N87 Cells Research Articles

Regulation of cathepsin X overexpression in H. pylori‐infected gastric epithelial cells and macrophages

Cathepsin X (CTSX) is strongly up-regulated in Helicobacter pylori-infected gastric mucosa and intestinal-type gastric cancer. The overexpression of CTSX is mediated predominantly by associated macrophages; depends on a functional type IV-secretion system; and leads to increased migration of gastric epithelial cells. In the present study, we analysed the role of CagA in CTSX overexpression and identified H. pylori-induced inflammatory factors and signalling pathways required for stimulating CTSX expression by H. pylori. Gastric epithelial cells were co-cultured with macrophages in Transwell chambers of 0.4 microm pore size, enabling exchange of fluids but retracting H. pylori. N87 gastric epithelial cells were infected with H. pylori P1 wild-type strain in the presence of inhibitors for p38, JNK, and ERK1/2 signal transduction pathways. Furthermore, cytokines and growth factors were tested for their regulatory function using inhibitory antibodies, and their gene expression was studied by quantitative RT-PCRs and western blots. CTSX is strongly up-regulated at both the mRNA and the protein levels by TNF-alpha, IL-1beta, IL-6, and IL-8, depending on cell type. All these cytokines were found to be increased by five- to ten-fold in macrophages by H. pylori infection of co-cultured N87 gastric epithelial cells. In macrophages, H. pylori up-regulated CTSX via ERK1/2 signalling pathways, and in N87 cells via JNK irrespective of p38 signalling. Our results suggest that H. pylori induced overexpression of CTSX in macrophages and epithelium through specific cytokines that are initiated by CagA-dependent pathways in a cell type-dependent manner.

Inhibition of Gastric Cancer Invasion and Metastasis by PLA2G2A, a Novel β-Catenin/TCF Target Gene

Elevated expression of the PLA2G2A phospholipase in gastric cancer (GC) is associated with improved patient survival. To elucidate function and regulation of PLA2G2A in GC, we analyzed a panel of GC cell lines. PLA2G2A was specifically expressed in lines with constitutive Wnt activity, implicating beta-catenin-dependent Wnt signaling as a major upstream regulator of PLA2G2A expression. The invasive ability of PLA2G2A-expressing AGS cells was enhanced by PLA2G2A silencing, whereas cellular migration in non-PLA2G2A-expressing N87 cells was inhibited by enforced PLA2G2A expression, indicating that PLA2G2A is both necessary and sufficient to function as an inhibitor of GC invasion in vitro. We provide evidence that antiinvasive effect of PLA2G2A occurs, at least in part, through its ability to inhibit the S100A4 metastasis mediator gene. Consistent with its invasion inhibitor role, PLA2G2A expression was elevated in primary gastric, colon, and prostrate early-stage tumors, but was decreased in metastatic and late-stage tumors. There was a strong association between PLA2G2A promoter methylation status and PLA2G2A expression, suggesting that the loss of PLA2G2A expression in late-stage cancers may be due to epigenetic silencing. Supporting this, among the non-PLA2G2A-expressing lines, pharmacologic inhibition of epigenetic silencing reactivated PLA2G2A in Wnt-active lines, but in non-Wnt-active lines, a combination of Wnt hyperactivation and inhibition of epigenetic silencing were both required for PLA2G2A reactivation. Our results highlight the complexity of PLA2G2A regulation and provide functional evidence for PLA2G2A as an important regulator of invasion and metastasis in GC.

Preferential chemosensitivity to gemcitabine by cyclin E overexpression in human gastric cancers

21020 Background: Exploration of molecular determinants for chemosensitivity is the key element of personalized cancer therapy. Cyclin E is a major G1-phase cyclin, together with the CDK2, which mediates phosphorylation and functional inactivation of Rb protein. Cyclin E overexpression has been demonstrated in a variety of cancers, including human gastric cancers (GC), while its biologic significance in drug therapy remains unclear. Previously, we have demonstrated that cyclin D1 overexpression plays an important role in differential chemosensitivity of human GC cells (Proc Am Assoc Cancer Res 2004; 45: abstract 4888). In this study, we examine the roles between cyclin E overexpression and chemosensitivity of human GC cells. Methods: Compared human gastric cancer cells (NCI-N87) with stably transfected cells (N87-CyE), which have 2-fold overexpression of cyclin E, the IC50 for gemcitabine (2'-2'-difluoro- deoxycytidine, dFdC) was more than 1-log lower in N87-CyE (17.0 ± 2.7 nM) than N87 cells (113.7 ± 6.5 nM) by MTT colorimetric cytotoxicity assay. In contrast, the N87-CyE cells are only slightly more chemosensitive to taxanes (paclitaxel and docetaxel), and confer largely identical chemosensitivity to 5-FU, cisplatin, and irinotecan (CPT-11). We applied RNA interference (RNAi) of cyclin E to N87 cells. The stably transfected N87-CyE/RNAi cells readily confer gemcitabine resistance with the IC50 for gemcitabine of 124.5 ± 1.6 nM. Results: Gemcitabine-induced apoptosis in N87, N87-CyE, or N87-CyE/RNAi cells was shown in either caspase-3 or PARP (poly ADP-ribose polymerase) cleavage assay by Western blotting, and Annexin-V-FITC apoptosis detection by flowcytometry. The threshold concentration of gemcitabine for caspase-3 and PARP cleavage was 10–25 nM for N87-CyE, and 100–200 nM for N87 or N87-CyE/RNAi, respectively. Conclusions: Our data demonstrate that preferential chemosensitivity to gemcitabine by cyclin E overexpression in gastric cancer cells. Gemcitabine-induced apoptosis is enhanced by cyclin E overexpression, while is reduced by RNAi of cyclin E. Further studies for potential clinical use of gemcitabine in personalized chemotherapy for cyclin E or cyclin D1-overexpressing GC are warranted (supported by the grants of NHRI-CN-CA9201S, Taiwan). No significant financial relationships to disclose.

H pylorireceptor MHC class II contributes to the dynamic gastric epithelial apoptotic response

To investigate the role of MHC class II in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class II and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. Pretreatment of N87 cells with the anti-MHC class II IgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class II resulted in a marked reduced caspase activation, while simple ligation of MHC class II did not. Crosslinking of MHC class II also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-MHC class II IgM blocked the recruitment of FADD to the cell surface. The results presented here demonstrate that the ability of MHC class II to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. Furthermore, while previous research has demonstrated that MHC class II signaling can be pro-apoptotic during extended ligation, we have shown that the crosslinking of this molecule has anti-apoptotic effects during the earlier time points of H pylori infection. This effect is possibly mediated by the ability of MHC class II to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by H pylori-infected gastric epithelial cells.

H pylorireceptor MHC class II contributes to the dynamic gastric epithelial apoptotic response

To investigate the role of MHC class II in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class II and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. Pretreatment of N87 cells with the anti-MHC class II IgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class II resulted in markedly reduced caspase activation, while simple ligation of MHC class II did not. Crosslinking of MHC class II also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-MHC class II IgM blocked the recruitment of FADD to the cell surface. The ability of MHC class II to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. The crosslinking of this molecule has anti-apoptotic effects during the earlier time points of H pylori infection. This effect is possibly mediated by the ability of MHC class II to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by H pylori-infected gastric epithelial cells.

Amplification of HER-2 in gastric carcinoma: association with Topoisomerase IIα gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab

BackgroundHER-2/neu gene amplification has predictive value in breast cancer patients responding to trastuzumab. We wanted to investigate the frequency and clinical significance of HER-2/neu amplification in gastric carcinoma. Patients and methodsThe frequency of HER-2/neu and Topoisomerase IIα gene amplification was studied in adenocarcinomas of the stomach (n=131) and the gastroesophageal junction (n=100) by chromogenic in situ hybridization (CISH). Sensitivity of a gastric cancer cell line N87 with HER-2/neu amplification to trastuzumab was studied by a cell viability assay and compared with that of a HER-2 amplified breast cancer cell line SKBR-3. Growth inhibition of N87 cells was also verified in vivo in N87 xenograft tumors. ResultsHER-2/neu amplification was present in 16 (12.2%) of the 131 gastric and in 24 (24.0%) of the 100 gastroesophageal adenocarcinomas. Co-amplification of Topoisomerase IIα was present in the majority of gastric (63%) and esophagogastric junction cancers (68%) with HER-2/neu amplification. HER-2/neu amplification was more common in the intestinal histologic type of gastric cancer (21.5%) than in the diffuse (2%) or the mixed/anaplastic type (5%, P=0.0051), but it was not associated with gender, age at diagnosis or clinical stage. Presence of HER-2/neu amplification was associated with poor carcinoma-specific survival (P=0.0089). HER-2/neu targeting antibody trastuzumab inhibited the growth of a p185HER-2/neu overexpressing gastric and breast carcinoma cell lines (N87 and SKBR-3) with equal efficacy. ConclusionsHER-2/neu amplification is common in the intestinal type of gastric carcinoma, and it is associated with a poor outcome. HER-2 might be a useful target in this disease, and this hypothesis deserves to be investigated in clinical trials.

Loss of beta-catenin expression in metastatic gastric cancer.

Beta-catenin (beta-catenin) participates in intercellular adhesion and is an integral part of the Wnt signaling pathway. The role of beta-catenin in the pathogenesis of gastric cancer and its metastasis is largely unknown. Immunohistochemistry and Western blot analysis were used to analyze the expression of beta-catenin in 87 human gastric cancers, in metastasis and cancer cell lines. The beta-catenin and the adenomatous polyposis coli (APC) genes were analyzed for gene mutations. Furthermore, methylation of the beta-catenin promoter in cell lines was assessed by treatment with 5'-azadeoxycytidine and sodium bisulfite genomic sequencing. beta-Catenin expression was present at either the cell membrane or the cytoplasm in 34 of 75 primary gastric cancers. Expression of beta-catenin was significantly more frequent in intestinal-type (P =.0049) and well-differentiated gastric cancers (P <.001). There were no quantitative differences between gastric cancers and the nonmalignant gastric tissues, as determined by Western blot analysis. One of 18 metastatic cancer lesions and four of five gastric cancer cell lines expressed beta-catenin protein. N87 cells, derived from the liver metastasis of a gastric cancer, did not express beta-catenin. Treatment with 5'-azadeoxycytidine restored beta-catenin protein levels in this cell line, which exhibited significantly more 5-methylcytosines in the beta-catenin promoter compared with the other cell lines. beta-Catenin expression is lost in a subgroup of primary gastric cancers, is frequently absent in metastases, and exhibits nuclear localization in cancers with either beta-catenin or APC gene mutations. Interestingly, the loss of beta-catenin expression in metastatic gastric cancers may result from hypermethylation of the beta-catenin promoter.

Inhibition of tumor growth by poly(ethylene glycol) derivatives of anti-ErbB2 antibodies.

Poly(ethylene glycol) (PEG) modification of substances with antitumor activity was shown to enhance penetration into growing solid tumors and extend antitumor effects. Accordingly, PEG was introduced as a modifier to two types of monoclonal antibodies (N12 and L26) specific to the ErbB2 (HER2) oncoprotein. These antibodies suppress the growth of tumors overexpressing ErbB2 (e.g. N87 human tumor) and the effect of PEG on their antitumor activity was evaluated. Methoxy-PEG-maleimide conjugated to sulfhydryl groups at the hinge region of the antibodies impaired their antibody binding to N87 tumor cells and did not enhance the antitumor inhibitory activity in tumor-bearing mice. A branched N-hydroxysuccinimide-activated PEG (PEG2), conjugated through amino groups of the protein, was used for binding to the whole antibody (Ab) or to its monomeric Fab' fragment. When tested against N87 cells in vitro, the binding activity and antitumor cytotoxic effects of Ab-PEG2 were mostly preserved. PEG2 modification did not seem to alter the tumor-inhibitory activity of the antibodies in vivo and the same pattern of tumor development was observed during the first few weeks following administration. However, the stimulating effects of PEG were observed at later stages of tumor growth since tumor development was either slowed down or completely arrested. Furthermore, a second tumor implanted into the same mice during this later stage was significantly or completely inhibited, as compared to results in mice injected with the unmodified antibody. The Fab'-PEG2 monomeric derivative was also shown to be effective in inhibiting the growth of a second tumor. The extended and prolonged enhancing effect of PEG on the antitumor activity of antibodies or Fab' fragments directed against ErbB2 may be of importance in the treatment of ErbB2-overexpressing neoplasms.

Growth inhibition of gastric cancer cell lines by the tyrphostin RG13022 and its effects on intracellular signalling

Overexpression of EGFr and c-erbB-2 is related to poor prognosis in a variety of cancers including gastric cancer. Thus: the ability to modulate the functional activity of these receptors is an attractive target for diagnostic intervention. In this study we examined the effect of a well characterised tyrosine kinase inhibitor (RG13022) on the cellular proliferation and EGF activated tyrosine kinase signalling pathway of two primary gastric cell lines: MKN45 and N87. RG13022 has a dose dependent, antiproliferative effect on both gastric cell lines when grown either in serum-free conditions or in the presence of FCS. Western blotting revealed RG13022 caused an inhibition of EGF stimulated tyrosine phosphorylation of EGFr in A431 cells and both EGFr and c-erbB-2 in MKN45 cells. No clear modulation of EGFr or c-erbB-2 phosphorylation was observed in N87 cells. In both A431 cells and N87 cells (which overexpress EGFr and c-erbB-2 respectively) exposed to EGF, MAP2 kinase immunoblot analysis resulted in the detection of a second protein band with reduced migration in SDS-PAGE. In N87 cells, this protein appeared to co-mi,orate with a strongly tyrosine phosphorylated protein, which suggests that it is a hyper-phosphorylated form of MAP2 kinase. However, treatment with RG13022, whilst inhibiting phosphorylation of this protein, did not prevent a shift in gel mobility (suggestive of activation) of MAP2 kinase in response to EGF. These findings demonstrate that the tyrphostin RG13022 inhibits cell proliferation of two primary gastric cancer cell lines. Investigation of intracellular signalling pathways suggests that alterations in intracellular signalling are responsible for the actions of RG 13022 in these cells. The biochemical analysis revealed that in N87 and A431, cells which overexpress c-erbB-2 and EGFr respectively, the tyrphostin affects the MAP2 kinase immunoreactivity and migration on SDS gels but fails to affect this protein in the MKN45 cell line. This data questions the usefulness of MAP2 kinase gel shift assays as markers of activation but supports the further development of tyrosine kinase inhibitors as potential inhibitors of gastric tumour proliferation.

Recombinant immunotoxin containing a disulfide-stabilized Fv directed at erbB2 that does not require proteolytic activation.

PE35/e23(dsFv)KDEL is a recombinant immunotoxin composed of a recombinant form of Pseudomonas exotoxin that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-erbB2 monoclonal antibody e23. In this molecule, the variable heavy (V H) domain is inserted near the carboxyl terminus of PE at position 607 and the variable light (V L) domain is connected to the V H domain by a disulfide bond engineered into the framework region. The disulfide bond forms between cysteines introduced at position 44 of V H and position 99 of V L [Reiter et al. (1994) J. Biol. Chem. 269, 18327-18331]. In contrast to other PE-derived Fv fusion proteins, this type of recombinant toxin does not need proteolytic activation of the toxin domain. PE35/e23(dsFv)KDEL is very cytotoxic toward erbB2 antigen-expressing N87 cells (IC50 = 0.8 ng/mL) despite the fact that it binds to the erbB2 protein only 25% as well as e23(dsFv)PE38KDEL, in which the dsFv moiety is located at the amino terminus of the toxin. The lower binding affinity is probably due to interference by domain III of PE with the amino terminus of e23(V H), possibly where the antigen binding sites are located. Nevertheless, the specificity of immunotoxin is still retained, and it is very stable at 37 degrees C. Because of its small size, stability, and activity without proteolytic processing, this immunotoxin may be advantageous for tumor treatment. PE35/e23(dsFv)KDEL was also used to gain information about whether reduction of the disulfide bonds connecting V H and V L occur in the endoplasmic reticulum (ER) or in a proximal compartment. To do this, we switched the ER retention sequence KDEL from the toxin--V H subunit to the V L subunit. Our results suggest that reduction of the disulfide bond connecting the dsFv heterodimer occurs before the immunotoxin reaches the ER, where translocation to the cytosol appears to occur.

Improved binding and antitumor activity of a recombinant anti-erbB2 immunotoxin by disulfide stabilization of the Fv fragment

e23(dsFv)-PE38KDEL is a recombinant immunotoxin composed of the Fv region of anti-erbB2 monoclonal antibody e23 connected to a truncated form of Pseudomonas exotoxin (PE38KDEL), in which the inherently unstable Fv heterodimer (composed of VH and VL) is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. We have now found that e23(dsFv)-PE38KDEL is considerably more cytotoxic to antigen-positive cell lines than the corresponding single-chain immunotoxin. The basis for the enhanced cytotoxic activity is that the e23 dsFv-immunotoxin binds to erbB2 with greater affinity than the single-chain counterpart. The dsFv-immunotoxin had 4-fold increased binding compared to the scFv and almost identical to the binding affinity of e23 Fab. e23(dsFv)-PE38KDEL was also considerably more stable at 37 degrees C than the single-chain immunotoxin. The therapeutic potential of the disulfide-stabilized immunotoxin was compared with its single-chain counterpart using two animal models of immunodeficient mice bearing subcutaneous tumor xenografts of human gastric tumor N87 cells or human A431 epidermoid carcinoma cells. The antitumor effect of e23(dsFv)-PE38KDEL was significantly better than that of the single-chain immunotoxin. e23(dsFv)-PE38KDEL caused complete regression of tumors at doses which caused no toxic effects in mice, whereas the single-chain immunotoxin did not cause complete regressions at the same doses.

Molecular cloning and expression of a cDNA encoding a protein detected by the ki antibody from an ovarian carcinoma (OVCAR‐3) cell line

MAb KI recognizes a cell-surface glycoprotein (MW approximately 40 kDa) present in ovarian carcinomas, malignant mesotheliomas, squamous-cell carcinomas and normal mesothelial cells. In this study, expression screening was used to isolate cDNA clones encoding an antigen recognized by MAb KI from a cDNA library made from a human ovarian carcinoma cell line (OVCAR-3). Subsequently, other clones were isolated by DNA hybridization using a cDNA probe derived from one of the initial clones. The sequence of all the clones was similar. The longest cDNA contains 2,444 base pairs, and encodes a polypeptide of 263 amino acids with a calculated molecular weight of 30,511 daltons. The nucleotide sequence and deduced amino-acid sequence of the protein show no homology to other sequences in current data bases. In vitro translation of RNA transcripts from the cDNA inserts yielded polypeptides of 29 and 30 kDa. Similar-sized proteins were obtained upon expression of the cDNA in Escherichia coli, and these proteins were reactive with MAb KI. The protein(s) expressed in E. coli were purified and used to make rabbit or mouse antisera. These antisera reacted strongly with a soluble cytosolic protein in OVCAR-3 cells, but not with the membrane-bound antigen. Soluble cytosolic proteins of a similar size, recognized with MAb KI, were found in OVCAR-3 and N87 (gastric cancer) cells but not in 10 other cancer cell lines. These data indicate that the cloned cDNA encodes a cytosolic protein that reacted with MAb KI. This soluble protein is expressed only in cells containing the CAKI surface glycoprotein, suggesting that the 2 proteins could be structurally related.