内源性活性氧水平及锰型超氧化物歧化酶的表达与胃癌细胞分化、增殖相关

检测了不同分化的胃癌细胞株内MnSOD基因的表达及胞内活性氧限（ROS)的水平。同时通过基因转染观察上调或下调MnSOD基因表达对SGC790l胃癌细胞胞内ROS水平及增殖能力的影响。用电穿孔法将人反义和正义MnSOD cDNA真核表达载体pHβA—SOD(-)／pHβA—SOD（ )转入790l细胞,用含G418的RPMIl640培养基筛选稳定表达克隆。然后用RT-PCR鉴定MnDSOD基因的表达。同时用RT-PCR方法检测正常胃粘膜组织及MKN-28、SGC790l、BGC823、HGC-27四株高、中、低、未分化胃癌细胞株内的MnSoD的mRNA表达。利用DCFH-DA荧光染色方法检测不同分化胃癌细胞株及790l转染细胞株内的ROS水平。四唑蓝比色法(MTT)绘制MKN-28、SGC790l、BGC823、HGC-27四株不同分化胃癌细胞及正义、反义、空载MnSOD转染790l细胞的生长曲线。发现不同分化胃癌细胞内的MnSOD普遍呈低表达且与分化程度平行,不同分化胃癌细胞株胞内ROS水平随着MnSOD表达的下调逐步上升,细胞增殖加快。较之MnSOD空载790l,正义、反义MnSOD转染的790l细胞中该基因的表达出现明显上调及下调,胞内ROS水平较对照细胞也相应有显著降低和升高。正义株增殖受抑,反义株增殖加快。表明胃癌细胞内MnSOD的表达与肿瘤的分化程度呈负相关。可通过改变胞内RoS水平改变MnSOD基因的表达,从而调节胃癌细胞的生长。     

Intestinal metabolite compound K of panaxoside inhibits the growth of gastric carcinoma by augmenting apoptosis via Bid-mediated mitochondrial pathway

Compound K (20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol, CK), an intestinal bacterial metabolite of panaxoside, has been shown to inhibit tumour growth in a variety of tumours. However, the mechanisms involved are largely unknown. We use human gastric carcinoma cell lines BGC823, SGC7901 and human gastric carcinoma xenograft in nude mice as models to study the mechanisms of CK in gastric cancers. We found that CK significantly inhibits the viabilities of BGC823 and SGC7901 cells in dose- and time-dependent manners. CK-induced BGC823 and SGC7901 cells apoptosis and cell cycle arrest in G2 phase by up-regulation of p21 and down-regulation of cdc2 and cyclin B1. Further studies show that CK induces apoptosis in BGC823 and SGC7901 cells mainly through mitochondria-mediated internal pathway, and that CK induces the translocation of nuclear Bid to mitochondria. Finally, we found that CK effectively inhibited the tumour formation of SGC7901 cells in nude mice. Our studies show that CK can inhibit the viabilities and induce apoptosis of human gastric carcinoma cells via Bid-mediated mitochondrial pathway.     

Enhancement of Metastatic and Invasive Capacity of Gastric Cancer Cells by Transforming Growth Factor-β1

Transforming growth factor-β (TGF-β),a multifunctional cytokine,exerts contradictory rolesin different kinds of cells.A number of studies have revealed its involvement in the progression of many typesof tumors.To investigate the effect of TGF-β on gastric carcinoma,SGC7901,BGC823 and MKN28 (aTGF-β-resistant cell line) adenocarcinoma clones were used.After pretreatment in serum-free medium withor without 10 ng/ml TGF-β1,their experimental metastatic potential,chemotaxis,and invasive and adhesiveability were measured.Furthermore,zymography for gelatinase was processed.Liver colonies were alsomeasured 4 weeks after inoculation of SGC7901,BGC823 and MKN28 in Balb/c nude mice,and an increasein the number of surface liver metastases was seen in SGC7901 (from 11.0±3.0 to 53.3±3.3) and BGC823(from 9.3±2.5 to 60.0±2.8) groups,whereas there was no difference between MKN28 groups (from 35.2±3.8 to 38.5±2.7).In vitro experiments showed that TGF-β1 increased the adhesion capacity of SGC7901and BGC823 cells to immobilized reconstituted basement membrane/fibronectin matrices and promoted theirpenetration through reconstituted basement membrane barriers.Zymography demonstrated that enhancedinvasive potential was partly due to the increased type Ⅳ collagenolytic (gelatinolytic) activity,but there wasno difference in type Ⅳ collagenolytic activity and other biological behaviors between MKN28 groups.Theseresults suggested that TGF-β1 might modulate the metastatic potential of gastric cancer cells by promotingtheir ability to break down and penetrate basement membrane barriers and their adhesive and motile activities.We speculated that TGF-β1 might act as a progression-enhancing factor in gastric cancer.Therefore blockageof TGF-β or TGF-β signaling might prevent gastric cancer cells from invading and metastasizing.     

RARβ在胃癌细胞生长调节中的作用

为探讨 RARβ受体介导全反式视黄酸 ( ATRA)抑制胃癌细胞生长的作用机理 ,用 Northern印迹测定 RARβ m RNA表达水平 ,脂质体介导的转染方法将含有 RARβ基因的表达载体转染MKN- 45细胞并稳定表达 ,MTT和软琼脂集落形成等实验测定细胞生长速率和生长状态 ,氯霉素乙酰转移酶活性 ( CAT)测定视黄酸应答元件βRARE的转录活性以及 AP- 1 ( activator protein- 1 )活性 .RARβ在 ATRA敏感细胞株 MGC80 - 3、BGC- 82 3和 SGC- 790 1中表达 ,而在 ATRA抗性细胞株 MKN- 45中不表达 .当 RARβ基因转染 MKN- 45细胞时 ,细胞变为 ATRA敏感 ,由此导致ATRA抑制 MKN- 45细胞生长和软琼脂集落形成 .ATRA可以加强诱导 MGC80 - 3、BGC- 82 3和SGC- 790 1细胞βRARE的转录活性 ,但对 MKN- 45细胞影响不大 ,不能抑制细胞 AP- 1活性 .当RARβ基因转染 MKN- 45细胞后 ,ATRA则能够诱导细胞 βRARE的转录活性 ,并抑制细胞的 AP-1活性 .RARβ表达与 ATRA抑制胃癌细胞生长密切相关 .ATRA诱导 βRARE转录活性和抑制AP- 1活性可能是其调控胃癌细胞生长的机制之一 .     

Estradiol mitigates ischemia reperfusion-induced acute renal failure through NMDA receptor antagonism in rats

Understanding the molecular mechanism of gastric cancer cell apoptosis is pivotal for the development of precise therapies targeting this disease. In the present study, we examined the effects of annexin A7 inhibition on the apoptosis of gastric cancer cells and the growth of tumour xenografts in vivo. Expression of annexin A7 in BGC823 cells was suppressed by small interference RNA, and cells apoptosis was assessed by flow cytometry. The mechanism by which annexin A7 mediates apoptosis in BGC823 cells was explored by determining the expression of key apoptosis regulators. In addition, by suppressing annexin A7 in BGC823 cells with small hairpin RNA, we studied the effects of annexin A7 inhibition on in vivo tumour growth. Our results showed that inhibiting annexin A7 expression induced more than fivefold increase in BGC823 cell apoptosis in vitro. This was in concord with a significant decrease of Bcl-2 expression and increases of Bax, Caspase-3, and Caspase-9. The activities of caspase-3 and caspase-9 were increased by 2.95?±?0.18 and 3.70?±?0.33 times, respectively, upon the annexin A7 downregulation in BGC823 cells. Importantly, suppressing annexin A7 showed the same apoptotic mechanism in vivo and significantly inhibited the growth of BGC823 xenografts in mice. These data suggest that annexin A7 likely protects gastric cells from apoptosis and targeting it may represent a valuable strategy in future therapeutic development.     

慢病毒siRNA靶向干扰YAP基因胃癌细胞株的建立

目的：构建并鉴定YAP基因短发夹干扰RNA（shRNA）慢病毒载体,建立稳定干扰YAP基因表达的胃癌细胞株SGC7901。方法：荧光定量PCR检测YAP基因在多种胃癌细胞株中的表达情况。构建重组靶向YAP基因的shRNA慢病毒表达质粒PGC-shRNA-YAP,用脂质体转染的方法将载体导入胃癌细胞。经杀稻瘟菌素筛选后,建立稳定表达siRNA的细胞株。荧光定量PCR检测干扰效率。结果：在胃癌细胞株SGC7901中,YAP基因显示高表达。测序验证PGC-shRNA-YAP重组质粒构建成功。将重组质粒稳定转染入胃癌细胞株SGC7901后能明显抑制YAPmRNA表达水平。结论：成功构建了PGC-shRNA-YAP慢病毒重组质粒,建立了靶向稳定干扰YAP基因表达的siRNA胃癌细胞株SGC7901。     

The direct effect of estrogen on cell viability and apoptosis in human gastric cancer cells

Epidemiology researches indicated that gastric cancer is a male-predominant disease; both expression level of estrogen and expression pattern of estrogen receptors (ERs) influence its carcinogenesis. But the direct effect of estrogen on gastric cancer cells is still unclear. This study aimed to explore the direct effect of β-estradiol (E2) on gastric cancer cells. SGC7901 and BGC823 were treated with a serial of concentrations of E2. The survival rates of both the cell lines were significantly reduced, and the reduction of viability was due to apoptosis triggered by E2 treatment. Caspase 3 was activated in response to the increasing E2 concentration in both SGC7901 and BGC823. Cleaved Caspase 3 fragments were detected, and the expression levels of Bcl-2 and Bcl-xL were reduced. Apoptosis was further confirmed by flow cytometry. The expression level of PEG10, an androgen receptor target gene, was reduced during E2 treatment. Both ERα and ERβ were expressed in these cell lines, and the result of bioinformatics analysis of gastric cancer from GEO datasets indicated that the expression levels of both ERα and ERβ were significantly higher in noncancerous gastric tissues than in gastric cancer tissues. Our research indicated that estrogen can reduce cell viability and promote apoptosis in gastric cancer cells directly; ERs expression level is associated with gastric cancer. Our research will help to understand the mechanism of gender disparity in gastric cancer.     

A forskolin derivative, FSK88, induces apoptosis in human gastric cancer BGC823 cells through caspase activation involving regulation of Bcl-2 family gene expression, dissipation of mitochondrial membrane potential and cytochrome c release

FSK88, a forskolin derivative, was extracted and purified from cultured tropical plant roots, Coleus forskohlii. Our previous studies have demonstrated that FSK88 can inhibit HL-60 cell proliferation and induce the differentiation of HL-60 cells to monocyte macrophages. In this study, we showed that FSK88 can induce apoptotic death of human gastric cancer BGC823 cells in a dose- and time-dependent manner. Results showed that FSK88-induced apoptosis was accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in BGC823 cells. Furthermore, treatment with caspase-3 inhibitor (z-DEVD-fmk) was capable of preventing the FSK88-induced caspase-3 activity and apoptosis. FSK88-induced apoptosis in human gastric cancer BGC823 cells was also accompanied by the up-regulation of Bax, Bad and down-regulation of Bcl-2. Theses results clearly demonstrated that the induction of apoptosis by FSK88 involved multiple cellular and molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family genes, mitochondrial membrane potential (Deltapsi(m)), cytochrome c, and caspase-3, participate in the FSK88-induced apoptotic process in human gastric cancer BGC823 cells.     

Anthraquinone G503 Induces Apoptosis in Gastric Cancer Cells through the Mitochondrial Pathway

G503 is an anthraquinone compound isolated from the secondary metabolites of a mangrove endophytic fungus from the South China Sea. The present study elucidates the anti-tumor activity and the underlying mechanism of G503. Cell viability assay performed in nine cancer cell lines and two normal cell lines demonstrated that the gastric cancer cell line SGC7901 is the most G503-sensitive cancer cells. G503 induced SGC7901 cell death via apoptosis. G503 exposure activated caspases-3, -8 and -9. Pretreatment with the pan-caspase inhibitor Z-VAD-FMK and caspase-9 inhibitor Z-LEHD-FMK, but not caspase-8 inbibitor Z-IETD-FMK, attenuated the effect of G503. These results suggested that the intrinsic mitochondrial apoptosis pathway, rather than the extrinsic pathway, was involved in G503-induced apoptosis. Furthermore, G503 increased the ratio of Bax to Bcl-2 in the mitochondria and decreased the ratio in the cytosol. G503 treatment resulted in mitochondrial depolarization, cytochrome c release and the subsequent cleavage of caspase -9 and -3. Moreover, it is reported that the endoplasmic reticulum apoptosis pathway may also be activated by G503 by inducing capase-4 cleavage. In consideration of the lower 50% inhibitory concentration for gastric cancer cells, G503 may serve as a promising candidate for gastric cancer chemotherapy.     

表达血管内皮生长因子-siRNA及双自杀基因yCDglyTK的联合基因载体构建及其应用研究

构建了新型联合基因载体pcDNA3.1(-)VEGF-siRNA/yCDglyTK,研究其在人胃癌细胞系SGC7901细胞中的表达和杀伤作用.构建靶向血管内皮生长因子(VEGF)的干扰质粒pGenesil-VEGF-siRNA,采用PCR法从中扩增siRNA表达框(含U6启动子),亚克隆至双自杀基因载体pcDNA3.1(-)CV-yCDglyTK,构建联合基因质粒pcDNA3.1(-)VEGF-siRNA/yCDglyTK;通过酶切、测序等鉴定重组质粒;以磷酸钙纳米颗粒为载体,将干扰质粒、双自杀基因质粒及联合基因质粒转染SGC7901细胞,RT-PCR、Western-blot验证目的基因表达;MTT法检测转染细胞对5-氟胞嘧啶(5-FC)的敏感性.结果表明:酶切及测序证实联合基因载体pcDNA3.1(-)VEGF-siRNA/yCDglyTK构建成功;SGC7901细胞转染联合基因质粒后,RT-PCR、Western-blot证实融合自杀基因表达,而VEGF基因表达下调;在前体药物5-FC作用下,转染联合基因组细胞存活率最低,与其他组比较有统计学差异.成功构建联合基因载体pcDNA3.1(-)VEGF-si...     

慢病毒siRNA靶向干扰YAP基因胃癌细胞株的建立

目的:构建并鉴定YAP基因短发夹干扰RNA(shRNA)慢病毒载体,建立稳定干扰YAP基因表达的胃癌细胞株SGC7901。方法:荧光定量PCR检测YAP基因在多种胃癌细胞株中的表达情况。构建重组靶向YAP基因的shRNA慢病毒表达质粒PGC-shRNA-YAP,用脂质体转染的方法将载体导入胃癌细胞。经杀稻瘟菌素筛选后,建立稳定表达siRNA的细胞株。荧光定量PCR检测干扰效率。结果:在胃癌细胞株SGC7901中,YAP基因显示高表达。测序验证PGC-shRNA-YAP重组质粒构建成功。将重组质粒稳定转染入胃癌细胞株SGC7901后能明显抑制YAPmRNA表达水平。结论:成功构建了PGC-shRNA-YAP慢病毒重组质粒,建立了靶向稳定干扰YAP基因表达的siRNA胃癌细胞株SGC7901。     

Melatonin inhibits cell growth and migration,but promotes apoptosis in gastric cancer cell line,SGC7901

Abstract

The pineal hormone, melatonin (MLT), has been shown to have therapeutic effects in patients with gastric cancer; however, the mechanisms for the anti-cancer effects are unknown. We investigated the effects of melatonin on cell proliferation, apoptosis, colony formation and cell migration in the gastric adenocarcinoma cell line, SGC7901, using MTT assay, Hoechst 33258 staining, flow cytometry, western blot, caspase-3 activity assay, soft agar colony formation assay, and scratch-wound assay. Our results showed that melatonin could inhibit cell proliferation, colony formation and migration efficiency, and it promoted apoptosis of SGC7901 cells. Our findings suggest that the anti-cancer effects of melatonin may be due to both inhibition of tumor cell proliferation and reduction of the metastatic potential of tumor cells.     

靶向survivin的siRNA对胃癌BGC-823细胞增殖和转移能力的影响

目的探讨沉默生存素（survivin）基因表达的干扰RNA对人胃癌BGC-823细胞增殖和成瘤能力的影响。方法应用已经在细胞上验证能够有效沉默survivin的小分子干扰RNA（shRNA-survivin-1）,并在体外实验的基础上,建立稳定表达干扰RNA细胞系,进一步探讨干扰RNA稳定表达对胃癌BGC-823细胞生长和裸鼠移植成瘤的影响。结果 shRNA-survivin-1有效沉默人胃癌BGC-823细胞survivin mRNA的表达,成功筛选shRNA-sur-vivin-1稳定表达细胞株BGC/siRNA-1细胞,实验表明,BGC/siRNA-1细胞的生长曲线缓慢上升,细胞增殖能力下降;BGC/siRNA-1细胞裸鼠移植成瘤体积与对照组相比,明显减小（P〈0.05）。结论 shRNA-survivin-1可以沉默survivin基因的表达,可以显著抑制胃癌BGC-823细胞的增殖,并降低胃癌BGC-823细胞的成瘤能力,本研究为靶向survivin的RNA干扰在胃癌的基因治疗提供了有力的理论依据和技术储备。 更多还原     

miR-375 targets the p53 gene to regulate cellular response to ionizing radiation and etoposide in gastric cancer cells

MicroRNAs (miRNAs) offer a new approach for molecular classification and individual therapy of human cancer due to their regulation of oncogenic pathways. In a previous report, elevated miR-375 was found in recurring gastric cancer, and it was predicted that miR-375 may be a regulator of p53 gene. However, its biological role and mechanism of actions remain unknown. In this study, we characterized the expression level of miR-375 in gastric cancer cell lines – BGC823, MGC803, SGC7901, AGS, N87, MKN45 – using RT-PCR. We found that exogenous expression of miR-375 promoted the growth of AGS cells in both liquid and soft agar media. In agreement with the previous report, overexpression of miR-375 in AGS cells reduced the p53 protein expression level. A luciferase assay demonstrated that miR-375 down-regulated p53 expression through an interaction with the 3′ UTR region of p53. In addition, the expression of miR-375 desensitizes cells to ionizing radiation and etoposide. Flow cytometry analyses showed that miR-375 abrogated the cell cycle arrest and apoptosis after DNA damage. These results demonstrate that miR-375 targets p53 to regulate the response to ionizing radiation and etoposide treatment.     

低氧模拟剂氯化钴对胃癌细胞BGC823中S100A4基因表达的影响

摘要: S100A4基因是肿瘤侵袭转移相关的重要基因, 该基因高表达与胃癌浸润、淋巴结转移及胃癌细胞体外侵袭力密切相关。为探讨S100A4基因高表达的机制, 文章应用低氧模拟剂氯化钴处理胃癌细胞BGC823, RT-PCR、免疫组化、免疫荧光及Western blotting方法分别检测BGC823细胞中S100A4 mRNA及蛋白表达情况。结果显示, 氯化钴处理胃癌BGC823细胞后, S100A4 mRNA及蛋白表达明显增加。提示低氧模拟剂氯化钴可促进胃癌细胞BGC823中S100A4 基因表达。     

Activation of LXRβ inhibits proliferation,promotes apoptosis,and increases chemosensitivity of gastric cancer cells by upregulating the expression of ATF4

Recently, great advances have been achieved in both surgery and chemotherapy for the treatment of gastric cancer, but there is still poor prognosis for this disease. The aim of this study is to investigate the role of liver X receptor β (LXRβ) in chemosensitivity of gastric cancer SGC7901 cells. From 171 patients with gastric cancer, the gastric cancer and paracancerous tissues were selected to measure the expression of LXRβ and ATF4. Gastric cancer cell lines were cultured and screened to figure out the proliferation and apoptosis of gastric cancer SGC7901 cells with the treatment of LXRβ agonist (GW3965), ATF4 short hairpin RNA (shRNA), and chemotherapy drug paclitaxel. The expression of apoptosis-related gene cleaved caspase-3 was detected by Western blot analysis. First, we found that the expressions of LXRβ and ATF4 in gastric cancer tissues and cells were significantly lower than those in their paracancerous tissues and gastric mucosal epithelial cells. In addition, activation of LXRβ and paclitaxel treatment suppressed proliferation of SGC7901 cells, and the expression of ATF4 was upregulated in a concentration-dependent manner. Furthermore, shRNA significantly inhibited the expression of ATF4 and blocked the chemosensitivity of SGC7901 cells to LXRβ activation. Our study demonstrates that the expression of LXRβ was low in gastric cancer. In addition, activation of LXRβ may inhibit the proliferation of gastric cancer cells, promote apoptosis, and increase chemosensitivity by upregulating the expression of ATF4.     

CXXC finger protein 4 inhibits the CDK18‐ERK1/2 axis to suppress the immune escape of gastric cancer cells with involvement of ELK1/MIR100HG pathway

Gastric cancer, is the fourth most common tumour type yet, ranks second in terms of the prevalence of cancer‐related deaths worldwide. CXXC finger protein 4 (CXXC4) has been considered as a novel cancer suppressive factor, including gastric cancer. This study attempted to investigate the possible function of CXXC4 in gastric cancer and the underlying mechanism. The binding of the ETS domain‐containing protein‐1 (ELK1) to the long non‐coding RNA MIR100HG promoter region was identified. Then, their expression patterns in gastric cancer tissues and cells (SGC7901) were detected. A CCK‐8 assay was used to detect SGC7901 cell proliferation. Subsequently, SGC7901 cells were co‐cultured with CD3+ T cells, followed by measurement of CD3+ T cell proliferation, magnitude of IFN‐γ+ T cell population and IFN‐γ secretion. A nude mouse model was subsequently developed for in vivo validation of the in vitro results. Low CXXC4 expression was found in SGC7901 cells. Nuclear entry of ELK1 can be inhibited by suppression of the extent of ELK1 phosphorylation. Furthermore, ELK1 is able to bind the MIR100HG promoter. Overexpression of CXXC4 resulted in weakened binding of ELK1 to the MIR100HG promoter, leading to a reduced proliferative potential of SGC7901 cells, and an increase in IFN‐γ secretion from CD3+ T cells. Moreover, in vivo experiments revealed that CXXC4 inhibited immune escape of gastric cancer cells through the ERK1/2 axis. Inhibition of the CXXC4/ELK1/MIR100HG pathway suppressed the immune escape of gastric cancer cells, highlighting a possible therapeutic target for the treatment of gastric cancer.     

ANKRD33 is overexpressed in gastric adenocarcinoma and predictive for poor prognosis

ABSTRACT

The aim of the current study was to investigate and discuss the function of ANKRD33 gene in the pathogenesis of gastric adenocarcinoma. The marked up-regulated expression of ANKRD33 gene in gastric adenocarcinoma tissues compared to normal tissues was found by bioinformatics analysis. Kaplan-Meier analysis revealed that high expression of ANKRD33 is correlated with lower overall survival of gastric adenocarcinoma patients. The results of qPCR revealed that mRNA expression level of ANKRD33 was dramatically higher in AGS, SGC7901, and BGC823 cell lines than that in the GES1 cells. Knockdown of ANKRD33 remarkably inhibited the viability, invasion, and migration of AGS and BGC823 cells. Furthermore, the ratio of p-AKT/AKT and p-mTOR/mTOR was significantly decreased in AGS cells which transfected with si- ANKRD33. All the above results illustrated that ANKRD33 would act as a tumor forwarder in gastric adenocarcinoma development and have a high potential to be a marker molecule in the diagnosis and treatment of gastric tumors.