胃泌素对胃癌细胞粘着斑激酶酪氨酸磷酸化的影响

背景与目的:胃泌素能够刺激胃癌细胞的生长和增殖,这一作用同酪氨酸激酶有关。本研究旨在阐明胃泌素对人胃癌细胞内粘着斑激酶(focaladhesion kinase,FAK)酪氨酸磷酸化的影响。方法:用人胃泌素受体(gastrinreceptor,GR)的真核表达载体pCR3.1/GR,转染人胃癌细胞株SGC7901,增强胃泌素受体表达;用胃泌素受体拮抗剂L-365260,抑制胃泌素和其受体结合;以不同浓度和作用时间的胃泌素刺激胃癌细胞,利用免疫沉淀和蛋白质印迹法检测上述情况下,FAK酪氨酸磷酸化的变化。结果:分别用0.1nmol/L、1nmol/L和10nmol/L的胃泌素作用后,转染pCR3.1/GR的SGC7901细胞内FAK酪氨酸磷酸化表达量分别为0.64±0.06、0.91±0.10和1.00±0.10,高于SGC7901细胞的0.40±0.05、0.52±0.07和0.62±0.06(P<0.01);转染pCR3.1/GR的SGC7901细胞内FAK酪氨酸磷酸化表达量分别为0.72±0.08、0.83±0.05、0.88±0.06和1.00±0.08,高于SGC7901细胞的0.59±0.05、0.65±0.07、0.58±0.03和0.47±0.10(P<0.01或P<0.05)。胃泌素受体拮抗剂L-365260使转染pCR3.1/GR的SGC7901细胞内FAK酪氨酸磷酸化表达量从1.00±0.07降至0.72±0.07(P<0.01),使SGC7901细胞内表达量由0.62±0.06降至0.45±0.05(P<0.01)。在此过程中,FAK蛋白表达量差异无统计学意义(P>0.05)。结论:FAK是胃泌素和其受体结合后发挥效应的关键下游信号分子,酪氨酸磷酸化是其活性形式。     

胃泌素及其受体拮抗剂对人胃癌细胞株MKN45增殖及HB-EGF表达的影响

目的探讨胃泌素-17及其受体拮抗剂丙谷胺对人胃癌细胞株MKN45增殖及肝素结合表皮生长因子样生长因子（HB-EGF）表达的影响。方法MTT法观察细胞增殖活力;Western blot测定肝素结合表皮生长因子样生长因子（HB-EGF）蛋白的表达。结果MTT结果显示胃泌素-17在10-6～10-9 mol/L时具有明显的促MKN45细胞增殖作用(P<0.05);丙谷胺在10-2～10-3 mol/L时显著抑制MKN45细胞增殖(P<0.05);胃泌素+丙谷胺组中,丙谷胺（10-2～10-3 mol/L）能阻断并抑制胃泌素对胃癌细胞MKN45的促增殖作用（P<0.05）。同时,Western blot结果显示胃癌细胞株MKN45中有HB-EGF蛋白的表达。胃泌素-17在10-6～10-9 mol/L时可显著增强MKN45细胞株中HB-EGF蛋白表达（P<0.05）;丙谷胺（10-2～10-3 mol/L）能阻断并抑制G-17（10-8 mol/L）诱导的HB-EGF蛋白表达上调（P<0.05）。 结论胃泌素-17促进人胃癌细胞MKN45增殖,其受体拮抗剂丙谷胺能阻断这一促进作用。胃泌素受体拮抗剂丙谷胺能抑制人胃癌细胞MKN45增殖。胃癌细胞株MKN45中有HB-EGF蛋白的表达,胃泌素-17促进HB-EGF蛋白表达上调,其受体拮抗剂丙谷胺能阻断并抑制这一促进作用。     

奥曲肽对胃癌细胞转录活化蛋白-1的抑制作用

背景与目的:生长抑素是一种多功能神经肽,其本身及其类似物能抑制多种内分泌肿瘤及某些胃肠肿瘤的生长,但是否能抑制胃癌的生长并不清楚。因此,本研究拟探讨生长抑素类似物奥曲肽对胃癌细胞生长的影响及其机制。方法:不同浓度的奥曲肽作用于人胃癌SGC-7901细胞24h后,用免疫印迹法检测奥曲肽对SGC-7901细胞中c-Fos、ERK蛋白表达的影响。建立人胃癌细胞裸鼠原位移植瘤模型,奥曲肽治疗8周,观察奥曲肽对胃癌生长的影响;免疫组化法检测裸鼠人胃癌组织c-Fos和ERK表达的变化。在此基础上,采用EMSA技术检测奥曲肽对胃癌细胞AP-1活化的影响。结果:1×10-5mol/L奥曲肽可明显降低胃癌细胞的3H-TdR的掺入;奥曲肽能抑制人胃癌细胞裸鼠原位移植瘤的生长,抑瘤率为62.3%。SGC-7901胃癌细胞经不同浓度奥曲肽作用后,其c-Fos和ERK的表达水平均有不同程度下降;组织标本检测亦有类似变化。EMSA分析显示,胃癌细胞有基础水平的AP-1活化,20%血清能刺激AP-1的结合力,奥曲肽能抑制这种刺激作用。结论:奥曲肽通过抑制胃癌c-Fos、ERK蛋白的表达而抑制AP-1的结合活性,从而抑制胃癌的生长。     

NS398对SGC7901胃癌细胞生长及CD44V6、MMP-9基因表达的影响

目的探讨COX-2特异性抑制剂NS398对SGC7901胃癌细胞体外生长及CD44V6、MMP-9基因表达的影响。方法体外培养SGC7901胃癌细胞,应用免疫细胞化学方法,检测SGC7901胃癌细胞中COX-2、CD44V6、MMP-9蛋白表达水平,酶联免疫吸附试验(ELISA)检测NS398对胃癌细胞PGE2分泌水平的影响,MTT法检测NS398对SGC7901胃癌细胞体外生长的抑制效应,半定量RT-PCR检测NS398作用后胃癌细胞株中CD44V6、MMP-9基因表达水平的变化。结果 SGC7901胃癌细胞中COX-2、CD44V6、MMP-9呈阳性表达,NS398可显著抑制SGC7901胃癌细胞PGE2的分泌,200μmol/L NS398抑制率达64.3%,NS398对胃癌细胞生长抑制作用呈时间及浓度依赖性;NS398作用24 h后胃癌细胞中CD44V6、MMP-9基因表达水平下降,200μmol/L NS398对CD44V6、MMP-9基因表达抑制率分别为45.7%、43.6%。结论 SGC7901胃癌细胞中COX-2、CD44V6、MMP-9呈阳性表达,NS398可有效抑制SGC7901胃癌细胞PGE2的分泌和胃癌细胞生长,并可抑制与胃癌转移相关的CD44V6、MMP-9基因的表达。     

siRNA下调KLF8对胃癌细胞SGC7901增殖能力的影响

目的:探讨KLF8基因特异性siRNA对胃癌细胞株SGC7901增殖能力的影响.方法:通过免疫组织化学、Western blot法、RT-PCR检测胃癌组织标本和胃癌细胞株KLF8的表达;通过基因重组方法构建KLF8的siRNA慢病毒载体,并感染人SGC7901细胞,通过Western blot验证siRNA干扰效率.通过MTT、平板克隆、流式细胞仪检测细胞增殖、细胞周期和凋亡,观察其对胃癌细胞SGC7901增殖能力的影响.结果:KLF8在胃癌组织和细胞株中的表达高于癌旁组织和正常胃上皮细胞,下调KLF8能能够明显抑制SGC7901细胞的增殖,促进细胞G0/G1期阻滞和诱导细胞凋亡.结论:KLF8在促进胃癌细胞SGC7901增殖中发挥重要作用,为胃癌靶向治疗提供理论依据.     

miRNA-141对胃癌细胞生长的抑制调节作用

目的:研究miRNA-141在胃癌细胞中的生物学功能。方法:定量PCR检测人胃黏膜细胞GES-1,胃癌细胞株BGC823、MGC803、SGC7901中miRNA-141的表达,通过miRNA-141 inhibitor、mimics分别抑制及促进miRNA-141的表达,观察癌细胞增殖、克隆形成、侵袭能力的变化。结果:胃癌细胞BGC823、MGC803、SGC7901中miRNA-141处于低表达状态。上调miRNA-141的表达后,miRNA-141明显抑制SGC7901细胞增殖（P<0.05）,抑制肿瘤细胞克隆形成（P<0.05）及癌细胞侵袭力（P<0.05）。结论:miRNA－141对胃癌细胞SGC7901具有负性调控作用,可以作为潜在的治疗靶点。     

多西紫杉醇对胃癌细胞作用及其机制的研究

目的:研究多西紫杉醇对人胃癌中分化细胞株SGC7901的作用效果及机制。方法:MTT法检测多西紫杉醇对胃癌细胞株SGC7901的增殖抑制作用。AnnexinV方法检测药物作用后胃癌细胞的凋亡。PI染色法检测凋亡晚期的胃癌细胞。采用流式细胞仪检测多西紫杉醇作用前后细胞凋亡相关分子Fas蛋白、Bcl2蛋白表达水平的变化。结果:0.92、3.7、14.8、59.2μg/mL多西紫杉醇作用于SGC7901细胞72h,抑制率分别为13.3%、21.6%、57.5%、61.0%。多西紫杉醇可诱导胃癌细胞株SGC7901发生细胞凋亡。多西紫杉醇使SGC7901凋亡分子Fas表达增加,作用前后分别为(26.5±7.2)%、(37.9±7.0)%;多西紫杉醇作用SGC7901前后Bcl2表达分别为(38.9±9.1)%、(31.2±5.6)%,差异无显著性。结论:多西紫杉醇对胃癌中分化细胞株SGC7901生长有明显的抑制作用,可诱导胃癌细胞系SGC7901凋亡,凋亡作用的发生可能与多西紫杉醇诱导Fas分子表达有关。     

反义MUC2体外抑制胃癌细胞侵袭活性的研究

目的：探讨应用反义脱氧寡核苷酸（ASODN）体外抑制粘蛋白MUC2对人胃癌细胞株SGC7901侵袭活性的影响。方法：采用人工合成MUC2ASODN经阳离子脂质体包裹后转染入胃癌细胞株SGC7901中,应用免疫组化及Boyden小室体外侵袭实验比较转染前后癌细胞侵袭能力的改变。结果：转染MUC2ASODN后,癌细胞穿膜细胞相对百分率较处理前明显下降;免疫组化S-P法染色提示转染MUC2ASODN后,SGC7901细胞的组织蛋白酶D及基质金属蛋白酶2表达水平明显降低。结论：使用人工合成MUC2ASODN能有效抑制胃癌细胞侵袭能力。     

甘氨酸延伸型胃泌素对人胃癌细胞株SGC-7901的体外作用

目的体外研究甘氨酸延伸型胃泌素(Glycin-extended gastrin, G-Gly)对人胃癌细胞株SGC-7901增殖以及对丝裂霉素（MMC）促凋亡作用的影响,探讨G-Gly在胃癌发生发展中的作用。方法应用MTT法检测不同浓度G-Gly对SGC-7901增殖的影响,应用流式细胞术检测细胞的凋亡率。结果G-Gly能促进SGC-7901的增殖,其增殖率在一定范围内与剂量有关,在浓度0.1 nmol/L时具有最大增殖效果。其效应不能被缩胆囊肽B受体（Cholecystokinin B receptor,CCKBR）的抗体所抑制。G-Gly能抑制MMC诱导的凋亡（P<0.01）。结论G-Gly能促进SGC-7901的增殖和抑制MMC诱导的凋亡,提示G-Gly能促进胃癌肿瘤的生长。     

cyclin D1反义寡核苷酸对胃癌细胞生长和G1期调控的影响

目的：研究cyclin D1反义寡核苷酸(ASODN)对胃癌细胞SGC7901和HS746T的生长、细胞周期和G1期调控的影响。方法：采用脂质体Lipofect AMINE2000包裹硫代修饰的cyclin D1 ASODN转染细胞，观察量效曲线和生长曲线，应用RT-PCR检测cyclin D1 mRNA的表达，流式细胞仪检测细胞周期，并用免疫组织化学技术检测胃癌细胞中cyclin D1、p21、p27、pRb蛋白的表达。结果：cyclln D1 ASODN对胃癌细胞产生剂量依赖性的抑制效应，0．2μmol/L cyclin D1 ASODN转染24h能显著抑制胃癌细胞生长，cyclin D1 mRNA表达分别是对照组的26．3％(SGC7901)和17．3％(HS746T)，G0／G1期比例明显增加，并使2种细胞中cyclin D1、pRb表达降低，p21表达升高，在HS746T中p27表达下降，但在SGC7901中p27表达无改变。结论：cyclin D1反义寡核苷酸能有效抑制胃癌细胞的生长，降低cyclin D1 mRNA表达水平，并能影响细胞周期及其调控的表达。     

Radiation sensitivity of merkel cell carcinoma cell lines

: Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions.     

桥接整合因子1通过AKT-mTOR通路诱导非小细胞肺癌H1975细胞周期阻滞

目的：研究桥接整合因子1（bridging intergrator 1,Bin1）基因过表达后对非小细胞肺癌细胞株H1975细胞周期的影响及其作用机制。方法：构建携带Bin1基因的CMV-MCS-GFP-SV40-Neomycin-Bin1质粒,并转染H1975细胞（Bin1+组）,另设置空白质粒转染组（Bin1-组）及空白对照组（Ctrl组）,利用RT-PCR和Western blotting分别检测3组细胞中Bin1在mRNA和蛋白质水平的表达情况。流式细胞术检测不同处理组H1975细胞周期的变化,Western boltting分别检测各组中AKT、mTOR磷酸化水平及细胞周期相关蛋白（周期蛋白D1、CDK4、Rb）的表达情况。结果：与Bin1-组、Ctrl组比较,Bin1+组H1975细胞中Bin1在mRNA、蛋白水平表达明显上调（均P<0.05）; H1975细胞阻滞在G1期\[（60.53±1.89）% vs（46.14±1.56）%、（47.33±2.07）%,均P<0.05\]; Bin1+组H1975细胞内p-AKT、p-mTOR表达下调（均P<0.05）,AKT、mTOR表达变化无统计学差异（P>0.05）;周期蛋白D1、CDK4的表达量均明显下调(P<0.05),Rb表达量明显增加（P<0.05）。结论：Bin1基因在H1975细胞株过表达后明显诱导细胞周期阻滞,其机制可能是通过抑制AKT-mTOR通路及其细胞周期相关蛋白实现的。     

Inhibition of S-adenosylhomocysteine hydrolase decreases cell mobility and cell proliferation through cell cycle arrest

S-adenosylhomocysteine hydrolase (AHCY) hydrolyzes S-adenosylhomocysteine to adenosine and l-homocysteine, and it is already known that inhibition of AHCY decreased cell proliferation by G2/M arrest in MCF7 cells. However, the previous study has not indicated what mechanism the cell cycle arrest is induced by. In this study, we aimed to investigate the different cell cycle mechanisms in both p53 wild-typed MCF7 and p53 mutant-typed MCF7-ADR by suppressing AHCY. We extensively proved that AHCY knockdown has an anti-proliferative effect by using the WST-1 assay, BrdU assay, and cell cytometry analysis and an anti-invasive, migration effect by wound-healing assay and trans-well analysis. Our study showed that down-regulation of AHCY effectively suppressed cell proliferation by regulating the MEK/ERK signaling pathway and through cell cycle arrests. The cell cycle arrest occurred at the G2/M checkpoint by inhibiting degradation of cyclinB1 and phosphorylation of CDC2 in MCF7 cells and at the G1 phase by inhibiting cyclinD1 and CDK6 in MCF7-ADR cells. Finally, we determined that AHCY regulates the expression of ATM kinase that phosphorylates p53 and affects to arrest of G2/M phase in MCF7 cells. The findings of this study significantly suggest that AHCY is an important regulator of cell proliferation through different mechanism in between MCF7 and MCF7-ADR cells as p53 status.     

Mechanisms of kaposis-sarcoma cell supernatant-induced vascular cell invasion

Kaposi's sarcoma (KS) is a highly angiogenic lesion frequently associated with acquired immune deficiency syndrome. Histologically the lesions appear to contain proliferative 'spindle shaped' cells with a mixed smooth muscle-endothelial-fibroblastic histotype and a conspicuous neovascularization, derived from host cell recruitment. Media conditioned by cultured KS cells (KS-CM) have angiogenic properties. KS-CM is able to promote endothelial and smooth muscle cell migration and invasion. The mechanisms of this KS-CM activity are still unknown. We hypothesize that KS-CM contains numerous factors with different roles in inducing the neo angiogenic process. We show that AIDS-IST-KS cell supernatants induce gelatinase A production and plasminogen activator (PA) up-regulation in vascular cells. KS-CM activity in vivo is heparin dependent. Also bFGF alone, a heparin dependent factor, alone can induce endothelial and smooth muscle cell invasion, MMP-2 production and PA activity. However, antibodies to bFGF do not block KS-CM activity and do not reduce the effect on PA up-regulation. This evidence suggests that heparin-binding factors other than bFGF may be present. Chromatography of KS-CM on heparin-sepharose demonstrates the presence of two heparin-binding fractions with chemotactic and gelatinase A inducing activity. The flow through was also active. KS-CM absorption on heparin-sepharose beads did not modify its induction of PA activity, further evidence for the presence of non heparin-binding factors as well.     

Landscape of immune cell infiltration in clear cell renal cell carcinoma to aid immunotherapy

The tumor microenvironment, comprised of tumor cells and tumor-infiltrating immune cells, is closely associated with the clinical outcome of clear cell renal cell carcinoma (ccRCC) patients. However, the landscape of immune infiltration in ccRCC has not been fully elucidated. Herein, we applied multiple computational methods and various datasets to reveal the immune infiltrative landscape of ccRCC patients. The tumor immune infiltration (TII) levels of 525 ccRCC patients using a single-sample gene were examined and further categorized into immune infiltration subgroups. The TII score was characterized by distinct clinical traits and showed a significant divergence based on gender, grade, and stage. A high TII score was associated with the ERBB signaling pathway, the TGF-β signaling pathway, and the MTOR signaling pathway, as well as a better prognosis. Furthermore, patients with high TII scores exhibited greater sensitivity to pazopanib. The low TII score was characterized by a high immune infiltration level of CD8⁺ T cells, T follicular helper cells, and regulatory T cells (Tregs). Moreover, the immune check point genes, including CTLA-4, LAG3, PD-1, and IDO1, presented a high expression level in the low TII score group. Patients in the high TII score group demonstrated significant therapeutic advantages and clinical benefits. The findings in this study have the potential to assist in the strategic design of immunotherapeutic treatments for ccRCC.     

肿瘤细胞自噬的诱导及其细胞周期分析

背景与目的:自噬作为Ⅱ型程序性死亡——自噬性死亡过程中的主要现象,与细胞的自噬性死亡有着密切的关系。研究者们对凋亡与细胞周期的关系进行了深入而细致的研究,但对自噬性细胞死亡与细胞周期的关系却知之甚少。本研究的目的是探讨不同方法诱导的细胞自噬与细胞周期之间的关系。方法:用Hanks’液替代培养基的饥饿诱导和用长春新碱诱导两种方法分别处理对数生长期的HeLa细胞、SW480细胞以及经过和未经过植物血凝素(phytohemagglutinin,PHA)刺激的健康人外周血淋巴细胞;应用激光共聚焦显微镜和透射电镜检测细胞自噬的发生,兔抗人微管相关蛋白1轻链3Ⅱ(microtubule-associatedprotein1lightchain3,MAP1-LC3-Ⅱ)/DNA双参数流式细胞术分析自噬细胞的细胞周期。结果:HeLa细胞和SW480细胞用饥饿和长春新碱两种方法诱导的细胞自噬在G1、S、G2/M期均可以发生,且自噬发生率随诱导时间的延长逐渐增加。处于静止期(未经PHA刺激)的外周血淋巴细胞没有自噬的发生,48h时LC3-Ⅱ表达率<2.62%(HanksL液饥饿诱导)或<6.16%(长春新碱诱导);经PHA刺激48h进入细胞周期的外周血淋巴细胞,2h时已有明显的自噬发生。结论:MAP1-LC3-Ⅱ/DNA双参数流式细胞术是对细胞自噬与细胞周期进行同步分析的一种新的简便可靠的方法;细胞自噬只发生在细胞进入周期后,而静止期细胞对自噬诱导因素不敏感。     

NDRG2 suppresses the proliferation of clear cell renal cell carcinoma cell A-498

Background

Recently, the anti-tumor activity of N-myc downstream-regulated gene 2 (NDRG2) was shown decreased expression in clear cell renal cell carcinoma (CCRCC), but the role of the down-expression of NDRG2 has not been described.

Methods

The NDRG2 recombinant adenovirus plasmid was constructed. The proliferation rate and NDRG2 expression of cell infected with recombinant plasmid were mesured by MTT, Flow cytometry analysis and western blot.

Results

The CCRCC cell A-498 re-expressed NDRG2 when infected by NDRG2 recombinant adenovirus and significantly decreased the proliferation rate. Fluorescence activated cell sorter analysis showed that 25.00% of cells expressed NDRG2 were in S-phase compared to 40.67% of control cells, whereas 62.08% of cells expressed NDRG2 were in G1-phase compared to 54.39% of control cells (P < 0.05). In addition, there were much more apoptotic cells in NDRG2-expressing cells than in the controls (P < 0.05). Moreover, upregulation of NDRG2 protein was associated with a reduction in cyclin D1, cyclin E, whereas cyclinD2, cyclinD3 and cdk2 were not affected examined by western blot. Furthermore, we found that p53 could upregulate NDRG2 expression in A-498 cell.

Conclusions

We found that NDRG2 can inhibit the proliferation of the renal carcinoma cells and induce arrest at G1 phase. p53 can up-regulate the expression of NDRG2. Our results showed that NDRG2 may function as a tumor suppressor in CCRCC.