Differential display of vincristine-resistance-related genes in gastric cancer SGC7901 cells

AIM: To isolate and clone the vincristine-resistine-relatedgenes in gastric cancer SGC7901 cell line and to clarify themultidrug-resistant molecular mechanism of gastric cancercells.METHODS: The modified differential-display polymerasechain reaction (DD-PCR) was used to examine thedifferences in the mRNA composition of Vincristine-resistantgastric cancer SGC 7901 cells (SGC7901/VCR), induced byvincristine sulfate versus SGC7901cells. The differentiallyexpressed cDNA fragments were confirmed byreverseNorthern analysis, sequencing, BLAST analysis andNorthern bolt analysis.RESULTS: DD-PCR identified that 54 cDNA fragments werepreferentially expressed in SGC 7901/VCR cells. When thesecDNA fragments were analyzed by reverseNorthern blot, 20were reproducibly expressed at a high level in SGC7901/VCR. Sequencing and BLAST analysis revealed that sevenof the genes were known genes: ADP-ribosylation factor 4,cytochrorne oxidase subunit Ⅱ, Ss-A/Ro ribonucleoprteinautoantigen 60kd subunit, ribosomal protein S13, galaectin-8 gene, oligophrenin 1 mRNA, and ribosomal protein L23mRNA; and thirteen of the genes were unknown genes. Thelength and abundance of the four unknown genes mRNAwere further confirmed by Northern blot analysis.CONCLUSION: The twenty differential known and unknowngenes may be related to the vincristine-resistant mechanismin human gastric cancer SGC7901 cell line.     

罗格列酮对人胃癌细胞系迁移侵袭的抑制及其机制

目的:探讨罗格列酮对人胃癌SGC7901、SGC7901/VCR细胞侵袭转移能力及LIMK1基因蛋白表达的影响.方法:罗格列酮(40 mg/L)作用SGC7901和SGC7901/VCR细胞24 h后,采用划痕实验和Transwell实验分别观察罗格列酮对细胞的迁移和侵袭能力.RT-PCR检测LIMKl mRNA和co...     

Dysregulation of mRNA profile in cisplatin-resistant gastric cancer cell line SGC7901

AIM To explore novel therapeutic target of cisplatin resistance in human gastric cancer.METHODS The sensitivity of SGC7901 cells and cisplatin-resistant SGC7901 cells(SGC7901/DDP) for cisplatin were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. High-quality total RNA which isolated from SGC7901/DDP cells and SGC7901 cells were used for mR NA microarray analysis. Results were analyzed bioinformatically to predict their roles in the development of cisplatin resistance and the expression of 13 dysregulated mR NAs we selected were validated by quantitative real-time polymerase chain reaction(qR T-PCR). RESULTS SGC7901/DDP cells highly resistant to cisplatin demonstrated by MTT assay. A total of 1308 m RNAs(578 upregulated and 730 downregulated) were differentially expressed(fold change ≥ 2 and P-value 0.05) in the SGC7901/DDP cells compared with SGC7901 cells. The expression of mR NAs detected by q RT-PCR were consistent with the microarray results. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway and protein-protein interaction analysis demonstrated that the differentially expressed mR NAs were enriched in PI3K-Akt, Notch, MAPK, ErbB, Jak-STAT, NF-kappa B signaling pathways which may be involved in cisplatin resistance. Several genes such as PDE3 B, VEGFC, IGFBP3, TLR4, HIPK2 and EGF may associated with drug resistance of gastric cancer cells to cisplatin.CONCLUSION Exploration of those altered mR NAs may provide more promising strategy in diagnosis and therapy for gastric cancer with cisplatin resistance.     

Study on biological characters of SGC7901 gastric cancer cell-dendritic cell fusion vaccines

AIM: To detect the biological characters of the SGC7901 gastric cancer cell-dendritic cell fusion vaccines. METHODS: The suspending living SGC7901 gastric cancer cells and dendritic cells were induced to be fusioned by polyethylene glycol. Pure fusion cells were obtained by selective culture with the HAT/HT culture systems. The fusion cells were counted at different time points of culture and their growth curves were drawn to reflect their proliferative activities. The fusion cells were also cultured in culture medium to investigate whether they could grow into cell clones. MTT method was used to test the stimulating abilities of the fusion cells on T lymphocytes' proliferations. Moreover, the fusion cells were planted into nude mice to observe whether they could grow into new planted tumors in this kind of immunodeficiency animals. RESULTS: The fusion cells had weaker proliferative activity and clone abilities than their parental cells. When they were cultured, the counts of cells did not increase remarkably, nor could they grow into cell clones in culture medium. The fusion cells could not grow into new planted tumors after planted into nude mice. The stimulating abilities of the fusion cells on T lymphocytes' proliferations were remarkably increased than their parental dendritic cells. CONCLUSION: The SGC7901 gastric cancer cell-dendritic cell fusion vaccines have much weaker proliferative abilities than their parental cells, but they keep strong abilities to irritate the T lymphocytes and have no abilities to grow into new planted tumors in immunodeficiency animals. These are the biological basis for their anti-tumor biotherapies.     

p38分裂原激活蛋白激酶信号转导途径与胃癌细胞长春新碱耐药相关

目的 研究p38分型原激活蛋白激酶（MAPK）信号转导途径在胃癌耐药形成中的意义。方法 用免疫共沉淀法及Western bolt法分别研究长春新碱处理胃癌细胞SGC7901及胃癌多药耐药SGC7901／VCR细胞内p38MAPK活性及量的变化。结果 胃癌多药耐药SGC7901／VCR细胞及亲本SGC7901细胞内均存在活性p38MAPK,长春新碱处理此两种细胞均可引起p38MAPK活性增强,后者反应迅速,可在2min内刺激p38MAPK活性增高,而前者反应较慢,至30min时才观察到p38MAPK活性增高。结论38MAPK信号转导途径可能与和长春新碱的抗肿瘤活性相关,肿瘤细胞内p38MAPK对长春新碱刺激的反应速度可能与胃癌长春新碱耐药相关。     

Effect of parthenolide on proliferation and apoptosis in gastric cancer cell line SGC7901

OBJECTIVE: To investigate the effect of parthenolide (PAR) on proliferation and apoptosis in gastric cancer cell line SGC7901. METHODS: Human gastric cancer cell line SGC7901 cells were incubated with various concentration of PAR. After various periods of incubation, the proliferation of SGC7901 cells was assessed by 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide methyl thiazolyl tetrazolium (MTT) assay. Apoptosis was measured by the annexin V‐fluorescein isothiocyanate fluoresceine isothiocyanate (FITC)/propidium iodide (PI) double labeled staining method and the morphology of the cell was observed under a fluorescent microscope. Mitochondrial potential was measured by flow cytometry after Rhodamine 123 staining. The expressions of cytochrome C and the Bcl‐2 family of proteins, including Bcl‐2, Bax, Bid and tBid were measured by Western blot. Caspase 3 and 8 activities were measured by enzyme‐linked immunosorbent assay. RESULTS: Treatment with PAR induced apoptosis as confirmed by annexin V‐FITC/PI assay. PAR‐induced apoptosis was associated with intracellular events including the decline of mitochondrial potential, increased release of cytochrome C from the mitochondria, decreased expression of Bcl‐2, increased expression of Bax, Bid and tBid and activation of caspase 3 and 8. CONCLUSION: These results suggest that possibly via activation of the mitochondrial pathway, PAR causes mitochondrial damage leading to the release of cytochrome C and by regulating the expression of the Bcl‐2 family of proteins and activating caspases which leads to results in apoptotic cell death in SGC7901 cells. Our results might be helpful in formulating new therapeutic approaches using Chinese herbal medicine.     

人端粒保护蛋白反义核酸抑制SGC7901胃癌细胞增殖

目的 研究人端粒保护蛋白（human protection of telomeres,hPOT1）对胃癌细胞生长增殖的作用.方法 利用本课题组先前所构建的hPOT1正、反义核酸真核表达载体,转染SGC7901胃癌细胞,G418筛选阳性克隆,用Western印迹法、MTT法、流式细胞仪、透射电镜等技术观察基因转染后SGC7901细胞hPOT1蛋白的表达、细胞生长曲线、细胞周期、细胞凋亡及细胞形态学的变化.结果 转染了hPOT1反义核酸真核表达载体（pcDNA-as-hPOT1）的SGC7901胃癌细胞hPOT1表达降低,生长减慢,阻滞于G2/M期,细胞凋亡增加.结论hPOT1反义核酸真核表达载体能显著抑制hPOT1蛋白的表达,hPOT1蛋白可能参与胃癌细胞的周期调控,并与细胞的生长增殖有关.     

应用基因转染方法调节HSP70在人胃癌细胞株SGC7901中的表达

目的　为探讨 ＨＳＰ７０ 在人胃癌中过度表达的意义, 调节ＨＳＰ７０ 在人胃癌细胞系ＳＧＣ７９０１ 中的表达．方法　应用脂质体介导的基因转染方法,将表达针对ＨＳＰ７０的反义ＲＮＡ 表达载体及ＨＳＰ７０ 表达载体转入ＳＧＣ７９０１ 细胞,通过ｈｙｇｒｏ ｍ ｙｃｉｎｅ 抗性筛选, 挑选阳性克隆;对阳性克隆从ＲＮＡ 水平（ 点杂交及ＲＮａｓｅ 保护试验） 及蛋白质水平（ Ｗｅｓｔｅｒｎｂｌｏｔ 及免疫组化） 进行研究．结果　ＨＳＰ７０ 在转染细胞中的表达得到了调节． 为进一步研究ＨＳＰ７０ 对胃癌形成与发展的作用创造了条件．结论　经过ＨＳＰ 反义或正义ＲＮＡ 转染的ＳＧＣ７９０１ 细胞具有不同的ＨＳＰ７０ 表达水平可用于胃癌发生机制的研究．     

白藜芦醇对胃癌细胞SGC7901增殖与凋亡的影响

目的:探讨白藜芦醇(Res)对胃癌细胞SGC7901增殖与凋亡的影响.方法:MTT法检测不同浓度(0,10,20,50,100,200 μmol/L)Res处理24,48,72 h对SGC7901细胞的抑制率.流式细胞仪采用AnnexinV和PI双染检测细胞的早期凋亡率.电镜扫描观察SGC7901细胞形态学改变,分光光度法检测caspase-3活性.结果:Res在20-300 μmol/L浓度范围内,以浓度和剂量依赖的方式抑制胃癌细胞的增殖(P＜0.01).经0,10,20,50,100,200 μmol/L Res处理24 h后,细胞凋亡率分别为1.17%,3.73%,8.75%,23.35%,63.97%和70.10%,晚期凋亡和坏死的细胞比例分别为5.66%,7.22%,9.86%,6.91%,12.51%及11.98%,各组之间差异不大.电镜下见到典型的细胞凋亡的形态学改变.100 μmol/L Res处理后6 h caspase-3活性增加,18 h达高峰,24 h后下降,48 h仍高于正常(0.135±0.036 vs 0.069±0.008,P＜0.05).经20,50,100 μmol/L Res处理18 h后的细胞的caspase-3活性分别为0.169±0.017,0.247±0.028,0.353±0.044(P＜0.01),较对照组(0.063±0.006)分别增加2.68倍、3.92倍和5.61倍.结论:Res通过诱导caspase-3活性增加以时间依赖和浓度依赖的方式抑制SGC7901细胞增殖,诱导细胞凋亡.     

幽门螺杆菌VacA重组蛋白对SGC7901胃癌细胞的作用

目的:研究幽门螺杆菌空泡毒素(VacA)编码基因在大肠杆菌中的表达产物对SGC7901胃癌细胞的作用.方法:常规培养pET32a-vacA-E.coliBL21(DE3)工程菌株,IPTG诱导重组蛋白表达,产物纯化后作用于胃癌细胞,倒置显微镜及电镜等检测细胞生长情况.结果:重组蛋白作用于胃癌细胞后,倒置显微镜下观察,细胞生长受抑制,其细胞空泡毒作用减弱甚至消失,电镜下可见10%-20%左右细胞呈现轻度空泡变,仅少数细胞空泡变明显,细胞表面微绒毛消失,少量细胞体积减小,并出现了早期核着边固缩等凋亡改变.结论:重组VacA蛋白对胃癌细胞的生长具有抑制作用,空泡毒作用减弱.     

钒化钠抑制人胃癌SGC7901细胞增殖的作用机理

目的通过观察钒化钠对人胃癌SGC7901细胞周期蛋白cyclinD1表达的影响,探讨其对人胃癌SGC7901细胞增殖抑制的作用机理。方法应用MTT法测定钒化钠对人胃癌SGC7901细胞的生长抑制作用;用Western-blot法检测钒化钠对人胃癌SGC7901细胞周期蛋白cyclinD1表达水平的变化。结果一定浓度的钒化钠使人胃癌SGC7901细胞增殖周期密切相关的cyclinD1蛋白表达降低。结论一定浓度的钒化钠能明显抑制人胃癌SGC7901细胞的生长,钒化钠的抗肿瘤机制可能与cyclinD1蛋白表达降低有关。     

Anti-proliferation effects of Twist gene silencing in gastric cancer SGC7901 cells

AIM:To study the role of Twist gene in gastric cancer by gene silencing,including the potential of induction of apoptosis,cell cycle arrest,and proliferation inhibition in human malignant gastric SGC7901 cells.METHODS:The expression level of Twist in gastric cancer samples was measured by immunohistochemistry.The effects of Twist gene silencing were detected at both m RNA and protein levels by RT-PCR and Western blot.We also evaluated the cell proliferation and apoptosis by CCK-8 assay and flow cytometry.We determined the activity of caspase-3 and caspase-9 with a caspase activity assay kit.Cell cycle distribution was analyzed by flow cytometry.Cell migration and invasion ability was evaluated by wound scratch assay and Boyden chamber assay.RESULTS:Twist protein was highly expressed in gastric cancer samples.Twist gene silencing significantly induced apoptosis,cell cycle arrest at G0/G1 phase,proliferation inhibition,and reduced the ability of migration and invasion in human gastric cancer SGC7901 cells.Meanwhile,both caspase-3 and caspase-9 were activated.CONCLUSION:The Twist gene could serve as a potential molecular target for gene therapy of gastric cancer with targeted small interfering RNA.     

维拉帕米逆转胃癌细胞系SGC7901/VCR多药耐药性的研究

目的研究维拉帕米对人胃癌耐药细胞系SGC7901/VCR多药耐药性的逆转效应.方法利用RT-PCR、免疫组化以及MTT等方法,观察维拉帕米对体外培养SGC7901/VCR细胞系耐药性的逆转作用.结果RT-PCR、免疫组化和MTT结果表明维拉帕米可以使SGC7901/VCR的MDRl基因表达水平和P-gP糖蛋白表达降低,细胞对丝裂霉素、替加氟等药物的敏感性明显增加.结论维拉帕米在体外有逆转SGC7901/VCR胞多药耐药的效应.     

Bax基因对人胃癌耐药细胞多药耐药性的逆转作用

目的 研究凋亡相关基因Ｂax对胃癌多药耐药细胞的多药耐药性的逆转作用。方法 构建含有人Ｂax cＤＮＡ全长的真核表达载体pＢＫ－Ｂax,经脂质体导入缺乏Ｂax蛋白表达的人 胃癌多药耐药细胞系ＳＧＣ７９０１/ＶＣＲ中。Ｗestern印迹观察Ｂax基因在转导细胞中的表达,ＭＴＴ法检测Ｂax转导细胞与空载体转导细胞对化疗药物的敏感性。结果 成功构建了Ｂax的真核表达载体,Ｂav转导细胞能稳定表达Ｂax蛋白,与空载体转导细胞相比。Ｂ     

Reversal of multidrug resistance in vincristine-resistant human gastric cancer cell line SGC7901/VCR by LY980503

AIM： To investigate the reversal effect of LY980503, a benflumetol derivative, on multidrug resistance in vincristine （VCR） -resistant human gastric carcinoma cell line SGC7901/VCR.
METHODS： Cells of a human gastric cancer cell line, SGC7901, and its VCR-resistant variant, SGC7901/VCR, were cultivated with LY980503 and/or doxorubicin （DOX）. The cytotoxicity of drugs in vitro was assayed by M-IF method. Based on the flow cytometric technology, the uptake of DOX was detected in these cells by measuring DOX -associated mean fluorescence intensity （MFI）.
RESULTS： SGC7901/VCR cells were 23.5 times more resistant to DOX in comparison with 5GC7901 cells. LY980503 at the concentrations of 2.0 μmol/L -10 μmol/ L had no obvious cytotoxicity to SGC7901 and SGC7901/ VCR cells. After simultaneous treatment with LY980503 at the concentrations of 2.0, 4.0 and 10 μmol/L, the ICso of DOX to SGC7901/VCR cells decreased from 1.6 ± 0.12 μmol/L to 0.55 ± 0.024, 0.25 ± 0.032 and 0.11 ± 0.015 μmol/L, respectively, thus, increasing the DOX sensitivity by 2.9-fold （P 〈 0. 05）, 6.4-fold （P 〈 0. 01） and 14.5-fold （P 〈 0. 01）, respectively. In the uptake study of DOX, simultaneous incubation of SGC7901/VCR cells with LY980503 significantly increased the DOX -associated MFI in SGC7901/VCR cells. No such results were found in parental SGC7901 cells.
CONCLUSION： LY980503 at non-cytotoxic concentrations can effectively circumvent resistance of SGC7901/VCR cells to DOX by increasing intracellular DOX accumulation.