Human Gastric Carcinoma Cell Research Articles

Alterations in metabolic pathways in gastric epithelial cells infected with Helicobacter pylori

Helicobacter pylori (H. pylori), which is a spiral-shaped Gram-negative microaerobic bacterium, is a causative pathogen. The entry of H. pylori into gastric epithelial cells involves various host signal transduction events, and its virulence factors can also cause a variety of biological responses. In this study, AGS human gastric carcinoma cells were infected with CagA-positive H. pylori strain ATCC43504, and then the metabolites in the AGS cells after the 2-, 6- and 12-h infections were analyzed by GC/MS-based metabolomic analysis. Among 67 metabolites detected, 11 metabolites were significantly altered by the H. pylori infection. The metabolite profiles of H. pylori-infected AGS cells were evaluated on the basis of metabolite pathways, and it was found that glycolysis, tricarboxylic acid (TCA) cycle, and amino acid metabolism displayed characteristic changes in the H. pylori-infected AGS cells. At 2 h post-infection, the levels of many metabolites related to TCA cycle and amino acid metabolism were lower in H. pylori-infected AGS cells than in the corresponding uninfected AGS cells. On the contrary, after 6-h and 12-h infections the levels of most of these metabolites were higher in the H. pylori-infected AGS cells than in the corresponding uninfected AGS cells. In addition, it was shown that the H. pylori infection might regulate the pathways related to isocitrate dehydrogenase and asparagine synthetase. These metabolite alterations in gastric epithelial cells might be involved in H. pylori-induced biological responses; thus, our findings are important for understanding H. pylori-related gastric diseases.

Perylenequione Derivatives with Anticancer Activities Isolated from the Marine Sponge-Derived Fungus, Alternaria sp. SCSIO41014.

Seven new secondary metabolites classified as two perylenequinone derivatives (1 and 2), an altenusin derivative (3), two phthalide racemates (4 and 5), and two phenol derivatives (6 and 7), along with twenty-one known compounds (8–28) were isolated from cultures of the sponge-derived fungus, Alternaria sp. SCSIO41014. The structures and absolute configurations of these new compounds (1–7) were determined by spectroscopic analysis, X-ray single crystal diffraction, chiral-phase HPLC separation, and comparison of ECD spectra to calculations. Altertoxin VII (1) is the first example possessing a novel 4,8-dihydroxy-substituted perylenequinone derivative, while the phenolic hydroxy groups have commonly always substituted at C-4 and C-9. Compound 1 exhibited cytotoxic activities against human erythroleukemia (K562), human gastric carcinoma cells (SGC-7901), and hepatocellular carcinoma cells (BEL-7402) with IC50 values of 26.58 ± 0.80, 8.75 ± 0.13, and 13.11 ± 0.95 μg/mL, respectively. Compound 11 showed selectively cytotoxic activity against K562, with an IC50 value of 19.67 ± 0.19 μg/mL. Compound 25 displayed moderate inhibitory activity against Staphylococcus aureus with an MIC value of 31.25 μg/mL.

Chibby is a weak regulator of β-catenin activity in gastric epithelium.

The canonical Wnt-β-catenin pathway is important in normal development. Mutations in β-catenin or proteins involved with regulating its phosphorylation or localization result in its nuclear accumulation where it activates its target genes and stimulates cell proliferation. This pathway is dysregulated in many different types of cancer, including gastric cancer (GC). Chibby (Cby) is a 14-kDa protein that inhibits β-catenin localization to the nucleus and represses β-catenin-induced transcriptional activity. In the current study, we examined the expression and function of Cby in normal and cancerous human gastric tissue. Reverse-transcription polymerase chain reaction and immunohistochemistry revealed that Cby is expressed in human stomach and localized to glandular elements. Immunohistochemical staining intensity of Cby was decreased in GC tissue when compared with normal gastric epithelium. In AGS cells, a human gastric carcinoma cell line, Cby expression was low. Stable AGS cell transfectants overexpressing Cby were prepared. Cby overexpression did not affect proliferation rates or β-catenin levels. However, confocal microscopy and subcellular fractionation studies revealed that Cby overexpression resulted in a small decrease in nuclear β-catenin. Moreover, Cby overexpression caused a molecular weight shift in nuclear β-catenin and resulted in decreased β-catenin signaling in AGS cells as measured by the TopFlash assay. However, Cby overexpression did not affect c-Myc protein levels. To conclude, Cby expression was decreased in GC samples and Cby expression altered β-catenin localization in cultured GC cells. However, Cby did not affect cell proliferation rates or β-catenin-induced protein expression. Cby may be involved in the early events in the pathogenesis of GC.

Long non-coding RNA NEAT1 promotes viability and migration of gastric cancer cell lines through up-regulation of microRNA-17.

Gastric carcinoma (GC) is a common cancer with heavy mortality and poor outcome at advanced stages and metastasis. Long non-coding RNA nuclear-enriched abundant transcript 1 (NEAT1) has been reported to be an oncogene in GC recently. However, the underlying mechanism is far from understood. We aimed to explore the role of NEAT1 in GC as well as the underlying mechanisms. The expression of NEAT1 in clinical human GC tissues and GC cell lines were assessed by quantitative reverse transcription PCR. Then, NEAT1 was non-physiologically expressed in GC cells (SGC-7901 and MKN45 cells), followed by estimation of cell viability, migration, invasion, apoptosis, activation of the phosphatidylinositol-3-kinase (PI3K)/AKT and glycogen synthase kinase 3β (GSK3β) pathways, and microRNA (miR)-17 level. Moreover, the effects of miR-17 inhibition on cell viability, migration, and activation of the PI3K/AKT and GSK3β pathways in GC cells overexpressing NEAT1 were also explored. NEAT1 was up-regulated in GC tissues and cell lines. Then, cell viability and migration of GC cells were markedly increased by NEAT1 overexpression, while the cell invasion and apoptosis were unchanged. The phosphorylated level of PI3K, AKT, and GSK3β were increased by NEAT1 overexpression. Subsequently, we found miR-17 level was positively correlated with NEAT1 expression, and NEAT1 functions through up-regulating miR-17. NEAT1 was up-regulated in GC tissues and cell lines. Its overexpression enhanced cell viability and migration through up-regulating miR-17, along with activation of the PI3K/AKT and GSK3β pathways.

Abstract 4537: Cryogenic biopsy device for preservation of labile biomarkers

Abstract Background: Preanalytical factors such as the handling of biospecimens between resection and analysis can have a significant impact on assay results which may adversely affect patient care. Global phosphoprotein analysis indicates that a 5 minute delay in time-to-stabilization (ischemia time) can impact 20% of phospho-serine and 50% of phospho-tyrosine residues in kinases. Consequently, inadequate tumor biopsy processing is a significant impediment to the use of phosphorylated biomarkers in oncology. We aim to develop new biopsy devices and methodologies to stabilize highly dynamic biomarkers and minimize the influence of preanalytical factors. Methods: A cryobiopsy device (CBD) was developed by integrating a 19G cryoablation needle with a 16G cutting cannula of a clinical biopsy device. The device enables in-situ freezing of the tissue sample before it is cut and removed from the tumor. We measured the stabilization of a highly labile biomarker, phosphorylated-MET (pMET), to compare between percutaneous biopsy samples acquired by the CBD (zero ischemic time) and by a clinical grade 18G biopsy device (Temno) (10-20 second ischemic time). The animal protocol was approved by the FNLCR's IACUC. Athymic nude mice (nu/nu NCr) were implanted with human gastric carcinoma cell line SNU-5. Tumors were biopsied 3 weeks after implantation (tumor size range 150-250mm3). The needle was passed through the skin into the tumor, a tissue sample was collected, and the biopsy needle was retracted. The tissue sample of the Temno device was rapidly frozen in a cryovial that was precooled in liquid nitrogen [1]. The already frozen tissue sample of the CBD was rapidly transferred to a second precooled cryovial. The vials were sealed and returned to liquid nitrogen. Frozen specimens were stored at −80°C until processing. Each tumor was biopsied once and the animal was euthanized. The methodology to determine the pMET/MET levels in the biopsy samples is detailed in [2]. Results: The pMET/MET ratio was not significantly different between samples acquired by the CBD and by the Temno device (mean±st dev): pY1234/35-MET/MET ratio: CBD 0.75±0.11; Temno 0.71±0.10 pY1356-MET/MET ratio: CBD 0.56±0.08; Temno 0.50±0.06 pY1235-MET/MET ratio: CBD 0.90±0.15; Temno 0.83±0.14 Conclusions: Past studies showed that tissue levels of highly labile biomarkers like pMET can change significantly in 1-2 minutes from the time of tissue harvesting [2]. The current study demonstrates that rapid freezing of the tissue within less than 20 seconds from tissue harvesting maintains the in-vivo levels of pMET similarly to tissue freezing before tissue harvesting. This result suggests that devices that rapidly freeze tissue samples after removal from the body, which are more suitable for clinical use, may be sufficient for preservation and accurate quantitation of highly labile biomarkers. 1. PMID: 18980982 2. PMID: 27001313 Supported by NCI Contracts HHSN261200800001E and HHSN261201200050C Citation Format: Erez Nevo, Melinda G. Hollingshead, Katherine Ferry-Galow, Jeevan P. Govindharajulu, Shoshan Nevo, Kay D. Gray, Ralph E. Parchment, James H. Doroshow, Apurva K. Srivastava. Cryogenic biopsy device for preservation of labile biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4537.

Generation and characterization of a paclitaxel-resistant human gastric carcinoma cell line.

The main aim of this study was to establish a novel paclitaxel (PTX)-resistant human gastric carcinoma cell line and to investigate its biological significance. A cell line, MGC803/PTX, was established by gradually increasing PTX density on the basis of MGC803 over a period of 10 months. In addition, a pair of resistant cell lines (SW620 and SW620/PTX) were added to further explain the resistant mechanism of PTX. The drug resistance index and stability of MGC803/PTX cells were detected using the Cell Counting Kit-8 method. The morphological features were observed using inverted microscopy. Apoptosis was measured by flow cytometry (FCM) and Hoechst 33258 fluorescence staining. The distribution of the cell cycle was determined by FCM, and protein expressions of P-gp, Bcl-2, Bax, and PARP were detected by western blot analysis. When characterizing the resistance in vitro, we found that MGC803/PTX cells were 10.3-fold more resistant to PTX compared with MGC803 cells. In addition, MGC803/PTX cells showed cross-resistance to 5-fluorouracil and adriamycin. FCM and Hoechst 33258 fluorescence staining indicated that MGC803/PTX cells had a significantly lower percentage of apoptotic cells after treatment with PTX compared with MGC803 cells. Other differences between parental cells and resistant cells included morphology, proliferation rate, doubling time, cell cycle distribution, and colony-formation rate. Western blot analysis indicated that P-gp, Bcl-2, and PARP protein were more abundant in MGC803/PTX and SW620/PTX cells compared with MGC803 and SW620 cells, whereas Bax protein levels were lower in resistant cells. Furthermore, MGC803/PTX cells showed obvious resistance to PTX in vivo. To our knowledge, this is the first report on the establishment of a PTX-resistant MGC803 cell line, which is an important tool to explore the resistance of anticancer drugs and to overcome tumor drug resistance.

Synthesis and anti-tumor activities of novel 7-O-amino acids chrysin derivatives

ObjectiveTo design and synthesize a series of chrysin derivatives and evaluate the antitumor activities with MTT assay, so as to investigate molecular structure-activity relationship with molecular docking. MethodsTarget products were synthesized with high yield by substitution reaction, hydrolysis reaction, esterification reaction, and saponification reaction in sequence, and activities of all compounds were evaluated with human gastric carcinoma cell lines MGC-803 and human breast carcinoma cell lines MCF-7 through standard MTT assay. Molecular docking results were calculated with Surflex Geom X programme of Sybyl X-2.0 version workstation. Results7-O-amino acids chrysin derivatives 6a–6l were synthesized and their inhibitory effects were evaluated by comparing the material chrysin with positive control drug 5-fluorouracil (5-FU). Among these derivatives, compound 5b (IC50 = 24.50 ± 2.26 µmol/L), 5k (IC50 = 24.30 ± 2.19 µmol/L), and 6f (IC50 = 24.61 ± 2.01 µmol/L) showed better inhibitory activities against MGC-803 cell lines, and compound 5g (IC50 = 13.15 ± 1.73 µmol/L) and 5j (IC50 = 12.34 ± 1.25 µmol/L) showed better inhibitory activities against MCF-7 cell lines than chrysin and 5-FU. Molecular docking scores showed a credible consistency compared with MTT results. ConclusionCompounds 5b, 5d, 5g, 5j, 5k, and 6f showed good antiproliferative effects on specific tumor cells, and compound 5g should be researched further when according to molecular docking.

Mancozeb selectively induces mitochondrial-mediated apoptosis in human gastric carcinoma cells through ROS generation

Mancozeb (Manganese ethylene bis-dithiocarbamate with zinc salt) is a dithiocarbamate fungicide used to control fungal disease in many fruit plants, flowers and the maintenance of field crops. The effect of mancozeb on cell viability of human gastric adenocarcinoma AGS, SNU-1 cells and human normal FHs 74 Int cells were investigated. This study demonstrated that mancozeb was able to inhibit cell proliferation by 56–82% at 5–10 μM concentrations after 48 h. Mancozeb treatment for 48 h resulted in 33% (P < 0.05) and 61% (P < 0.001) increase in apoptotic cells at 5 and 10 μM concentrations in AGS cells, respectively. Treatment with mancozeb did not cause cell cycle arrest, while modulated the expression level of cleaved caspase-3, and cleavage of poly-(ADP-ribose) polymerase. Furthermore, treatment with mancozeb caused a rapid stimulation of reactive oxygen species (ROS) and loss of mitochondrial transmembrane potential. The results also showed that mancozeb-induced apoptosis was accompanied by up-regulation of Bax and down-regulation of Bcl-2 and Bcl-xL. Overall, our data suggested that mancozeb caused ROS generation which induced significant (P < 0.05) apoptosis in AGS cells that was attenuated with pretreatment of NAC. More importantly, same concentration of mancozeb did not show any considerable effect on cell growth, death, cell cycle arrest and ROS generation in normal FHs 74 Int cells. Overall, for the first time these results suggest that mancozeb has selective anticancer activity at lower concentrations against gastric cancer cells.

In vitro effects of two major phenolic compounds from the family Lamiaceae plants on the human gastric carcinoma cells.

Phenolic compounds of essential oils from the family Lamiaceae are commonly used substances in the food industry because of their flavouring, antimicrobial and antioxidant properties. In this context, it has become important to have healthy and safe products for consumers who are exposed to these phenolic compounds. The present study was aimed to investigate the toxic effects of carvacrol, thymol and their mixture on human gastric carcinoma (AGS) cells. Cells were analyzed after 24 h of exposure to different concentrations of carvacrol, thymol and their mixture by the ATP cell viability, 2',7' dichlorodihydrofluorescein diacetate (H2DCF-DA), reducte glutatione/oxide glutathione((GSH)/GSSG-Glo) and comet assays. Apoptosis induction was studied by acridine orange/ethidium bromide staining and western blotting. Carvacrol, thymol and their mixture induced cytotoxicity, genotoxicity, apoptosis, increased reactive oxygen species (ROS) and decreased GSH levels after 24 h of their exposure in a dose-dependent manner. A close negative relationship was found between cell viability and ROS generation. We examined dose-dependent cytotoxic effects of carvacrol, thymol and their mixture in human AGS cells. Increased intracellular ROS causes oxidative stress in cells. The results indicated that these compounds should be used carefully in the food industry.

Effects of isoimperatorin on proliferation and apoptosis of human gastric carcinoma cells

Resistance to apoptosis is an characteristic of cancer cells that serves a critical function in tumor development and represents a target for antitumor therapy. Isoimperatorin (ISOIM), a coumarin compound, exhibits antitumor functions in multiple types of tumor cells. However, its antitumor effects and molecular mechanisms with respect to gastric cancer have not been elucidated. The present study assessed the anti-proliferative and apoptotic effects of ISOIM on human BGC-823 gastric cancer cells and elucidated its underlying molecular mechanisms. Cell proliferation was evaluated using MTT assays. Analysis of cell morphology was performed by hematoxylin and eosin, Hoechst 33258 and acridine orange/ethidium bromide staining. In addition, cell cycle and apoptosis was evaluated using flow cytometry analysis; expression of apoptosis-associated proteins was studied by western blotting. The results of the present study revealed that ISOIM significantly inhibited cell proliferation by arresting the cell cycle at the G2/M phase and induced apoptosis by increasing Bcl-2-associated X (Bax) expression with a concomitant decrease in Bcl-2 expression, resulting in a decreased Bcl-2/Bax ratio compared with the control. In addition, ISOIM treatment also resulted in cytochrome c translocating from the mitochondria to the cytosol. Furthermore, caspase-3 was significantly activated in response to treatment with ISOIM, suggesting that apoptosis in BGC-823 cells is induced in the mitochondrial pathway. Taken together, the results of the present study indicate that ISOIM may significantly induce apoptosis in BGC-823 cells and that the pro-apoptotic mechanisms of ISOIM could be associated with the mitochondrial pathway.

A novel polysaccharide derived from algae extract inhibits cancer progression via JNK, not via the p38MAPK signaling pathway.

Cancer has long been one of the most malignant diseases worldwide. Processes in cancer cells are often mediated by Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and other signaling pathways. Traditional therapies are often problematic. Recently, a novel polysaccharide derived from algae extract was investigated due to the increasing interest in biological activities of compounds from marine organisms. The effect of this novel polysaccharide on human MKN45 gastric carcinoma cells was determined previously. The current aimed to determine whether the polysaccharide affects other types of cancer, and the deeper mechanisms involved in the process. Human MCF-7 breast cancer cells were used to investigate the novel polysaccharide for its role in the cell growth and migration, and determine the mechanisms affected. MTT assay, nuclear staining and fluorescence activated cell sorting analysis demonstrated that the novel polysaccharide reduced the viability of MCF-7 cells by inducing cell apoptosis and arresting the cells at G2/M phase. Results of western blot analysis demonstrated that phosphorylation of JNK and expression of p53, caspase-9 and caspase-3 were upregulated in the polysaccharide-treated MCF-7 cells. SP600125, an inhibitor of JNK, maintained MCF-7 cell viability, prevented cell apoptosis and cycle arrest, and downregulated the polysaccharide-induced protein phosphorylation/expression. However, a migration assay demonstrated that the novel polysaccharide did not change the migration of MCF-7 cells, as well as the expression of p38 MAPK, and matrix metalloproteinase-9 and -2. Taken together, the current study demonstrated that the novel polysaccharide suppressed cancer cell growth, induced cancer cell apoptosis and cell cycle arrest via JNK signaling, but had no effect on cancer cell migration and p38 MAPK signaling.

Autophagy inhibition enhanced 5‑FU‑induced cell death in human gastric carcinoma BGC‑823 cells.

The exact molecular mechanism of 5-fluorouracil (5-FU) in human gastric cancer cells remains to be elucidated. Cultured BGC‑823 human gastric carcinoma and AGS cell lines were treated with 5‑FU. Autophagosome formation was investigated through multiple approaches, including the quantification of green fluorescent protein‑microtubule‑associated protein1A/1B‑light chain3 (LC3) puncta, LC3 conversionand electron microscopy observations. Additionally, autophagy was inhibited using 3‑methyladenine (3‑MA) and beclin‑1 ablation, to determine its role in 5‑FU‑mediated cell death. In addition, the present study assessed alterations in sirtuin expression following 5‑FU treatment with reverse transcription‑quantitative polymerase chain reaction. 5‑FU treatment induced apoptosis and inhibited proliferation in BGC‑823 and AGS gastric cancer cells. It is of note that the 5‑FU treatment only promoted autophagy in BGC‑823 cells. Additionally, inhibition of autophagy by either 3‑MA or beclin‑1 ablation increased 5‑FU‑induced cell death in BGC‑823 cells. The present study quantified changes in sirtuin (SIRT1, SIRT3, SIRT5, and SIRT6) expression following 5‑FU treatment and using a specific inhibitor, sirtinol, the present study investigated their involvement in 5‑FU‑mediated autophagy. Autophagy inhibition through manipulation of sirtuin proteins may increase the therapeutic efficacy of the 5‑FU chemotherapeutic drug against gastric carcinoma.

Induction of caspase-mediated apoptosis using Alnus japonica extracts in AGS human gastric carcinoma cells

Alnus japonica has been used as a traditional oriental medicine for many diseases such as fever, haemorrhage and alcoholism. In this study, A. japonica extracts were evaluated for their in vitro antioxidant potentials and anticancer effects in AGS human gastric carcinoma cell line. The antioxidant properties of A. japonica extracts were evaluated using several biochemical assays, including FRAP (ferric reducing antioxidant power) assay, ABTS (2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)), DPPH (2,2-diphenyl-1-picrylhydrazyl), alkyl and hydroxyl radical scavenging activity assay. Our study showed that ethanol extract of A. japonica (AJE) has a more potent antioxidant activity than its water extract. In addition, AJE extract inhibited the cell growth and induced the cell death by increasing reactive oxygen species (ROS) production in AGS cells. Moreover, AJE extract specifically triggered the apoptosis mediated through the activation of caspase-8, 7, 3, and poly-ADP ribose polymerase (PARP). Thus, these results suggest that AJE extract could be potentially useful as a new promising strategy in the therapy for gastric carcinoma cancer.

Hydroxysafflor-Yellow A Induces Human Gastric Carcinoma BGC-823 Cell Apoptosis by Activating Peroxisome Proliferator-Activated Receptor Gamma (PPARγ).

BackgroundAnti-tumor properties of hydroxysafflor-yellow A (HSYA) have been recently revealed, as a series of apoptotic factors were confirmed to be regulated by HSYA and associated with peroxisome proliferator-activated receptor Gamma (PPARγ). In this study, we investigated the cell apoptosis mechanism of HSYA via activated PPARγ signal in human gastric carcinoma cells.Material/MethodsBGC-823 cells were cultured and divided into 5 independent groups: Tumor, HSYA, HSYA+PPARγ inhibitor (GW9662), and PPARγ agonist (RGZ), RGZ+GW9662. Cell proliferative activity was measured by MTT. Apoptosis and cell cycle were detected by flow cytometry. The nuclear translocation of PPARγ was detected by immunofluorescence staining chemistry, and mRNA levels of PPARγ and caspase-3 were measured by real-time qPCR.ResultsCompared to the RGZ group, the HSYA group (100 μM) showed a similar inhibitory effect on the proliferation process of BGC-823 cells, inducing their apoptosis. As a result, the transition of BGC-823 cells from G0/G1 phase to S phase was blocked. HSYA was also found to promote the nuclear translocation of PPARγ, leading to increased expression of PPARγ and caspase-3. The regulatory effect of HSYA on BGC-823 cells could be further inhibited by PPARγ inhibitor in group GW9662.ConclusionsWe report the inhibitory effect of HSYA on the proliferation of BGC-823 cells, which results in activating PPARγ-dependent cell cycle blocking and cell apoptosis, suggesting that PPARγ is a specific type of HSYA that can induce apoptosis of BGC-823 cells.

Concomitant modulation of PTEN and Livin in gastric cancer treatment.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Livin are important in the development of gastric cancer (GC). PTEN and Livin are involved in the regulation of tumor cell proliferation, migration and apoptosis. The modulation of PTEN or Livin has been investigated extensively in various cancer models. However, no studies have been performed to evaluate the combined effect of concurrently modulating these two genes on the development of GC. In the present study, the BGC823 human gastric carcinoma cell line was transfected with a dual gene modified vector (pCL-neo-PTEN-siLivin) in parallel with single gene modified vectors (pCL‑neo‑PTEN or pRNAT‑U6.1‑siLivin), and an empty control vector. Dual gene modulation (pCL‑neo‑PTEN‑siLivin) had a more marked effect on the inhibition of cell proliferation, induction of apoptosis, and reduction of cell penetration in Matrigel, compared with either single gene alone or empty vector transfection. In a xenograft nude mouse model, the inoculation of pCL‑neo‑PTEN‑siLivin‑transfected BGC823 cells led to a markedly reduced tumor burden, compared with that in all other inoculation groups. In conclusion, the overexpression of PTEN concomitant with Livin gene silencing was confirmed as a feasible and effective invitro and invivo gene modulation method, which may represent a potential therapeutic strategy for the treatment of GC.

Integrin-linked kinase silencing induces a S/G2/M phases cell cycle slowing and modulates metastasis-related genes in SGC7901 human gastric carcinoma cells.

Integrin-linked kinase has been implicated in metastasis of human tumors. Recent studies have also shown that the down-regulation of integrin-linked kinase has anti-tumor potential by inhibiting the metastatic potential of several types of cultured human cancer cells. However, the mechanism by which integrin-linked kinase regulates metastasis in human gastric carcinoma is not fully clear. We investigated the effect of integrin-linked kinase deletion on metastasis-associated markers in human gastric carcinoma SGC-7901 cell lines. We generated cell lines depleted for integrin-linked kinase. Cell adhesion and invasion were measured by the MTS assay and transwell assay. The cell cycle was measured by flow cytometry. Expression of metastasis-related genes was assessed by reporter assay, quantitative RT-PCR and western blotting. Our data showed an inhibitory effect on cell adhesion and invasion after silencing of integrin-linked kinase. The cell cycle was slowed in S/G2/M phases. Metastasis-related genes E-cadherin, MMP-2/9 and cystatin B, as well as the signaling molecules p-Akt, NF-κB, and AP-1 activation, were also modulated. Our results indicate that integrin-linked kinase plays an important role in metastasis of human gastric carcinoma cells. Down-regulation of integrin-linked kinase resulted in the impairment of the metastatic potential of gastric tumor cells by regulating metastasis-related gene expression, by inhibiting the Akt pathway as well as the activity of transcription factors. Integrin-linked kinase could be used as a potential therapeutic target.

Re-assembled Casein Micelles for Oral Delivery of Chemotherapeutic Combinations to Overcome Multidrug Resistance in Gastric Cancer

Multidrug resistance (MDR) mediated by ATP-dependent efflux transporters continues to be a dominant obstacle towards curative cancer therapy. We have previously demonstrated the ability of $\beta $-casein micelles ($\beta $-CM) to orally deliver a combination of individually encapsulated hydrophobic cargo of drugs and chemosensitizers designed to overcome MDR in gastric cancer. Herein we investigated the potential of re-assembled casein micelles (rCM) to serve as a target-activated delivery platform comprising a synergistic duo of a chemotherapeutic drug (paclitaxel) and a P-glycoprotein-specific transport inhibitor (tariquidar). The high binding affinity of paclitaxel and tariquidar to rCM was demonstrated by spectrofluorometry. Furthermore, solubilization of the drugs and suppression of crystal growth was shown by light microscopy and dynamic light scattering. A remarkable antitumor activity and complete MDR reversal were demonstrated in the in vitro cytotoxicity assay against MDR human gastric carcinoma cells overexpressing P-glycoprotein. The structure and cytotoxic activity of drug-loaded rCM were completely retained after freeze-drying and reconstitution as in $\beta $-CM. Hence, our findings highlight the great potential of casein-based nanovehicles as efficient platforms for oral delivery and local target-activated release of synergistic hydrophobic drug combinations to treat gastric cancer, and overcome of cancer chemoresistance, and for the possible treatment of non-malignant gastric disorders.

Potential mechanism of specific small interfering RNA against ubiquitin-specific protease 22 gene on proliferation of human gastric carcinoma cells

Objective To explore the effects of ubiquitin-specific protease 22 (USP22) on the proliferation of human gastric carcinoma cells and the potential mechanism. Methods The specific small interfering RNA (siRNA) fragments of USP22 were designed, and the highest efficient siRNA was selected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting. The proliferation of SGC-7901 cells was tested using methyl thiazol tetrazolium (MTT) assay after USP22 silencing, and the cell cycle and apoptosis were tested by flow cytometry. The altered expression of cell cycle and apoptosis related proteins was detected by Western blotting. Results The efficiency of USP22 siRNA was more than 80%. The proliferation of SGC-7901 cells was significantly suppressed after USP22 silencing, and the cell cycle was arrested in G0/G1 phase [(52.87±1.41)% vs. (73.73±1.78)%, t=15.491, P=0.000]. Meanwhile, the apoptosis rate was significantly increased by USP22 silencing [(19.63±0.82)% vs. (4.54±0.46)%, t=58.051, P=0.000]. The results of Western blotting revealed that the expression of B cell lymphoma/leukemia-2 associated X protein (bax) was significantly increased (t=10.630, P=0.000), whereas that of B-cell lymphoma/leukemia-2 (bcl-2) and pro-cysteinyl aspartate-specific protease (Caspase)-3 was significantly reduced (t=6.674, 7.269; P=0.000). Furthermore, the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3β (GSK3β) was significantly reduced (t=13.512, 6.846; P=0.003). Conclusion Cell growth of SGC-7901 cells was inhibited by USP22 silencing via reducing the activity of Akt. Key words: Ubiquitin-specific protease 22; Gastric carcinoma; Proliferation; Apoptosis; Protein kinase B

Parthenolide facilitates apoptosis and reverses drug-resistance of human gastric carcinoma cells by inhibiting the STAT3 signaling pathway.

In the present study, SGC-7901/DDP cells were treated with different concentrations of parthenolide (PN) (2.5–15 µmol/l), cisplatin (DDP) (1.25–15 µg/ml) and PN+DDP. The proliferation inhibition rates were measured using an MTT assay, and the synergies of PN and DDP were analyzed. The effect of PN and DDP on SGC-7901/DDP cell proliferation demonstrated a time- and concentration-dependent association, and a synergy between PN and DDP was identified. DAPI staining and flow cytometry results indicated that 15 µmol/l PN significantly induced SGC-7901/DDP apoptosis and G1 phase arrest compared with the untreated control group. Western blotting analysis results indicated that among the apoptosis-associated proteins, there were dose-dependent increases in the protein expression of apoptosis regulator BAX, cellular tumor antigen p53, cleaved caspase-3 and cleaved capase-9, and decreases in apoptosis regulator Bcl-2 and Bcl-xL protein expression levels. Among the cell cycle-associated proteins, cyclin D1 expression was significantly decreased, cyclin-dependent kinase inhibitor 1 expression was significantly increased, and signal transducer and activator of transcription 3 (STAT3) activation was inhibited. Scratch and Transwell assay results revealed that PN significantly inhibited SGC-7901/DDP cell migration, and invasion. The present study demonstrated that PN induces SGC-7901/DDP apoptosis, inhibits SGC-7901/DDP proliferation, migration and invasion, and enhances the drug sensitivity of the cells to DDP. The underlying mechanisms may be associated with inhibition of the STAT3 signaling pathway and regulation of the downstream apoptotic protein and cyclin expression levels.

Resveratrol induced apoptosis in human gastric carcinoma SGC-7901 cells via activation of mitochondrial pathway.

Resveratrol is a natural polyphenolic compound and its anticancer effect has been receiving considerable attention. Previous studies showed that resveratrol could inhibited the growth of human gastric carcinoma cells and apoptosis induction was an important mechanism. However, whether mitochondrial pathway was involved in resveratrol-induced apoptosis in human gastric cancer was not very clear. The cells were examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, Annexin V/PI staining assay, mitochondrial membrane depolarization, cell morphological assessment, cytochrome c release assay, and Western blotting assay. In this study, we found that resveratrol induced apoptosis in human gastric carcinoma SGC-7901 cells. Cleaved PARP was observed and caspase-3 was activated by resveratrol. Next, the mitochondrial membrane potential of cells dissipated after the cells were treated by resveratrol. Moreover, we found that pro-caspase 9 was downregulated and cytochrome c released from mitochondrial to the cytosol. We also found that the expression ratio of Bax/Bcl-2 was increased in the treated cells. We finally showed that resveratrol inhibited the proliferation of SGC-7901 xerograph in vivo. Collectively, our findings demonstrate that resveratrol triggers apoptosis via mitochondrial pathway in SGC-7901 cells, which provide more basis for resveratrol acting as antitumor agents in cancer therapy.