Human Gastric Cancer AGS Cells Research Articles

Escin Suppresses Migration and Invasion Involving the Alteration of CXCL16/CXCR6 Axis in Human Gastric Adenocarcinoma AGS Cells

Escin, a natural mixture of triterpene saponins isolated from horse chestnut, has been reported to possess anticancer activity in many human cancer cells. However, the effect of escin on the metastasis has not been studied. The present study examined the effect of escin on the migration and invasion of AGS human gastric cancer cells. To examine the effects of escin on metastatic capacities of gastric cancer cells, AGS cells were cultured in the presence of 0–4 μmol/L escin. Escin inhibited cell migration and invasion in AGS cells. However, escin did not affect the viability of these cells at these concentrations. The chemokine receptor and its ligands play an important role in cancer metastasis. Escin decreased the production of soluble C-X-C motif chemokine (CXCL)16 but increased the expression of trans-membranous CXCL16. The expression of C-X-C chemokine receptor (CXCR)6 was not affected by escin treatment. Exogenous CXCL16 reversed escin-induced migration inhibition. In addition, escin inhibited the phosphorylation of focal adhesion kinase and Akt. These results demonstrate that escin inhibited the migration and invasion of AGS cells, which is associated with altered CXCL16/CXCR6 axis. These findings suggest that escin has potential as an antimetastatic agent in gastric cancer.

Inhibition of the JAK2/STAT3 pathway reduces gastric cancer growth in vitro and in vivo.

Signal Transducer and Activator of Transcription-3 (STAT3) is constitutively activated in many cancers where it promotes growth, inflammation, angiogenesis and inhibits apoptosis. We have shown that STAT3 is constitutively activated in human gastric cancer, and that chronic IL-11-driven STAT3 transcriptional activity induces gastric tumourigenesis in the gp130757FF mouse model of gastric cancer development. Here we show that treatment of human AGS gastric cancer cells with the Janus Kinase (JAK) inhibitor WP1066 dose-, and time-dependently inhibits STAT3 phosphorylation, in conjunction with reduced JAK2 phosphorylation, reduced proliferation and increased apoptosis. In addition, application of intraperitoneal WP1066 for 2 weeks, reduced gastric tumour volume by 50% in the gp130757FF mouse coincident with reduced JAK2 and STAT3 activation compared with vehicle-treated, littermate controls. Gastric tumours from WP1066- treated mice had reduced polymorphonuclear inflammation, coincident with inhibition of numerous proinflammatory cytokines including IL-11, IL-6 and IL-1β, as well as the growth factors Reg1 and amphiregulin. These results show that WP1066 can block proliferation, reduce inflammation and induce apoptosis in gastric tumour cells by inhibiting STAT3 phosphorylation, and that many cytokines and growth factors that promote gastric tumour growth are regulated by STAT3-dependent mechanisms. WP1066 may form the basis for future therapeutics against gastric cancer.

Identification of anti-cancer active components of Taraxacum coreanum on human gastric cancer AGS cells

Anti-cancer effects were compared amongst Taraxacum coreanum extract, its fractions, and 7 ingredients (β-sitosterol, daucosterol, taraxasteryl acetate, chrysoeriol, diosmetin, luteolin, and luteoloside). Exposure to the ethyl acetate fraction (50 and 100 μg/mL) of T. coreanum extract and luteolin (10 and 50 μM) for 24 h induced the cleavage of poly (ADP-ribose) polymerase (PARP), caspase-3, and caspase-8, in a dose-dependent manner. These findings demonstrate that luteolin is the main active component of T. coreanum extract activating caspases-3 and -8 which contribute to apoptotic cell death.

Tangeretin, a citrus polymethoxyflavonoid, induces apoptosis of human gastric cancer AGS cells through extrinsic and intrinsic signaling pathways

Tangeretin, a natural polymethoxyflavone present in citrus peel oil, is known to have anticancer activities in breast cancer, colorectal carcinoma and lung carcinoma, yet, the underlying mechanisms of tangeretin in human gastric cancer AGS cells have not been investigated to date. In the present study, the apoptotic mechanisms of tangeretin in AGS cells were explored. It was observed that tangeretin increased the apoptotic rates of AGS cells following treatment with tangeretin for 48 h in a dose-dependent manner by Annexin V-FITC and PI double staining. In addition, characteristic apoptotic morphology such as nuclear shrinkage and apoptotic bodies was observed after Hoechst 33258 staining. Flow cytometric assay showed that treatment of AGS cells with tangeretin decreased the mitochondrial membrane potential (MMP) in a dose-dependent manner, which indicated that mitochondrial dysfunction was involved in the tangeretin-induced apoptosis. Caspase-3, -8 and -9 activities were increased by tangeretin in a dose-dependent manner. Western blotting showed that the protein levels of pro-apoptotic proteins including cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax, Bid, tBid, p53, p21/cip1, Fas and FasL were significantly upregulated by tangeretin. In addition, PFT-α (a p53 inhibitor) reduced the apoptotic rates and the expression of p53, p21, caspase-3 and caspase-9 induced by tangeretin, indicating that tangeretin-induced apoptosis was p53-dependent. In conclusion, these results suggest that tangeretin induces the apoptosis of AGS cells mainly through p53-dependent mitochondrial dysfunction and the Fas/FasL-mediated extrinsic pathway.

Targeting Cadherin-17 Inactivates Ras/Raf/MEK/ERK Signaling and Inhibits Cell Proliferation in Gastric Cancer

Cadherin-17 (CDH17), one member of 7D-cadherin superfamily, was overexpressed in gastric cancer (GC) and was associated with poor survival, tumor recurrence, metastasis, and advanced tumor stage. So far the cellular function and signaling mechanism of CDH17 in GC remains unclear. In this study, we showed that over 66% of GC cell lines (20/30) were CDH17 positive. Tissue microarray (TMA) assay showed that 73.6% Chinese GC tissues (159/216) were CDH17 positive, while 37% respective adjacent normal tissues were CDH17 positive. Knockdown of CDH17 inhibited cell proliferation, migration, adhesion and colony formation, and also induced a cell cycle arrest and apoptosis in AGS human GC cells. On the other side, overexpression of CDH17 facilitated MGC-803 GC tumor growth in nude mice. Antibody array and Western blotting assay demonstrated that knockdown of CDH17 in AGS cells down-regulated integrin β series proteins, further inactivated the Ras/Raf/MEK/ERK pathway and led to p53 and p21 accumulation, which resulted in proliferation inhibition, cell-cycle arrest and apoptosis induction. Collectively, our data firstly demonstrate the capacity of CDH17 to regulate the activity of Ras/Raf/MEK/ERK pathway for cell proliferation in GC, and suggest that CDH17 can serve as an attractive therapeutic target for future research.

Stereospecific anticancer effects of ginsenoside Rg3 epimers isolated from heat-processed American ginseng on human gastric cancer cell

BackgroundResearch has been conducted with regard to the development of methods for improving the pharmaceutical effect of ginseng by conversion of ginsenosides, which are the major active components of ginseng, via high temperature or high-pressure processing. MethodsThe present study sought to investigate the anticancer effect of heat-processed American ginseng (HAG) in human gastric cancer AGS cells with a focus on assessing the role of apoptosis as an important mechanistic element in its anticancer actions. Results and ConclusionHAG significantly reduced the cancer cell proliferation, and the contents of ginsenosides Rb1 and Re were markedly decreased, whereas the peaks of less-polar ginsenosides [20(S,R)-Rg3, Rk1, and Rg5] were newly detected. Based on the activity-guided fractionation of HAG, ginsenoside 20(S)-Rg3 played a key role in inducing apoptosis in human gastric cancer AGS cells, and it was generated mainly from ginsenoside Rb1. Ginsenoside 20(S)-Rg3 induced apoptosis through activation of caspase-3, caspase-8, and caspase-9, as well as regulation of Bcl-2 and Bax expression. Taken together, these findings suggest that heat-processing serves as an increase in the antitumor activity of American ginseng in AGS cells, and ginsenoside 20(S)-Rg3, the active component produced by heat-processing, induces the activation of caspase-3, caspase-8, and caspase-9, which contributes to the apoptotic cell death.

Evaluation of DNA Binding, Cleavage, and Cytotoxic Activity of Cu(II), Co(II), and Ni(II) Schiff Base Complexes of 1-Phenylindoline-2,3-dione with Isonicotinohydrazide.

One new series of Cu(II), Co(II), and Ni(II) Schiff base complexes was prepared through the condensation reaction between 1-phenylindoline-2,3-dione with isonicotinohydrazide followed by metalation, respectively. The Schiff base ligand(L), (E)-N′-(2-oxo-1-phenylindolin-3-lidene)isonicotinohydrazide, and its complexes were found soluble in DMF and DMSO solvents and characterized by using the modern analytical and spectral techniques such as elemental analysis, conductivity, magnetic moments, IR, NMR, UV-visible, Mass, CV, and EPR. The elemental analysis data of ligand and their complexes were well agreed with their calculated values in which metal and ligand stoichiometry ratio 1 : 2 was noted. Molar conductance values indicated that all the complexes were found to be nonelectrolytes. All the complexes showed octahedral geometry around the central metal ions. Herein, we better characterized DNA binding with the complexes by UV-visible and CD spectroscopy and cyclic voltammetry techniques. The DNA cleavage experiments were carried out by Agarose gel electrophoresis method and the cytotoxicity experiments by MTT assay method. Based on the DNA binding, cleavage, and cytotoxicity studies, Cu and Ni complexes were found to be good anticancer agents against AGS-human gastric cancer cell line.

한국산 및 일본산 봄배추와 이를 이용하여 제조한 김치의 품질특성과 기능성

We examined the quality characteristics and functionalities of Korean and Japanese spring Baechu cabbages and the kimchi prepared with them. To study the physiochemical properties of the cabbages and the kimchis, we measured their water content, pH, acidity, microbial counts, and springiness. On the third week of the kimchi fermentation at 5°C, their sensory properties and in vitro DPPH radical scavenging and anticancer activities using AGS human gastric cancer cells were determined. The Japanese Baechu contained 97.1% water, and the Korean Baechu, 92.4%. The comparison of the textures of the raw Baechu and the brined Baechu showed that the Korean Baechu had higher springiness scores than the Japanese Baechu. After four-week fermentation, the springiness score of the kimchi with Korean Baechu was 53.5%, significantly higher than the 41.4% of the kimchi with Japanese Baechu. The kimchi prepared with Korean Baechu had a low total bacterial count but higher Lactobacillus sp. and Leuconostoc sp. counts than the kimchi with Japanese Baechu. The kimchi prepared with Korean Baechu had the highest overall acceptability score in the sensory evaluation test. The DPPH radical scavenging activity of the kimchi with Korean Baechu was 83.2%, and that of the kimchi with Japanese Baechu, 46.1%. When the AGS human gastric cancer cells were treated with the kimchis, the kimchi prepared with Korean Baechu showed a 45% cancer cell growth inhibition rate, and the kimchi with Japanese Baechu, 26%, at 1 mg/mL of methanol extracts. At the 2 mg/mL concentration, the kimchis with Korean Baechu and Japanese Baechu showed 97% and 74% inhibition, respectively. The Korean Baechu showed better quality than the Japanese Baechu, and the kimchi prepared with the Korean Baechu showed better kimchi quality and functionality than the Japanese Baechu.

Glycogen synthase kinase 3 beta inhibits microRNA-183-96-182 cluster via the β-Catenin/TCF/LEF-1 pathway in gastric cancer cells

Glycogen synthase kinase 3 beta (GSK3β) is a critical protein kinase that phosphorylates numerous proteins in cells and thereby impacts multiple pathways including the β-Catenin/TCF/LEF-1 pathway. MicroRNAs (miRs) are a class of noncoding small RNAs of ∼22 nucleotides in length. Both GSK3β and miR play myriad roles in cell functions including stem cell development, apoptosis, embryogenesis and tumorigenesis. Here we show that GSK3β inhibits the expression of miR-96, miR-182 and miR-183 through the β-Catenin/TCF/LEF-1 pathway. Knockout of GSK3β in mouse embryonic fibroblast cells increases expression of miR-96, miR-182 and miR-183, coinciding with increases in the protein level and nuclear translocation of β-Catenin. In addition, overexpression of β-Catenin enhances the expression of miR-96, miR-182 and miR-183 in human gastric cancer AGS cells. GSK3β protein levels are decreased in human gastric cancer tissue compared with surrounding normal gastric tissue, coinciding with increases of β-Catenin protein, miR-96, miR-182, miR-183 and primary miR-183-96-182 cluster (pri-miR-183). Furthermore, suppression of miR-183-96-182 cluster with miRCURY LNA miR inhibitors decreases the proliferation and migration of AGS cells. Knockdown of GSK3β with siRNA increases the proliferation of AGS cells. Mechanistically, we show that β-Catenin/TCF/LEF-1 binds to the promoter of miR-183-96-182 cluster gene and thereby activates the transcription of the cluster. In summary, our findings identify a novel role for GSK3β in the regulation of miR-183-96-182 biogenesis through β-Catenin/TCF/LEF-1 pathway in gastric cancer cells.

17β-Estradiol Inhibits Mesenchymal Stem Cells-Induced Human AGS Gastric Cancer Cell Mobility via Suppression of CCL5- Src/Cas/Paxillin Signaling Pathway

Gender differences in terms of mortality among many solid organ malignancies have been proved by epidemiological data. Estrogen has been suspected to cast a protective effect against cancer because of the lower mortality of gastric cancer in females and the benefits of hormone replacement therapy (HRT) in gastric cancer. Hence, it suggests that 17β-estradiol (E2) may affect the behavior of cancer cells. One of the key features of cancer-related mortality is metastasis. Accumulating evidences suggest that human bone marrow mesenchymal stem cells (HBMMSCs) and its secreted CCL-5 have a role in enhancing the metastatic potential of breast cancer cells. However, it is not clear whether E2 would affect HBMMSCs-induced mobility in gastric cancer cells. In this report, we show that CCL-5 secreted by HBMMSCs enhanced mobility in human AGS gastric cancer cells via activation of Src/Cas/Paxillin signaling pathway. Treatment with specific neutralizing antibody of CCL-5 significantly inhibited HBMMSCs-enhanced mobility in human AGS gastric cancer cells. We further observe that 17β-estradiol suppressed HBMMSCs-enhanced mobility by down-regulating CCL5-Src/Cas/paxillin signaling pathway in AGS cells. Collectively, these results suggest that 17β-estradiol treatment significantly inhibits HBMMSCS-induced mobility in human AGS gastric cancer cells.

A new tetrahydrofuran lignan diglycoside from Viola tianshanica Maxim.

A new lignan glycoside, tianshanoside A (1), together with a known phenylpropanoid glycoside, syringin (2) and two known lignan glycosides, picraquassioside C (3), and aketrilignoside B (4), were isolated from the whole plant of Viola tianshanica Maxim. The structure of the new compound was elucidated by extensive NMR (1H, 13C, COSY, HSQC, HMBC and ROESY) and high resolution mass spectrometry analysis. The three lignans 1, 3, and 4 did not exhibit significant cytotoxicity against human gastric cancer Ags cells or HepG2 liver cancer cells. This is the first report of the isolation of a lignan skeleton from the genus Viola L.

Type II cGMP-dependent protein kinase inhibits epidermal growth factor-induced phosphatidylinositol-3-kinase/Akt signal transduction in gastric cancer cells

Our previous study revealed that Type II cGMP-dependent protein kinase (PKG II) inhibits epidermal growth factor (EGF)-induced MAPK/ERK and MAPK/JNK-mediated signal transduction through the inhibition of the phosphorylation/activation of the EGF receptor (EGFR). As EGFR also mediates several other signal transduction pathways besides MAPK-mediated pathways, the present study was designed to investigate whether PKG II was able to inhibit EGF/EGFR-induced phosphatidylinositol-3-kinase (PI3K)/Akt-mediated signal transduction. The AGS human gastric cancer cell line was infected with adenoviral constructs encoding a cDNA of PKG II (Ad-PKG II) to increase the expression of PKG II, and treated with 8-pCPT-cGMP to activate the enzyme. Western blotting was used to detect the phosphorylation/activation of the key components of the signal transduction pathway, including EGFR, PI3K, Akt, mTOR and NF-κB. The levels of apoptosis-related proteins, including Bax, Bcl-2, caspase 9 and DNA fragment factor (DFF), were also determined by western blotting. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining was used to detect the apoptosis of the AGS cells. The results revealed that EGF treatment increased the phosphorylation (activation) of EGFR, PI3K, Akt and mTOR, and increased the nuclear localization (activation) of NF-κB. EGF treatment also reduced the apoptosis of the AGS cells and increased the expression of the anti-apoptotic protein, Bcl-2, but had no effect on the expression of the pro-apoptotic protein, Bax, and did not alter the levels of caspase 9 and DFF. Increasing the PKG II activity of AGS cells by infecting them with Ad-PKG II and stimulating them with 8-pCPT-cGMP inhibited the EGF-induced activation of EGFR, PI3K, Akt, mTOR and NF-κB; caused an increase in caspase 9 breakdown (activation) and DFF levels; and reversed the anti-apoptotic effect of EGF. The results suggest that PKG II may also inhibit EGF-induced signal transduction of PI3K/Akt-mediated pathways, and further confirm that PKG II is able to block the activation of EGFR.

Berberine counteracts enhanced IL-8 expression of AGS cells induced by evodiamine

AimsAlthough showing an anti-tumor activity, evodiamine also up-regulated IL-8 production of human gastric cancer AGS cells. This study aimed to assess this effect and to examine whether co-administration with berberine counteracts it. Main methodsMTT assay was used to assess the cell proliferation and adhesive ability. Flow cytometry was performed to measure the cell cycle distribution. Wound healing assay was used to detect the migration ability of cells. IL-8 production was determined by ELISA. Levels of mRNA expression of IL-8, VCAM-1 and ICAM-1 were measured by real-time PCR. Molecular pathways involved were evaluated by ELISA and western-blotting methods. Key findingsEvodiamine triggered proliferative inhibition and cell cycle arrest, and decreased migration of AGS cells. IL-8 expression and the adhesive ability of AGS cells to HUVECs were significantly increased by evodiamine, but were inhibited after being co-treated with berberine in AGS cells. As IL-8 was neutralized, increased adhesion of AGS cells to HUVECs induced by evodiamine was abolished. Berberine significantly suppressed the up-regulation of VCAM-1 and the down-regulation of ICAM-1 induced by evodiamine. Evodiamine provoked IL-8 secretion via ERK1/2, SAPK/JNK, JAK2 and AP-1 pathways which could be counteracted by berberine. SignificanceAlthough showing anti-proliferative and anti-migratory activities in AGS cells, evodiamine displayed a potential tendency to promote metastasis of gastric cancer cells by increasing IL-8 secretion and adhesion molecules. However, berberine could counteract the side-effect and simultaneously keep anti-proliferative and anti-migratory properties of evodiamine on AGS cells, which reduces the risk to use evodiamine in therapy of gastric cancers.

Capsosiphon fulvescens glycoprotein reduces AGS gastric cancer cell migration by downregulating transforming growth factor-β1 and integrin expression

Seaweeds are commonly used as functional foods and drugs. A glycoprotein (GP) from the green alga Capsosiphonfulvescens (Cf) has been reported to have antitumor activity toward various cancer cells. We previously observed that Cf-GP induced different pathways of apoptosis in AGS human gastric cancer cells. Transforming growth factor (TGF)-β1 plays an important role in cancer cell migration. Increased TGF-β1 levels increase the expression of the small GTPases and activate the FAK/PI3K/AKT pathways, resulting in the upregulation of integrin receptor proteins, which mediate the attachment of cells to surrounding tissues, cells or extracellular matrix. Thus, the inhibition of TGF-β1 signaling would downregulate integrin expression and thereby effectively decrease cell growth and migration. In the present study, we determined the effect of Cf-GP treatment on the proliferation, migration and apoptosis of AGS human gastric cancer cells. To investigate the mechanism by which Cf-GP exerts its anticancer actions, we examined the effect of Cf-GP on the expression levels of TGF-β1, FAK, PI3K, AKT, the small GTPases and integrins in AGS cells. Our findings indicate that Cf-GP inhibits AGS cell proliferation and migration by downregulating integrin expression via the TGF-β1-activated FAK/PI3K/AKT pathways. These results suggest that Cf-GP may be an important factor in the development of functional foods and therapeutic agents.

Atrial natriuretic peptide modulates the proliferation of human gastric cancer cells via KCNQ1 expression

Atrial natriuretic peptide (ANP) and brain NP (BNP) belong to the NP family that regulates mammalian blood volume and blood pressure. ANP signaling through NP receptor A (NPR-A)/cyclic guanosine 3′5′-monophosphate (cGMP)/ cGMP-dependent protein kinase (PKG) activates various downstream effectors involved in cell growth, apoptosis, proliferation and inflammation. Evidence has shown the critical role of plasma K+ channels in the regulation of tumor cell proliferation. However, the role of ANP in the proliferation of gastric cancer cells is not clear. In the present study, the expression of NPR-A in the human gastric cancer cell line, AGS, and the effect of ANP on the proliferation of AGS cells were investigated using western blotting, immunofluorescence, qPCR and patch clamp assays. The K+ current was also analyzed in the effect of ANP on the proliferation of AGS cells. NPR-A was expressed in the human gastric cancer AGS cell line. Lower concentrations of ANP promoted the proliferation of the AGS cells, although higher concentrations decreased their proliferation. Significant increases in the levels of cGMP activity were observed in the AGS cells treated with 10−10, 10−9 and 10−8 M ANP compared with the controls, but no significant differences were observed in the 10−7 and 10−6 M ANP groups. The patch clamp results showed that 10−9 M ANP significantly increased the tetraethylammonium (TEA)- and 293B-sensitive K+ current, while 10−6 M ANP significantly decreased the TEA- and 293B-sensitive K+ current. The results showed that 10−10 and 10−9 M ANP significantly upregulated the expression of potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) at the protein and mRNA levels, although 10−7 and 10−6 M ANP significantly downregulated the expression of KCNQ1. The data indicated that lower and higher concentrations of ANP have opposite effects on the proliferation of AGS cells through cGMP-dependent or -independent pathways. KCNQ1 upregulation and downregulation by lower and higher concentrations of ANP, respectively, have separate effects on the promotion and inhibition of proliferation.

Apoptosis and G2/M arrest induced by Allium ursinum (ramson) watery extract in an AGS gastric cancer cell line

BackgroundThe present study was designed to determine whether Allium ursinum L (ramson) could inhibit the proliferation of human AGS gastric cancer cells. Furthermore, we attempted to determine whether this inhibition could occur by targeting regulatory elements of the cell cycle.MethodsFlow cytometry was used to observe apoptosis and the cell cycle in AGS cell lines treated or not treated with ramson watery extract. Proteins related to the cell cycle were detected by Western blotting. Caspase activity was measured using a colorimetric assay kit according to the manufacturer’s instructions.ResultsRamson watery extract induced apoptosis and G2/M phase arrest in AGS cells. Western blotting showed that cyclin B was inhibited by ramson watery extract. However, G1 phase-related proteins remain unchanged after treatment.ConclusionOur results indicate that ramson effectively sup pressed proliferation and induced apoptosis and G2/M arrest in AGS cells by regulating elements of the cell cycle.

Inhibition of AGS human gastric cancer cell invasion and proliferation by Capsosiphon fulvescens glycoprotein

Seaweeds are increasingly being used as foodstuffs and therapeutics. Capsosiphon fulvescens (C. fulvescens) is a green sea alga which has demonstrated anti-tumor activity in various cancer cell lines. In cancer cells, homeostasis is not maintained, enabling mutations to develop and growth to continue unchecked. Overexpression of matrix metalloproteinase (MMP) and tight junction (TJ) proteins is important for cancer cell proliferation, invasion and metastasis. In addition, these proteins are closely associated with cell membrane permeability. In the current study, C. fulvescens glycoprotein (Cf-GP) was found to inhibit TJ proteins and invasion of AGS human gastric cancer cells. Cf-GP-mediated inhibition of cell proliferation and invasion was confirmed, as well as changes in TJ protein levels. In addition, MMP-2 and −9 activities were inhibited, as indicated by increased transepithelial electrical resistance. Inhibition of MMP protein expression was also found to correlate with tissue inhibitor of metalloproteinase 1 and Cf-GP treatment, as revealed by western blot analysis and RT-PCR. In conclusion, these results indicate that Cf-GP inhibits cancer cell invasion and therefore demonstrates a potential therapeutic strategy to decrease cancer metastasis.

Tu1900 MiR-1246 and Mir-302 Are Novel Targets of Epigenetic Therapy With Dznep and SAHA in Human Cancer Cells

Introduction Epigenetic therapy with DNA methylation inhibitors and histone deacetylase (HDAC) inhibitors holds clinical promise for the treatment of human malignancies. MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous silencers of various target genes and play critical roles in cancer. We have proposed that epigenetic activation of tumor suppressor miRNAs can be a novel therapeutic approach for human cancers (Cancer Cell 9: 435, 2006). Recently, 3-deazaneplanocin A (DZNep) was discovered to inhibit a polycomb group protein, enhancer of zeste homologue 2 (EZH2), which has activity for trimethylation of histone H3K27. To investigate the molecular mechanisms underlying the effect of epigenetic therapy with DZNep and the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) against human cancer, miRNA expression profiles were analyzed. Materials and Methods AGS human gastric cancer cells and HepG2 human liver cancer cells were treated with DZNep and SAHA. MiRNA expression profiles were analyzed by microarray analysis and TaqMan quantitative RT-PCR. Cell proliferation was analyzed by cell counting, and target genes of miRNAs were analyzed by Western blotting. Levels of apoptosis and the cell cycle were analyzed by flow cytometry. Results The results of microarray and quantitative RT-PCR analyses revealed that miR-1246 was up-regulated by treatment with DZNep in both AGS and HepG2 cells, and that miR-302 was up-regulated by treatment with SAHA in AGS cells. Treatment with DZNep or SAHA reduced the cell proliferation activity of both AGS and HepG2 cells, and this reduction was more marked when both drugs were used in combination. The anti-apoptotic factor DYRK1A, which was recently identified as one of the targets of miR-1246, was significantly suppressed by treatment with DZNep and SAHA, resulting in apoptosis of AGS andHepG2 cells. In addition, treatment of AGS cells with DZNep and SAHA suppressed CDK2 and BMI-1, which were recently identified as the targets of miR-302, inducing cell cycle (G1/S) arrest of AGS cells. Conclusions These findings suggest that DZNep is a promising drug for the treatment of gastric and liver cancers via activation of miR-1246. DZNep and SAHA may synergistically suppress the proliferation of gastric cancer cells via activation of miR-1246 and miR-302. MiR-1246 and miR-302 are novel targets of epigenetic therapy with DZNep and SAHA in human cancer cells.

Abstract 391: Short hairpin RNA targeting autotaxin reduces the human gastric carcinoma AGS cell proliferation, migratory and invasive capability in vitro and causes tumor regression in vivo.

Abstract To investigate the effect of short hairpin RNA(shRNA) targeting autotaxin on the migratory and invasive capability of human gastric cancer cell AGS and the growth xenografts in nude mice. The pSUPER-ATX and pSUPER-mock (non-specific), which were constructed in corresponding to the ATX-shRNA and negative control mock-shRNA synthesized on basis of gene sequence, were transfected with blank plasmid pSUPER-control into human gastric cancer cell AGS by Lipofectamine. At 24 h, 48 h and 72 h post-transfection, the cells were harvested and analyzed. The endogenous ATX mRNA and protein of different group AGS cells were detected by RT-PCR and western blot assays. The cell proliferation was measured by MTT assay. In vitro Transwell and Matrigel assay were used to detect the cell migratory and invasive capability. Tumor xenograft model was generated by subcutaneous injection of AGS cells into the dorsum of nude mice. The growth of xenograft tumor was monitored and measured; change of the tumor morphology and mice organs were observed by HE staining. The expressions of autotaxin, MMP-2 and MMP-9 protein in xenograft tumor tissue were detected by immunohistochemistry and western blot. in vitro, both the mRNA and protein level of ATX in pSUPER-ATX group were significantly down-regulated (P<0.01), and the cell proliferation, migration and invasive capability were significantly decreased as well. In vivo, no obvious damage of the organs was found. The pSUPER-ATX significantly suppressed the tumor volume and weight since 7th week after cell transplantation, when compared to the pSUPER-mock, pSUPER-control and WT groups, the inhibition rates were about 50%(P<0.05). However, no significant differences of these parameters were found among the WT, pSUPER-mock and pSUPER-control group. Furthermore, autotaxin, MMP-2/MMP-9 were expressed at significantly lower level in pSUPER-ATX group than in other three groups (P<0.05), and a significant correlation between ATX and MMP-2 expression was displayed (r=0.869, P<0.01). The specific shRNA targeting ATX down-regulated the endogenous ATX of human gastric cancer AGS cells, and could inhibite the AGS cell proliferation, migratory and invasive capability. Moreover, the shRNA targeting autotaxin inhibited the growth of human gastric cancer xenograft in nude mice. Citation Format: Zhouxun Chen, Yingpeng Huang, Xian Shen, Henning Dralle, Cuong Hoang-Vu, Oliver Gimm. Short hairpin RNA targeting autotaxin reduces the human gastric carcinoma AGS cell proliferation, migratory and invasive capability in vitro and causes tumor regression in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 391. doi:10.1158/1538-7445.AM2013-391 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

HPLC fraction of ethylacetate extract from Orostachys japonicus induces apoptosis in human gastric cancer cells

Orostachys japonicus is traditionally used as anti‐inflammatory, anti‐febrile, hemostatic, anti‐dote, and anti‐cancer agent in Korea. The soluble faction of ethyl acetate (EtOAc) showed the highest anti‐cancer activity on AGS human gastric cancer cells. Further factions of EtOAc extracts were performed by HPLC. The aim of the present study was to investigate the apoptotic activity of further fraction of EtOAc fraction of O. japonicus in AGS cells. ASG cells were cultured in several concentration of further fraction of EtOAc fraction. The apoptotic effect of further fractions of EtOAc fraction was measured using MTS assay, annexin V‐FITC, PI‐labeling, and western blotting. In MTS assay, the survival rates were decreased in dose‐dependent manner. O. japonicus‐induced apoptosis was confirmed by assays with annexin V‐FITC, PI‐labeling. The increase in apoptosis level induced was associated with down‐regulation of Bcl‐2 and activation of Bax, caspase ‐3, ‐8 and ‐9.