细小病毒H-1感染对胃癌细胞凋亡相关基因表达的影响

背景:细小病毒H鄄1对肿瘤细胞和转化细胞具有选择性杀伤和抑制生长的作用,是很好的基因治疗用载体。但肿瘤细胞对细小病毒H鄄1杀伤作用敏感或耐受的分子机制目前尚不十分清楚。目的:从mRNA水平观察细小病毒H鄄1感染对胃癌细胞凋亡相关基因表达的影响,探讨细小病毒H鄄1对胃癌细胞细胞毒作用的相关机制。方法:选取对细小病毒H鄄1敏感的人胃癌细胞株HGC鄄27和不敏感的人胃癌细胞株BGC鄄823,于细小病毒H鄄1感染48h后提取细胞总RNA。应用不同荧光染料标记的dUTP逆转录制备荧光探针,与含有8000点人类体细胞基因序列的基因表达谱芯片杂交,再经计算机荧光扫描以分析敏感细胞株HGC鄄27凋亡相关基因表达谱的改变。采用逆转录聚合酶链反应(RT鄄PCR)对敏感细胞株HGC鄄27部分凋亡相关基因,如SARP1、BCL鄄10、CL鄄20、NCDRP和RAIDD等的表达作进一步检证,并比较其与不敏感细胞株BGC鄄823相应凋亡相关基因的表达差异。结果:基因表达谱芯片检测结果显示,HGC鄄27细胞感染细小病毒H鄄148h后,所检测的64对凋亡相关基因中有15对基因表达水平下调至原水平的50%以下,仅有1对基因表达水平上调至2倍以上。RT鄄PCR扩增结果显示,感染细小病毒H鄄148h后,敏感细胞株HGC鄄27的SARP1、BCL鄄10、CL鄄20和NCDRP基因表达明显下调,RAIDD基因     

细小病毒H-1非结构蛋白NS1基因对人胃癌细胞的抑制作用研究

前期研究表明细小病毒H-1（H-1PV）对胃癌细胞生长具有一定抑制作用,非结构蛋白NSl可能是其细胞毒作用的效应蛋白。胃癌细胞CD44^＋群体中可能含有肿瘤干细胞。目的：探讨H-1PV非结构蛋白NSl基因对不同分化程度的人胃癌细胞的影响。方法：以双酶切和质粒测序鉴定真核表达质粒pcDNA3．1-NSl,采用脂质体法将重组质粒转染高分化胃癌MKN28细胞、中分化胃癌SGC7901细胞和低分化胃癌MKN45细胞,并设置空质粒转染对照。G418筛选稳定转染的细胞克隆．以RT-PCR法检测NSl基因表达、裸鼠体内成瘤实验检测NSl基因对不同分化程度胃癌细胞的作用．流式细胞术分析CD44表达。结果：重组质粒pcDNA3．1-NSl转染的三种胃癌细胞均稳定表达NSl基因。以10^6／300μl浓度的肿瘤细胞皮下接种裸鼠3周后,MKN28细胞空质粒转染组和重组质粒pcDNA3．1-NSl转染组均观察到肿瘤生长;SGC7901、MKN45细胞空质粒转染组形成瘤体,而重组质粒pcDNA3．1-NSl转染组未见瘤体。转染重组质粒pcDNA3．1-NSl后,MKN28细胞不表达CD44,SGC7901和MKN45细胞CD44表达明显降低。结论：H-1PV非结构蛋白NSl基因表达对中低分化的胃癌细胞有较好的抗肿瘤活性．可能与其降低胃癌干细胞表型CD44的表达有关。     

表没食子儿茶素没食子酸酯抗消化道肿瘤作用的实验研究

背景：表没食子儿茶素没食子酸酯(EGCG)具有抗肿瘤作用，但是其有效浓度和可能作用机制尚不十分清楚。目的：研究EGCG对不同消化道肿瘤细胞株的杀伤作用及其抗肿瘤作用的靶位点，为其应用于临床肿瘤治疗奠定理论基础。方法：采用四唑蓝(MTT)比色法检测8株消化道肿瘤细胞株对EGCG敏感性的差异；采用流式细胞仪检测EGCG对敏感肿瘤细胞株细胞周期分布的影响；采用聚合酶链反应(PCR)-酶联免疫吸附测定(ELISA)检测EGCG对敏感肿瘤细胞株端粒酶活性的影响。结果：EGCG对8株消化道肿瘤细胞株均具有剂量依赖性杀伤作用。SW1116和MKN45细胞对EGCG最敏感，半数抑制浓度(IC50)分别为51．7μmol／L和55．9μmol／L；BGC823和SGC7901细胞次之，IC50分别为68．5μmol／L和79．1μmol／L；AGS细胞对低浓度EGCG不敏感，对高浓度EGCG敏感，IC50为83．8μmol／L；MKN28细胞对EGCG中等敏感，IC50为119．8μmol／L；HGC27和LoVo细胞对EGCG不敏感，IC50分别为183μmol／L和194．6μmol／L。3种不同浓度的EGCG作用48h后，敏感肿瘤细胞株MKN45的细胞周期分布与对照组相比均无明显改变。EGCG可抑制MKN45细胞的端粒酶活性，其作用呈剂量和时间依赖性。结论：EGCG对不同消化道肿瘤细胞株具有不同程度、呈剂量依赖性的杀伤作用；对敏感肿瘤细胞株MKN45的细胞周期分布无明显影响。EGCG的抗肿瘤作用可能与其抑制肿瘤细胞的端粒酶活性有关。     

hCTB006联合伊立替康对胃癌细胞BGC823、SGC7901协同杀伤的作用机制

目的:研究人死亡受体(death receptor5,DR5)激动型抗体hCTB006联合伊立替康对不同分化程度胃癌细胞BGC823、SGC7901的体外抑瘤作用,并对其诱导胃癌细胞凋亡的机制进行初步探讨.方法:实验将BGC823、SGC7901细胞分为伊立替康组、hCTB006组和两者联合应用组,应用ATPlite法研究各组药物的体外抑瘤作用;采用ELISA法研究伊立替康处理胃癌细胞前后DR5表达的变化.采用Western blot技术检测药物处理前后BGC823、SGC7901细胞X-联锁凋亡抑制蛋白(X-chromosome-linked inhibitory of apoptosis protein,XIAP)的表达变化情况.结果:BGC823对hCTB006中度敏感,对SGC7901不敏感,伊立替康对胃癌细胞的增殖抑制作用呈浓度依赖性,与hCTB006联合用药后对BGC823具有良好的协同抑制作用,但对SGC7901协同作用不明显.ELISA测得伊立替康处理后胃癌细胞DR5的表达量无明显变化.但伊立替康和hCTB006联合用药可使胃癌细胞BGC823内XIAP水平明显降低,而对SGC7901细胞内XIAP没有明显作用.结论:伊立替康联合hCTB006后对低分化胃癌细胞BGC823的增殖有明显协同抑制作用,而对中度分化的SGC7901细胞呈拮抗作用,这种诱导胃癌细胞凋亡的机制可能与其DR5的表达无关,而与联合作用后细胞抑凋亡蛋白XIAP的表达水平有关.     

NS-398对人胃癌细胞株增殖及COX-2表达的影响

目的 体外观察选择性环氧化酶2(COX-2)抑制剂NS-398对人胃癌细胞株SGC7901细胞增殖及COX-2表达的影响。方法 采用噻唑蓝(MTT)法观察NS-398对SGC7901细胞增殖的影响，流式细胞仪(FCM)研究NS-398对SGC790l细胞凋亡的作用．免疫细胞化学观察COX-2蛋白的表达。结果 体外NS-398能减少SGC790l细胞株COX-2的表达．对SGC7901有细胞毒作用．可增加细胞凋亡率。结论 体外NS-398对SGC7901细胞增殖有抑制作用。可能与抑制COX-2表达及诱导细胞凋亡有关。     

miR-204在胃癌组织中的表达及对胃癌细胞凋亡的影响

目的探讨miR-204在胃癌组织中的表达与临床病理参数的关系及其对胃癌细胞凋亡的影响。方法接受手术治疗的胃癌患者53例,收集手术切除的胃癌组织作为胃癌组标本,并收集其癌旁(3 cm)正常组织作为对照组标本,同时培养人胃癌细胞株(BGC823、SGC7901)和永生化胃上皮细胞株(GES-1)。采用逆转录-聚合酶链反应(RT-PCR)检测胃癌组标本、对照组标本、BGC823、SGC7901、GES-1细胞株中的miR-204表达,分析胃癌组组织miR-204相对表达量与临床病理参数的关系,对部分BGC823、SGC7901细胞株进行转染miR-204,对比转染miR-204组与未转染miR-204组的BGC823、SGC7901细胞株的细胞增殖和细胞凋亡情况。结果胃癌组标本中的miR-204相对表达量明显低于对照组标本中miR-204的相对表达量(P0.05)。BGC823细胞株和SGC7901细胞株中的miR-204相对表达量明显低于GES-1细胞株中的miR-204相对表达量(P0.05)。胃癌组组织标本中miR-204相对表达量与患者的年龄、性别无关(P0.05),与TNM分期、淋巴结转移、分化程度有关(P0.05)。第3天的BGC823、SGC7901细胞株中,未转染组的吸光度高于转染miR-204组(P0.05),未转染组的细胞凋亡百分比显著低于转染miR-204组(P0.05)。结论 miR-204在胃癌组织以及胃癌细胞株中呈现低表达,且其表达与TNM分期、淋巴结转移以及分化程度有关,转染miR-204后能抑制胃癌细胞株的细胞增殖,并能促进其细胞凋亡。     

runt相关转录因子3及其甲基化在人胃癌细胞中的表达及调节因素的初步研究

目的检测不同分化程度胃癌细胞株runt相关转录因子3（RUNX3）基因及其甲基化表达，观察RUNX3 mRNA再表达对胃癌细胞株生物学特性的影响。方法5-氮杂脱氧胞苷（5-AZA-dC）化学处理法和甲基化敏感性聚合酶链反应（MSP法）检测胃癌细胞RUNx3基因的甲基化状态；采用四甲基偶氮唑盐（MTT）实验检测5-氟尿嘧啶（5-FU）对5-AZA-dC处理前、后细胞株的生长抑制率，流式细胞术检测转化生长因子p1（TGF-p1）诱导凋亡的作用。结果①RUNX3在人胃癌细胞SGC7901、MKN45、BGC823中表达，而在MKN28中表达沉默，DNA异常甲基化仅存在于MKN28细胞；②5-AZA-dC处理后，MKN28细胞生长速度显著慢于未处理组，不同浓度5-FU对5-AZA-dC处理后MKN28的生长抑制率显著高于未处理组（P〈0．05）；③TGF-β1诱导5-AZA-dC处理前后MKN28细胞的早期凋亡具有时效性（P〈0．05），两者联用具有协同凋亡作用（P〈0．05）。结论DNA异常甲基化是MKN28细胞RUNX3表达沉默的重要机制；RUNX3甲基化可被去甲基化剂5-AZA-dC有效逆转；RUNx3mRNA的再表达可增强MKN28细胞对5-FU的敏感性和对TGF-β1诱导细胞凋亡的能力。     

核糖核酸干扰下调骨桥蛋白表达对胃癌细胞生物学行为的影响

目的探讨骨桥蛋白（OPN）下调对胃癌细胞株MKN28和SGC7901生物学行为的影响。方法参考相关文献，设计并合成OPN的小干扰RNA（siRNA），通过荧光标记测定2株细胞的转染效率。Western印迹法检测OPN蛋白下调情况。实时定量聚合酶链反应（RT-PCR）检测siRNA对OPNmRNA的下调率及随时间的变化。流式细胞仪检测转染前、后细胞周期和凋亡变化。四甲基偶氮唑蓝（MTT）法连续7d检测OPN干扰时肿瘤细胞增殖的变化，并采用混合模型进行统计分析。Transwell实验检测转染前、后细胞的运动侵袭能力并行t检验进行分析。结果2株细胞中OPN siRNA的转染效率均在90％以上。干扰后2株细胞中OPN蛋白表达均下调。SGC7901细胞在siRNA转染后72hOPN mRNA表达下降最低，达47％；MKN28细胞在siRNA转染后48hOPN mRNA表达下降最低，达40％。OPN被干扰后，SGC7901和MKN28细胞的增殖能力明显减弱（混合模型分析与未干扰组比较，P〈0．01）。OPN被干扰后MKN28和SGC7901细胞周期受到影响，其中SGC7901分裂期细胞比例由18．78％降至17．02％，MKN28分裂期细胞比例则由4．96％降至0．39％。2株细胞都未发现下调OPN后诱发细胞凋亡。OPN下调后2株肿瘤细胞的运动侵袭能力均下降，穿过人工基底膜的细胞数明显减少（SGC7901：t=5．172，P〈0．01；MKN28：t=11．365，P〈0．01）。结论siRNA能下调MKN28和SGC7901中OPN表达，OPN可能与MKN28和SGC7901细胞的增殖和运动侵袭相关。     

小干扰RNA SOX5对胃癌细胞增殖和凋亡的影响

[目的]探讨Y染色体上的性别决定区相关高迁移率族盒蛋白-5(SOX5)的小干扰RNA(siRNA)对胃癌细胞BGC823增殖及凋亡的影响。[方法]通过qRT-PCR和Western-blot分别检测胃癌细胞系(SGC7901,BGC823,AGS,HGC27)及正常胃上皮细胞GES-1中SOX5 mRNA和蛋白的表达情况;取对数生长周期的胃癌细胞BGC823,通过Lipofiectamine2000将设计合成的SOX5的特异性siRNA以及NC转染BGC823细胞,作为siSOX5组和NC组,不做任何处理的细胞作为空白对照组。qRT-PCR检测各组细胞中SOX5 mRNA的表达情况;Western-blot检测各组SOX5及CyclinD1、P21、Bax、Bcl-2蛋白的表达;MTT检测转染12 h、24 h、48 h、72 h后,各组细胞的增殖情况;流式细胞仪检测转染72 h后各组细胞的凋亡情况。[结果]与正常胃上皮细胞GES-1相比,胃癌细胞SGC7901,BGC823,AGS,HGC27中SOX5 mRNA和蛋白表达升高(P0.05);转染siSOX5后,siSOX5组SOX5 mRNA及蛋白表达降低(P0.05);MTT结果显示:与空白对照组和NC组相比,siSOX5组细胞增殖受到抑制,且CyclinD1蛋白表达水平显著下降,p21蛋白表达显著升高(P0.05);流式细胞检测结果显示:与空白对照组和NC组相比,siSOX5组细胞凋亡率升高,且Bax蛋白表达升高,Bcl-2蛋白表达下降(P0.05)。[结论]干扰SOX5能够降低胃癌BGC823细胞中SOX5的表达,抑制BGC823细胞的增殖并促进凋亡。     

RNA干扰FLOT2基因表达下调NF-κB信号对胃癌细胞凋亡诱导作用研究

目的 探讨RNA干扰FLOT2基因表达对胃癌细胞凋亡及NF-κB信号的影响。方法 Western blotting检测人胃癌BGC823、SGC7901和MKN28细胞中FLOT2蛋白表达。BGC823细胞分为空白组、阴性组和si-FLOT2组,参照脂质体Lipofectamine~(TM)2000将siRNA转染细胞,细胞转染48 h,Western blotting检测FLOT2、NF-κB p65、IKK-β、p-IKK-β和Bax的蛋白表达。流式细胞术检测细胞凋亡率。结果 BGC823、SGC7901和MKN28胃癌细胞中FLOT2蛋白表达均显著高于人正常胃黏膜上皮细胞GES1(P0.05)。转染si-FLOT2的BGC823细胞FLOT2蛋白表达显著低于空白组(P0.05)。与空白组比较,si-FLOT2组细胞凋亡率显著升高,NF-κB p65、IKK-β和p-IKK-β蛋白表达显著降低,Bax蛋白表达显著升高(P0.05)。结论 RNA干扰FLOT2基因表达可诱导胃癌细胞凋亡,机制可能与下调NF-κB信号通路有关。     

Investigation of the sensitivities of distinct gastric cancer cells to parvovirus H‐1 induced cytotoxicity

OBJECTIVE: To investigate the sensitivities of distinct gastric cancer cells to parvovirus H‐1 induced cytotoxicity and the possible mechanism(s). METHODS: There were six distinct differentiated gastric cancer cell lines: HGC27 (undifferentiated), BGC823 (undifferentiated), MKN45 (poorly differentiated), AGS (poorly differentiated), SGC7901 (moderately differentiated) and MKN28 (well differentiated). The cell cycle distributions were measured by flow cytometry and the differential sensitivities of the six distinct gastric cancer cells after H‐1 virus infection were detected by MTT assay. RT‐PCR was used to detect viral NS1 gene expression in all six gastric cancer cell lines. RESULTS: The S phase ratios of HGC27, BGC823, MKN45, AGS, SGC7901 and MKN28 were 24.72%, 30.15%, 27.10%, 29.03%, 31.82% and 33.73%, respectively. HGC27 cells were sensitive to H‐1 virus induced cytotoxicity, followed by SGC7901 cells. MKN45 and AGS cells were moderately sensitive and MKN28 cells were insensitive. However, BGC823 cells were resistant to H‐1 virus induced cytotoxicity. The expressions of viral NS1 were higher in HGC27, BGC823, MKN45 and SGC7901 cells, and lower in AGS and MKN28 cells. CONCLUSIONS: The sensitivities of the distinct gastric cancer cells to H‐1 virus induced cytotoxicity were markedly different. In general, the poorly differentiated cells showed an enhanced sensitivity to H‐1 virus attack compared with well‐differentiated ones. The enhanced sensitivity of poorly versus well‐differentiated gastric cancer cells to H‐1 virus is related in part to the enhanced capacity of the former for NS1 protein production and accumulation. The undifferentiated BGC823 cells were resistant to H‐1 virus triggered cytotoxicity. It may further verify that not all tumor cells are sensitive to H‐1 virus lytic effects.     

癌性锚蛋白重复序列在胃癌细胞凋亡中的作用

目的 探讨癌性锚蛋白重复序列(Gankyrin)在人胃癌细胞的表达,以及在尼美舒利诱导细胞凋亡过程中的改变.方法 培养不同分化程度的人胃癌细胞系[MKN28(高分化)、AGS(低分化)、MKN45(低分化)和SGC7901(中分化)],以尼美舒利处理细胞,应用四甲基偶氮唑盐试验和流式细胞术检测细胞活力及细胞凋亡,实时PCR和Western印迹法检测Gankyrin基因和蛋白表达.结果 在4种不同分化程度的人胃癌细胞系中,均存在不同水平的Gankyrin基因和蛋白表达.尼美舒利以时间-剂量依赖方式抑制AGS、FYGC7901细胞增殖.与对照组相比,尼美舒利400 μmol/L处理48 h可诱导AGS、SGC7901细胞显著凋亡(细胞凋亡率分别为0.57%±0.19%比23.30%±2.50%和0.88%±0.17%比16.80%±1.55%,P均<0.01).在AGS细胞凋亡过程中,Gankyrin基因和蛋白表达水平下降,以尼美舒利作用后24 h(0.0035±0.0014)和36 h(0.0980±0.0160)改变最为显著(对照组为0.4690±0.1190,P值均<0.01).结论 在人胃癌细胞中存在Gankyrin基因和蛋白表达.Gankyrin可能参与尼美舒利诱导的AGS胃癌细胞凋亡.     

Comparative study of the chitooligosaccharides effect on the proliferation inhibition and radiosensitization of three types of human gastric cancer cell line

Objective:To observe the chitooligosaccharides(COS) effect on the proliferation inhibition and radiosensitivity of three types of human gastric cancer cell line.Mothods:CCK-8 assay was employed to obtain the inhibition ratio of COS on BGC823 cells,MKN45 cells and SGC7901 cells at 48 h after treatment and the proliferation-inhibition curve was drawn with the inhibition ratio of COS on three types of cells.The clonogenic assay was used to detect the cell viability of 0,1,2,4,6 and 8 Gy(6 dose grades) in RAY group and RAY+COS group after X-ray,and the cell survival curve was used to analyze the sensitization enhancement ratio of COS.Flow cytometry was employed to detect cell cycle and apoptosis rate in control group,RAY group and RAY+COS group after 48 h treatment.Results:COS inhibited the proliferation of three types of cells.The inhibition rate was positively correlated with the concentration of COS,and the susceptibility of MKN45 cells,SGC7901 cells and BGC823 cells to COS decreased in turn.The cell viability decreased gradually with the increasing radiation dose in RAY group and RAY+COS group(P0.01).The cell viabilities of RAY+COS group were lower than those of RAY group at all the dose grades under X-ray exposure(P0.01),and the sensitization enhancement ratios of COS on BGC823 cells,MKN45 cells and SGC7901 cells were 1.06,1.28 and 1.15 respectively.In controlled trials,apoptosis rate and percentage in the G_2/M phase of three types of cells in RAY+COS group were higher than those in control group and RAY group,and percentage in the S phase and the G_0/G_1 phase in RAY+COS group were lower than those in the other two groups(P0.01).Conclusions:COS can inhibit the proliferation of three types of human gastric cancer cells and enhance the radiosensitivity by inducing apoptosis and G_2/M phase arrest.     

Effect of NF-κB, survivin, Bcl-2 and Caspase3 on apoptosis of gastric cancer cells induced by tumor necrosis factor related apoptosis inducing ligand

AIM: To study the effect of NF-κB, survivin, Bd-2 and Caspase3 on tumor necrosis factors related apoptosis inducing ligand (TRAIL) induced apoptosis of gastric cancer cells. METHODS: Gastric cancer cells of SGC-7901, MKN28, MKN45 and AGS lines were cultured in PRMI-1640 medium and the apoptosis rates of the cells of 4 lines were observed after treatment of tumor necrosis factors related apoptosis indudng ligand (TRAIL) with a flow cytometer. The expression of NF-κB, survivin, Bcl-2 and Caspase3 in gastric cancer cells of 4 lines was analyzed with Western blot. RESULTS: After the gastric cancer cells were exposed to TRAIL 300 ng/ml for 24 hours, the apoptosis rate was 36.05%, 20.27%, 16.50% and 11.80% in MKN28, MKN45,AGS and SC-C-7901cells respectively. Western blot revealed that the expressions of NF-EB and survivin were lower in MKN28 cells than in MKN45, AGS and SGC-7901 cells. In contrast, the expression of Caspase3 was higher in MKN28 cells than in MKN45, AGS and SGC-7901 cells. CONCLUSION: There is a selectivity of TRAIL potency to induce apoptosis in gastric cancer cells of different cell lines.The anticancer potency of TRAIL is associated with the decreased expression of NF-κB and survivin and increased expression of Caspase3 of gastric cancer cells.     

H3K27me3在胃癌中的表达及其临床意义

目的:研究H3K27me3在胃癌细胞和组织中的表达,并分析与临床病理因素的关系,探讨H3K27me3在胃癌发生发展中的作用和意义.方法:应用Western blot方法检测胃癌细胞系SGC7901、BGC823、AGS和正常胃黏膜上皮细胞GES-1中H3K27me3的表达;免疫组织化学方法检测61例胃癌组织及20例正常胃黏膜组织中H3K27me3的表达.结果:与正常胃黏膜细胞GES-1相比,H3K27me3在胃癌细胞SGC7901、BGC823、AGS中高表达;H3K27me3在胃癌组织中阳性表达率为80.3%,并与肿瘤大小、浸润深度、淋巴结转移、血管侵犯、临床分期、TNM分期有关(P=0.049,0.030,0.034,0.025,0.003,0.031),而与患者的性别、年龄、病变部位、分化程度、神经侵犯之间无相关性.结论:H3K27me3在胃癌中高表达,并与肿瘤的侵袭转移有关,可能是胃癌患者重要的预后因子.     

&beta;-elemene enhances the radiosensitivity of gastric cancer cells by inhibiting Pak1 activation

AIM: To explore the potential of β-elemene as a radiosensitizer for gastric cancer cells and the underlying mechanisms. METHODS: SGC7901, MKN45, MKN28, N87, and AGS human gastric cancer cell lines were used to screen for radioresistant gastric cancer cell lines. A 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay was used to determine the effects of β-elemene and IPA-3 on cell viability in MKN45 and SGC7901 gastric cancer cell lines. A clonogenic survival assay and annexin V-FITC/PI apoptosis detection assay were used to evaluate cellular radiosensitivity and radiation-induced cell death, respectively. A proteomic method, isobaric tags for relative and absolute quantitation (iTRAQ), was employed to screen the proteins regulated by β-elemene pretreatment prior to ionizing radiation (IR) in SGC7901 gastric cancer cell line. IPA-3 was used as a specific small molecule inhibitor of p21-activated protein kinase 1 (Pak1) to target Pak1 signaling. Protein levels of PAK1IP1 (p21-activated protein kinase-interacting protein 1), total Pak1 (t-Pak1), phospho-Pak1 (T423), phospho-ERK1/2 (Thr202/Tyr204), and cleaved caspase-3 (17 kDa) were assessed by western blotting. RESULTS: MKN45 and SGC7901 gastric cancer cell lines were relatively more resistant to IR. β-elemene pretreatment decreased clonogenic survival following IR in MKN45 and SGC7901 gastric cancer cell lines. Additionally, β-elemene pretreatment prior to IR increased radiation-induced cell death compared with IR alone in MKN45 (10.4% ± 0.9% vs 34.8% ± 2.8%, P < 0.05) and SGC7901 (11.6% ± 0.9% vs 46.7% ± 5.2%, P < 0.05) human gastric cancer cell lines, respectively, consistent with the level of cleaved caspase-3 (17 kDa). Through iTRAQ analysis and western blot validation, we found that β-elemene upregulated PAK1IP1 and downregulated phospho-Pak1 (T423) and phospho-ERK1/2 in SGC7901 gastric cancer cells. IR increased the level of phospho-Pak1 (T423). Pretreatment with β-elemene decreased radiation-induced Pak1 and ERK1/2 phosphorylation. Inhibition of Pak1 using IPA-3 decreased clonogenic survival following IR. In addition, IPA-3 increased radiation-induced cell death in MKN45 (13.4% ± 0.3% vs 26.6% ± 1.0%, P < 0.05) and SGC7901 (16.0% ± 0.6% vs 37.3% ± 1.7%, P < 0.05) gastric cancer cell lines, respectively, consistent with the level of cleaved caspase-3 (17 kDa). Western blotting showed that IPA-3 decreased radiation-induced Pak1 and ERK1/2 phosphorylation. CONCLUSION: This is the first demonstration that β-elemene enhances radiosensitivity of gastric cancer cells, and that the mechanism involves inhibition of Pak1 signaling.     

扶正抗癌冲剂对体外胃癌细胞的抑制作用研究

目的研究中药扶正抗癌冲剂对体外胃癌细胞有无直接抑制或杀伤作用．方法采用体外培养的ＭＫＮ４５，ＭＫＮ２８及ＳＧＣ７９０１三种胃癌细胞株，以扶正抗癌冲剂配制药液（１６１５ｍｇ／ｍｌ含量）的不同稀释度，对３种胃癌细胞体外作用观察，并设丝裂霉素阳性对照组及单纯培养液加生理盐水阴性对照组．结果扶正抗癌冲剂对３种胃癌细胞株均有一定抑制作用，对低分化ＭＫＮ４５胃癌细胞抑制作用最明显，在１∶２稀释度４８ｈ抑制作用最强，抑制率达７４７％，持续到１２０ｈ抑制率仍在７１０％，与正常对照组比较有显著差异（Ｐ＜００５）．在电镜下观察被抑制的ＭＫＮ４５胃癌细胞发生线粒体肿胀、内质网水肿、胞浆脂肪空泡样变性、核仁缩小等变化．结论扶正抗癌冲剂对ＭＫＮ４５低分化胃癌细胞有较强的抑制和杀伤作用，并能使胃癌细胞结构改变．     

Effect of NF-κB, survivin, Bcl-2 and Caspase3 on apoptosis of gastric cancer cells induced by tumor necrosis factor related apoptosis inducing ligand

AIM: To study the effect of NF-κB, survivin, Bcl-2 and Caspase3 on tumor necrosis factors related apoptosis inducing ligand (TRAIL) induced apoptosis of gastric cancer cells.METHODS: Gastric cancer cells of SGC-7901, MKN28,MKN45 and AGS lines were cultured in PRMI-1640 medium and the apoptosis rates of the cells of 4 lines were observed after treatment of tumor necrosis factors related apoptosis inducing ligand (TRAIL) with a flow cytometer. The expression of NF-κB, survivin, Bcl-2 and Caspase3 in gastric cancer cells of 4 lines was analyzed with Western blot.RESULTS: After the gastric cancer cells were exposed to TRAIL 300 ng/ml for 24 hours, the apoptosis rate was 36.05%, 20.27%, 16.50% and 11.80% in MKN28, MKN45,AGS and SGC-7901cells respectively. Western blot revealed that the expressions of NF-κB and survivin were lower in MKN28 cells than in MKN45, AGS and SGC-7901 cells. In contrast, the expression of Caspase3 was higher in MKN28 cells than in MKN45, AGS and SGC-7901 cells.CONCLUSION: There is a selectivity of TRAIL potency to induce apoptosis in gastric cancer cells of different cell lines.The anticancer potency of TRAIL is associated with the decreased expression of NF-κB and survivin and increased expression of Caspase3 of gastric cancer cells.     

核糖体蛋白L5在胃癌中的表达及功能

目的:探讨核糖体蛋白L5(ribosomal protein L5,RPL5) 在胃癌细胞中的表达及对胃癌细胞生长的影响．方法:Western blot检测RPL5在胃癌细胞系中的表达, 构建RPL5特异性siRNA载体,转染细胞,Western blot进行鉴定,MTT方法和流式细胞术检测转染细胞的生长变化．结果:RPL5在胃癌细胞系AGS、MKN45、SGC7901、 MGC803中的表达均明显强于在GES-1和正常胃黏膜上皮中的表达．成功构建RPL5特异siRNA载体U6- RPL5A和U6-RPL5B,转染AGS细胞,进行稳定筛选,发现U6-RPL5A能显著抑制RPL5的表达,其相应的细胞系AGS-U6-RPL5A的生长速度减慢．细胞周期检测结果显示AGS-U6-RPL5A细胞中处于增殖期的细胞减少了约5％．结论:对RPL5功能的进一步深入研究可能会有助于胃癌的诊断和治疗．