Gastric Carcinoma Cell Line MKN-45 Research Articles

Mechanobiology Platform Realized Using Photomechanical Mxene Nanocomposites: Bilayer Photoactuator Design and In Vitro Mechanical Forces Stimulation.

Mechanotransduction is the process by which cells convert external forces and physical constraints into biochemical signals that control several aspects of cellular behavior. A number of approaches have been proposed to investigate the mechanisms of mechanotransduction; however, it remains a great challenge to develop a platform for dynamic multivariate mechanical stimulation of single cells and small colonies of cells. In this study, we combined polydimethylsiloxane (PDMS) and PDMS/Mxene nanoplatelets (MNPs) to construct a soft bilayer nanocomposite for extracellular mechanical stimulation. Fast backlash actuation of the bilayer as a result of near-infrared irradiation caused mechanical force stimulation of cells in a controllable manner. The excellent controllability of the light intensity and frequency allowed backlash bending acceleration and frequency to be manipulated. As gastric gland carcinoma cell line MKN-45 was the research subject, mechanical force loading conditions could trigger apoptosis of the cells in a stimulation duration time-dependent manner. Cell apoptotic rates were positively related to the duration time. In the case of 6 min mechanical force loading, apoptotic cell percentage rose to 34.46% from 5.5% of the control. This approach helps apply extracellular mechanical forces, even with predesigned loading cycles, and provides a solution to study cell mechanotransduction in complex force conditions. It is also a promising therapeutic technique for combining physical therapy and biomechanics.

Inhibition of Membrane-Associated Catalase, Extracellular ROS/RNS Signaling and Aquaporin/H2O2-Mediated Intracellular Glutathione Depletion Cooperate during Apoptosis Induction in the Human Gastric Carcinoma Cell Line MKN-45.

The human gastric carcinoma cell line MKN-45 is a prototype of bona fide tumor cells, as it is protected from the NADPH oxidase-1 (NOX-1)-driven HOCl- and nitric oxide (NO)/peroxynitrite apoptosis-inducing signaling pathways by a membrane-associated catalase. The use of inhibitors/scavengers shows that inhibition of membrane-associated catalase is sufficient for the activation of NO/peroxynitrite or HOCl signaling. However, this signaling is not sufficient for apoptosis induction, as intracellular glutathione peroxidase/glutathione counteracts these signaling effects. Therefore, intrusion of extracellular tumor cell-derived H2O2 through aquaporins is required for the full apoptosis-inducing effect of extracellular reactive oxygen/nitrogen species. This secondary step in apoptosis induction can be prevented by inhibition of aquaporins, inhibition of NOX1 and decomposition of H2O2. Pretreatment with inhibitors of glutathione synthase or the cysteine-glutamine antiporter (xC transporter) abrogate the requirement for aquaporin/H2O2-mediated glutathione depletion, thus demonstrating that intracellular glutathione is the target of intruding H2O2. These data allow definition of mechanistic interactions between ROS/RNS signaling after inhibition of membrane-associated catalase, the sensitizing effects of aquaporins/H2O2 and the counteraction of the xC transporter/glutathione synthase system. Knowledge of these mechanistic interactions is required for the understanding of selective apoptosis induction in tumor cells through reestablishment of apoptosis-inducing ROS/RNS signaling.

Pyruvate Kinase M2 Knockdown Suppresses Migration, Invasion, and Epithelial-Mesenchymal Transition of Gastric Carcinoma via Hypoxia-Inducible Factor Alpha/B-Cell Lymphoma 6 Pathway.

Gastric carcinoma is a common malignant cancer. Pyruvate kinase M2 (PKM2) is highly expressed in cancers, including gastric carcinoma. However, its function and molecular mechanism in gastric carcinoma remains unclear. Here, we aimed to explore the function and the underlying mechanism of PKM2 on malignant phenotypes in gastric carcinoma. In this study, the mRNA levels and protein levels of PKM2 in gastric carcinoma cell lines and normal gastric mucosa epithelial cell lines were detected using quantitative real-time PCR and western blot, respectively. PKM2 was downregulated by siRNA transfection. HIF-1α or BCL-6 was upregulated by corresponding overexpression plasmid. Cell viability was detected using CCK-8 assay. Cell invasion and migration were determined using transwell assay. Higher expression of PKM2 was observed in human gastric carcinoma cell lines MKN-45 and SGC-7901 than in the normal gastric mucosa epithelial cell line GES-1. PKM2 knockdown suppressed cancer cell invasion and migration and inhibited the epithelial-mesenchymal transition (EMT) phenotype by inhibiting E-cadherin and promoting vimentin and N-cadherin expression. Also, we observed that PKM2 knockdown suppressed the hypoxia-inducible factor alpha (HIF-1α) and B-cell lymphoma 6 (BCL-6) signaling pathway. HIF-1α overexpression reversed the function of PKM2 silencing on cell invasion, migration, EMT, and BCL-6 expression. BCL-6 overexpression also reversed the function of PKM2 silencing on cell invasion, migration, and EMT but did not affect HIF-1α expression. Taken together, data from our study suggest that PKM2 knockdown impeded cell migration, invasion, and EMT of gastric carcinoma cells via the HIF-1α/BCL-6 pathway.

Effect of trametes robiniophila on the apoptosis of human gastric carcinoma MKN-45 and SGC-7901 cells and the expressions of matrix metalloproteinase 2 and matrix metalloproteinase 9

Objective To analyze the effect of trametes robiniophila on the apoptosis and the expressions of invasion and metastasis related matrix metalloproteinase 2 (MMP-2) and MMP-9 in human gastric carcinoma poorly differentiated cell line MKN-45 and medium differentiated cell line SGC-7901. Methods Human gastric carcinoma cell lines MKN-45 and SGC-7901 incubated with trametes robiniophila at the different concentrations [0 (the negative control group), 5, 10, and 20 mg/ml] for 24 h. When bifluorescently stained with acridine orange-ethidium bromide (AO-EB), morphology was observed by using microscopy. Flow cytometry was used to test the apoptosis ratio after 24, 48, and 72 h. Reverse transcription polymerase chain reaction (RT-PCR) was applied to analyze the expressions of mRNA of MMP-2 and MMP-9 after 24 h, and the absorbance values of the bands after gel electrophoresis were used as their expression levels. Results Under the fluorescence microscope, the cells of the negative control group were green (normal cells), and the membrane was even in size and integrity. The proportion of apoptotic and necrotic cells was increased with the concentration enrichment of trametes robiniophila. The apoptotic cells were yellow, and their cell membrane lost integrity. Apoptotic bodies were found in cancer cells, and cell membrane showed bud projection. The nuclei of the apoptotic cells showed brightly condensed chromatin or fragmented. Chromatin was strongly stained and located in karyotheca. The necrotic cells were dyed red with integrity of size. The apoptotic ratio of MKN-45 cell line induced by trametes robiniophila after 24 h was (6.5±0.8)%, (14.6±1.0)%, (18.0±1.1)%, and (23.1±1.2)%, respectively (F = 333.972, P < 0.01) in negative control group and 5, 10, and 20 mg/ml trametes robiniophila group. After 48 h, the apoptotic ratio was (7.3±1.2)%, (18.3±1.6)%, (24.5±1.3)%, and (27.2±1.7)%, respectively (F = 528.432, P=0.001); after 72 h, the apoptotic ratio was (7.5±0.9)%, (50.2±1.6)%, (58.0±1.9)%, and (69.0±1.4)%, respectively(F = 3 814.238, P < 0.01). After SGC-7901 cell line induced by trametes robiniophila for 24 h, the apoptotic ratio was (12.9±1.0)%, (19.4±1.2)%, (22.0±1.7)%, and (23.0±1.9)%, respectively (F = 120.190, P < 0.01) in negative control group and 5, 10, and 20 mg/ml trametes robiniophila group. For 48 h, the apoptotic ratio was(10.2±1.3)%, (40.9±1.4)%, (51.6±1.9)%, and (66.2±1.9)%, respectively (F = 1 281.342, P < 0.01). For 72 h, the apoptotic ratio was (27.4±1.8)%, (49.7±1.4)%, (65.1±1.4)%, and (69.0±2.0)%, respectively (F = 1 112.767, P < 0.01). The induction of apoptosis showed time and dose dependence (both P < 0.01). There was a trendency that the apoptotic ratio of SGC-7901 cell line was higher than that of the MKN-45 cell line at the same condition. RT-PCR showed that mRNA relative expression level of MMP-2 was 0.64±0.02, 0.49±0.01, 0.36±0.02, and 0.32±0.01, respectively (F = 274.321, P < 0.01) of negative control group and 5, 10, and 20 mg/ml trametes extract group after the effect of trametes extract on gastric cancer MKN-45 cell line. The relative expression level of MMP-9 in MKN-45 cell line was 0.71±0.01, 0.54±0.02, 0.47±0.02, and 0.39±0.02, respectively (F = 203.948, P < 0.01). The expression level of MMP-2 in SGC-7901 cell line was 0.64±0.01, 0.42±0.02, 0.34±0.20, and 0.29±0.01, respectively (F = 305.877, P < 0.01), while the mRNA expression level of MMP-9 was 0.65±0.15, 0.47±0.01, 0.44±0.01, and 0.39±0.02, respectively (F = 265.259, P < 0.01). Compared with the negative control group, the expression levels of MMP-2 and MMP-9 of the two cell lines were both decreased, after incubated with trametes robiniophila at the different concentrations (all P < 0.05), and with the addition of concentration, the expression levels of MMP-2 and MMP-9 were decreased. Conclusion Trametes robiniophila can induce the apoptosis of human gastric carcinoma cell lines MKN-45 and SGC-7901 in vitro and can inhibit the expressions of MMP-2 and MMP-9 and the effect of trametes robiniophila may be related with the differentiation degree. Key words: Stomach neoplasms; Apoptosis; Neoplasm invasiveness; Neoplasm metastasis; Trametes robiniophila

RETRACTED ARTICLE: Lidocaine inhibits growth, migration and invasion of gastric carcinoma cells by up-regulation of miR-145

BackgroundGastric cancer receives considerable attention not only because it is the most common cancer all through the world, but also because it’s on the top third leading reason for cancer-related death. Lidocaine is a well-documented local anesthetic that has been reported to suppress cancer development. The study explored the effects of lidocaine on the growth, migration and invasion of the gastric carcinoma cell line MKN45 and the mechanism behind.MethodsThe effect of lidocaine on viability, proliferation and apoptosis of MKN45 cells were analyzed by Cell Counting Kit-8 assay, BrdU staining assay and flow cytometry, respectively. Moreover, cell migration and invasion were both examined by Transwell assay. The expression of apoptosis-, migration-, and invasion-related proteins were detected by western blot. The relative expression of miR-145 was determined by qRT-PCR. Moreover, the impact which lidocaine brought on MEK/ERK and NF-κB pathways were examined by western blot.ResultsLidocaine inhibited viability, proliferation, migration, and invasion of MKN45 cells, while enhanced apoptosis. Moreover, miR-145 expression was enhanced by lidocaine; and transfection with miR-145 inhibitor increased cell viability, proliferation, migration, and invasion, but inhibited apoptosis. The up-regulation of miR-145 was partly contributed to the inhibitory effect of lidocaine on gastric cancer cell line MKN45. Finally, lidocaine inactivated MEK/ERK and NF-κB pathways via up-regulation of miR-145.ConclusionsOur results suggested that lidocaine decreased growth, migration and invasion of MKN45 cells via regulating miR-145 expression and further inactivation of MEK/ERK and NF-κB signaling pathways.

Trametes robiniophila may induce apoptosis and inhibit MMPs expression in the human gastric carcinoma cell line MKN-45.

Gastric carcinoma (GC) is one of the most common malignant tumors and is mainly treated by invasive surgeries. The present study aimed to investigate the treatment potential of Trametes robiniophila on GC using the human GC cell line MKN-45. Cells were incubated with Trametes robiniophila at a concentration of 0, 5 and 10 mg/ml for 24 h. The apoptosis of the cell line was examined with acridine orange/ethidium bromide staining and flow cytometry. The expression of B-cell lymphoma (Bcl)-2, Fas, caspase-3, matrix metalloproteinase (MMP)-2 and MMP-9 was analyzed using reverse transcription-polymerase chain reaction and western blotting. With increasing drug concentrations, the proportion of apoptotic and necrotic cells increased. For a certain concentration, the apoptotic ratio also increased with increasing response times. Compared with the control group, the Bcl-2, MMP-2 and MMP-9 expression levels in the MKN-45 cell line decreased, while the expression levels of Fas and caspase-3 increased (P<0.05), and the expression patterns were strengthened with increasing drug concentrations. The present study revealed that Trametes robiniophila had treatment potential on GC, and it may act on gastric cells through apoptotic induction and MMPs expression inhibition. Based on the present results, Trametes robiniophila may be considered as an alternative approach for noninvasive therapy of GC. However, future studies should be performed to clarify this further.

Complementary PTM Profiling of Drug Response in Human Gastric Carcinoma by Immunoaffinity and IMAC Methods with Total Proteome Analysis.

Gaining insight into normal cellular signaling and disease biology is a critical goal of proteomic analyses. The ability to perform these studies successfully to extract the maximum value and discovery of biologically relevant candidate biomarkers is therefore of primary importance. Many successful studies in the past have focused on total proteome analysis (changes at the protein level) combined with phosphorylation analysis by metal affinity enrichment (changes at the PTM level). Here, we use the gastric carcinoma cell line MKN-45 treated with the c-Met inhibitor SU11274 and PKC inhibitor staurosporine to investigate the most efficient and most comprehensive strategies for both total protein and PTM analysis. Under the conditions used, total protein analysis yielded few changes in response to either compound, while analysis of phosphorylation identified thousands of sites that changed differentially between the two treatments. Both metal affinity and antibody-based enrichments were used to assess phosphopeptide changes, and the data generated by the two methods was largely complementary (non-overlapping). Label-free quantitation of peptide peak abundances was used to accurately determine fold-changes between control and treated samples. Protein interaction network analysis allowed the data to be placed in a biologically relevant context, and follow-up validation of selected findings confirmed the accuracy of the proteomic data. Together, this study provides a framework for start-to-finish proteomic analysis of any experimental system under investigation to maximize the value of the proteomic study and yield the best chance for uncovering actionable target candidates.

Identification of miRNA-338-5P as an effector of the tumor suppressor activity of immunized anticancer bioactive peptide-3 (TUM2P.1036)

Abstract Gastric cancer remains one of the leading causes of global cancer mortality. Cancer stem cells (CSC) have been studied in many solid tumors, including gastric cancer and they are promising for the development of anticancer drugs that can target all CSC subsets within a tumor to prevent recurrence. Previously, we identified a novel anti-cancer bioactive peptide (ACBP), which isolated and purified from goat liver following immunization with human gastric cancer protein extract, and designated as ACBP-3. ACBP-3 inhibited cell proliferation and clone formation of gastric cancer stem cells (CSCs). In present study, we investigated the mechanism of ACBP-3 inhibition of gastric CSCs in vitro and in vivo. We employed a microRNA array and screened for the genes of which had increased expression by ACBP-3 in CD44 positive gastric carcinoma cell lines MKN45 and MKN74. MiRNA-338-5p showed the greatest difference in the expression level. In vivo, ACBP-3 alone significantly inhibited MKN45 tumor growth in a xenograft nude mouse model. The tumor tissues were harvested after treatment, and ACBP-3 treatment induced expression of miRNA-338-5p, Bac, and Bim as quantified by RT-PCR and western blot. Overexpression of miRNA-338-5p reduced cell proliferation and induced apoptosis in both MKN45 and MKN74 cell lines in vitro. We concluded that miRNA-338-5p is the effector of tumor suppressor activity of ACBP-3 in gastric cancer stem cells. ACBP-3 could be a potential anti-cancer agent.

Primary analysis and screening of microRNAs in gastric cancer side population cells.

To explore the microRNA (miRNA) profiles and to determine the key miRNAs within the side population (SP) cells of the gastric cancer cell line MKN-45. We used fluorescence-activated cell sorting and Hoechst 33342 labeling to obtain SP cells from the human gastric carcinoma cell line MKN-45. The miRNA expression profiles of the SP and major population (MP) cells were examined using a miRNA gene chip, and key miRNAs were obtained according to aberrant expression and the miRNAs' possible targets as predicted by bioinformatics. Using a significance criterion of a 1.5-fold or greater difference in expression level, we observed an increase in the expression of 34 miRNAs and a decrease in the expression of 34 miRNAs when comparing SP to MP cells. Using quantitative real-time reverse transcription-polymerase chain reaction to test for differentially expressed miRNAs combined with bioinformatics results, we found that the downregulated miRNAs, such as hsa-miR-3175 and hsa-miR-203, and the upregulated miRNAs, including hsa-miR-130a, hsa-miR-324-5p, hsa-miR-34a, and hsa-miR-25-star, may be important in maintaining and regulating the characteristics of SP cells. There are key miRNAs expressed within the SP cells of the gastric cancer cell line MKN-45, and include hsa-miR-3175, hsa-miR-203, hsa-miR-130a, hsa-miR-324-5p, hsa-miR-34a, and hsa-miR-25-star.

Tumor suppressor ING4 overexpression contributes to proliferation and invasion inhibition in gastric carcinoma by suppressing the NF-κB signaling pathway

There is growing evidence that inhibitor of growth 4 (ING4) plays a pivotal role in development and progression of multiple different tumors; however, its precise function in gastric carcinoma remains to be elucidated. In the present study, we investigated ING4 level in gastric carcinoma tissues and cells, and preliminarily elucidated the role of ING4 in the proliferation and invasion of gastric carcinoma. The results demonstrated that expressions of ING4 mRNA and protein in gastric carcinoma tissues and cells were significantly lower than those in normal tissues and cells (P < 0.05). ING4 level in gastric carcinoma cells stably expressing ING4 was markedly higher than those in untreated group and empty vector pcDNA3.1 group (P < 0.05). Elevated ING4 level resulted in the inhibition of proliferation and invasion in three of gastric carcinoma cell lines MKN-28, SGC-7901 and MKN-45. Most notably, increased ING4 level evidently evoked the down-regulation of p65, p-IκBα, MMP-9 and uPA proteins and the up-regulation of IκBα protein. Our results presented herein suggest that ING4 level elevation mediated proliferation and invasion inhibition may be tightly associated with the suppression of NF-κB signaling pathway.

Developing a molecular imaging agent for Met using onartuzumab (MetMAb).

11083 Background: Developing an imaging agent to assess Met expression would aid in diagnosis and monitoring tumor response to Met-targeted therapies. Onartuzumab (MetMAb), a Met selective humanized one-armed monoclonal antibody, has been studied in Phase I-II clinical trials in which it was generally well tolerated and has shown the most benefit in patients with MET positive tumors. Methods: Studies to assess Met-binding were executed using the human gastric carcinoma cell line MKN-45 which exhibits a high level of Met expression. Murine PET studies and biodistribution assays were performed using MKN-45 xenografts. Results: Plasma shed Met concentration is directly related to total tumor burden (p &lt; 0.001). The absence of a positive correlation between shed Met and %ID in blood indicates that binding of tracer to shed Met present in plasma is unlikely. There are positive correlations between tumor mass, Met abundance, and phosphoMet content and uptake of 89Zr-df-onartuzumab in MKN-45 mouse xenografts. Lastly, tumor mass, Met, pMet and 89Zr-df-onartuzumab uptake were all significantly decreased by drug treatment. Conclusions: MKN-45 tumor uptake of 89Zr-df-onartuzumab correlated significantly with tumor mass and Met abundance. Blood tracer uptake did not positively correlate with the presence of plasma shed Met. The amounts of Met, pMet, as well as 89Zr-df-onartuzumab image intensity correlated significantly with tumor size (all Spearman p values &lt; 0.001).Tumor volumes and Met content were significantly decreased in TKI treated versus control mice, which correlated with imaging results.89Zr-df-onartuzumab has potential utility for imaging Met to identify patients for treatment with Met-targeted therapeutics and to identify the emergence of Met-driven acquired resistance to other molecularly targeted cancer therapies.

RETRACTED ARTICLE: Characterization of cancer stem-like cells in the side population cells of human gastric cancer cell line MKN-45

Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer. Many kinds of cell lines and tissues have demonstrated the presence of SP cells, including several gastric cancer cell lines. This study is aimed to identify the cancer stem-like cells in the SP of gastric cancer cell line MKN-45. We used fluorescence activated cell sorting (FACS) to sort SP cells in the human gastric carcinoma cell line MKN-45 (cells labeled with Hoechst 33342) and then characterized the cancer stem-like properties of SP cells. This study found that the SP cells had higher clone formation efficiency than major population (MP) cells. Five stemness-related gene expression profiles, including OCT-4, SOX-2, NANOG, CD44, and adenosine triphosphate (ATP)-binding cassette transporters gene ABCG2, were tested in SP and MP cells using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Western blot was used to show the difference of protein expression between SP and MP cells. Both results show that there was significantly higher protein expression in SP cells than in MP cells. When inoculated into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, SP cells show higher tumorigenesis tendency than MP cells. These results indicate that SP cells possess cancer stem cell properties and prove that SP cells from MKN-45 are gastric cancer stem-like cells.

Aquaporin 5 promotes the proliferation and migration of human gastric carcinoma cells

Aquaporin 5 (AQP5) promotes the progression and invasion of several cancers, but its role in the tumorigenesis of human gastric carcinoma (GC) has not been clearly defined. Here, we investigated the potential functions of AQP5 in the proliferation and migration of human GC. RT-PCR and western blotting were used to detect the expression of AQP5 in human GC cell lines. Immunohistochemistry was applied to evaluate the expression of AQP5 in human GC tissues and corresponding normal tissues. Following ectopic overexpression of AQP5 or inhibition of AQP5 by its inhibitor, acetazolamide (AZA), cell proliferation and migration of AGS cells were analyzed by MTT assay, colony formation assay, and wound healing assay. Heterogeneous expression of AQP5 mRNA and protein was observed in human GC cell lines MKN45, MKN28, AGS, and SGC7901. AQP5 was up-regulated in GC tissues in comparison to corresponding normal tissues. AQP5 protein was mainly localized in the cell membrane. Overexpression of AQP5 was correlated with enhanced lymph node metastasis. In vitro, overexpression of AQP5 notably enhanced, while inhibition of AQP5 by AZA significantly attenuated the proliferation and migration of AGS cells. Our data indicate that AQP5 may play an important role in the tumorigenesis and progression of human GC and suggest that AQP5 is a potential therapeutic target against GC.

P-0031 Pathobiological Significance of Arhi Expression in Human Gastric Carcinoma and The Precancerous Lesions as Well as Gastric Cancer Cell Lines

IntroductionAplesia Ras homologue member I (ARHI, DIRAS3) is a maternally imprinted tumor suppressor gene which is frequently lost in breast and ovarian cancers, and results in autophagic cell death of human ovarian cancer cells in vitro. There has been no report in PMC about ARHI in human gastric carcinomas and gastric cancer cell lines so far. MethodsThe ARHI expression in 309 cases of gastric carcinomas (GCs), with corresponding normal gastric mucosa and the precancerous lesions was investigated immunohistochemically, and the prognostic value of ARHI expression was analyzed. ARHI mRNA and protein expressions in gastric cancer cell lines BGC823, MGC803, SGC7901, MKN45, NCI-N87 and normal gastric epithelial cell line GES-1 were also evaluated using quantitative real time PCR(qrtPCR) and immunofluorescence (IF) methods. ResultsThe positive expression of ARHI protein in intestinal metaplasia (76.1%, 86/113), dysplasia (50.0%, 8/16) and gastric carcinoma (29.8%, 92/309) were down-regulated compared with corresponding normal gastric mucosa (87.4%, 270/309), P<0.05. The rates of ARHI up-regulated and down-regulated expression were 9.7% (30) and 67% (207), the median patients survival time of up-regulated group (62.3 months) was longer than that of down-regulated group (36.7 months). The results of qrtPCR showed that mRNA level of ARHI in the gastric carcinoma cell lines BGC-823, MGC-803 SGC-7901, MKN-45, and NCI-N87 are 0.41, 0.41, 1.39, 1.53, 0.88 fold of gastric cell line GES, respectively. ARHI protein expression detected by IF and Western blot in the cell lines mentioned above accord with the ARHI mRNA expression by qrtPCR in the study. ConclusionWe report, for the first time, that tumor suppressor ARHI expression is mainly down regulated in human gastric carcinoma and gastric dysplasia, suggesting ARHI gene plays important roles in gastric carcinogenesis, in which ARHI related autophagy may be involved. Further study is needed in this field.

P-0030 CypA Plays Roles in the Proliferation of Gastric Carcinoma Cells

ABSTRACT Introduction Cyclophilin A (CypA) is a member of the peptidyl-prolyl isomerases, the primary intracellular receptor of the immunosuppressant cyclosporin A (CsA). CypA plays an important role in life activities and participate in a variety of disease processes. Recent evidence points to possible roles for CypA overexpression in tumorigenesis. However gastric carcinoma was rarely involved in CypA research so far. Methods In this research, using immunohistochemistry assay we confirmed the overexpression of CypA in gastric carcinoma tissues at protein level. The mRNA level of CypA in gastric carcinoma cell lines are also detected by Real-Time PCR. Results Compared with normal gastric epithelial cell line GES-1, the mRNA levels of CypA in gastric carcinoma cell lines SGC-7901, MKN-45, BGC-823 and MGC-803 were 1.29, 1.46, 1.54 and 1.55 fold high respectively (Fig.1). To investigate the roles CypA plays in the occurrence and development of gastric carcinoma, we constructed 4 RNA interference vectors: pRNA-CypA shRNA 1-4 and confirmed the positive clone by PCR with pRNA primer, then sequenced the plasmid DNA for additional verification. BGC-823 cells were grown to 70% confluency in 24-well plates and transfected with 0.5ug pRNA-CypA shRNAs using Lipofectamine LTX (Invitrogen). At 48 and 96 hours following transfection the mRNA level of CypA in these cells were detected by Real-Time PCR. The results showed us that after transfection with pRNA-CypA shRNA3, BGC-823 cells yielded ∼80% and ∼30% CypA knockdown at 48h and 96h respectively compared with untreated BGC-823 cells; with pRNA-CypA shRNA2 yielded ∼27% and ∼35% CypA knockdown at 48h and 96h respectively; pRNA-CypA shRNA1 and pRNA-CypA shRNA4 had little RNA interference effects. A transient increase of CypA mRNA level occurred in transfection, and reduced to the normal level at 96h after transfection. We collected the BGC-823 cells at 48h with the best RNAi efficiency by pRNA-CypA shRNA3 plasmid to perform flow cytometric analysis of cell cycle. From the cell cycle distribution, cell populations of S & G2/M phase (M2 & M3) in untreated BGC-823cells, BGC-823cell transfected with pRNA plasmid and CypA knockdown (transfected with pRNA-CypA-shRNA3 plasmid) BGC-823cell were 45.3%, 49.7% and 33.0% respectively. CypA knockdown resulted in an augmentation of cell populations in G0/G1 phase (M1) accompanied with a decrease in S & G2/M phase (M2 & M3). Cell proliferation decreased obviously in CypA knockdown BGC-823 cells, and cell cycle arrested in G0/G1 phase (M1). These results establish the relevance of CypA to gastric carcinoma proliferation in vitro. Conclusion Our results establish the relevance of CypA to gastric carcinoma proliferation in vitro. In further study we will do more experiments to reveal mechanisms how CypA plays roles in occurrence and development of gastric carcinoma and the pathways though which the signals of CypA transfer.

Cysteine-rich protein 61 suppresses cell invasion via down-regulation of matrix metalloproteinase-7 expression in the human gastric carcinoma cell line MKN-45

Cysteine-rich protein 61 (CYR61) is a member of the CCN (CYR61/CTGF/NOV) family, which is associated with progression in a variety of human cancers. Our previous study confirmed that the expression levels of CYR61 protein were decreased in gastric carcinoma compared to non-tumoral mucosa as determined by proteome analysis. Histological research also showed that the reduction in CYR61 expression was significantly correlated with cellular invasiveness and inversely correlated with matrix metalloproteinase-7 (MMP-7/matrilysin) expression in human gastric carcinoma. We examined the cause of CYR61 down-regulation in a human gastric carcinoma cell line, MKN-45. Lower expression of CYR61, but no genetic or epigenetic alterations of the gene, were observed. We then examined the correlation between CYR61 protein and MMP-7 expression and cellular invasiveness in MKN-45 cells. CYR61 was secreted from CYR61 expression-vector-transfected 293T cells, and the supernatant was added to MKN-45 cells. The expression level of MMP-7 was reduced by treatment of the supernatant, including CYR61, in a dose-dependent manner. An invasion assay showed that the cellular invasiveness of MKN-45 was significantly suppressed by the transfection of CYR61 expression vector compared to transfection with a control vector. Taken together, these results raise the possibility that CYR61 suppresses cell invasion at least partly via the down-regulation of MMP-7 expression in human gastric carcinoma cells.

Direct repression of Sonic Hedgehog expression in the stomach by Cdx2 leads to intestinal transformation

Shh (Sonic Hedgehog) is a morphogen involved in gastric fundic gland differentiation in the adult. Shh expression is reduced in Helicobacter pylori-associated intestinal metaplastic change of the gastric epithelium and mice that lack Shh show intestinal transformation of the gastric mucosa. Similarly, in the stomach of Cdx2 (caudal-type homeobox 2)-transgenic mice, the gastric mucosa is replaced by intestinal metaplastic mucosa. The aim of the present study was to use Cdx2-transgenic mice to investigate: (i) Shh expression in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach; and (ii) the relationship between Shh and Cdx2. We determined that Shh mRNA levels were dramatically reduced in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach compared with the normal (wild-type) mouse stomach. This was not due to hypermethylation of the Shh promoter, but instead we showed that Cdx2 directly bound to the TATA box region of the Shh promoter. Cdx2 also down-regulated transcription of the Shh gene in the human gastric carcinoma cell lines AGS, MKN45 and MKN74. In conclusion, Cdx2 reduced Shh expression by binding to the unmethylated Shh promoter in the intestinal metaplastic mucosa of Cdx2-transgenic mouse stomach.

Transgenic Cdx2 induces endogenous Cdx1 in intestinal metaplasia of Cdx2‐transgenic mouse stomach

Cdx1 and Cdx2, which are transcription factors regulating normal intestinal development, have been studied as potential key molecules in the pathogenesis of the precancerous intestinal metaplasia of the human stomach. However, the regulation of Cdx1 expression in the intestinal metaplasia is poorly understood. Cdx2-expressing gastric mucosa of Cdx2-transgenic mouse stomach was replaced by intestinal metaplastic mucosa. The aim of this study was to investigate the following: (a) Cdx1 expression in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach; and (b) the relationship between Cdx1 and Cdx2. A mouse model of intestinal metaplasia, the Cdx2-transgenic mouse, was used to investigate Cdx1 gene expression by RT-PCR. DNA methylation profile analysis was performed by bisulfite sequencing, and the interaction of Cdx2 with the Cdx1 promoter was examined by chromatin immunoprecipitation assay, electrophoretic mobility shift assay, and luciferase reporter assays. Cdx2 mRNA was expressed in the Cdx2-transgenic mouse stomach. However, endogenous Cdx2 mRNA was not expressed in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. On the other hand, endogenous Cdx1 mRNA and protein were expressed in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. The Cdx1 promoter was unmethylated in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. Chromatin immunoprecipitation assay and electrophoretic mobility shift assay showed that Cdx2 was bound to the Cdx1 promoter region in the intestinal metaplasia and the normal intestine. Cdx2 upregulated and siRNA-Cdx2 downregulated the transcriptional activity of the Cdx1 gene in the human gastric carcinoma cell lines AGS, MKN45, and MKN74. In conclusion, transgenic Cdx2 induced endogenous Cdx1 through the binding of Cdx2 to the unmethylated Cdx1 promoter region in the intestinal metaplasia of the Cdx2-transgenic mouse stomach.

Aberrant expression of cortactin and fascin are effective markers for pathogenesis, invasion, metastasis and prognosis of gastric carcinomas

Tumor metastasis depends on cell adhesiveness, motility and deformability, resulting from quantitative alterations and rearrangement of various actin-binding cytoskeletal components, such as cortactin and fascin. To clarify the involvement of cortactin and fascin expression in tumorigenesis and progression of gastric carcinoma, we performed immunohistochemistry (IHC) on tissue microarray containing gastric carcinomas, adenomas and adjacent non-neoplastic mucosa (ANNM) using the antibodies against cortactin (Ab-466, -421) and fascin as well as a comparison of their expression with clinicopathological parameters of the tumors. Gastric carcinoma cell lines MKN28, AGS, MKN45, KATO-III and HGC-27 were studied for both proteins by IHC. Cortactin-466 was found to be highly expressed in adenoma, compared with ANNMs and carcinoma (p<0.05), and more frequently in ANNMs than in carcinoma (p<0.05). Cortactin-421 expression was higher in gastric carcinomas than in adenoma and ANNMs (p<0.05). There was increased fascin expression in gastric carcinoma and adenoma than in ANNMs (p<0.05). Most of the gastric carcinoma cell lines showed expression of cortactin and fascin at different levels. Cortactin-466 expression was inversely correlated with tumor size, depth of invasion, lymphatic and venous invasion, lymph node metastasis and UICC staging (p<0.05). The converse was observed for cortactin-421 and fascin (p<0.05). There was stronger positivity of both cortactins in intestinal- versus diffuse-type carcinomas (p<0.05). Univariate analysis indicated the cumulative survival rate of patients with positive cortactin-466 expression to be higher than without its expression even stratified according to the depth of invasion (p<0.05). However, it was the converse for fascin (p<0.05). Multivariate analysis showed that age, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren's classification were independent prognostic factors for carcinomas (p<0.05). It was suggested that aberrant expression of cortactin and fascin possibly contributes to the pathogenesis, growth, invasion and metastasis of gastric carcinomas. Thus, they may be objective and effective markers to indicate the pathobiological behaviors and prognosis of gastric carcinomas.

Inhibition of human gastric carcinoma cell growth by treatment of N3-o-toluyl-fluorouracil as a precursor of 5-fluorouracil

N(3)-o-toluyl-fluorouracil (TFU), the pro-drug of 5-fluorouracil (5-FU), is the metabolite of N(1)-acetyl-N(3)-o-toluyl-fluorouracil (atofluding). We aimed to evaluate the efficacy of TFU as a precursor of 5-FU on the growth inhibition of human gastric carcinoma cell lines SGC-7901 and MKN-45. Growth of SGC-7901 and MKN-45 cells was remarkably suppressed by treatment with TFU in the presence of liver microsomal enzymes in vitro, suggesting that TFU may be converted to 5-FU by the enzymes. Similar treatment of TFU induced apoptosis of the cells, which was deduced from typical apoptotic features such as morphology, the formation of characteristic ladder pattern of DNA migration and the accumulation of sub-G1 phase. Cancer cells xenografts in nude mice were employed to evaluate the efficacy of TFU in vivo. Growth of human gastric carcinoma cells was significantly delayed by oral administration of TFU with low side effects. Apoptosis in xenografts was also observed by means of TUNEL staining method. These results suggest that the treatment of TFU in the presence of liver microsomal enzymes and the oral administration of TFU in mice induced anti-proliferation and apoptosis in gastric carcinoma cells. This suggests that TFU may be a promising pro-drug of 5-FU for cancer treatments.