应用蛋白质组学技术筛选胃癌耐药相关蛋白质

胃癌多药耐药性是临床胃癌化疗失败最主要的原因之一,但其分子机制仍然不太清楚.为了寻找新的胃癌耐药相关的蛋白质,揭示胃癌多药耐药的分子机制,以胃癌细胞SGC7901和长春新碱诱导的耐药胃癌细胞SGC7901/VCR为研究对象,应用二维凝胶电泳(two-dimensionalelectrophoresis,2-DE)技术分离两种细胞的总蛋白质,图像分析识别差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(matrix-assistedlaserdesorption/ionizationtimeofflightmassspectrometry,MALDI-TOF-MS)及电喷雾电离串联质谱(electrosprayionizationtandemmassspectrometry,ESI-Q-TOF)对差异表达的蛋白质点进行鉴定,蛋白质印迹和实时RT-PCR验证部分差异蛋白质在两株细胞中的表达水平,反义核酸转染技术分析HSP27(heatshockprotein27,HSP27)高表达与SGC7901/VCR耐药的相关性.得到了分辨率较高、重复性较好的两株细胞系的二维凝胶电泳图谱,质谱分析共鉴定了24个差异蛋白质点,蛋白质印迹和实时RT-PCR验证了部分差异蛋白的表达水平,反义寡核苷酸抑制HSP27表达能增加SGC7901/VCR对长春新碱的敏感性.研究结果不仅提示这些差异蛋白质如HSP27,Sorcin等可能与胃癌的多药耐药相关,而且为揭示胃癌细胞的多药耐药性产生机制提供了线索.     

Proteomics-based strategy to delineate the molecular mechanisms of RhoGDI2-induced metastasis and drug resistance in gastric cancer

Rho GDP dissociation inhibitor 2 (RhoGDI2) was initially identified as a regulator of the Rho family of GTPases. Our recent works suggest that RhoGDI2 promotes tumor growth and malignant progression, as well as enhances chemoresistance in gastric cancer. Here, we delineate the mechanism by which RhoGDI2 promotes gastric cancer cell invasion and chemoresistance using two-dimensional gel electrophoresis (2-DE) on proteins derived from a RhoGDI2-overexpressing SNU-484 human gastric cancer cell line and control cells. Differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 47 differential protein spots were identified; 33 were upregulated, and 14 were downregulated by RhoGDI2 overexpression. Upregulation of SAE1, Cathepsin D, Cofilin1, CIAPIN1, and PAK2 proteins was validated by Western blot analysis. Loss-of-function analysis using small interference RNA (siRNA) directed against candidate genes reveals the need for CIAPIN1 and PAK2 in RhoGDI2-induced cancer cell invasion and Cathepsin D and PAK2 in RhoGDI2-mediated chemoresistance in gastric cancer cells. These data extend our understanding of the genes that act downstream of RhoGDI2 during the progression of gastric cancer and the acquisition of chemoresistance.     

激光捕获显微切割技术结合18O标记定量蛋白质组技术在胃癌标志物筛查中的应用研究

建立一种更加精确地分离鉴定胃癌特异肿瘤标志物的定量蛋白质组学技术．首先采用激光捕获显微切割技术(LCM)纯化胃腺癌细胞及胃黏膜良性上皮细胞,将裂解的样本总蛋白经过1D SDS-PAGE预分离,然后采用¹⁸O/¹⁶O分别标记两种样本酶切后的多肽混合物．结合纳升级液相色谱(Nano-HPLC-MS/MS)定量地鉴定胃癌细胞和胃黏膜良性上皮细胞的差异表达蛋白．共筛选出78个差异表达蛋白,其中42个蛋白质在胃癌组织中表达上调,36个蛋白质下调．Western blot 技术验证了其中几个差异蛋白(moesin, periostin, annexin A2, annexin A4)的表达,与蛋白质组学研究的结果一致．LCM技术结合¹⁸O稳定同位素标记的定量蛋白质组学技术,为研究胃癌发生机制、筛选胃癌的分子标志物提供了新的思路,亦为诸如胃癌等复杂体系蛋白质的分离鉴定提供了新的技术选择．     

Differential proteomic analysis of noncardia gastric cancer from individuals of northern Brazil

Gastric cancer is the second leading cause of cancer-related death worldwide. The identification of new cancer biomarkers is necessary to reduce the mortality rates through the development of new screening assays and early diagnosis, as well as new target therapies. In this study, we performed a proteomic analysis of noncardia gastric neoplasias of individuals from Northern Brazil. The proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. For the identification of differentially expressed proteins, we used statistical tests with bootstrapping resampling to control the type I error in the multiple comparison analyses. We identified 111 proteins involved in gastric carcinogenesis. The computational analysis revealed several proteins involved in the energy production processes and reinforced the Warburg effect in gastric cancer. ENO1 and HSPB1 expression were further evaluated. ENO1 was selected due to its role in aerobic glycolysis that may contribute to the Warburg effect. Although we observed two up-regulated spots of ENO1 in the proteomic analysis, the mean expression of ENO1 was reduced in gastric tumors by western blot. However, mean ENO1 expression seems to increase in more invasive tumors. This lack of correlation between proteomic and western blot analyses may be due to the presence of other ENO1 spots that present a slightly reduced expression, but with a high impact in the mean protein expression. In neoplasias, HSPB1 is induced by cellular stress to protect cells against apoptosis. In the present study, HSPB1 presented an elevated protein and mRNA expression in a subset of gastric cancer samples. However, no association was observed between HSPB1 expression and clinicopathological characteristics. Here, we identified several possible biomarkers of gastric cancer in individuals from Northern Brazil. These biomarkers may be useful for the assessment of prognosis and stratification for therapy if validated in larger clinical study sets.     

Identification of target proteins of N-acetylglucosaminyl transferase V in human colon cancer and implications of protein tyrosine phosphatase kappa in enhanced cancer cell migration

To gain a better understanding of the mechanism underlying colon cancer and to search for potential markers of colon cancer prognosis, a comparative proteomic analysis of colon cancer WiDr cells was conducted using 2-DE and lectin blot, followed by identification based on ESI-MS. Through these approaches 14 proteins were identified as candidate target proteins for N-acetylglucosaminyl transferase V (GnT-V) that would be expected to be implicated in the progression of colon cancer. We selected protein tyrosine phosphatase kappa (PTPkappa) as a model protein to validate this approach to the discovery of novel biomarkers in colon cancer. PTPkappa underwent an aberrant glycosylation in GnT-V-overexpressing WiDr cells, and the aberrantly glycosylated PTPkappa was vulnerable to proteolytic cleavage. The enhanced cleavage of PTPkappa in GnT-V-overexpressing cells was responsible for the mitigation of the homophilic binding capacity, resulting in an increase in cancer cell migration.     

Identification of the heterogeneous nuclear ribonucleoprotein A2/B1 as the antigen for the gastrointestinal cancer specific monoclonal antibody MG7

MG7 is an early gastrointestinal cancer specific monoclonal antibody. It can detect gastric cancer with high sensitivity and specificity. However, the target antigen for MG7 has not been identified. Western blot analysis revealed that the MG7 antibody reproducibly recognized two approximately 35 kDa proteins in the total cell lysates of human gastric carcinoma cell lines KATO III and MKN-45. Using a proteomic approach, we identified these MG7 immunoreactive proteins as the human heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). Western blot analysis of nuclear and cytosolic fraction of KATO III cells using either MG7 or hnRNP A2/B1 antibodies confirmed that the target antigen is located exclusively in the nucleus. With the use of archival samples, we also found that the level of hnRNP A2/B1 protein was increased in gastric cancer tissues (4 out of 5 patients), when compared to their corresponding matching normal stomach tissue.     

Anti-tumor activity of the X-linked inhibitor of apoptosis (XIAP) inhibitor embelin in gastric cancer cells

This study investigated the anticancer effects of embelin in human gastric cancer cells and the underlying molecular mechanisms. Gastric cancer cells were treated with embelin and 5-FU for methyl-thiazolyl-tetrazolium bromide cell viability assay and flow cytometric detection of cell viability and apoptosis. Protein pathway array (PPA) and Western blot were used to investigate differentially expressed proteins in embelin-treated gastric cancer cells. Embelin reduced gastric cancer cell viability, induced apoptosis, and enhanced 5-FU antitumor activity in gastric cancer cells. Mechanistically, embelin induced cell cycle arrest at the S and G2/M phases. Molecularly, embelin downregulated expression of X-linked inhibitor of apoptosis and cell cycle-regulatory proteins, such as CDK1, CDC25B, CDC25C, cyclinB1, and CDK2. PPA analysis showed that embelin modulated several pathways that are associated with cell growth and apoptosis, such as PI3K/AKT, JAK/STAT, p38 MAPK, and p53. The data from the current study implied that reduction of gastric cancer cell viability after treatment with embelin was through cell cycle arrest at the S and G2/M phases and apoptosis.     

Melatonin inhibits the migration of human gastric carcinoma cells at least in part by remodeling tight junction

The recurrence and metastasis is one of the major reasons for malignant tumor treatment failure. Melatonin, a naturally occuring hormone, could reduce the recurrence and metastasis of various tumors. However, the underlying molecular mechanisms of melatonin on tumor metastasis inhibition have not been fully elucidated. In the present study, we explored the impact of melatonin on the migratory capability of human gastric carcinoma cells using wound healing assay, and further investigated if the inhibition on migration ability of melatonin was embodied by relocating tight junction proteins zo-1 and occludin onto the cells surface to remodel tight junction structure. Immunofluorescence assay and Western blot analysis were performed to detect the expression and cell location of the tight junction proteins. The migration distance was decreased as the cells were treated with melatonin. And melatonin increased the membrane location of tight junction proteins, zo-1 and occludin, showed by immunofluorescence staining and Western blot analysis. The results we got show that melatonin makes tight junction proteins anchored more on the cells membrane to remodel cells tight junction, which increase cells adhesion and decrease motility, resulting in the inhibition of gastric cancer cells migration and metastasis ability.     

A serum glycomics approach to breast cancer biomarkers

Because the glycosylation of proteins is known to change in tumor cells during the development of breast cancer, a glycomics approach is used here to find relevant biomarkers of breast cancer. These glycosylation changes are known to correlate with increasing tumor burden and poor prognosis. Current antibody-based immunochemical tests for cancer biomarkers of ovarian (CA125), breast (CA27.29 or CA15-3), pancreatic, gastric, colonic, and carcinoma (CA19-9) target highly glycosylated mucin proteins. However, these tests lack the specificity and sensitivity for use in early detection. This glycomics approach to find glycan biomarkers of breast cancer involves chemically cleaving oligosaccharides (glycans) from glycosylated proteins that are shed or secreted by breast cancer tumor cell lines. The resulting free glycan species are analyzed by MALDI-FT-ICR MS. Further structural analysis of the glycans can be performed in FTMS through the use of tandem mass spectrometry with infrared multiphoton dissociation. Glycan profiles were generated for each cell line and compared. These methods were then used to analyze sera obtained from a mouse model of breast cancer and a small number of serum samples obtained from human patients diagnosed with breast cancer or patients with no known history of breast cancer. In addition to the glycosylation changes detected in mice as mouse mammary tumors developed, glycosylation profiles were found to be sufficiently different to distinguish patients with cancer from those without. Although the small number of patient samples analyzed so far is inadequate to make any legitimate claims at this time, these promising but very preliminary results suggest that glycan profiles may contain distinct glycan biomarkers that may correspond to glycan "signatures of cancer."     

Proteomics-Based Identification and Analysis of Proteins Associated with Helicobacter pylori in Gastric Cancer

Helicobacter pylori (H. pylori) is a spiral-shaped Gram-negative bacterium that causes the most common chronic infection in the human stomach. Approximately 1%-3% of infected individuals develop gastric cancer. However, the mechanisms by which H. pylori induces gastric cancer are not completely understood. The available evidence indicates a strong link between the virulence factor of H. pylori, cytotoxin-associated gene A (CagA), and gastric cancer. To further characterize H. pylori virulence, we established three cell lines by infecting the gastric cancer cell lines SGC-7901 and AGS with cagA⁺ H. pylori and transfecting SGC-7901 with a vector carrying the full-length cagA gene. We detected 135 differently expressed proteins from the three cell lines using proteome technology, and 10 differential proteins common to the three cell lines were selected and identified by LC-MS/MS as well as verified by western blot: β-actin, L-lactate dehydrogenase (LDH), dihydrolipoamide dehydrogenase (DLD), pre-mRNA-processing factor 19 homolog (PRPF19), ATP synthase, calmodulin (CaM), p64 CLCP, Ran-specific GTPase-activating protein (RanGAP), P43 and calreticulin. Detection of the expression of these proteins and genes encoding these proteins in human gastric cancer tissues by real-time PCR (RT-qPCR) and western blot revealed that the expression of β-ACTIN, LDH, DLD, PRPF19 and CaM genes were up-regulated and RanGAP was down-regulated in gastric cancer tissues and/or metastatic lymph nodes compared to peri-cancerous tissues. High gene expression was observed for H. pylori infection in gastric cancer tissues. Furthermore, the LDH, DLD and CaM genes were demethylated at the promoter -2325, -1885 and -276 sites, respectively, and the RanGAP gene was highly methylated at the promoter -570 and -170 sites in H. pylori-infected and cagA-overexpressing cells. These results provide new insights into the molecular pathogenesis and treatment targets for gastric cancer with H. pylori infection.     

Wnt/beta-catenin 信号通路相关蛋白在胃癌组织中的表达和 肿瘤转移的关系

目的：探讨了Wnt信号通路相关蛋白在胃癌组织中的表达及与肿瘤转移的关系。方法：选取2011 年6 月到2012 年6 月我 院胃癌术后47 例肿瘤标本作为研究对象,并选取同一患者的正常胃组织作为对照研究。采用实时荧光定量PCR 和Western blot 对胃癌组织和正常胃组织Wnt 信号通路相关蛋白进行分析,并分析了肿瘤转移和非转移患者Wnt信号通路相关蛋白的变化。结 果：与正常胃组织比较,胃癌组织中Wnt1、Wnt3、Wnt3a、beta-catenin、CyclinD1 和c-Myc 等分子的mRNA 水平明显上调,差异有显 著统计学意义（P<0.05）。胃癌组织中总beta-catenin 和核内beta-catenin蛋白较正常胃组织明显增加,而磷酸化beta-catenin 较正常组明显 下降、差异有显著统计学意义（P<0.05）。与非转移组比较,转移组患者胃癌组织中Wnt1、Wnt3、Wnt3a 等分子mRNA水平显著上 调,差异有统计学意义（P<0.05）。结论：Wnt信号通路异常激活在胃癌发生和癌细胞转移中发挥着重要的作用,为临床治疗提供了 一定靶点。     

Induction of Apoptosis and Effect on the FAK/AKT/mTOR Signal Pathway by Evodiamine in Gastric Cancer Cells

Evodiamine isolated from Evodia rutaecarpa has been known to have anti-tumor activity against various cancer cell types. Although there have been reports showing the inhibitory effect of evodiamine on cell survival of gastric cancer cell, it is not clearly explained how evodiamine affects the expression and modification of proteins associated with apoptosis and upstream signal pathways. We confirmed the cytotoxic activity of evodiamine against AGS and MKN45 cells by a WST assay, cell morphological change, and clonogenic assay. The apoptotic cells were evaluated by Annexin V/PI analysis and Western blot and the expressions of apoptosis-related molecules were confirmed by Western blot. Evodiamine promoted apoptosis of AGS gastric cancer cells through both intrinsic and extrinsic signal pathways in a time- and dose-dependent manner. Evodiamine attenuated the expression of anti-apoptotic proteins, including Bcl-2, XIAP, and survivin, and elevated that of the pro-apoptotic protein Bax. Evodiamine also suppressed the FAK/AKT/mTOR signal pathway. Based on these results, we expect that the results from this study will further elucidate our understanding of evodiamine as an anti-cancer drug.     

早期胃癌患者外周血清差异蛋白表达谱的建立及其生物学分析

目的:分析早期胃癌患者外周血中低丰度蛋白的表达差异,以筛选诊断早期胃癌血清多肽或蛋白标志物。方法:应用高通量的AAH-BLG-1000蛋白芯片分别检测3例早期胃癌患者和3例正常对照成人的外周血清,建立早期胃癌患者外周血清差异蛋白表达谱,分析其相关生物学信息,以筛选早期胃癌的血清肿瘤标志物。结果:与正常对照组比较,早期胃癌患者外周血清中有10种蛋白表达显著上调(P0.05),52种蛋白表达显著下调(P0.05)。生物信息学分析发现差异蛋白质集中于血管生成、信号调控,免疫调节、酶联受体蛋白信号,细胞凋亡等生物进程,而差异蛋白中的VEGI、CD40L、SMAD7、PLUNC、NTN、LTβR和HEVM7个差异蛋白的特征性改变有望成为早期胃癌血清学的肿瘤标志物。结论:应用蛋白芯片技术所得的早期胃癌患者血清中的差异表达蛋白有望成为诊断早期胃癌的血清肿瘤标志物。     

Inhibition of matrine against gastric cancer cell line MNK45 growth and its anti-tumor mechanism

Anti-tumor activity and mechanism of matrine is evaluated and investigated. MTT assay showed that the matrine was able to inhibit gastric cancer cell line MNK45 in a dose-dependent manner. The concentration required for 50% inhibition (IC50) was found to be 540 μg/ml. This anti-tumor function was achieved through modulation of the NF-κB, XIAP, CIAP, and p-ERK proteins expression in cell line MNK45. By western blot analysis, we found that expression of NF-κB, XIAP, CIAP, and p-ERK proteins in cell line MNK45 would vary with varying concentration of matrine. These protein interactions possibly play a pivotal role in the regulation of apoptosis, for which further detailed analyzes are need. These results overall indicate that matrine can be used as an effective anti-tumor agent in therapy of gastric cancer.     

MicroRNA-137 Contributes to Dampened Tumorigenesis in Human Gastric Cancer by Targeting AKT2

MiRNAs play important roles in tumorigenesis. This study focused on exploring the effects and regulation mechanism of miRNA-137 on the biological behaviors of gastric cancer. Total RNA was extracted from tissues of 100 patients with gastric cancer and from four gastric cancer cell lines. Expression of miR-137 was detected by real-time PCR from 100 patients. The effects of miR-137 overexpression on gastric cancer cells’ proliferation, apoptosis, migration and invasion ability were investigated in vitro and in vivo. The target gene of miR-137 was predicted by Targetscan on line software, screened by dual luciferase reporter gene assay and demonstrated by western blot. As a result, the expression of miR-137 was significant reduced in gastric cancer cell line HGC-27, HGC-803, SGC-7901 and MKN-45 as well as in gastric cancer tissues compared with GES-1 cell or matched adjacent non-neoplastic tissues (p<0.001). The re-introduction of miR-137 into gastric cancer cells was able to inhibit cell proliferation, migration and invasion. The in vivo experiments demonstrated that the miR-137 overexpression can reduce the gastric cancer cell proliferation and metastasis. Bioinformatic and western blot analysis indicated that the miR-137 acted as tumor suppressor roles on gastric cancer cells through targeting AKT2 and further affecting the Bad and GSK-3β. In conclusion, the miR-137 which is frequently down-regulated in gastric cancer is potentially involved in gastric cancer tumorigenesis and metastasis by regulating AKT2 related signal pathways.     

Combined serum IgG response to Helicobacter pylori VacA and CagA predicts gastric cancer

Helicobacter pylori is a major factor for the development of gastric cancer. The aim of this study was to define serum antibody patterns associated with H. pylori infection in patients with gastric cancer using a Western blot technique. Serum samples collected from 115 patients with gastric cancer and 110 age- and gender-matched patients without gastrointestinal diseases were tested for IgG antibodies to H. pylori antigens (outer membrane proteins and whole cell preparations). No significant differences were found between patients with and without gastric cancer using outer membrane proteins (82% and 73%, P>0.05) or whole cell antigens (84% and 76%, P>0.05), respectively. The significant differences between patients with and without gastric cancer were associated with bands of 94 kDa (54% and 20%, P<0.001) and 30 kDa (65% and 44%, P<0.01). A combination of antibodies to 85 kDa (VacA) and 120 kDa (CagA) was significantly (P<0.01) more frequent in gastric cancer patients than in patients without gastric cancer. The detection of antibodies to 94- and 30-kDa bands, in association with the determination of serum antibodies to CagA+/VacA+, may have a prospective value in assessment of the risk of developing of gastric cancer.     

Influence of gastrin on the expression of cyclooxygenase-2, hepatocyte growth factor and apoptosis-related proteins in gastric epithelial cells.

BACKGROUND: Several studies have shown a link between gastrin and gastric cancer, both in humans and animals, especially infected with Helicobacter pylori (H. pylori). However, the exact role of hypergastrinemia in gastric carcinogenesis remains still undetermined. The aim of the present study was to evaluate the interaction between gastrin, cyclooxygenase-2 (COX-2), hepatocyte growth factor (HGF) and apoptosis-related proteins (Bax, Bcl-2, caspase-3, survivin) in cultured gastric epithelial cancer cells. MATERIAL AND METHODS: In the present study, gastric cultured cancer cells (KATO III cells) were exposed to increasing concentrations of gastrin (1-1000 nM). Cells incubated with culture medium alone, without added gastrin, served as controls. Using RT-PCR and Western blot, we examined the mRNA and protein expression for COX-2, HGF and apoptosis-related proteins (Bax, Bcl-2, caspase-3 and survivin). In addition, the gene expression of gastrin and gastrin receptor (CCK-2) as well as the release of gastrin in culture medium in the unstimulated cells were examined by RT-PCR and RIA, respectively. The apoptosis rate in cells was measured by flow cytometric analysis. RESULTS: The present study shows that the gastric cultured epithelial cells exhibit the expression of gastrin and CCK-2 receptors and release of gastrin into the culture medium. The epithelial gastric cancer cells incubated with gastrin showed a concentration-dependent increase of COX-2 and HGF expression. Although no significant changes in apoptosis rate were observed, the exposure of these cells was associated with a dose-dependent increase in the expression of antiapoptotic proteins Bcl-2 and survivin. CONCLUSIONS: This study demonstrates that 1) gastrin stimulates the gene and protein expression of COX-2 and HGF in human cultured gastric cancer cells and 2) gastrin shows antiapoptotic activity through the upregulation of Bcl-2 and survivin.     

Salvianolic acid B suppresses EMT and apoptosis to lessen drug resistance through AKT/mTOR in gastric cancer cells

The drug resistance of tumor cells greatly reduces the efficacy of chemotherapy drugs in gastric cancer. Salvianolic acid B (Sal-B) is considered as a chemopreventive agent which suppresses oxidative stress and apoptosis. Therefore, the study aims to clarify the mechanism of Sal-B in drug-resistant gastric cancer cells. CCK8 assay analyzed cell viabilities after GES1, AGS and AGS/DDP cells were respectively treated by Sal-B of different concentration or after AGS/DDP cells were disposed by cisplatin (DDP) in different concentration. The colony formation, ROS generation, apoptosis, migration, invasion and EMT marker proteins were respectively analyzed through formation assay, ROS kits, TUNNEL staining, Wound healing, Transwell assays and Western blot. The results demonstrated that Sal-B acted alone or in synergy with DDP to reduce cell viabilities, initiate ROS generation, promote cell apoptosis, as well as decrease migration, invasion and EMT in AGS and AGS/DDP cells. AKT activator and mTOR activator significantly reversed the above effects of Sal-B. Collectively, Sal-B regulated proliferation, EMT and apoptosis to reduce the resistance to DDP via AKT/mTOR pathway in DDP-resistant gastric cancer cells. Sal-B could be a potential anti-drug resistance agent to chemotherapy in gastric cancer.