MKN45 Gastric Carcinoma Cells Research Articles

The preparation of a cold-water soluble polysaccharide from Grifola frondosa and its inhibitory effects on MKN-45 cells.

In this study, a novel water soluble polysaccharide (named GFP-4) was extracted from Grifola frondosa at 4 oC, and its preliminary structure and inhibitory effects on human gastric carcinoma MKN-45 cells through the Fas/FasL death receptor apoptosis pathway were investigated. High-performance gel permeation chromatography (HPGPC), fourier-transform infrared spectroscopy (FT-IR), and ion chromatography (IC) results showed that GFP-4 was a 1.09 × 106 Da neutral hetero polysaccharide with pyranose rings, and α- and β-type glycosidic linkages that contained galactose, glucose, and mannose at a molar ratio of 1.00:3.45:1.19. MTT results indicated that GFP-4 significantly inhibited the proliferation of MKN-45 cells in a concentration-dependent manner. The H&E staining and Hoechst 33342/PI double staining results showed that GFP-4-treated MKN-45 cells were subjected to underwent typical apoptotic morphologic changes such as nuclear pyknosis, chromatin condensation, and an increase of membrane permeability. Annexin V-FITC/PI double staining, cell cycle analysis, and western blot results revealed the GFP-4 induced MKN-45 cells apoptosis through the Fas/FasL-mediated death receptor pathway with cells arrested at the G0/G1 phase. These data indicate that GFP-4 is a promising candidate for treating gastric cancer and provide a theoretical basis for the future development and utilization of G. frondosa clinically.

Light-Induced Radiosynthesis of 89Zr-DFO-Azepin-Onartuzumab for Imaging the Hepatocyte Growth Factor Receptor.

Methods that provide rapid access to radiolabeled antibodies are vital in the development of diagnostic and radiotherapeutic agents for PET or radioimmunotherapy. The human hepatocyte growth factor receptor (c-MET) signaling pathway is dysregulated in several malignancies, including gastric cancer, and is an important biomarker in drug discovery. Here, we used a photoradiochemical approach to produce 89Zr-radiolabeled onartuzumab (a monovalent, antihuman c-MET antibody), starting directly from the fully formulated drug (MetMAb). Methods: Simultaneous 89Zr-radiolabeling and protein conjugation was performed in one-pot reactions containing 89Zr-oxalate, the photoactive chelate desferrioxamine B (DFO)-aryl azide (DFO-ArN3), and MetMAb to give 89Zr-DFO-azepin-onartuzumab. As a control, 89Zr-DFO-benzyl Bn-isothiocyanate Bn-NCS-onartuzumab was prepared via a conventional two-step process using prepurified onartuzumab and DFO-Bn-NCS. Radiotracers were purified by using size-exclusion methods and evaluated by radiochromatography. Radiochemical stability was studied in human serum, and immunoreactivity was determined by cellular binding assays using MKN-45 gastric carcinoma cells. PET imaging at multiple time points (0-72 h) was performed on female athymic nude mice bearing subcutaneous MKN-45 xenografts. Biodistribution experiments were performed after the final image was obtained. The tumor specificity of 89Zr-DFO-azepin-onartuzumab was assessed in vivo by competitive inhibition (blocking) studies. Results: Initial photoradiosynthesis experiments produced 89Zr-DFO-azepin-onartuzumab in less than 15 min, with an isolated decay-corrected radiochemical yield (RCY) of 24.8%, a radiochemical purity of approximately 90%, and a molar activity of approximately 1.5 MBq nmol-1 Reaction optimization improved the radiochemical conversion of 89Zr-DFO-azepin-onartuzumab to 56.9% ± 4.1% (n = 3), with isolated RCYs of 41.2% ± 10.6% (n = 3) and radiochemical purity of more than 90%. Conventional methods produced 89Zr-DFO-Bn-NCS-onartuzumab with an isolated RCY of more than 97%, radiochemical purity of more than 97% and molar activity of approximately 14.0 MBq nmol-1 Both radiotracers were immunoreactive and stable in human serum. PET imaging and biodistribution studies showed high tumor uptake for both radiotracers. By 72 h, tumor and liver uptake (percentage injected dose [%ID]) reached 15.37 ± 5.21 %ID g-1 and 6.56 ± 4.03 %ID g-1, respectively, for 89Zr-DFO-azepin-onartuzumab (n = 4) and 21.38 ± 11.57 %ID g-1 and 18.84 ± 6.03 %ID g-1, respectively, for 89Zr-DFO-Bn-NCS-onartuzumab (n = 4). Blocking experiments gave a statistically significant reduction in tumor uptake (6.34 ± 0.47 %ID g-1) of 89Zr-DFO-azepin-onartuzumab (n = 4). Conclusion: The experiments demonstrated that photoradiosynthesis is a viable alternative approach for producing 89Zr-radiolabeled antibodies directly in protein formulation buffer, reducing protein aggregation and liver uptake.

Cytotoxicity of two water-soluble polysaccharides from Codonopsis pilosula Nannf. var. modesta (Nannf.) L.T.Shen against human hepatocellular carcinoma HepG2 cells and its mechanism

Two water-soluble polysaccharides named CPP1a and CPP1c were isolated from C. pilosula Nannf. var. modesta L.T.Shen by hot-water extraction and purified by graded alcohol precipitation and DEAE-52 cellulose column. The structure of CPP1c with higher yield has been characterized while its antitumor activities has not been elucidated. In this study, we firstly analyzed the chemical structure of CPP1a. The results of instrumental analysis combined with chemical analysis showed that CPP1a was composed of →1)- β‑l‑Rhap‑(4→, →1)- β‑Arap‑(5→, →1)- β‑d‑GalpA‑(4→, →1)- β‑d‑Galp‑(6→, terminal‑β‑d‑Glcp in a molar ratio of 1:12:1:10:3 and its relative and absolute molecular weight were 1.01 × 105 Da and 1.03 × 105 Da respectively. Further, the cytotoxicity assay indicated that CPP1a and CPP1c were more sensitive to HepG2 cells than cervical carcinoma Hela cells and gastric carcinoma MKN45 cells. Both of CPP1a and CPP1c could influence cell morphology, inhibit the migration and induce apoptosis by affecting the G2/M phase of HepG2 cells. Preliminary mechanism studies confirmed that CPP1a and CPP1c could induce apoptosis through up-regulating the ratio of Bax/Bcl-2 and activating caspase-3. According to previous research, we might speculate that the reason for the stronger cytotoxicity and pro-apoptotic effect of CPP1c than that of CPP1a can be attributed to its high uronic acid content.

TMEM119 silencing inhibits cell viability and causes the apoptosis of gastric cancer SGC-7901 cells.

Gastric cancer is the second major cause of death associated with cancer and ranks among the top four cancers diagnosed worldwide. Previous findings identified the association of transmembrane proteins (TMEMs) with tumorigenesis of various types of cancer, including breast, liver and kidney cancer. However, the expression and the biological function of TMEMs, especially TMEM119, and its possible molecular mechanism in gastric cancer remain less understood. CCK-8 and flow cytometric analysis was employed to examine the viability and apoptosis of gastric adenocarcinoma SGC-7901 and AGS cells, gastric carcinoma MKN45 cells, as well as gastric epithelial cell lines GES-1 after transfection with TMEM119-siRNA (siTMEM119), respectively. Quantitative PCR, western blot analysis and immunohistochemistry was performed to detect the expression levels of TMEM119, Bax, Bcl-2 and caspase-3. The results showed that, TMEM119 was elevated with the highest expression detected in SGC-7901 cells compared to AGS cells, MKN45 cells, as well as GES-1. TMEM119 silencing in the gastric cancer cell line, SGC-7901, significantly inhibited cell viability and induced apoptosis. The downregulation of TMEM119 exhibited reduced levels of Bcl-2 and higher levels of Bax and caspase-3 in SGC-7901 cells. These results suggest that TMEM119 is useful in the treatment of gastric cancer.

A novel polysaccharide derived from algae extract inhibits cancer progression via JNK, not via the p38MAPK signaling pathway.

Cancer has long been one of the most malignant diseases worldwide. Processes in cancer cells are often mediated by Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and other signaling pathways. Traditional therapies are often problematic. Recently, a novel polysaccharide derived from algae extract was investigated due to the increasing interest in biological activities of compounds from marine organisms. The effect of this novel polysaccharide on human MKN45 gastric carcinoma cells was determined previously. The current aimed to determine whether the polysaccharide affects other types of cancer, and the deeper mechanisms involved in the process. Human MCF-7 breast cancer cells were used to investigate the novel polysaccharide for its role in the cell growth and migration, and determine the mechanisms affected. MTT assay, nuclear staining and fluorescence activated cell sorting analysis demonstrated that the novel polysaccharide reduced the viability of MCF-7 cells by inducing cell apoptosis and arresting the cells at G2/M phase. Results of western blot analysis demonstrated that phosphorylation of JNK and expression of p53, caspase-9 and caspase-3 were upregulated in the polysaccharide-treated MCF-7 cells. SP600125, an inhibitor of JNK, maintained MCF-7 cell viability, prevented cell apoptosis and cycle arrest, and downregulated the polysaccharide-induced protein phosphorylation/expression. However, a migration assay demonstrated that the novel polysaccharide did not change the migration of MCF-7 cells, as well as the expression of p38 MAPK, and matrix metalloproteinase-9 and -2. Taken together, the current study demonstrated that the novel polysaccharide suppressed cancer cell growth, induced cancer cell apoptosis and cell cycle arrest via JNK signaling, but had no effect on cancer cell migration and p38 MAPK signaling.

Anticancer effect of cucurbitacin B on MKN-45 cells via inhibition of the JAK2/STAT3 signaling pathway

The aim of the present study was to investigate the effect of cucurbitacin B on MKN-45 gastric carcinoma cells. Cell proliferation was determined using a cell counting kit-8 assay, and commercial cell cycle and apoptosis analysis kits were used to determine the cell cycle by flow cytometry. The mRNA expression of genes which mediate cell cycle checkpoints and apoptosis was detected using reverse transcription-quantitative polymerase chain reaction, and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine apoptosis rate. Western blot analysis was used to detect the protein expression levels of JAK2/STAT3 signaling pathway-associated proteins. The presented data show that cucurbitacin B significantly inhibited the proliferation of MKN-45 cells in a dose- and time-dependent manner. In accordance with these findings, cucurbitacin B blocked the progression of the cell cycle from G0/G1 to S phase, which was confirmed by the mRNA expression analysis. Cucurbitacin B treatment significantly suppressed the expression of cyclin D1, cyclin E, cyclin-dependent kinase 4 (CDK4) and CDK2, while increasing the expression of p27. Cucurbitacin B also promoted cell apoptosis, as was determined by TUNEL assay and evaluation of mRNA expression. Further experiments suggested that the beneficial effect of cucurbitacin B on blocking the proliferation and inducing the apoptosis of MKN-45 cells may have been associated with suppression of the JAK2/STAT3 signaling pathway. Thus, the present results indicate that cucurbitacin B suppresses proliferation and promoted apoptosis of MKN-45 cells, which may be mediated by inhibition of the JAK2/STAT3 signaling pathway. Cucurbitacin B therefore may warrant further investigation as a feasible therapy for gastric carcinoma.

A novel polysaccharide derived from algae extract induces apoptosis and cell cycle arrest in human gastric carcinoma MKN45 cells via ROS/JNK signaling pathway.

In recent years, interest in biological activities of compounds from marine organisms has intensified. Cancer is the most principal enemy for human life and health. For the first time, to the best of our knowledge, we investigated a novel algae-derived polysaccharide for its role in inducing apoptosis and cell cycle arrest in human gastric carcinoma MKN45 cells. We found that the novel polysaccharide suppressed MKN45 cell proliferation, induced cell apoptosis and arrested the cells at G2/M phase. Furthermore, we observed that the generation of reactive oxygen species (ROS) and the phosphorylation of Jun N-terminal kinase (JNK), p53, caspase-9 and-3 were induced in the polysaccharide-treated MKN45 cells. In addition, pretreatment with N-acetyl-cysteine (NAC) and SP600125, the inhibitor of ROS and JNK, induced MKN45 cell proliferation, prevented the cell apoptosis and released the cells from cycle arrest. Finally, we found that pretreatment with NAC prevented the JNK, p53, caspase-9 and-3 protein phosphorylation induced by the polysaccharide, however, pretreatment with SP600125 did not affect the generation of ROS, suggesting that ROS is upstream of JNK. Taken together, the novel polysaccharide induced cancer cell apoptosis and arrested cell cycle via ROS/JNK signaling pathway.

Glycomic and sialoproteomic data of gastric carcinoma cells overexpressing ST3GAL4.

Gastric carcinoma MKN45 cells stably transfected with the full-length ST3GAL4 gene were characterised by glycomic and sialoproteomic analysis. Complementary strategies were applied to assess the glycomic alterations induced by ST3GAL4 overexpression. The N- and O-glycome data were generated in two parallel structural analyzes, based on PGC-ESI-MS/MS. Data on glycan structure identification and relative abundance in ST3GAL4 overexpressing cells and respective mock control are presented. The sialoproteomic analysis based on titanium-dioxide enrichment of sialopeptides with subsequent LC-MS/MS identification was performed. This analysis identified 47 proteins with significantly increased sialylation. The data in this article is associated with the research article published in Biochim Biophys Acta “Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer” [1].

Inhibitory effects of XAV939 on gastric carcinoma MKN-45 cells proliferation and Wnt/β-catenin signaling pathway in vitro

Objective To investigate the effects of XAV939 on proliferation and and Wnt/β-catenin signaling pathway of MKN-45 cells in vitro.Methods MKN-45 cells were cultured with different concentrations of XAV939.The inhibition of cell proliferation was detected by methyl thiazol tetrazolium (MTT).The expression levels of β-catenin and cell cycle protein 1 (Cyclin D1) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR),and the protein expression of Axin 1,β-catenin,p-β-catenin and Cyclin D1 by Western blotting.Results Inhibitory rate of MKN-45 cells treated with XAV939 for 48 h was 22.5％,39.1％,52.5％,and 62.1％ respectively (P ＜0.05).Different concentrations of XAV939 could down-regulate β-catenin and Cyclin D1 mRNA level (0.727 ±0.031,0.467 ±0.051,0.353 ±0.035,0.293 ±0.031,P＜0.05; 0.690±0.161,0.487 ±0.231,0.360 ±0.257,0.2 1 6 ± 0.197,P ＜ 0.05),down-regulate β-catenin and Cyclin D1 protein levels,but significantly up-regulate Axin 1 and p-β-catenin expression levels.Conclusion XAV939 can inhibite proliferation of MKN-45 cells,which may be associated with down-regulation of Wnt/β-catenin signaling pathway. Key words: Gastric carcinoma; Wnt/β-catenin signaling pathway; Cyclin D1

Bile acid induces MUC2 expression and inhibits tumor invasion in gastric carcinomas.

Bile acids might induce mucin expression and regulate tumor behavior in esophageal and colon cancers. However, little is known about the effect of bile acids on tumor invasiveness of gastric carcinoma (GC). The aim of the current study was to elucidate the mechanisms by which bile acids regulate tumor invasion in GC. We investigated bile acid-induced MUC2 expression and cell invasion and migration in the cultured GC cell lines, SNU-216, and MKN45. In addition, immunohistochemical analysis of MUC2 and Snail was performed on 389 archival paraffin-embedded tissues of GC to evaluate the correlation of their expression with prognosis. Deoxycholic acid (DCA), a secondary bile acid, had no effect on the viability of SNU-216 and MKN45 GC cells at low concentrations (0-100 μM), but decreased viability at a higher concentration (200 μM). MKN45 cells showed higher MUC2 expression than SNU-216 cells under basal conditions. DCA treatment upregulated MUC2 mRNA expression in both SNU-216 and MKN45 cells. Expression of Snail and MMP9 was markedly decreased by DCA treatment, and E-cadherin expression was subsequently increased. DCA significantly inhibited invasion and migration of SNU-216 and MKN45 cells. In human GC, MUC2 expression showed a negative correlation with Snail expression (P = 0.021) and a significantly positive correlation with better prognosis (P = 0.023). Taken together, our data suggest that DCA induced MUC2 expression in GC cells and inhibited tumor invasion and migration. Additionally, MUC2-expressing GCs showed low rates of Snail expression and were associated with a favorable prognosis.

Impact of miR-148a on the proliferation of gastric carcinoma MKN45 cells and its mechanism

To investigate the influence of miR-148a on cell proliferation of human gastric cancer cell lines MKN45. Expression level of miR-148a was detected by qRT-PCR in the carcinoma tissues and the tissues adjacent to carcinoma of 60 patients with gastric cancer. Lentivirus packaging was used to establish MKN45 gastric cancer cell line expressing stable miR-148a as transfection group,and the untransfected MKN45 cell line as the control group. Gastric cancer MKN45 cell proliferation was detected by CCK8 method. The target gene of miR-148a was predicted by targetscan. Expression of target gene was examined by Western blotting. Expression of miR-148a in gastric cancer tissues of 54 cases(90.0%, 54/60) decreased, and among them, 37 tissue samples(61.7%, 37/60) decreased by 2 times. mir-148a expression in cancer tissues of patients with lymph node metastasis, vascular invasion and TNM stage III(-IIII( was down-regulated more obviously(P<0.05). From the third day after transfection, the growth of MKN45 cells was significantly inhibited(P<0.01). Gene CDC25B might be the target gene of miR-148a according to the results of targetscan. Western blot showed that CDC25B expression in transfection group was lower as compared to control group. MiR-148a expression is down-regulated in gastric cancer tissues and inhibits gastric cancer cell proliferation. CDC25B may be the target gene of miR-148a that plays a role in tumor suppressor.

Oxidized S100A4 inhibits the activation of protein phosphatase 5 through S100A1 in MKN-45 gastric carcinoma cells

S100 proteins bind to numerous target proteins, as well as other S100 proteins and activate signaling cascades. S100 proteins can be modified by various post-translational modifications, such as phosphorylation, methylation and acetylation. In addition, oxidation is important for modulating their activities. Previous studies have shown that S100A1 interacts with S100A4 in vitro and in vivo. Due to this potential cross‑talk among the S100 proteins, the aim of the present study was to examine whether S100A4 modulates the activity of S100A1. S100A4 was readily oxidized and formed disulfide-linked dimers and oligomers. Although non-oxidized S100A4 bound to protein phosphatase 5 (PP5), the Cu-oxidized S100A4 failed to bind PP5. Instead, the Cu-oxidized S100A4 directly interacted with S100A1 and prevented PP5 activation. Hydrogen peroxide induced S100A4 oxidation in MKN-45 gastric adenocarcinoma cells and decreased S100A1‑PP5 interaction, resulted in the inhibition of PP5 activation by S100A1. These data indicate that oxidized S100A4 regulates PP5 activity in a unique manner under oxidative stress conditions.

Expression of ST3GAL4 Leads to SLex Expression and Induces c-Met Activation and an Invasive Phenotype in Gastric Carcinoma Cells

Sialyl-Lewis X (SLex) is a sialylated glycan antigen expressed on the cell surface during malignant cell transformation and is associated with cancer progression and poor prognosis. The increased expression of sialylated glycans is associated with alterations in the expression of sialyltransferases (STs). In this study we determined the capacity of ST3GAL3 and ST3GAL4 sialyltransferases to synthesize the SLex antigen in MKN45 gastric carcinoma cells and evaluated the effect of SLex overexpression in cancer cell behavior both in vitro and in vivo using the chicken chorioallantoic membrane (CAM) model. The activation of tyrosine kinase receptors and their downstream molecular targets was also addressed. Our results showed that the expression of ST3GAL4 in MKN45 gastric cancer cells leads to the synthesis of SLex antigens and to an increased invasive phenotype both in vitro and in the in vivo CAM model. Analysis of phosphorylation of tyrosine kinase receptors showed a specific increase in c-Met activation. The characterization of downstream molecular targets of c-Met activation, involved in the invasive phenotype, revealed increased phosphorylation of FAK and Src proteins and activation of Cdc42, Rac1 and RhoA GTPases. Inhibition of c-Met and Src activation abolished the observed increased cell invasive phenotype. In conclusion, the expression of ST3GAL4 leads to SLex antigen expression in gastric cancer cells which in turn induces an increased invasive phenotype through the activation of c-Met, in association with Src, FAK and Cdc42, Rac1 and RhoA GTPases activation.

Effects of KIAA0101 expression on proliferation and invasion of gastric carcinoma MKN-45 cells

To investigate the expression of KIAA0101 protein in gastric carcinoma cells, and to explore the effects of its down-regulation on the cell proliferation, cell cycle and invasion. Western blot was used to detect KIAA0101 protein expression in three gastric carcinoma cell lines including MKN-28, SGC-7901 and MKN-45. KIAA0101 siRNA and control siRNA were utilized to transfect MKN-45 cells, respectively. CCK-8 was used to analyze the changes of cell proliferation, and flow cytometry to examine the changes of cell cycle distribution. Finally, Boyden chamber was used to detect the ability of cell invasion. Relative level of KIAA0101 protein in MKN-45 cells was significantly higher than those in MKN-28 and SGC-7901 cells, and there was significant difference among the three cell lines (P < 0.05). The result of CCK-8 study demonstrated that, compared with untreated group and control siRNA group, the proliferation of MKN-45 cells in KIAA0101 siRNA group was significantly inhibited (P < 0.05). Additionally, the result of cell cycle analysis revealed that the percentage of cell number in G(0)/G(1) phase in KIAA0101 siRNA group [(61.47 ± 0.89)%] was significantly higher than those in untreated group [(47.43 ± 0.85)%] and control siRNA group [(48.43 ± 0.73)%; F = 271.653, P = 0.000]. Further, Boyden chamber assay showed that the cell numbers migrated to Matrigel in KIAA0101 siRNA group (61.51 ± 4.76) were significantly lower than those in untreated group (138.74 ± 10.16) and control siRNA group (132.93 ± 11.25; F = 65.949, P = 0.000). Down-regulation of KIAA0101 expression leads to an inhibition of cell proliferation, cell cycle and cell invasion. It may provide a novel target for the treatment of patients with gastric carcinoma.

Xanthatin Induces G2/M Cell Cycle Arrest and Apoptosis in Human Gastric Carcinoma MKN-45 Cells

Xanthatin, a natural bioactive compound of sesquiterpene lactones, was isolated and purified from air-dried aerial part of Xanthium sibiricum Patrin ex Widder. In the present study, we demonstrated the significant antiproliferative and proapoptotic effects of xanthatin on human gastric carcinoma MKN-45 cells. MTS assay showed that xanthatin produced obvious cytotoxicity in MKN-45 cells with IC50 values of 18.6, 9.3, and 3.9 µM for 12, 24, and 48 h, respectively. Results of flow cytometry analysis indicated that the antiproliferative activity induced by xanthatin might be executed via G2/M cell cycle arrest and proapoptosis in MKN-45 cells. Western blot analysis elucidated that: a) xanthatin downregulated expression of Chk1 and Chk2 and phosphorylation of CDC2, which are known as key G2/M transition regulators; b) xanthatin increased p53 activation, decreased the bcl-2/bax ratio and the levels of downstream procaspase-9 and procaspase-3, which are key regulators in the intrinsic apoptosis pathway; c) xanthatin blocked phosphorylation of NF-κB (p65 subunit) and of IκBα, which might contribute to its proapoptotic effects on MKN-45 cells. In conclusion, our results suggest that xanthatin may have therapeutic potential against human gastric carcinoma.

Activator protein-1 involvement in proliferation inhibition by gene silencing of Twist in gastric cancer cells

To study the role of the Twist gene in growth of gastric cancer cell line MKN45 and the possible mechanisms involved. Human gastric carcinoma MKN45 cells were stably transfected with Twist antisense plasmid using the lipofectamine transfection technique. Expression of Twist in Twist antisense plasmid transfected cells (TwistAS), non-transfected cells (NT) and non-specific Twist antisense plasmid transfected cells (CON) were examined by Western blotting. Cell growth ability in vitro was evaluated by MTT and clone formation assays. Xenograft cancer models were established by nude mouse transfer method. Activator protein-1 (AP-1) DNA binding activity was measured by electrophoretic mobility shift assay (EMSA). The expression of c-Jun and c-Fos were examined by Western blotting. The mRNA level of cyclin D1 was detected by RT-PCR. TwistAS inhibited cell growth and proliferation. In vitro, the cloning efficiency of TwistAS cells (8.0 ± 0.6%) was significantly lower compared to that in NT (26.5 ± 1.1%) and CON (22.7 ± 1.2%). In vivo, the average tumour weight was lighter in the TwistAS group (425.3 ± 20.8 mg) compared with the CON group (1217.0 ± 50.2 mg) and the NT group (1120.6 ± 75 mg). TwistAS inhibited AP-1 activity in MKN45 cells (15.3 ± 3.2% versus 50.2 ± 3.6% and 52.4 ± 3.8%). TwistAS inhibited the expression of c-Fos in MKN45 cells (20.4 ± 3.8% versus 72.5 ± 3.6% and 75.3 ± 4.0%) but not c-Jun (p < 0.05). cyclin D1 mRNA level was significantly lower in TwistAS cells (40.5 ± 3.8%) than that in CON (132 ± 5.4%) and NT cells (130 ± 5.2%). This study demonstrated that down-regulation of the Twist gene suppressed the proliferation of MKN45 gastric cancer cells by negatively regulating the AP-1 activity resulting in the cyclin D1 mRNA level decreasing.

Abstract A141: Analysis of cellular kinase activity of resistance-related MET mutants using transmembrane-domain (TMD) activated kinase variants.

Abstract Kinases are important targets in oncology. However, the frequent emergence of resistance (gatekeeper) mutants in target kinases seriously limits the patient's response time to a given drug. Secondary drugs need to be developed that specifically target mutant enzymes. The discovery of such kinase inhibitors requires relevant assay systems. In order to efficiently analyze whether novel inhibitors target gatekeeper mutants of a given kinase in a cellular context we set out to establish a suitable cellular test system for the receptor tyrosine kinase MET. Our aim was to minimize variations such as cellular background and ligand dependent activation while putting the focus on the mutation of interest. In an approach to uncouple kinase activity from ligand activation, we screened a set of artificial transmembrane domains (TMD) to identify a TMD that induces constitutive MET autophosphorylation in transient transfections. Subsequently, Rat1 cells were transduced to express the tag-labelled intracellular wild-type (wt) MET domain harbouring the activating TMD while lacking the extracellular domain. Characterization of TMD-MET-wt in Rat1 cells showed constitutive MET expression and phosphorylation. ELISA studies to analyze the inhibition of MET phosphorylation by cognate MET inhibitors revealed comparable IC50 values to those obtained with wild-type MET naturally over expressed in MKN45 gastric carcinoma cells. Moreover, expression of TMD-MET-wt in Rat1 cells led to cell transformation as assessed by growth in soft agar as well as in vivo as a subcutaneous xenograft. Interestingly, the same artificial TMD induced constitutive activity of various gatekeeper mutants of MET when transduced into Rat1 cells. This observation allowed for the comparative analysis of various small molecule MET inhibitors on multiple MET mutants in an identical cellular background and without the need of ligand-induced kinase activation. Using a straight forward ELISA-based approach, we generated profiles of various MET inhibitors on cellular MET mutant phosphorylation and compared these with inhibition profiles obtained in biochemical MET activity assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A141.

Impact of MUC1 Mucin Downregulation in the Phenotypic Characteristics of MKN45 Gastric Carcinoma Cell Line

BackgroundGastric carcinoma is the second leading cause of cancer-associated death worldwide. The high mortality associated with this disease is in part due to limited knowledge about gastric carcinogenesis and a lack of available therapeutic and prevention strategies. MUC1 is a high molecular weight transmembrane mucin protein expressed at the apical surface of most glandular epithelial cells and a major component of the mucus layer above gastric mucosa. Overexpression of MUC1 is found in approximately 95% of human adenocarcinomas, where it is associated with oncogenic activity. The role of MUC1 in gastric cancer progression remains to be clarified.MethodologyWe downregulated MUC1 expression in a gastric carcinoma cell line by RNA interference and studied the effects on cellular proliferation (MTT assay), apoptosis (TUNEL assay), migration (migration assay), invasion (invasion assay) and aggregation (aggregation assay). Global gene expression was evaluated by microarray analysis to identify alterations that are regulated by MUC1 expression. In vivo assays were also performed in mice, in order to study the tumorigenicity of cells with and without MUC1 downregulation in MKN45 gastric carcinoma cell line.ResultsDownregulation of MUC1 expression increased proliferation and apoptosis as compared to controls, whereas cell-cell aggregation was decreased. No significant differences were found in terms of migration and invasion between the downregulated clones and the controls. Expression of TCN1, KLK6, ADAM29, LGAL4, TSPAN8 and SHPS-1 was found to be significantly different between MUC1 downregulated clones and the control cells. In vivo assays have shown that mice injected with MUC1 downregulated cells develop smaller tumours when compared to mice injected with the control cells.ConclusionsThese results indicate that MUC1 downregulation alters the phenotype and tumorigenicity of MKN45 gastric carcinoma cells and also the expression of several molecules that can be involved in tumorigenic events. Therefore, MUC1 should be further studied to better clarify its potential as a novel therapeutic target for gastric cancer.

Therapeutic Effects of Ca2+ on Peritoneal Dissemination of Gastric Carcinoma in Vivo

It is well known that oral calcium supplementation has inhibited the gastric carcinogenesis in animals treated with chemical carcinogen and sodium chloride. However, the therapeutic effects of calcium ion (Ca2+) for peritoneal dissemination of gastric carcinoma in vivo remain unknown. Here, we showed that Ca2+ could be effective for inhibiting the peritoneal metastasis of gastric carcinoma MKN-45 cells along with apoptosis in vivo. Significantly prolonged survival was obtained for the peritoneal dissemination mouse models of MKN-45 cells after the intraperitoneal injection (i.p.) of Ca2+. These results suggest that Ca2+ could be a new candidate i.p. drug against peritoneal dissemination of gastric cancer in human.

Bispecific Adapter-Mediated Retargeting of a Receptor-Restricted HSV-1 Vector to CEA-Bearing Tumor Cells

The safety and efficacy of viral therapies for solid tumors can be enhanced by redirecting the virus infection to tumor-specific cell-surface markers. Successful retargeting of herpes simplex virus type 1 (HSV-1) has been achieved using vectors that carry a modified envelope glycoprotein D (gD) engineered to interact directly with novel receptors. In addition, soluble bridging molecules (adapters) have been used to link gD indirectly to cell-specific receptors. Here, we describe the development of an adapter connecting gD to the common tumor antigen carcinoembryonic antigen (CEA). The adapter consisted of a CEA-specific single-chain antibody fused to the gD-binding region of the gD receptor, herpes virus entry mediator (HVEM). We used this adapter in combination with a vector that is detargeted for recognition of the widely expressed gD receptor nectin-1, but retains an intact binding region for the less common HVEM. We show that the adapter enabled infection of HSV-resistant Chinese hamster ovary (CHO) cells expressing ectopic CEA and nectin-1/CEA-bearing human gastric carcinoma cells that are resistant to the vector alone. We observed cell-to-cell spread following adapter-mediated infection in vitro and reduced tumor growth in vivo, indicating that this method of vector retargeting may provide a novel strategy for tumor-specific delivery of tumoricidal HSV.