Normal Gastric Epithelial GES-1 Cells Research Articles

Propofol-Induced miR-493-3p Inhibits Growth and Invasion of Gastric Cancer through Suppression of DKK1-Mediated Wnt/β-Catenin Signaling Activation.

Gastric cancer (GC) is one of the most common malignant tumors and also one of the most deadly tumors. In recent years, studies have shown that propofol can inhibit the proliferation and metastasis of many tumor cells. In the present study, we aimed to investigate the underlying mechanism of propofol inhibition of the growth and invasion of GC cells. Human gastric cancer cell line SGC-7901 and human normal gastric epithelial cell GES-1 were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum at 37°C with 5% CO2. Propofol of different concentrations (0, 2, 5, and 10 μg/mL) was used to treat SGC-7901, and miR-493-3p inhibitor was transfected into SGC-7901. The cell proliferation of SGC-7901 was analyzed by MTT as well as colony formation assay. The qRT-PCR was used to assess the expression of mRNA for key genes. We examined the protein expression of DKK1 and relative markers with western blot. Putative binding places of miR-493-3p on the 3'-untranslated area of DKK1 were predicted using bioinformatics and dual-luciferase method. Propofol prohibited phenotypic features of GC, according to our findings. Furthermore, research into the underlying mechanisms of propofol's suppressive effects in GC cell proved that propofol therapy improved the degrees of expression of the potential tumor suppressor miR-493-3p. The inhibiting properties of propofol on GC cell development, migration, and invasion were abolished when propofol-induced miR493-3p was silenced with anti-miR-493-3p. We also found that this drug reversed epithelial-mesenchymal transformation in SGC-7901 cells via inducing miR-493-3p. Propofol-induced miR-493-3p decreases GC cell development via targeting DKK1 and hence inhibits Wnt/β-catenin signaling, according to these findings. Propofol-induced miR-493-3p decreased GC cell development via targeting DKK1 and hence inhibited Wnt/β-catenin signaling, according to these findings.

Novel Phenylmethylenecyclohexenone Derivatives as Potent TrxR Inhibitors Display High Antiproliferative Activity and Induce ROS, Apoptosis, and DNA Damage.

The natural product piperlonguminine (PL) has been shown to exert potential anticancer activity against several types of cancer via elevation of reactive oxidative species (ROS). However, the application of PL has been limited due to its poor water solubility and moderate activity. To improve PL's potency, we designed and synthesized a series of 17 novel phenylmethylenecyclohexenone derivatives and evaluated their pharmacological properties. Most of them exerted antiproliferative activities against four cancer cell lines with IC50 values lower than PL. Among these, compound 10 e not only showed good water solubility and exerted the most potent antiproliferative activity against HGC27 cells (IC50 =0.76 μM), which was 10-fold lower than PL (IC50 =7.53 μM), but also exhibited lower cytotoxicity in human normal gastric epithelial cells GES-1 compared with HGC27 cells. Mechanistically, compound 10 e inhibited thioredoxin reductase (TrxR) activity, increased ROS levels, and diminished mitochondrial transmembrane potential (MTP) in HGC27 cells. Furthermore, 10 e also induced G2 /M cell-cycle arrest, and triggered cancer cell apoptosis through the regulation of apoptotic proteins. Finally, 10 e promoted DNA damage in HGC27 cells via the activation of the H2AX(S139ph) and p53 signaling. In conclusion, 10 e, with prominent tumor selectivity and water solubility, could be a promising candidate for the treatment of cancer and, as such, warrants further investigation.

LncRNA SUMO1P3 regulates the invasion, migration and cell cycle of gastric cancer cells through Wnt/β-catenin signaling pathway

Objective To investigate the regulatory effect of long non-coding RNA (lncRNA) SUMO1P3 on invasion, migration and cell cycle of gastric cancer (GC) cells through Wnt/β-catenin signaling pathway. Methods Tumor tissues and adjacent normal tissues from the GC patients were collected, and human normal gastric epithelial cells GES1 and GC cells SGC-7901, MKN45, HGC-27 and AGS were selected for study. The expression of SUMO1P3 in GC tissues and cells were detected by RT-qPCR. The effects of SUMO1P3 on the proliferation, invasion and migration of SGC-7901 and MKN45 cells were detected by CCK-8, transwell and wound healing assay respectively, and the effects of SUMO1P3 on apoptosis and cycle progression of SGC-7901 and MKN45 cells were detected by flow cytometry. The expressions of Wnt/β-catenin pathway-related and cell cycle-related proteins were detected by Western blot. Results The expression of SUMO1P3 was significantly upregulated in GC tissues and cell lines. Downregulation of SUMO1P3 significantly inhibited the SGC-7901 and MKN45 cell proliferation, invasion, migration, and cycle progression and promoted the cell apoptosis, while overexpression of SUMO1P3 showed the opposite effect. Further study showed that downregulation of SUMO1P3 significantly reduced the expressions of Wnt1, β-catenin, c-myc, and Cyclin D1 in SGC-7901 and MKN45 cells. Conclusion SUMO1P3 may promote invasion, migration, and cycle progression of SGC-7901 and MKN45 cells by enhancing the Wnt/β-catenin pathway.

Effects of Long Non-coding RNA Plasmacytoma Variant Translocation 1 Gene on Inflammatory Response and Cell Migration in Helicobacter Pylori Infected Gastric Epithelial Cell Line

Objective To investigate the mechanism of long non-coding RNA plasmacytoma variant translocation 1 (PVT1) in gastric cancer caused by helicobacter pylori (HP) infection. Methods The expression of PVT1 was detected by quantitative real-time polymerase chain reaction in HP-infected normal gastric epithelial cells GES-1. Gastric cancer cell line SGC-7901 was transfected with PVT1 small interfering RNA and co-cultured with HP,and then the inflammatory cytokines such as tumor necrosis factor-α (TNF-α),interleukin (IL) -1β,IL-6 and IL-8 were detected. After PVT1 was knocked down,the effects of PVT1 on the proliferation and migration of gastric cancer cells were examined by cell scratch assay. RNA-pulldown combined with mass spectrometry was used to detect the protein binding to PVT1,and the result of mass spectrometry was verified by RNA-pulldown combined with Western blot. Results In HP-infected normal gastric epithelial cells GES-1,quantitative real-time polymerase chain reaction showed that PVT1 was significantly up-regulated (t=7.160,P=0.019). PVT1 was knocked down in gastric cancer cells,and then infected with HP. The expressions of inflammatory factors including TNF-α (t=3.899,P=0.011),IL-1β (t=14.610,P=0.000),and IL-8 (t=6.557,P=0.001) were significantly inhibited. Although PVT1 knockdown had no significant effect on the proliferation ability of gastric cancer cells,it inhibited the migration of cells. PVT1 might interact with RPS8 protein. Conclusion PVT1 may act as a pro-inflammatory factor and regulate gastric cancer caused by HP infection.

Downregulation of microRNA‐92b‐3p suppresses proliferation, migration, and invasion of gastric cancer SGC‐7901 cells by targeting Homeobox D10

To investigate the effect and mechanism of microRNA-92b-3p (miR-92b-3p) targeting Homeobox D10 (HOXD10) on proliferation, migration, and invasion of gastric cancer, we detected the expression of miR-92b-3p and HOXD10 in SGC-7901 cells. The effects of miR-92b-3p or HOXD10 on proliferation, migration, invasion, and matrix metalloproteinase (MMP)-2/9 expression in SGC-7901 cells were measured by the Cell Counting Kit-8 assay, Transwell assay, and Western blot, respectively. The results showed that miR-92b-3p expression was increased, and HOXD10 expression was decreased in SGC-7901 cells, compared with human normal gastric epithelial cells GES-1. Functional experiments demonstrated that cell proliferation, migration, invasion, and expression of MMP-2/9 in SGC-7901 cells were significantly inhibited by miR-92b-3p silencing and HOXD10 overexpression. Moreover, HOXD10 was a potential target gene of miR-92b-3p as evidenced by the TargetScan software and double luciferase reporter assay. In the rescue experiment, knockdown of HOXD10, accompanied by higher expression of MMP-2/9, could significantly eliminate the inhibitory effects of miR-92b-3p silencing on cell proliferation, migration, and invasion. In conclusion, miR-92b-3p is highly expressed in gastric cancer SGC-7901 cells, and interfering with its expression might inhibit SGC-7901 cell proliferation, migration, and invasion via downregulating MMP-2/9 expression and targeting HOXD10.

Effect of euphorbia factor L1 on oxidative stress, apoptosis, and autophagy in human gastric epithelial cells

BackgroundEuphorbia factor L1 (EFL1), a lathyrane-type diterpenoid from the medicinal herb Euphorbia lathyris L. (Euphorbiaceae), has been reported for many decades to induce gastric irritation, but the underlying mechanisms remain unclear. PurposeThe objective of this study was to investigate EFL1-induced cytotoxicity and the potential mechanisms of action on the human normal gastric epithelial cell GES-1. MethodsGES-1 cells were treated with EFL1 (12.5–200 μM) for different time intervals, and cell survival, LDH release, intracellular reactive oxygen species (ROS), malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity were detected. Mitochondrial membrane potential (MMP) assay, DAPI staining, DNA fragment assay, and annexin V-FITC/PI staining were performed. The interaction between EFL1 and Bcl-2, cytochrome c, caspase-9, caspase-3, PI3K, AKT, and mTOR proteins was simulated by molecular docking. The mRNA and protein expression of apoptosis and autophagy factors were detected by RT-qPCR and Western blotting. ResultsEFL1 decreased survival, increased LDH leakage, and induced abnormal production of ROS, MDA and SOD in GES-1 cells. Mitochondria-mediated apoptosis was characterized by decreased MMP, condensed nuclei, fragmented DNA, and increased apoptosis rate. EFL1 interacted with proteins via hydrogen bonding. The mRNA, total or phosphorylated protein expression of Bcl-2, mitochondrial cytochrome c, PI3K, AKT, mTOR and p62 were downregulated; in contrast, those of cytoplasmic cytochrome c, cleaved caspase-9, cleaved caspase-3, LC3-ll and Beclin-1 were upregulated. ConclusionThese findings indicated that EFL1 decreased the survival of GES-1 cells through EFL1-induced oxidative stress, activation of the mitochondria-mediated apoptosis as well as autophagy via inhibition of the PI3K/AKT/mTOR pathway.

Expression of miR-124 in gastric adenocarcinoma and the effect on proliferation and invasion of gastric adenocarcinoma SCG-7901 cells.

Expression of miR-124 in gastric adenocarcinoma cell line SGC-7901 and its effect on biological functions was investigated. Expression of miR-124 in cancer tissues and paracancerous tissues of gastric adenocarcinoma patients was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RT-qPCR was used to detect the expression of miR-124 in human normal gastric epithelial cells GES-1 and gastric adenocarcinoma SGC-7901 cells. Cells in miR-124 group were transfected with miR-124 agomir, cells in NC group were transfected with agomir-negative control sequence and cells in the control group were not transfected. MTT assay was used to detect cell proliferation, and Transwell invasion assay to detect cell invasion ability, and the effect of transfected miR-124 agonist on the proliferation and invasive ability of gastric adenocarcinoma cells was evaluated. RT-qPCR results showed that miR-124 expression was significantly downregulated in gastric adenocarcinoma tumor tissues compared with paracancerous tissues. Compared with cells of normal human gastric epithelial cell line GES-1, the expression of miR-124 human gastric adenocarcinoma SGC-7901 cells was significantly downregulated. At 12 h, there was no significant difference in OD at 490 nm in the three groups (P>0.05). OD (490) in the three groups showed a gradual upward trend. After transfection, proliferation curves of the three groups showed an upward trend, proliferation rate of miR-124 group was significantly lower than that of NC and control groups (P<0.05). The number of invading cells in miR-124 group was significantly lower than that in NC group and control group, but there was no significant difference in the number of cell invasion between the NC and control groups. miR-124 can inhibit the proliferation and invasion of gastric adenocarcinoma cells. Downregulation of miR-124 expression in gastric adenocarcinoma may be closely related to the development of gastric adenocarcinoma.

Long noncoding RNA NEAT1‐modulated miR‐506 regulates gastric cancer development through targeting STAT3

Accumulating evidence has indicated that long noncoding RNA NEAT1 exerts critical roles in cancers. So far, the detailed biological role and mechanisms of NEAT1, which are responsible for human gastric cancer (GC), are still largely unknown. Here, we observed that NEAT1 and STAT3 expressions were significantly upregulated in human GC cells including BGC823, SGC-7901, AGS, MGC803, and MKN28 cells compared with normal gastric epithelial cells GES-1, while miR-506 was downregulated. We inhibited NEAT1 and observed that NEAT1 inhibition was able to repress the growth, migration, and invasion of GC cells. Conversely, overexpression of NEAT1 exhibited an increased ability of GC progression in BGC823 and SGC-7901 cells. Bioinformatics analysis, dual luciferase reporter assays, RIP assays, and RNA pull-down tests validated the negative binding correlation between NEAT1 and miR-506. In addition, it was found that miR-506 can modulate the expression of NEAT1 in vitro. STAT3 was predicted as a messenger RNA (mRNA) target of miR-506, and miR-506 mimics can suppress STAT3 mRNA expression. Subsequently, it was observed that downregulation of NEAT1 can restrain GC development by decreasing STAT3, which can be reversed by miR-506 inhibitors. Therefore, it was hypothesized in our study that NEAT1 can be recognized as a competing endogenous RNA to modulate STAT3 by sponging miR-506 in GC. In conclusion, we implied that NEAT1 can serve as an important biomarker in GC diagnosis and treatment.

MiR-505 acts as a tumor suppressor in gastric cancer progression through targeting HMGB1.

Gastric cancer (GC) is a frequent type of malignant tumor worldwide. GC metastasis results in the majority of clinical treatment failures. MicroRNAs (miRNA) are identified to exhibit crucial roles in GC. Our current study aimed to explore the biological roles of miR-505 in GC progression. It was observed that miR-505 was robustly decreased in GC cells compared with human normal gastric epithelial GES-1 cells. Overexpression of miR-505 was able to repress GC progression in AGS and BGC-823 cells. In addition, high-mobility group box 1 (HMGB1) has been identified as a crucial oncogene in several cancer types. By carrying out bioinformatics analysis, HMGB1 was predicted as a direct target of miR-505. Meanwhile, HMGB1 was found to be significantly increased in GC cells and it was confirmed in our study that miR-505 can directly target HMGB1 in vitro. miR-505 mimics can inhibit HMGB1 messenger RNA and protein expression dramatically. Subsequently, knockdown of HMGB1 can inhibit GC cell proliferation, colony formation, and induce cell apoptosis. Furthermore, HMGB1 silence suppressed GC cell migration and invasion greatly in vitro. Finally, it was validated that miR-505 can inhibit GC progression by targeting HMGB1 in vivo. Taken these together, it was indicated that miR-505/HMGB1 axis was involved in the development of GC. miR-505 can serve as a potential prognostic indicator in GC therapy.

Steroidal dimer by001 inhibits proliferation and migration of esophageal cancer cells via multiple mechanisms.

To investigate the potential inhibitory effects of structurally novel steroidal dimer by001 in esophageal cancer in vitro. The cytotoxicity of by001 on esophageal, gastric, neuroblastoma and prostate cancer cells was examined MTT assay and colony formation assay. By001 induced apoptosis and production of intracellular reactive oxygen species on esophageal cancer cells Ec109, TE-1 and human normal gastric epithelial cells GES-1 was detected by flow cytometry. The effect of by001 on mitochondrial membrane potential was detected by fluorescence microscope through JC-1 staining. The level of intracellular reactive oxygen species was measured by fluorescence microscope and flow cytometry via DCFH-DA staining. The effect of by001 on members of Bcl-2 family, Fas, LC3, PARP and caspases was determined by Western blot. The effect of by001 on migration was measured by transwell assay. By001 effectively inhibited proliferation of esophageal, gastric, neuroblastoma and prostate cancer cells in a time- and concentration-dependent manner in vitro. By001 reduced the number and the size of colonies at low micromolar concentrations, elevated cellular ROS levels and caused mitochondrial dysfunction in esophageal cancer cells. Molecular mechanistic studies showed that by001 triggered apoptosis through regulating members of Bcl-2 family and Fas. These findings suggested that by001 may inhibited proliferation of esophageal cancer cells through mitochondria and death receptor-mediated apoptotic pathways, autophagy induction, as well as suppressed migration of esophageal cancer cells.

Expression of superoxide dismutase 2 in gastric cancer and downregulation of its expression on biological characteristics

Objective To examine the superoxide dismutase 2 (SOD2) expression in gastric cancer (GC) and the effect of downregulated SOD2 expression on proliferation and reactive oxygen species (ROS) level of GC cells. Methods The SOD2 expression of GC patient tissue and paired adjacent tissue was detected by immunohistochemical staining; The SOD2 expression of normal gastric epithelial cell GES1 and GC cells MKN1, MKN45, NUGC3, NUGC4, AGS, N87 was analyzed by Western blotting; SOD2 siRNA (siSOD2) was transfected into MKN1 and NUGC4 cells to downregulate SOD2 expression and set non-target control siRNA (siNTC) as the negative control; Cell proliferation and cellular ROS were determined using counting kit-8 (CCK-8) assay and cellular ROS detection assay kit. Results The positive percentage of SOD2 expression was 72.7% (48/66) in GC patient tissue and was 40.9% (27/66)in paired adjacent tissue. The difference was statistically significant (χ2=13.617, P=0.000). The SOD2 expression of GC cells was higher than that in gastric epithelial cell GES1 (tMKN1=23.062, PMKN1=0.002; tMKN45=5.648, PMKN45=0.030; tNUGC3=26.613, PNUGC3=0.001; tNUGC4=17.103, PNUGC4=0.003; tAGS=39.475, PAGS=0.001; tN87=37.677, PN87=0.001). The proliferation in GC cells MKN1 and NUGC4 with siSOD2 transfection was lower than that in control groups in 72 h, 96 h (MKN1, t72 h=6.329, P72 h=0.001; t96 h=10.487, P96 h=0.000), (NUGC4, t72 h=5.551, P72 h=0.003; t96 h=6.304, P96 h=0.001). The ROS level of siSOD2 and siNTC groups in MKN1 was 195.341±10.71, 99.439±9.311; the ROS level of siSOD2 and siNTC groups in NUGC4 was 181.561±14.502, 97.876±12.867. The ROS level of GC cells with siSOD2 transfection was significantly higher than the control group (MKN1, t=11.295, P=0.001), (NUGC4, t=23.476, P=0.000). Conclusion SOD2 expression is highly upregulated in GC patients and GC cells. Downregulation of SOD2 expression can decrease GC cell proliferation and increase cellular ROS. Key words: Gastric cancer; Superoxide dismutase 2; Reactive oxygen species; Proliferation

DS2, a newly synthetic ent-kaurane diterpenoid analog, inhibits proliferation and migration of human gastric cancer cell

Objective: To investigate the effects and the underlying mechanism of DS2, a newly synthetic analog of natural ent-kaurane diterpenoid, on the proliferation and migration capabilities of human gastric cancer cells. Methods: MTT assay, colony formation assay and flow cytometry were used to measure the effects of DS2 on growth, apoptosis and cell cycle of several human gastric cancer cell lines. The function of DS2 in the migration was further detected by wound healing and transwell assays. The expression of migration related proteins were determined by western blot. Results: DS2 inhibited the growth of MGC-803, SGC-7901 and HGC-27 cells in a dose dependent manner. After treatment of DS2 at a concentration of 6.25 μmol/L for 24 h, the survival rates of MGC-803, SGC-7901 and HGC-27 cells were 53.87±3.05%, 55.91±6.97% and 32.41±2.64%, respectively. However, for the normal gastric epithelial cell GES-1, no obvious growth inhibition was observed. In addition, DS2 caused significant G(2)/M arrest and induced apoptosis in MGC-803 cells. Furthermore, compared with the negative control, the colony formation, wound healing rate as well as the number of migrating cells of MGC-803 were significantly decreased in a dose dependent manner after DS2 treatment. DS2 induced the expression of E-cadherin, whereas β-catenin and N-cadherin levels were downregulated in MGC-803. Conclusion: The new compound DS2 has a strong anti-cancer activity, and this study will help us to design and synthesize better diterpenoids derivatives.

Dual targeting of survivin and X-linked inhibitor of apoptosis protein suppresses the growth and promotes the apoptosis of gastric cancer HGC-27 cells.

Gastric cancer can be a fatal tumor and therefore represents one of the primary challenges in modern oncology. Survivin and X-linked inhibitor of apoptosis protein (XIAP) are members of the IAP family, which exerts a strong inhibitory effect on cellular apoptosis. In previous studies, the expression levels of survivin and XIAP have been demonstrated to influence the prognosis of patients with gastric cancer; therefore, the present study investigated the effect of silencing survivin and XIAP on the biological activity of the gastric cancer HGC-27 cell line. It was demonstrated that the expression levels of survivin and XIAP were significantly increased in gastric cancer tissues, compared with the adjacent non-tumor tissues. Furthermore, it was observed that the expression levels of survivin and XIAP were similarly elevated in gastric cancer HGC-27 cells, compared with normal gastric epithelial GES-1cells. Furthermore, small interfering RNA-mediated surviving- or XIAP-knockdown, in addition to the dual knockdown of survivin and XIAP, inhibited the proliferation and promoted the apoptosis of HGC-27 cells. Simultaneous inhibition of XIAP and survivin expression was more effective, compared with inhibition of XIAP or survivin alone. These results indicated that the dual knockdown of survivin and XIAP may be an effective strategy for treating gastric cancer in the future.

MicroRNA‑153 functions as a tumor suppressor in gastric cancer via targeting Kruppel‑like factor 5

Various microRNAs (miRs) have been demonstrated to serve important roles in gastric cancer (GC). miR-153 in particular has been reported to serve a suppressive role in GC; however, the underlying mechanism remains unclear. In the present study Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to examine the mRNA and protein expression of Kruppel-like factor 5. An MTT, wound healing and transwell assay were used to study cell proliferation, migration and invasion, respectively. In the present study, quantitative polymerase chain reaction data indicated that miR-153 was significantly downregulated in GC tissues compared with the adjacent non-tumor tissues. In addition, the reduced expression of miR-153 was significantly associated with GC aggressiveness and poor prognosis of patients. The expression of miR-153 was also reduced in GC cell lines, including KATO III, NCI-N87, SNU-16 and SNU-5, when compared with normal gastric epithelial GES-1 cells. Overexpression of miR-153 in the GC SNU-5 cells by miR-153 mimic transfection significantly inhibited the cell proliferation, migration and invasion. Furthermore, KLF5 was identified as a target gene of miR-153 in SNU-5 cells by bioinformatics prediction. It was observed that KLF5 was significantly upregulated in GC tissues and cell lines, and its expression was negatively regulated by miR-153 in SNU-5 cells. Overexpression of KLF5 impaired the suppressive effects of miR-153 on the proliferation, migration and invasion of SNU-5 cells. In conclusion, the present study demonstrated that miR-153 serves a tumor suppressive role in GC, at least partly, through directly targeting KLF5, thus highlighting the clinical significance of miR-153 in GC.

XIST promotes gastric cancer (GC) progression through TGF-β1 via targeting miR-185.

LncRNAs and microRNAs can play significant roles in various cancers, including gastric cancer (GC). In our study, we investigated the role of lncRNA XIST in GC. We observed that XIST was increased in MGC803 and BGC823 cells compared to human normal gastric epithelial GES-1 cells. It was also shown that miR-185 was decreased in GC cell lines. Silencing XIST can inhibit the growth of GC cells and bioinformatics analysis was performed to confirm the correlation between XIST and miR-185. Interestingly, a negative correlation was indicated between XIST and miR-185 in GC cells. In addition, TGF-β1 was predicted as a target gene of miR-185. miR-185 can modulate TGF-β1 expression negatively in vitro. Moreover, we found that sh-XIST inhibited GC development via decreasing TGF-β1 by upregulating miR-185 in vitro. Therefore, we speculated that XIST can act as a competing endogenous lncRNA (ceRNA) to regulate TGF-β1 by sponging miR-185 in GC. Taken these together, it was indicated that XIST/miR-185/TGF-β1 axis participated in the development of GC. XIST could act as a potential prognostic biomarker in GC development.

Decreased expression of hsa_circ_0001895 in human gastric cancer and its clinical significances.

Circular RNAs are a special class of endogenous RNAs characterized by jointing 3' and 5' ends together via exon or intron circularization. Recent studies found that circular RNAs are involved in the development of some human diseases. However, little is known about their roles in human gastric cancer. In this study, we chose hsa_circ_0001895 as a targeted circRNA to investigate its clinical significances in gastric cancer patients. Hsa_circ_0001895 expression levels in five gastric cancer cell lines and 257 specimens of tissues were measured by real-time quantitative reverse transcription polymerase chain reaction. Then, the potential relationship between hsa_circ_0001895 expression levels and patients' clinicopathological factors was investigated. A receiver operating characteristic curve was constructed for evaluating the diagnostic value of hsa_circ_0001895. Hsa_circ_0001895 expression levels in five detected gastric cancer cell lines (AGS, BGC-823, HGC-27, MGC-803, and SGC-7901) were all significantly downregulated than those in normal gastric epithelial GES-1 cells. Besides, compared with healthy control tissues, it was downregulated not only in 69.8% (67/96) gastric cancer tissues but also in gastric precancerous lesions. Moreover, hsa_circ_0001895 expression levels were significantly correlated with cell differentiation, Borrmann type, and tissue carcino-embryonic antigen expression. Our results suggested that hsa_circ_0001895 may play crucial roles during gastric cancerogenesis and is a potential biomarker for clinical prognosis prediction.

The contribution of toll-like receptor 2 on Helicobacter pylori activation of the nuclear factor-kappa B signaling pathway in gastric epithelial cells

Helicobacter pylori (H. pylori) is a spiral shaped gram-negative bacterium that induces immune responses in the gastric mucosa. Toll-like receptors (TLRs) play important roles in mediating inflammatory cytokines by recognition of conserved molecular patterns on bacteria. Changes in the expression of toll-like receptor (TLR) 2, TLR4 and the relative inflammatory cytokines were analyzed in normal gastric epithelial GES-1 cells following treatment with H. pylori or Escherichia coli lipopolysaccharide (E. coli LPS) in order to investigate the contribution of TLRs in recognizing and mediating the inflammatory response to H. pylori, and study the differences in TLRs’ performance between H. pylori and E. coli. Specific inhibitors for the declines in TLR2 and TLR4 were also employed. The results showed that H. pylori infection increased TLR2 expression in GES-1 cells, but TLR4 remained unchanged regardless of H. pylori or TLR2 small interfering RNA treatment. Furthermore, the secretion of cyclooxygenase-2 (COX-2) induced by H. pylori was inhibited by declines in TLR2, but not in TLR4. In conclusion, TLR2 plays an even more important role than TLR4 not only in recognizing H. pylori, but also in the induction of inflammatory cytokines initiated by H. pylori. However, both TLR2 and TLR4 are necessary in mediating the inflammatory response to E. coli LPS.

The natural secolignan peperomin E induces apoptosis of human gastric carcinoma cells via the mitochondrial and PI3K/Akt signaling pathways in vitro and in vivo

BackgroundPeperomin E (PepE) is a type of secolignan that is a major component of the plant Peperomia dindygulensis. It has been shown to exert anticancer effects in various cancer cell lines; however, the effects of PepE on human gastric cancer remain unexplored. PurposeThe aim of this study was to investigate the effectiveness of PepE as a treatment of gastric cancer and to identify the underlying mechanisms of its anticancer activity. Study designThe efficacy of PepE was examined using human gastric carcinoma SGC-7901, BGC-823, MKN-45 cell lines and normal gastric epithelial GES-1 cell line as an in vitro model and SGC-7901 xenograft mice as an in vivo model. MethodsCell viability assays were used to examine the anticancer effect of 0–204.8µM concentrations of PepE in vitro. Additionally, flow cytometry and western blotting were used to elucidate the mechanism with a particular focus on apoptosis. SGC-7901 cells were injected into BALB/c mice, which were then treated with 5 or 15mg/kg/day dose of PepE. The in vivo activity of PepE was investigated by measuring tumors and conducting immunohistochemistry experiments. The safety of PepE was investigated by measuring blood biochemical parameters and conducting histopathological analysis. Taxol was used throughout as a positive control. ResultsThe results showed that PepE exhibited antiproliferative effects against gastric cancer cells and induced their apoptosis in a dose dependent manner with lower toxicity against normal gastric epithelial cells. Mechanistic evaluations indicated that PepE induced apoptosis by reducing the mitochondrial membrane potential (MTP), inducing cytochrome C release from mitochondria, reducing the ratio of Bcl-2/Bax and Bcl-xl/Bad, increasing activation of caspase-3, and decreasing the levels of PI3K and pAkt. The apoptotic effect of PepE on SGC-7901 cells was partially blocked by an Akt activator SC79. PepE potently inhibited in vivo tumor growth with no obvious toxicity following subcutaneous inoculation of SGC-7901 cells in nude mice. ConclusionsThese findings indicate that PepE can inhibit cell proliferation and induce apoptosis of gastric cancer cells through mitochondrial and PI3K/Akt signaling pathways with relative safety and may be a novel effective chemotherapeutic agent against gastric cancer.

Evolution of a gastric carcinoma cell-specific DNA aptamer by live cell-SELEX.

Aptamers have emerged as promising molecular probes for disease diagnosis and therapy. In the present study, the entire live cell-SELEX method was used to generate gastric cancer cell‑specific aptamers. Human gastric carcinoma AGS cells were used as target cells for positive selections and human normal gastric epithelial GES-1 cells as the negative cells for counter selections. The selection procedure was monitored by gel electrophoresis and flow cytometric analysis. By successive invitro evolutions and subsequent cloning and sequencing, a gastric carcinoma cell‑specific DNA aptamer termed cy-apt20 with minimal recognition to the controls was identified from the final enriched ssDNA pool. Flow cytometry binding assays revealed that cy-apt20 had a >70%binding rate to AGS cells and <30% binding affinity to non-gastric cancer cells. Furthermore, the targeting recognition of AGS cells was established by using minimal doses of FITC-cy-apt20 that continued for a long period of time. As visualized by fluorescence imaging, the majority of AGS cells were stained by FITC-cy-apt20. The fluorescence intensity of AGS cells was ~6-fold over that of non-AGS cells. The present study demonstrated that the entire live cell-SELEX was simple, but effective in generating gastric cancer cell‑specific aptamers, and that the aptamer cy-apt20 has great potential to be used for the study and diagnosis of gastric cancer.

Anti-cancer effect of rubropunctatin against human gastric carcinoma cells BGC-823

The Monascus pigment, rubropunctatin, was extracted and purified from red mold rice (RMR) and its cytotoxic activities against human gastric adenocarcinoma BGC-823 cells were studied both in vitro and in vivo. Rubropunctatin inhibited the proliferation of BGC-823 cells with an inhibitory concentration (IC₅₀) of 12.57 μM, while it exhibited no significant toxicity to normal gastric epithelial cell GES-1 at the same concentration. Treatment of BGC-823 cells with rubropunctatin resulted in a dose- and time-dependent apoptosis, as validated by the increase in the percentage of cells in sub-G1 phase and phosphotidylserine externalization. The in vivo experimental data demonstrated that rubropunctatin could offer similar therapeutic benefits in comparison with the same dose of taxol. After five times of intravenous injection, tumor weight in BGC-823-bearing nude mice reduced 23.5% at the dose of 8 mg/kg and 37.7% at the dose of 32 mg/kg, respectively. The expressions of 30 genes related to induction of apoptosis were found up-regulated significantly. The two most expressed genes were tumor necrosis factor (TNF) and DNA-damage inducible transcript 3. TNF was considered as a major mediator of apoptosis induced by rubropunctatin. This is the first report describing the anti-proliferative effect of rubropunctatin and its apoptosis mechanism on BGC-823 cells. Rubropunctatin has potential to be developed as a new natural anti-cancer agent.