KATO-III Cells Research Articles

Abstract 1735: Evaluation of a novel flow cytometry-based system for the detection of circulating tumor cells

Abstract Background: The presence and number of circulating tumor cells (CTCs) in the blood of solid tumor patients is predictive of clinical outcome. To date the CellSearch system is the only FDA-approved CTCs enumeration system for advanced breast, prostate and colon cancers. However, sensitivity issues due to EpCAM-based enrichment and limited capability for subsequent molecular analysis need to be addressed for the use of CTCs in the clinical setting. We have developed a flow cytometry-based CTCs detection system independent of EpCAM expression which allows enumeration and characterization of CTCs for multiple molecular analyses. Here we report the results of preclinical study with this novel flow cytometry-based CTCs detection system. Materials and Methods: EpCAM-positive gastric cancer cell line KATO-III and lung cancer cell line PC-9 and EpCAM-negative lung cancer cell line A549 were used for this study. Cells were spiked into 1 mL or 4 mL of peripheral blood from healthy donors. Samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells followed by fixation and labeling with Alexa Fluor 700-CD45, PE-EpCAM and/or FITC-cytokeratin (CK) antibodies. The number of cancer cells in the enriched sample was counted using a novel FISHMAN-R flow cytometry system. CTCs enumeration and a head-to-head comparison with CellSearch system were carried out in a blinded manner at two different sites, Shizuoka cancer center (SCC) and National cancer center Tokyo (NCC) in parallel. Results: Various numbers of KATO-III and PC-9 cells were spiked into 1 mL of blood for a pilot study and sensitivity in detection was evaluated. Recovery rate ranged from 108.8 to 160.0% for KATO-III and from 70.0 to 74.7% for PC-9 cells, respectively. Good correlation was observed between the observed number and the expected number of cancer cells by regression analysis demonstrating a slope of 1.07 and a correlation coefficient (R2) of 0.99 for KATO-III and a slope of 0.75 and R2 of 1.00 for PC-9 cells, respectively. Then we proceeded to the spike-in experiment in 4 mL of blood and this study was performed at two different sites. Recovery rate ranged from 89.0 to 120.0% at SCC and from 88.0 to 130.0% at NCC, respectively, suggesting the robustness of our detection system. In the following head-to-head comparison study with CellSearch system in a blinded manner, the average recovery rate was 90.3% at SCC, 87.6% at NCC and 62.0% by CellSearch system, respectively. EpCAM-negative lung cancer cell line A549 cells were also spiked into 4 mL of peripheral blood and enumerated by our system and CellSearch system. Markedly higher recovery rate was observed with our system (91.9%) than with CellSearch system (28.8%), suggesting superior sensitivity of our system in capturing EpCAM-negative tumor cells. Conclusions: These data imply the potential of our detection system and further evaluation with clinical samples should be conducted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1735. doi:1538-7445.AM2012-1735

Characterization of a neuronostatin receptor in a human kidney cell line

Neuronostatin (NST), a recently discovered peptide hormone derived from the somatostatin preprohormone (JBC 283:31949), was shown previously to induce c‐Fos expression in the human gastric cancer cell line (KATOIII). Preliminary data from our lab suggests that the orphan G‐protein coupled receptor, GPR107, is a cognate receptor for neuronostatin. In this study we further explored neuronostatin activity in a human embryonic kidney cell line (HEK 293). GPR107 expression was detected in untreated HEK 293 cells; we therefore hypothesized that HEK 293 cells would respond to treatment with neuronostatin with an increase in c‐Fos expression. Indeed, NST induced a concentration dependent increase in c‐Fos expression after a 60 minute exposure, as determined by real time PCR. These data suggest that HEK 293 cells are a useful model to study neuronostatin signaling. In ongoing experiments, we are attempting to compromise the production of GPR107 in HEK 293 cells using siRNA and, by assaying for responsiveness to neuronostatin, confirm findings in KATOIII cells and in vivo models that have identified GPR107 as the cognate receptor for endogenously produced neuronostatin. Supported by NIJ HL66023.

Role of p53β in the inhibition of proliferation of gastric cancer cells expressing wild-type or mutated p53.

p53 is a tumor suppressor gene whose mutation is highly associated with tumorigenesis. The present study investigated the role of p53β in the inhibition of proliferation of gastric cancer cell lines expressing wild-type or mutated p53. Wild-type p53 is expressed in MKN45 cells, but deleted in KATOIII cells, whereas mutated p53 is expressed in SGC7901 cells. The mRNA expression levels of p53β and Δ133p53 were detected in MKN45, SGC-7901 and KATOIII gastric cancer cell lines using nested polymerase chain reaction (PCR). The mRNA expression levels of p53, p53β and B-cell lymphoma 2-associated X protein (Bax) were detected in the MKN45 and SGC-7901 cells following treatment with cisplatin by reverse transcription-PCR. The inhibition of cellular proliferation following treatment with cisplatin was measured by MTT assay. The results of the present study demonstrated that both p53β and Δ133p53 mRNA were expressed in the MKN45 cells, whereas only p53β mRNA was expressed in the SGC7901 cells. No expression of p53β or Δ133p53 mRNA was detected in the KATOIII cells. Following treatment with cisplatin, the number of both MKN45 and SGC-7901 cells was significantly reduced (P<0.001). In the MKN45 cells, p53β, p53 and Bax mRNA expression levels gradually increased with the dose of cisplatin, and the expression of p53β was positively correlated with the expression of p53 (tr=6.358, P<0.05) and Bax (tr=8.023, P<0.05). In the SGC-7901 cells, the expression levels of p53β, p53 and Bax mRNA did not alter with the dose of cisplatin, and the expression of p53β was positively correlated to the expression of p53 (tr=26.41, P<0.01) but not that of Bax. The present study identified the different roles of the p53β isoform in gastric cancer cells with different p53 backgrounds. Enhanced knowledge regarding the p53 status is required for the development of specific biological therapies against gastric cancer.

Effect of a combination of aurora kinase A inhibitor MLN-8237 and cisplatin on gastrointestinal tumors.

58 Background: Gastric cancers respond poorly to current chemotherapeutic protocols indicating active intrinsic mechanisms against drug induced cell death. We have previously reported that AURKA overexpression induces pro-survival (AKT and β-catenin) and inhibits apoptotic (p53 and p73) pathways. Methods: We evaluated the effect of AURKA specific inhibitor (MLN-8237) alone and/or in combination with cisplatin (CDDP) in gastrointestinal (GI) cancer cells. Following treatment with MLN-8237 and/or CDDP, cell survival, cell viability, western blot and qRT-PCR analyses were done on GI cancer cell lines. Additionally, tumor xenografted mice were treated with MLN (30mg/kg) and/or CDDP (2mg/kg) for 21 days and tumor volume was measured every alternate day. Results: The ATP-Glo cell viability and cell survival assay data indicated that in comparison to single agent treatments alone; the combination treatment with MLN-8237 (0.5µM) and CDDP (2.5µM) significantly increased inhibition of cellular viability and survival (p&lt;0.05) in AGS, KATO-III, MKN28, FLO-1, OE-19 and OE33 cell lines. Similarly, the western blot data exhibited higher levels of p73, Puma, Noxa, cleaved caspase 3 and cleaved PARP with MLN (0.5µM) and CDDP (2.5µM) combination treatment, as compared to single agent treatments in AGS, FLO-1 and OE33 cell lines. The tumor growth results for OE33 mouse xenograft study demonstrate synergistic antitumor effect with MLN-8237 and CDDP combination in the following order of effectiveness: Control &gt; CDDP &gt; MLN-8237 &gt; and MLN-8237/CDDP (p&lt;0.01). Moreover, the qRT-PCR data from tumor xenograft samples treated with MLN-8237/CDDP combination showed significantly higher mRNA levels of p73 downstream transcriptional target genes (P21, PUMA, NOXA and BAX) (p&lt;0.05). Conclusions: Our in vitro and in vivo data indicate that inhibition of AURKA with MLN-8237 in combination with CDDP enhances the antitumor activity of MLN-8237 against GI cancer cells. Therefore, our study demonstrates that MLN-8237 is an effective anti-tumor agent which can be potentially combined with CDDP for better therapeutic outcome in GI cancer patients.

A WWOX-binding molecule, transmembrane protein 207, is related to the invasiveness of gastric signet-ring cell carcinoma

Using the PCR-based subtractive messenger RNA hybridization assay described in this paper, we isolated a hitherto uncharacterized gene, transmembrane protein 207 (TMEM207), which was selectively expressed in collagen gel-invading cultured signet-ring cell carcinoma KATO-III cells. TMEM207 has a C-terminal proline-rich PPxY motif, which binds to the WW domain-containing oxidoreductase, WWOX. Enforced expression of TMEM207 significantly increased Matrigel invasion activity of KATO-III cells in vitro without affecting cell growth. In contrast, expression of TMEM207 with mutations in the PPxY motif did not significantly increase Matrigel invasion activity of KATO-III cells. Immunohistochemical staining showed that TMEM207 was strongly expressed in 7 of 30 gastric signet-ring cell carcinoma tissue specimens. Notably, TMEM207 expression was associated with the depth of cancer invasion and the presence of lymph node metastasis. The results of co-immunoprecipitation followed by western immunoblotting showed that TMEM207 is bound to WWOX in a PPxY motif-dependent manner. Small interfering RNA-mediated downregulation of WWOX also significantly increased Matrigel invasion activity of KATO-III cells. Notably, exogenous expression of TMEM207 impaired the WWOX-mediated repression of Matrigel invasion activity of another cultured signet-ring cell carcinoma cell line, NUGC-4 cells. Recent studies have highlighted the fact that WWOX acts as a tumor suppressor factor in various malignant tumors, including gastric cancer. On the basis of these findings and the results of the present study, we think that overexpression of TMEM207 may facilitate invasive activity and metastasis of gastric signet-ring cell carcinoma, which possibly occur through binding to WWOX and attenuation of its function.

Interleukin-8 associates with adhesion, migration, invasion and chemosensitivity of human gastric cancer cells

To investigate the relationship between Interleukin-8 (IL-8) and proliferation, adhesion, migration, invasion and chemosensitivity of gastric cancer (GC) cells. The IL-8 cDNA was stably transfected into human GC cell line MKN-45 and selected IL-8-secreting transfectants. The expression of IL-8 in human GC cell line KATO-III was inhibited by RNA interference. The expressions of mRNA and protein of IL-8 in GC cells were detected by real-time reverse transcription-polymerase chain reaction or enzyme-linked immunosorbent assay (ELISA). The overexpression of IL-8 resulted in an increased cell adhesion, migration and invasion, and a significant resistance to oxaliplatin in MKN-45 cells. Inhibition of IL-8 expression with small interfering RNA decreased the adhesion, migration and invasion functions and oxaliplatin resistance in KATO-III cells. IL-8 increased NF-κB and Akt activities and adhesion molecules ICAM-1, VCAM-1, and CD44 expression in GC cells. Overexpression of IL-8 promotes the adhesion, migration, invasion, and chemoresistance of GC cells, indicating that IL-8 is an important therapeutic target in GC.

Blockade of Chloride Ion Transport Enhances the Cytocidal Effect of Hypotonic Solution in Gastric Cancer Cells

Cancer cells that are exfoliated into the peritoneal cavity during surgery are viable and have the potential to produce peritoneal recurrence. Although peritoneal lavage with distilled water is applied in some cancer surgeries to kill tumor cells, there is no consensus regarding the optimal methodology and its effects. Three human gastric cancer cell lines, MKN28, MKN45, and Kato-III, were exposed to distilled water, and the resultant morphologic changes were observed using a microscope. Analysis of cell volume changes was performed using a flow cytometer. To investigate the cytocidal effects of the water, re-incubation of the cells was performed after exposing them to hypotonic solution. Additionally, the effects of 5-nitro-2-3-phenylpropylamino)-benzoic acid (NPPB), a Cl(-) channel blocker, and R(+)-[(dihydroindenyl)oxy] alkanoic acid (DIOA), a blocker of the K(+)/Cl(-) co-transporter, on the cells during their exposure to hypotonic solution were analyzed. After the cells had been exposed to the distilled water, a rapid increase in cell volume occurred followed by cell rupture. In the MKN45 and Kato-III cells, treatment with NPPB increased cell volume by inhibiting regulatory volume decrease and enhanced the cytocidal effects of the hypotonic solution, whereas no such effects were observed in the MKN28 cells. On the other hand, treatment of the MKN28 cells with DIOA inhibited RVD and enhanced the cytocidal effects of hypotonic shock. These findings support the efficacy of peritoneal lavage with distilled water during surgery for gastric cancer and suggest that the regulation of Cl(-) transport enhances the cytocidal effects of hypotonic shock.

Abstract B236: Pazopanib, a novel multitargeted kinase inhibitor, shows potent in vitro antitumor activity in gastric cancer cell lines harboring FGFR2 amplification, which is a potential therapeutic target in gastric cancer.

Abstract Background: Identification of critical genes which play pivotal role in controlling tumor growth and survival will establish the basis for developing therapeutic targets. However, these approaches have not been extensively studied in gastric cancer (GC). The fibroblast growth factor receptor (FGFR) tyrosine kinase family has long been implicated in cancer. Among them, recent studies have shown that activating somatic mutations in FGFR2 are correlated with sensitivity to FGFR2 kinase inhibition in the cell lines bearing such FGFR2 mutations, implicating FGFR2 as a novel therapeutic target. Pazopanib is an orally bioavailable, ATP competitive, multi-targeted kinase inhibitor mainly targeting VEGFR2 and PDGFR tyrosine kinases, which results in selectively inhibiting VEGF-induced angiogenesis. However, the biologic sequences of pazopanib activities beyond anti-angiogenesis are poorly defined. Method and Results: Using our in vitro platform for modeling the genotype-correlated drug sensitivity in a panel of 37 GC cell lines, we performed this preclinical study to test the efficacy of pazopanib, whose clinical activity is not well recognized in gastric cancer. In growth inhibition assay, pazopanib showed differential effect on cell growth according to the genomic changes. Treatment of the KATO-III, OCUM-2M, SNU-16 and HSC-39 gastric cancer cell lines harboring FGFR2 amplification with pazopanib resulted in a marked decrease in cell survival with IC50 in ranges of 0.1 ∼ 2.0 μM, respectively, while same treatment of the cell lines without FGFR2 amplification had no growth inhibitory effect. In growth inhibition and colony formation assay using ectopic FGFR2-expressing model, treatment with the indicated concentrations of pazopanib significantly inhibited cell growth and colony formation by the FGFR2-expressing NIH 3T3 cells with WT FGFR2 and mutant FGFR2 (S252W), as compared with the NIH-3T3 cell with vector. Pazopanib also selectively suppressed the constitutive FGFR2 signaling effectively abrogated the baseline phosphorylation of downstream effectors of growth factor receptors, such as Erk and Akt in FGFR2-amplified cells. In cell cycle analysis, the FGFR2-amplified cells underwent cell cycle arrest at the G1-S phase after pazopanib treatment, whereas no significant effect on cell cycle progression in cells without FGFR2 amplification treated with pazopanib. Also, only in the FGFR2-amplified cells did pazopanib increase a substantial fraction of sub-G1. Conclusion: Taken together, our findings show activation of FGFR2 signaling by amplification may be a critical mediator of cell proliferation in the small subset of GC patients and may sensitize these cancer cells to pazopanib. Therefore these results suggest that pazopanib may provide genotype-correlated clinical benefit beyond the setting of highly vascular tumor like RCC, which should be further explored as a novel therapeutic target in prospective clinical trials. 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 B236.

Analysis of the Cytotoxicity of Green Pigment Produced on the Surface of Roasted and Retorted Chestnuts Using Immune Cells and Gastrointestinal Cancer Cells

Roasted and retorted (RR) chestnuts develop green pigment spots on their surface during storage. The purpose of this study was to evaluate the cytotoxicity of the green pigment using RAW 264.7, MOLT-4, KATOⅢ and HT-29 cells. The pigment scraped from RR chestnuts (GP), whole RR chestnuts with green pigment spots (GC), whole RR chestnuts without green pigment (WC) and roasted and frozen stored chestnuts (FC) were extracted in 10% DMSO. MOLT-4 cells were less resistant to the cytotoxicity of the chestnut extracts than the RAW 264.7 cells. The GP extracts did not show different responses against H2O2-induced oxidative stress and LPS-induced NO production compared to the other extracts. The chestnut extracts did not have proliferative activity on either of the KATOⅢ or HT-29 cells (p>0.05). Our results from the comparison of the green pigment produced on the surface of the RR chestnuts to chestnuts that do not develop the green pigment suggest that the pigment may not be harmful in terms of either cytotoxicity towards immune cells or proliferation of gastrointestinal cancer cells.

The 3′ flanking region of the human ABO histo-blood group gene is involved in negative regulation of gene expression

Gene expression is driven by promoters, enhancers, silencers, and other cis-regulatory elements upstream and downstream of the gene. Previous studies of the regulation of human ABO gene transcription have focused mainly on the 5′ region, including the core promoter and the region proximal to it. However, as the involvement of the 3′ flanking region in transcriptional regulation has not yet been examined, we focused on this issue. The 3′ region approximately 2.2 kb downstream of the ABO gene was PCR-amplified and inserted into a cloning vector, followed by sequence determination and preparation of luciferase reporter vectors. Transient transfections into KATOIII and K562 cells were performed using various reporter plasmids containing the 3′ region. The 3′ region of the ABO gene, which was characterized by a high degree of sequence repetition, was effectively cloned by a single-copy cloning method. Transfections in KATOIII and K562 cells showed that negative elements were demonstrable within the 3′ region. These observations suggest that negative regulatory elements seem to be present in the 3′ region of ABO in both epithelial and erythroid lineages. As we had observed a negative region just upstream of the ABO promoter, transcription from ABO could be negatively regulated by repressive regions just upstream of the promoter and downstream of the gene. Further studies of the enhancer will be required for elucidating the molecular basis of ABO gene expression.

Heparin-binding epidermal growth factor expression in KATO-III cells after Helicobacter pylori stimulation under the influence of strychnos Nux vomica and Calendula officinalis

Previous studies have shown the stimulating effect of Helicobacter pylori on the gene expression of heparin-binding epidermal growth factor (HB-EGF) using the gastric epithelial cell line KATO-III. Strychnos Nux vomica (Nux vomica) and Calendula officinalis are used in highly diluted form in homeopathic medicine to treat patients suffering from gastritis and gastric ulcers. To investigate the influence of Nux vomica and Calendula officinalis on HB-EGF-like growth factor gene expression in KATO-III cells under the stimulation of H. pylori strain N6 using real-time PCR with and without addition of Nux vomica and Calendula officinalis as a 10c or 12c potency. Baseline expression and stimulation were similar to previous experiments, addition of Nux vomica 10c and Calendula officinalis 10c in a 43% ethanolic solution led to a significant reduction of H. pylori induced increase in gene expression of HB-EGF (reduced to 53.12+/-0.95% and 75.32+/-1.16% vs. control; p<0.05), respectively. Nux vomica 12c reduced HB-EGF gene expression even in dilutions beyond Avogadro's number (55.77+/-1.09%; p<0.05). Nux vomica 12c in a 21.5% ethanol showed a smaller effect (71.80+/-3.91%, p<0.05). This effect was only be observed when the drugs were primarily prepared in ethanol, not in aqueous solutions. The data suggest that both drugs prepared in ethanolic solution are potent inhibitors of H. pylori induced gene expression.

Abstract 200: Methylation of GATA4 and GATA5 transcription factor genes in gastric cancers

Abstract GATA factors comprise a small family of transcription regulatory proteins containing two conserved zinc finger DNA binding domains for the consensus sequence (WGATAR). Among the six GATA factors, GATA-1/2/3 factors are prominently expressed in the hematopoietic cell lineage, while GATA-4/5/6 factors show a partly overlapping expression pattern in the heart and endoderm-derived organs. We previously reported that GATA4 and GATA5 were silenced by their promoter hypermethylation in gastrointestinal, lung and ovarian cancers. However, it remains unknown the mechanism underlying loss of GATA transcription factors in gastric carcinogenesis. To clarify the role of GATA, we examined GATA4/5 methylation and their downstream target genes in gastric cancer (GC). By using methylation specific PCR, the methylation frequency of GATA5 (30, 39%) was significantly higher than that of GATA4 (10, 13%) in 77 GC tissues (P&amp;lt;0.01). However, methylation of GATA6 was not observed in GCs examined. We transiently over-expressed GATA4 or GATA5 in GC cell lines lacking their expression by aberrant methylation. Both GATA4 and GATA5 suppressed colony formation and cell proliferation in MKN74 cells. After GATA4/5 over-expression in MKN74 and KATO-III cells, enhanced expression of numerous genes including ones indicating tumor-suppressive functions, such as TFF1, disabled-2 and IQGAP2, was observed by microarray and RT-PCR analyses. The target genes of GATA4 and GATA5 were closely similar in MKN74 cells. We also preformed siRNA transfection of GATA4 and GATA5 in AGS and HSC43 cells which basally expressed them. Reduced expression of above predicted GATA target genes was detected in these two cell lines after siRNA transfection. These data suggest that loss of GATA4 and GATA5 expression may contribute to gastric carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 200.

Abstract 284: FGFR2 is required for growth and survival in a rare subset of FGFR2 amplified colorectal cancer

Abstract FGFR2 amplification has been reported in subsets of gastric cancer and breast cancer. We describe FGFR2 amplification in a cell line derived from a colorectal carcinoma arising in a young patient with ulcerative colitis. The cell line has features of endocrine differentiation, representing a rare (&amp;lt; 1%) subset of colorectal cancer. FGFR2 amplification in this cell line results in an overexpressed and highly activated FGFR2 kinase. The FGFR selective inhibitor PD173074 blocked FGFR2 phosphorylation and inhibited Erk, Akt, and S6 ribosomal protein phosphorylation. FGFR2 inhibition resulted in profound cell death as measured by robust accumulation of cells in subG1 by flow cytometry and PARP cleavage by western analysis. A structurally unrelated FGFR2 inhibitor, MK2461, also caused cell death and inhibited downstream signaling pathways. RTK arrays revealed coactivation of EGFR family kinases, and a functional role for these kinases is being investigated. Interestingly, an FGFR2 isoform in this cell line migrated at 160kD, in contrast to the 140kD isoform observed in SNU16 and other cell lines. However, C-terminal FGFR2 antibodies did not react with the 160kD FGFR2 isoform, suggesting that it has a C-terminal deletion similar to the FGFR2 isoform in OCUM2M and KatoIII cell lines. We are currently defining the frequency of FGFR2 overexpression and activation in colon cancer tissue microarrays. The strong growth inhibition and induction of cell death observed in vitro suggests that colorectal cancer tumor cells harboring FGFR2 amplification / activation may show similar response to FGFR inhibitor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 284.

Nuclear or cytoplasmic localization of Bag-1 distinctly correlates with pathologic behavior and outcome of gastric carcinomas

Bag-1 is an antiapoptotic protein with its altered expression and localization in malignancies. To clarify the role of Bag-1 in gastric carcinogenesis, its expression was examined by immunohistochemistry and in situ hybridization on a tissue microarray containing gastric carcinomas, adjacent nonneoplastic mucosa (NNM), adenomas, intestinal metaplasia (IM), or gastritis. Gastric carcinoma tissue and cell lines were studied for Bag-1 expression by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). The results demonstrated that Bag-1 proteins were differentially expressed in the nucleus or cytosol of MKN28, AGS, MKN45, KATO-III, or HGC-27 cell lines, despite similar levels of messenger RNA (mRNA) expression. The Bag-1 mRNA overexpression was detectable in 73.3% of 15 gastric carcinomas without significant difference in its encoding products' levels. The nuclear Bag-1 expression gradually decreased from gastritis, IM, adenoma to carcinoma (P < .05), and negatively correlated with lymphatic invasion or lymph node metastasis, cytoplasmic Bag-1 expression, negative parafibromin expression, and poor prognosis (P < .05). Cytoplasmic Bag-1 was weakly immunoreactive in carcinomas, compared with gastritis (P < .05), and positively associated with invasive depth and poor prognosis of the carcinoma (P < .05). The positive rate of Bag-1 mRNA expression was higher in adjacent IMs than carcinomas or adjacent NNM (P < .05). Bag-1 mRNA was expressed more in carcinomas from female patients than the male counterparts (P < .05). There was a positive correlation of Bag-1 mRNA expression with invasive depth and venous invasion (P < .05). Our study indicated that aberrant expression and subcellular distribution of Bag-1 might play an important role in the malignant transformation of gastric epithelial cells and should be considered as a biomarker for gastric carcinogenesis, subsequent progression, and prognosis.

Nucleolin on the Cell Surface as a New Molecular Target for Gastric Cancer Treatment

Nucleolin is an abundant non-ribosomal protein found in nucleolus and a major component of silver-stained nucleolar organizer region (AgNOR), a histopathological marker of cancer which is highly elevated in cancer cells. We recently reported that nucleolin on the cell surface of mouse gastric cancer cells acts as a receptor for tumor necrosis factor-alpha-inducing protein (Tipalpha), a new carcinogenic factor of Helicobacter pylori. In this study, we first examined the localization of nucleolin on cell surface of five gastric cancer cell lines by cell fractionation and flow cytometry: We found that large amounts of nucleolin were present on surface of MKN-45, KATOIII, MKN-74, and AGS cells, with smaller amounts on surface of MKN-1 cells. The membrane fraction of normal epithelial cells of mouse glandular stomach did not contain much nucleolin, suggesting that translocation of nucleolin to the cell surface occurs during carcinogenesis, making for easier binding with Tipalpha. AS1411, a nucleolin targeted DNA aptamer, inhibited growth of gastric cancer cell lines in this order of potency: MKN-45>KATOIII>AGS>MKN-74=MKN-1, associated with induction of S-phase cell cycle arrest. Fluorescein isothiocyanate (FITC)-AS1411 was more rapidly incorporated into MKN-45 and AGS than into MKN-1 cells, based on varying amounts of cell surface nucleolin. We think that AS1411 first binds to nucleolin on the cell surface and that the binding complex is then incorporated into the cells. All results indicate that nucleolin on the cell surface is a new and promising therapeutic target for treatment of gastric cancer.

CD44 and CD133 as Cancer Stem Cell Markers for Gastric Cancer

Purpose: Currently, the two most influential gastric stem cell marker candidates are CD44 and CD133. The aim of this study was to make a comparison and determine the appropriate marker for use in gastric cancer stem cell research. Materials and Methods: We analyzed the expressions of CD44, CD133, and CD24 from the gastric cancer cell lines MKN45, MKN74, KATO-III, NCI-N87, SNU-1, SNU-216, SNU-601, SNU-638, and SNU-688 using flow cytometry. In addition, we measured the change in viability after applying 5 fluorouracil (5-FU) to the MKN45, MKN74, KATO-III, and NCI-N87 cell lines using a Cell Counting Kit 8. Results: CD133 expression was above moderate in the KATO-III, SNU-216, SNU-601 cell lines, whereas it was below 1% in the remaining cell lines. CD44 was expressed at levels above 5% in all gastric cancer cell lines. The effect of 5-FU on viability and CD133 or CD44 expression in the cell lines were not related. Conclusions: Expression of CD133 positive cells was insufficient in the gastric cancer cell lines. Therefore, of the cell lines tested, CD44 was the most appropriate tumor maker for research on gastric cancer stem cells.

Abstract B81: AUY922, a heat shock protein 90 inhibitor, actively inhibits human gastric cancer cells via down regulation of growth factor receptors, and acts synergistically with relevant chemotherapeutic agents

Abstract Heat shock protein 90 (Hsp90) is the most abundant cellular chaperone and has nearly 100 pivotal client proteins, which are commonly dysregulated in cancers. The positive rate of Hsp90beta in gastric cancer tissue was approximately 30%, and was higher than non-cancerous gastric mucosa. Hsp90 inhibitors should have great potentials in the exploration for treatment strategy in gastric cancer (GC). AUY922, kindly provided by Novartis Pharma AG, is a novel resorcinylic isoxazole amide Hsp90 inhibitor. A panel of human GC cell lines (N87, AGS, SNU-1, SNU-16, KATO-III from ATCC) was used in this study. Using 72-hour MTT colorimetric assay, we demonstrated that AUY922 has highly active growth inhibitory effects as a single agent of IC50 in the ranges between 8.1 ± 0.2 and 14.7 ± 0.8 nM (except the c-met-overexpressing KATO-III cells with a IC50 of 158.1 ± 5.1 nM), which is more active than 17-AAG (17-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethyl-amino-17-demethoxygeldanamycin) of IC50 between 17.2 ± 0.6 and 61.3 ± 1.9 nM, and IC50 between 10.5 ± 0.5 and 24.3 ± 1.0 nM, respectively. By MTT colorimetric assay and median effect analysis, AUY922 has significant synergistic cytotoxicity with cisplatin, oxaliplatin, 5-fluorouracil (5-FU), and irinotecan, while acts antagonistically with paclitaxel and docetaxel in GC cells. Several important growth factor receptors are highly expressed in GC, namely 50-60% of EGFR (epidermal growth factor receptor, HER1), 15–22% of HER-2/neu, and 32–58% of c-met (hepatocyte growth factor receptor, HGF receptor), respectively. By immunoblotting analysis, AUY922 effectively down-regulates growth factor receptors (c-met, EGFR, and HER-2/neu) with a concentration of 25nM, 25nM, and 125nM in KATO-III GC cells, respectively. The c-met-overexpressing KATO-III cells are highly resistant to AUY922. In KATO-III cells, knocking-down of c-met by siRNA, then examining by 48-hour MTT colorimetric assay may partly abrogate the AUY922 resistance and increase sensitivity to AUY922 by decreasing IC50 from 790.0 ± 10.6 nM to 350.8 ± 5.4 nM. Our data indicate that AUY922 actively inhibits human GC cells via down regulation of growth factor receptors, and acts synergistically with various relevant chemotherapeutic agents. It is reasonable to use AUY922 alone, or to combine AUY922 with relevant chemotherapeutic agents (cisplatin, oxaliplatin, 5-FU, or irinotecan) in future development of clinical trials for gastric cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B81.

Abstract C145: The nonredundant oncogenic role of miR-17-5p in gastric cancer in vitro

Abstract MicroRNAs are non-coding RNA molecules that consist of 19 to 25 nucleotides. Overexpression of miR-17-92 cluster is reported in many human cancers including gastric cancers, the second most common cause of cancer mortality worldwide. While the data in the literature predominantly supports the oncogenic role of miR-17-92 cluster, some studies suggested tumor suppressor role of the miR-17-92 cluster. Given that function of a given microRNA is context-dependent, the exact function of miR-17-92 cluster in gastric cancer cells should be experimentally documented in order to validate this microRNA as a novel therapeutic target. MiR-17-5p is one member of the miR-17-92 cluster, the other members of which are miR-18a, miR-19a, miR-20a, miR-19b and miR-92. Although knockdown of mir-17-5p is reported to inhibit the growth of neuroblastoma and breast carcinoma cells, the intracluster redundancy has not been fully evaluated in gastrointestinal cancers. We tested a functional role of miR-17-5p in gastric cancer cells, given our patient data that miR-17-5p is significantly overexpressed in gastric cancer patients, compared with healthy volunteers. MiR-17-5p knock-down was performed in KATO-III human gastric cancer cell line, using commercially available lentiviral vectors targeting specifically miR-17-5p. Knock-down of miR-17-5p significantly decreased the proliferation rate of KATO-III cells. A smaller number of KATO-III colonies were formed in soft agar after miR-17-5p knock-down, suggesting a critical role of miR-17-5p in anchorage-independent cell growth. On the other hand, there was no change in the sensitivity of KATO-III cells to either fluorouracil or cisplatin after miR-17-5p knock-down, consistent with our patient data showing no association of miR-17-5p expression with chemotherapy response. These data suggests the non-redundant oncogenic function of miR-17-5p in vitro, leading to increased growth, but not decreased apoptosis of gastric cancer cells. Thus, inhibiting miR-17-5p is a promising therapeutic strategy against gastric cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C145.

Downregulation of SFRP5 expression and its inverse correlation with those of MMP-7 and MT1-MMP in gastric cancer

BackgroundAs negative regulators in Wnt signaling, Secreted Frizzled-Related Proteins (SFRPs) are downregulated in a series of human cancers; and specifically, some matrix metalloproteinases (MMPs), including MMP-2, MMP-7, MMP-9 and MT1-MMP, are frequently overexpressed in gastric cancer. The aim of this study is to determine the expression status of SFRP5 in gastric cancer and explore the correlation between both the expression of SFRP5 and that of these MMPs in this cancer.MethodsExpression of SFRP5, MMP-2, MMP-7, MMP-9 and MT1-MMP was determined by real-time PCR, RT-PCR or Western blotting. The methylation status of SFRP5 was detected by Methylation-specific PCR (MSP). Cell lines with SFRP5 methylation were demethylated by a DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (DAC). KatoIII cells were transfected with pcDNA3.1 SFRP5 vector to strengthen SFRP5 expression. To abrogate SFRP5 expression in MKN1 cells, SFRP5 RNAi plamid was used to transfect them.ResultsSFRP5 expression was remarkably downregulated in 24 of 32 primary gastric cancer specimens, and even was not detectable in 5 of 8 gastric cancer cell lines. MMP-7 and MT1-MMP mRNA showed a stronger expression in these 24 specimens compared to the other 8 specimens. They also showed higher levels in gastric cancer cell lines AGS and NCI-N87 which had no SFRP5 expression, compared to MKN1 with strong SFRP5 expression. However, they were significantly downregulated, with SFRP5 expression restored in AGS and NCI-N87; and were considerably upregulated with it abrogated in MKN1.ConclusionThe results indicate there are frequent occurrences of downregualtion of SFRP5 expression in gastric cancer, primarily due to SFRP5 methylation. It seems to be responsible for the upregulation of MMP-7 expression and MT1-MMP expression on the ground that they are inversely correlated with SFRP5 expression.

DNA methyltransferase inhibitor 5‐aza‐CdR enhances the radiosensitivity of gastric cancer cells

The National Comprehensive Cancer Network guidelines recommend radiotherapy as a standard treatment for patients with a high risk of recurrence in gastric cancer. Because radiation is harmful to the surrounding organs, a radiation sensitizer might therefore be useful to decrease the side effects of patients with advanced gastric carcinoma. The aim of the current study was to clarify the effect of a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (CdR), on radiation sensitivity in gastric cancer cells. Five gastric cancer cell lines, OCUM-2M, OCUM-12, KATO-III, MKN-45, and MKN-74, were used. The effects of 5-aza-CdR with irradiation on the growth activity, cell-cycle distribution, apoptosis, and apoptosis-associated gene expression were examined. 5-aza-CdR sensitized three of five gastric cancer cell lines to radiation. A combination of irradiation and 5-aza-CdR significantly (P<0.05) decreased the growth activity compared with irradiation alone in OCUM-2M, OCUM-12, and MKN-45 cells, but not in KATO-III and MKN-74 cells. The percentage of cells in G2-M phase and the apoptotic rate with irradiation in combination with 5-aza-CdR were increased in OCUM-2M, OCUM-12, and MKN-45 cells compared with irradiation alone, but not in KATO-III and MKN-74 cells. 5-aza-CdR increased the expression of p53, RASSF1, and death-associated protein kinases (DAPK) genes compared with the control or irradiation alone. These findings suggest that 5-aza-CdR might therefore be useful as a radiation sensitizer to treat some types of gastric carcinoma. The arrest at G2-M phase and increased apoptotic rate might be partly mediated by enhanced expression of the p53, RASSF1, or DAPK gene families by 5-aza-CdR.