用。方法:CNE-2Z细胞随机分4组:对照组、单纯加热组、单纯照射组和联合处理组。采用平板克隆形成实验观察各温度(39.5℃、41.5℃、43.5℃、45.5℃)对CNE-2Z细胞的放射增敏作用,Western Blotting检测核转录因子κB(NF-κB)的表达。结果:实验所采用的4种高温均显示出对鼻咽癌细胞系CNE-2Z的放射增敏作用。其存活分数SF_2值按顺序排列为SF_(2,37℃)>SF_(2,39.5℃)>SF_(2,41.5℃)>SF_(2,43.5℃)>SF_(2,45.5℃)。NF-κB的表达,从第40分钟起逐渐增加,至第4小时达最高。结论:温和性高温对鼻咽癌细胞系CNE-2Z具有放射增敏作用。在41.5℃、1 h作用下,放射诱导的NF-κB的细胞核内表达受抑,可能是增敏CNE-2Z对射线作用的机制。

检测细胞周期分布的改变以探讨温和性高温对鼻咽癌细胞系CNE-2Z放射增敏作用的机制

目的 检测细胞周期分布的改变以探讨温和性高温对鼻咽癌细胞系CNE-2Z放射增敏作用的机制.方法 实验分对照组、单纯加热组、单纯照射组和联合处理组.采用流式细胞仪分析G2/M期分布比例及凋亡百分比,Western-Blotting检测CyclinB1的表达,改良的荧光染色法观察核碎片形态.结果 联合处理组G2/M期分布比例较高,CyclinB1表达较高,并产生更多的核碎片,放射诱导的凋亡百分比并不增加.结论 G2/M期分布比例增高,CyclinB1表达增加是温和性高温对鼻咽癌细胞系CNE-2Z放射增敏作用的机制.

2Gy多分割不同顺序照射对鼻咽癌CNE-2细胞生物效应的研究

目的探讨总剂量为2 Gy时部分分割和顺序照射对鼻咽癌CNE-2细胞的杀伤效应的差异。方法采用克隆形成法和四唑盐(MTT)比色法,检测2 Gy剂量照射后鼻咽癌CNE-2细胞的细胞生存率和具有代谢活性细胞比例。结果在多个等分割照射中,较小分割(≤0.5 Gy)多次照射的细胞存活率均明显低于单次照射组(2 Gy),差异有统计学意义(P<0.05),而较大分割(1 Gy)照射与单次照射组细胞生存率差异无统计学意义;在2个部分分割和3个部分分割照射中,随着S分割剂量的增大,各S-L和S-S-L照射组细胞生存率呈逐渐下降趋势,至S分割为0.4 Gy(S-L)或者0.5Gy(S-S-L)组时细胞生存率最低;大部分S-L照射组细胞生存率明显低于L-S照射组,大部分S-S-L照射组细胞生存率和相对细胞生存率明显低于L-S-S照射组,差异有统计学意义(P<0.05)。结论总剂量为2 Gy照射时,分割剂量大小和照射顺序是影响鼻咽癌CNE-2细胞放射治疗生物效应的重要因素。

人鼻咽癌CNE－2Z细胞的不同转移潜能克隆株在严重联合免疫缺陷小鼠体内的检验和比较研究

将人类鼻咽癌不同克隆株的细胞悬液移植在严重联合免疫缺陷（ＳＣＩＤ）小鼠和ＢＡＬＢ／ｃ（ｕｎ／ｕｎ）裸小鼠的颈背侧皮下，５６ｄ后处死全部动物进行观察。结果发现，在ＳＣＩＤ小鼠体内移植后ＣＮＥ２Ｌ２为高转移克隆株，其淋巴结转移率为１００％，肺转移率为７１％；而ＣＮＥ２Ｌ４为低转移克隆株，其肺转移率为１３％，淋巴结未见癌转移。这是从１个细胞母系中新筛选出的１个高转移和１个低转移的癌细胞克隆株。实验结果还显示，同ＢＡＬＢ／ｃ（ｕｎ／ｕｎ）裸小鼠相比，ＳＣＩＤ小鼠的恶性表型的表达能力高。另外，皮下移植时肿瘤细胞的数量可能与转移表型的表达有相关性，移植的瘤细胞数越多，转移率越高，反之亦然。

MicroRNA-34a对人鼻咽癌CNE-2细胞裸鼠皮下移植瘤的生长抑制作用及分子机制研究

目的:探讨MicroRNA-34a(miR-34a)对鼻咽癌裸鼠皮下移植瘤的生长抑制作用及其可能的分子机制。方法:体外培养人鼻咽癌CNE-2细胞系,利用mi R-34a重组质粒及Scrambled mi RNA重组质粒分别构建miR-34a及阴性对照mi RNA稳定转染细胞株,q RT-PCR检测细胞内mi R-34a表达。选取6周龄雄性BLAB/c裸鼠15只,随机分为3组:mi R-34a组(5只),Scrambled mi RNA组(5只)及空白对照组(5只)。于裸鼠背部近右后肢皮下注射重组CNE-2细胞悬液,建立移植瘤模型,每7 d测定实验组与对照组肿瘤体积变化,饲养35 d后处死,完整取出肿瘤,测定肿瘤重量及终体积。提取肿瘤组织RNA及蛋白,采用qRT-PCR检测肿瘤组织中miR-34a的表达;分别采用q RT-PCR及免疫组化法检测细胞周期调控基因CDK6及抗凋亡基因Bcl-2 m RNA及蛋白的表达。结果:mi R-34a稳定转染CNE-2细胞后可显著提高细胞内mi R-34a表达量(P<0.05)。15只裸鼠均成功构建皮下移植瘤模型,饲养21 d后,与Scrambled mi RNA组及空白对照组相比,mi R-34a组肿瘤体积显著减小(P<0.05)。饲养35 d后,mi R-34a组、Scrambled mi RNA组及空白对照组肿瘤平均重量分别为(0.81±0.13)g、(1.47±0.21)g、(1.58±0.37)g,肿瘤平均终体积分别为(351.37±98.19)mm3、(798.75±91.04)mm3、(849.62±101.32)mm3,mi R-34a组肿瘤平均重量及肿瘤平均终体积均显著小于其余两组(P<0.05)。mi R-34a组中mi R-34a的表达量显著高于其余两组(P<0.05);mi R-34a组中CDK6及Bcl-2 m RNA及蛋白表达量显著低于其余两组(P<0.05)。结论:miR-34a可能通过下调CNE-2细胞内CDK6及Bcl-2的表达水平从而抑制CNE-2细胞裸鼠皮下移植瘤的生长,发挥其抗肿瘤作用。

Interaction among Rb/p16, Rb/E2F1 and HDAC1 Proteins in Gallbladder Carcinoma

The mechanism and interaction among Rb/p16, Rb/E2F1 and HDAC1 proteins in gallbladder carcinoma were investigated. By using the immunoprecipitation method, the interactions among Rb, p16, E2F1, HDAC1 proteins in gallbladder carcinoma cell line （Mz-ChA-1） were studied. It was found that there were Rb and E2F1 proteins in the precipitates with anti-HDAC1, and there were HDAC1 and E2F1 proteins in the precipitate with anti-Rb. It was concluded that there are specific interactions among Rb, HDAC1 and E2F1 proteins in gallbladder carcinoma, indicating the existence of the direct Rb/E2F1/HDAC1 signal transduction pathway. There is no direct relationship between p16 proteins with Rb, HDAC1, and E2F1 proteins.

Review of the treatment of metastatic non small cell lung carcinoma:A practical approach

In recent years,as we have a better knowledge and understanding of the biology of non small cell lung carcinoma(NSCLC),which leads us to targeting biomarkers driving the NSCLC carcinogenesis and metastatic potential,we now have an increased number of options to offer our patients with NSCLC.We also realize the importance of distinguishing squamous and non squamous histology to guide our treatment decisions of NSCLC.The palliative care concomitant with therapies from the very start of the treatment also showed an impact on survival.This review examines the treatment options in all lines of therapy for metastatic NSCLC that have been approved in Canada,the United States,or Europe.

Differential Expression of Genes in HepG2 Cells Caused by UC001kfo RNAi as Shown by RNA-seq

The differential expression of genes in HepG2 cells caused by UC001 kfo RNAi was investigated using RNA-seq. HepG2 cells were infected by Lenti-sh UC001 kfo lentivirus particles. The expression of UC001 kfo m RNA in the HepG2-sh UC001 kfo cell line was detected by real-time PCR. RNA-seq technology was used to identify the difference in the expression of genes regulated by lnc RNA UC001 kfo in the HepG2 cell line. Gene ontology and signaling pathway analysis were performed to reveal the biological functions of the genes encoding of significantly different m RNAs. The results showed that m RNAs were differentially expressed between the HepG2-sh UC001 kfo cell line and the HepG2 cell line. The UC001 kfo m RNA was significantly down-regulated in the stable cell line HepG2-sh UC001kfo（P〈0.001）. Additionally, we found 19 signaling pathways or functional classifications encompassing 30 genes that played a role in cancer characteristics, cell adhesion, invasion and migration. The results also showed that the expression of many genes associated with cancer cell invasion and metastasis was decreased with the down-regulation of the lnc RNA UC001 kfo. Lnc RNA UC001 kfo may play a role in regulating cancer cell invasion and metastasis. It was suggested that m RNAs were differentially expressed in the HepG2 cell line after the down-regulation of lnc RNA-UC001 kfo. Some took part in the extracellular matrix, cell adhesion, motility, growth, and localization. The genes encoding of differentially expressed m RNAs may participate in cell invasion and metastasis.

Inhibitory effects of apogossypolone on subcutaneous implants of human LNCaP prostatic carcinoma cells

Objective:The aim of this study was to investigate the inhibitory effect of apogossypolone (ApoG2) on subcutaneous implants of human LNCaP prostatic carcinoma cells, and explore its mechanism. Methods:To establish human LNCaP prostatic carcinoma cell line subcutaneous xenograft models and observe the inhibitory effect of ApoG2 on the tumor model. Immunohistochemistry was employed to observe the expression of Bcl-2, PCNA, CD31, caspase-3 and-8 in tumor tissues. The microvessel density was calculated. Results:ApoG2 could obviously inhibit the growth of subcutaneous prostatic carcinoma implant. ApoG2 decreased the expression of PCNA and CD31, and increased the expression of caspases-3,-8 in tumor tissues. Conclusion:ApoG2 has an inhibitory effect on prostatic carcinoma implants.

Inhibitory effect of metformin on the proliferation of human hepatoma HepG2 cells and its potential mechanism

Objective： This work aimed to study the inhibitory effect and the related mechanism of metformin （MET） on the proliferation of human hepatoma HepG2 cells. Methods： Human hepatoma HepG2 cells were treated with MET （0, 2, 10, and 50 mM）. The inhibitory effect of MET on the proliferation of HepG2 cells was determined by MTT method. The apoptosis of HepG2 cells was detected by flow cytornetry. The expression of cyclin D1 in HepG2 cells was examined by Western blot. ROS-DHE fluorescence probe was used to stain the reactive oxygen species （ROS） generated by HepG2 cells after treat- ment. Results： MET could inhibit the proliferation of HepG2 cells in a dose and time dependent manner. MET promoted the apoptosis of HepG2 cells. In addition, MET suppressed the expression of cell cycle protein cyclin D1 and induced the produc- tion of ROS in HepG2 cells. Conclusion： MET can inhibit the proliferation of human hepatoma HepG2 cells and induce cell apoptosis. Meanwhile, MET has the ability to decrease the expression of cyclin D1 and induce ROS generation, which may be involved in the mechanism of inhibiting hepatoma cells proliferation.

基于CRISPR/Cas9技术构建与鉴定敲除ACE2基因的Huh7肝癌细胞株

利用CRISPR-Cas9基因编辑技术敲除人Huh7肝癌细胞的血管紧张素转化酶2(ACE2)基因,构建ACE2基因敲除细胞株,为研究ACE2在肝细胞癌的作用提供细胞模型。首先,对ACE2结构域进行鉴定,利用在线网站设计两条破坏所有结构域、靶向作用于ACE2外显子的sgRNA。其次,构建重组载体并转染肝癌细胞Huh7,嘌呤霉素筛选出单克隆细胞株。最后,免疫印迹鉴定敲除效果。结构域鉴定结果显示,在340-520号氨基酸位置存在Zn结合位点和激活位点,根据sgRNA靶点设计原则,采用片段敲除的方式,针对ACE2的第9、第10外显子设计两对sgRNA,并成功构建PX459-ACE2-sgRNA重组质粒。嘌呤霉素成功筛选出单克隆细胞株,并测序证实了发生片段敲除,ACE2蛋白在敲除细胞株中不表达;成功构建ACE2敲除的Huh7细胞株,为日后研究ACE2在肝细胞癌的发生机制奠定基础。