Crebanine N-oxide, a natural aporphine alkaloid isolated from Stephania hainanensis, induces apoptosis and autophagy in human gastric cancer SGC-7901 cells

Objective: To investigate the cytotoxic effects and the potential mechanisms of crebanine N-oxide in SGC-7901 gastric adenocarcinoma cells. Methods: The cytotoxicity of crebanine N-oxide was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay and cellular morphology was observed under a microscope. Cell apoptosis was determined by flow cytometry using propidium iodide staining. The expression levels of apoptotic-related proteins, cleaved caspase-3, cytochrome C, p53 and Bax, and autophagyrelated proteins p62, beclin1 and LC3 were detected by Western blotting assays. Results: Crebanine N-oxide treatment significantly inhibited the proliferation of SGC-7901 cells in a dose-dependent and timedependent manner via induction of G2-phase cell cycle arrest, apoptosis, and autophagy in SGC-7901 cells.Conclusions: Crebanine N-oxide could inhibit the growth of gastric cancer cells by promoting apoptosis and autophagy and could be used as a potential agent for treating gastric cancer.

ATP1B1真核表达质粒的构建及其对胃腺癌SGC-7901细胞的作用

目的构建Na+,K+-ATP酶β1亚基(ATP1B1)真核表达质粒,为利用ATP1B1进行肿瘤基因治疗奠定基础。方法以白细胞文库为模板扩增ATP1B1 cDNA,定向插入到pEGFP-C3真核表达质粒中,构建pEGFP-ATP1B1重组质粒;经酶切分析和测序鉴定后,通过脂质体介导转染胃腺癌SGC-7901细胞,利用real-time PCR检测转染后SGC-7901细胞的ATP1B1基因表达,并进行其ATP酶活性检测;MTT法测定转染后SGC-7901细胞的增殖活性。结果重组质粒经鉴定证实含有ATP1B1 cDNA序列,转染pEGFP-ATP1B1的SGC-7901细胞ATP1B1基因表达增高达129.2%,ATP酶活性增强为(2.95±0.210)%,细胞生长增殖显著受抑。结论成功构建了ATP1B1真核表达质粒,为进一步利用ATP1B1研究恶性肿瘤基因治疗奠定基础。

叉头框蛋白D3在胃贲门腺癌组织中的表达及其对SGC-7901细胞生物学行为的影响

目的:探讨叉头框蛋白D3(forkhead box protein D3,FOXD3)在胃贲门腺癌(gastric cardia adenocarcinoma,GCA)中的表达及其对SGC-7901细胞生物学行为的影响。方法:从河北医科大学第四医院生物标本库中选取2014年6月至2016年12月手术切除的49例GCA组织及相应癌旁组织标本,qRT-PCR检测FOXD3在GCA组织、癌旁组织以及在5种胃癌细胞系中(BGC-823、SGC-7901、HGC-27、MGC-803及NCI-N87)的表达。向SGC-7901细胞转染pc-DNA3.1-FOXD3或pc-DNA3.1,采用细胞增殖实验、克隆形成实验、划痕愈合实验和Transwell小室侵袭实验分别检测FOXD3过表达对SGC-7901细胞增殖、克隆形成、迁移和侵袭的影响,qRT-PCR及WB法检测细胞转染前后上皮-间质转化(epithelial-mesenchymal transition,EMT)相关分子mRNA及蛋白的表达情况,流式细胞术检测转染前后细胞周期改变。结果:GCA组织中FOXD3 mRNA的表达量明显降低,其表达水平与患者临床分期和淋巴结转移密切关联;FOXD3在胃癌细胞系中的表达均低于正常细胞(均P<0.01)。FOXD3过表达能明显抑制SGC-7901细胞的增殖、克隆形成、迁移和侵袭能力(均P<0.01),提高SGC-7901细胞中E-cadherin的表达水平,减少N-cadherin、β-catenin和vimentin的表达水平(均P<0.01),使细胞周期阻滞在G0/G1期(P<0.01)。结论:FOXD3在GCA组织中的表达明显下调,其过表达可以抑制胃癌细胞的生物学行为,FOXD3可作为抑癌基因为肿瘤治疗提供新思路。

Bcl-2基因沉默对胃腺癌SGC-7901细胞5-Fu敏感性的影响

目的探讨B淋巴细胞瘤-2(Bcl-2)基因沉默增强胃腺癌SGC-7901细胞对5-氟尿嘧啶(5-FU)敏感性的价值。方法设立人胃腺癌SGC-7901细胞研究组和对照组,采用逆转录聚合酶链反应(RTPCR)检测两组人胃腺癌SGC-7901细胞中Bcl-2 m RNA的表达。研究组采用脂质体法转染化学合成的Bcl-2 si RNA序列至人胃腺癌SGC-7901细胞,对照组不进行干预。比较转染前后两组Bcl-2 m RNA的表达水平。两组均添加不同浓度5-FU,采用Annexin V标记和流式细胞仪检测48 h后两组不同浓度5-FU细胞株的增殖抑制率和凋亡率。结果两组转染前Bcl-2 m RNA相对表达量比较,差异无统计学意义(P>0.05)。与转染前比较,研究组转染后的Bcl-2 m RNA相对表达量降低(P<0.05);与对照组比较,研究组转染后的Bcl-2m RNA相对表达量降低(P<0.05)。与对照组比较,研究组培养48 h的细胞增殖抑制率和凋亡率较高(P<0.05)。结论 Bcl-2基因沉默可增强胃腺癌SGC-7901细胞对5-FU的敏感性,抑制癌细胞的增殖,并促进癌细胞的凋亡。Bcl-2基因沉默可能是改善胃腺癌5-FU疗效的有效方法。

Inhibitory effects of dobutamine on human gastric adenocarcinoma

AIM:To explore the inhibitory effects of dobutamine on gastric adenocarcinoma cells.METHODS:Dobutamine was used to treat gastric adenocarcinoma cells(SGC-7901)and cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.The effects of dobutamine combined with cisplatin on cell viability were also analyzed.Cell migration was studied using the wound healing assay,and cell proliferation was analyzed using the colony formation assay.A cell invasion assay was carried out using Transwell cell culture chambers.The cell cycle and cell apoptosis were analyzed by flow cytometry.Western blot and immunocytochemistry were performed to determine the expression of Yes-associated protein(YAP)in treated cells.RESULTS:Dobutamine significantly inhibited cell growth,migration,cell colony formation,and cell invasion into Matrigel.Dobutamine also arrested the cell cycle at G1/S phase,and increased the rate of apoptosis of gastric adenocarcinoma cells.The expression ofYAP was detected mainly in the nucleus in the absence of dobutamine.However,reduced expression of phosphorylated YAP was mainly found in the cytosol following treatment with dobutamine.CONCLUSION:Dobutamine has significant inhibitory effects on gastric adenocarcinoma cells and may be used in neoadjuvant therapy not only for gastric cancer,but also for other tumors.

丹参酮Ⅰ对人胃腺癌细胞SGC-7901 EIF2a和EIF3a基因表达的调控作用

目的分析丹参酮Ⅰ对人胃腺癌细胞SGC-7901真核翻译起始因子2a(EIF2a)、真核翻译起始因子3a(EIF3a)基因表达的调控作用。方法体外培养SGC-7901人胃腺癌细胞株,经丹参酮Ⅰ干预后,采用实时荧光定量聚合酶链反应(RT-PCR)法测定EIF2a和EIF3a mRNA水平;采用四甲基偶氮唑蓝(MTT)比色法测定丹参酮Ⅰ对SGC-7901细胞株增殖的影响,采用流式细胞术检测细胞周期及细胞凋亡率。结果不同质量浓度丹参酮Ⅰ干预后SGC-7901细胞株EIF2a mRNA和EIF3a mRNA降低,与空白对照组比较,差异有统计学意义(P<0.05);EIF2a mRNA与EIF3a mRNA比较,丹参酮Ⅰ1μg/mL>5μg/mL>10μg/mL(P<0.05)。丹参酮Ⅰ1,5,10μg/mL组细胞培养不同时间A值低于空白对照组(P<0.05),抑制率高于空白对照组(P<0.05);不同时间A值比较,丹参酮Ⅰ1μg/mL>5μg/mL>10μg/mL;不同时间抑制率比较,丹参酮Ⅰ1μg/mL<5μg/mL<10μg/mL(P<0.05)。丹参酮Ⅰ不同质量浓度组G1期细胞比例高于空白对照组(P<0.05),S期细胞比例低于空白对照组(P<0.05);G1期细胞比例丹参酮Ⅰ不同质量浓度组对比,丹参酮Ⅰ1μg/mL<5μg/mL<10μg/mL;S期细胞比例丹参酮Ⅰ不同质量浓度组对比,丹参酮Ⅰ1μg/mL>5μg/mL>10μg/mL(P<0.05),丹参酮Ⅰ1,5,10μg/mL组细胞凋亡率均高于空白对照组(P<0.05),不同质量浓度组丹参酮Ⅰ细胞凋亡率比较,丹参酮Ⅰ1μg/mL<5μg/mL<10μg/mL(P<0.05)。结论丹参酮Ⅰ可通过下调人胃腺癌细胞SGC-7901 EIF2a和EIF3a基因表达,抑制癌细胞增殖,促进肿瘤细胞凋亡。

Growth inhibitory effect of 4-phenyl butyric acid on human gastric cancer cells is associated with cell cycle arrest

AIM: To investigate the growth effects of 4-phenyl butyric acid (PBA) on human gastric carcinoma cells and their mechanisms. METHODS: Moderately-differentiated human gastric carcinoma SGC-7901 and lowly-differentiated MGC-803 cells were treated with 5, 10, 20, 40, and 60 μmol/L PBA for 1-4 d. Cell proliferation was detected using the MTT colorimetric assay. Cell cycle distributions were examined using flow cytometry.RESULTS: The proliferation of gastric carcinoma cells was inhibited by PBA in a doseand time-dependent fashion. Flow cytometry showed that SGC-7901 cells treated with low concentrations of PBA were arrested at the G0/G1 phase, whereas cells treated with high concentrations of PBA were arrested at the G2/M phase. Although MGC-803 cells treated with low concentrations of PBA were also arrested at the G0/G1 phase, cells treated with high concentrations of PBA were arrested at the S phase. CONCLUSION: The growth inhibitory effect of PBA on gastric cancer cells is associated with alteration of the cell cycle. For moderately-differentiated gastric cancer cells, the cell cycle was arrested at the G0/G1 and G2/M phases. For lowly-differentiated gastric cancer cells, the cell cycle was arrested at the G0/G1 and S phases.

吴茱萸碱对人胃腺癌细胞SGC-7901作用的研究

目的研究吴茱萸碱(Evo)对人胃腺癌(SGC-7901)细胞的作用,并初步探讨其作用机制。方法 MTT法测定Evo的抗肿瘤活性;电镜观察Evo对细胞形态学的影响;琼脂糖凝胶电泳检测Evo对细胞DNA电泳行为的影响;流式细胞技术检测Evo对SGC-7901细胞的抑制作用和细胞周期的影响。结果一定浓度范围内Evo对SGC-7901细胞的增殖有剂量、时间依赖性抑制作用;透射电镜下可见细胞核染色质边集、核碎裂及凋亡小体;一定浓度的Evo可使细胞基因组DNA电泳出现阶梯状条带;流式细胞仪检测Evo作用后,G0/G1期和S期细胞减少,大量细胞停滞在G2/M期,凋亡细胞增多。结论 Evo对SGC-7901细胞的抑制作用与诱导其凋亡使细胞停滞于G2/M期密切相关。

三氧化二砷对人胃腺癌细胞株SGC-7901细胞核及线粒体作用的实验研究

目的:研究三氧化二砷(As2O3)对人胃腺癌细胞株SGC-7901的生物效应及其对线粒体和半胱氨酸蛋白酶家族-3(caspase-3)的作用。方法:通过MTT比色实验检测不同浓度As2O3对该细胞株的生长抑制作用;经Hoechst 33258染色后用荧光显微镜观察细胞核的形态变化;经过细胞线粒体膜电位检测区分凋亡细胞与正常细胞,并经流式细胞仪分析;caspase-3吸光度检测法测定As2O3组caspase-3的活化程度。结果:As2O3明显抑制SGC-7901人胃腺癌细胞的生长,抑制作用的强度呈时间依赖性(方差分析,P<0.01);Hoechst 33258染色后荧光显微镜观察细胞核固缩碎裂边集呈强蓝色荧光;线粒体膜电位检测法,流式细胞仪检测法,caspase-3吸光度检测法均检测到胃腺癌细胞的凋亡。结论:As2O3破坏线粒体跨膜电位和激活caspase-3活性可能是As2O3诱导人胃腺癌SGC-7901细胞凋亡的重要机制。

COX-2通过NF-κB通路上调胃癌细胞P-gp表达的实验研究

目的探讨紫杉醇(TAX)作用下环氧化酶-2(COX-2)诱导胃癌细胞株SGC-7901多药耐药P蛋白(P-gp)表达可能的信号通路。方法 MTT法检测TAX、COX-2抑制剂NS-398和核因子-κB(NF-κB)信号通路抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)不同剂量对胃腺癌细胞株SGC-7901生长的影响;Western blotting检测TAX作用于SGC-7901细胞株以及联合NS-398、PDTC对COX-2、NF-κB p65和P-gp表达的影响。结果 TAX、NS-398和PDTC均对胃癌细胞株SGC-7901的生长具有细胞毒作用,并呈剂量依赖性。TAX(0.1、0.3、0.5μmol/L)可诱导COX-2、p65和P-gp表达,随剂量增加,3种蛋白表达显著增加;与NS-398(5、10μmol/L)联用时,随剂量增加和时间延长,3种蛋白表达减少;与PDTC(0.2μmol/L)联用时,随作用时间延长,p65和P-gp表达减少。结论在TAX作用下,COX-2可能通过激活NF-κB通路而引起胃癌SGC-7901细胞株P-gp表达增加。

G5-BGG/pDNA的制备及其对SGC-7901细胞的抑制作用

非病毒载体具有质量可控、携带能力高和免疫原性低等优点,但存在转染效率低的致命缺陷,限制了广泛应用。本研究采用计算机模拟设计了新型功能性基团1,3-间苯二甲酰胍(BGG),并通过酰胺键将其修饰于第5代(G5)聚酰胺-胺树枝状大分子表面,得到新型非病毒载体材料G5-BGG。G5-BGG可将基因压缩成荷正电的基因复合物。体外定性和定量转染试验表明,G5-BGG作为基因药物载体可显著提高报告基因在人胃腺癌细胞(SGC-7901)中的转染效率,同时具有良好的生物相容性。G5-BGG负载肿瘤坏死因子相关凋亡诱导配体(pTRAIL)基因时可在细胞水平上诱导SGC-7901细胞凋亡,抑制肿瘤细胞生长,具有良好的抗肿瘤作用。本研究表明G5-BGG在胃癌基因治疗领域具有潜在的应用前景。

Src激酶抑制剂PP2对人胃癌细胞生物学行为的影响

目的:探讨Src激酶抑制剂PP2对人胃癌SGC-7901细胞生长、迁移、侵袭和诱导血管形成等生物学行为的影响。方法:通过Western印迹法检测PP2作用对SGC-7901细胞中Src激酶活化的影响;分别采用MTT法、划痕实验、Transwell小室法和体外诱导血管形成分析实验观察PP2对SGC-7901细胞生长、迁移、基质侵袭和诱导血管形成等生物学行为的影响。结果:MTT法检测结果提示,15μmol/L PP2作用人胃癌SGC-7901细胞12 h时能明显抑制细胞的增殖;5和10μmol/L PP2均能抑制SGC-7901细胞的迁移、侵袭和诱导血管的形成,且其抑制能力随PP2浓度的增加而逐渐增强。结论:Src激酶抑制剂PP2具有抑制人胃癌SGC-7901细胞生长、迁移、侵袭和诱导血管形成的能力,可能对胃癌具有一定的治疗效果。

RNA干扰对胃癌细胞株c-Met基因的研究

目的研究c-Met-siRNA体外对人胃腺癌SGC-7901细胞株增殖、凋亡和细胞周期等生物学功能的影响。方法设计合成有效的C-MET mRNA的siRNA,体外通过Lipofectamine2000将合成的siRNA转染入人胃癌SGC-7901细胞株,同时设置未转染组、阴性对照组、阳性对照组作为对照,RT-PCR检测转染后C-MET mRNA表达水平的变化,通过Western blot检测蛋白质的表达水平,采用MTT法检测细胞的生长状况。结果 c-Met-siRNA转染人胃癌SGC-7901细胞后,c-Met的mRNA和蛋白表达水平显著降低,细胞的生长受到明显抑制。结论 c-Met-siRNA能成功下调靶基因的表达,并能显著抑制胃癌SGC-7901细胞生长,是潜在的胃癌治疗新方法。