Human Gastric Carcinoma AGS Cells Research Articles

The synergistic effect of Levilactobacillus brevis IBRC-M10790 and vitamin D3 on Helicobacter pylori-induced inflammation.

Owing to the emergence and spread of multidrug resistance mechanisms in Helicobacter pylori, achieving a successful eradication has become exceedingly difficult. Thus, this study for the first time determines the effect of a combination of vitamin D3 and probiotic on the pathogenesis and treatment of H. pylori. We established an in vitro experimental system using AGS human gastric carcinoma cells and explored the synergistic effect of Levilactobacillus brevis IBRC-M10790 and vitamin D3 on H. pylori. Live and pasteurized L. brevis, L. brevis-derived membrane vesicles (MVs), and L. brevis cell-free supernatant (CFS), as well as their combination with vitamin D3 were used during this study. We assessed the anti-inflammatory and anti-oxidative effects of these combinations using RT-qPCR and ELISA, respectively. We further performed an adhesion assay to evaluate the influence of L. brevis and vitamin D3 on the adherence rate of H. pylori to AGS cells. Our results demonstrated that L. brevis and vitamin D3 possess anti-inflammatory and anti-oxidative effects against H. pylori infection in AGS cells. The combination of vitamin D3 with the probiotic strain (particularly live L. brevis and its CFS) can more efficiently reduce the expression of pro-inflammatory cytokines IL-6, IL-8, IFN-γ, and TNF-α in the AGS cells. Moreover, vitamin D3 and L. brevis exhibited an additive impact preserving the integrity of the epithelial barrier by increasing the expression of the tight junction protein ZO-1. Furthermore, this combination can potentially reduce H. pylori adherence to AGS cells. This study indicates the advantage of combining vitamin D3 and probiotic to attenuate H. pylori-induced inflammation and oxidative stress. Consequently, probiotic and vitamin D3 co-supplementation can be considered as a novel therapeutic approach to manage and prevent H. pylori infection.

Potentially Bioaccessible Phenolics from Mung Bean and Adzuki Bean Sprouts Enriched with Probiotic-Antioxidant Properties and Effect on the Motility and Survival of AGS Human Gastric Carcinoma Cells.

Gastric digests from mung (MBS) and adzuki (ABS) bean sprouts enriched with probiotic Lactobacillus plantarum 299v were tested for their antioxidant potential, as well as antiproliferative and antimotility properties, in human stomach cancer cells (AGS). The digest of ABS contained quercetin and kaempferol derivates, while kaempferol and apigenin derivates were dominant in MBS. Compared to the controls, the probiotic-rich sprouts had a higher antioxidant potential—by 13% and 9%, respectively. Adzuki bean sprouts decreased the viability of AGS already at low concentrations (25% motility inhibitions). MBS and ABS displayed dose-independent cytostatic effects. The ABS extracts decreased the proliferation of AGS more effectively than the MBS extracts—0.2‰ ABS exerted c.a. 70% of inhibitions. Moreover, the phytochemicals from the probiotic-rich sprouts considerably reduced this activity. The increased vinculin level, the apoptotic shape of cell nuclei, and the reduced cell motility and proliferation indicate that the extracts exhibited cytostatic and cytotoxic activity.

Effects of Helicobacter pylori on the glutathione-related pathway in gastric epithelial cells

Virulence factors of Helicobacter pylori (H. pylori) are diverse, so various biological responses happen in a host infected with H. pylori. The aim of this study is to conduct the metabolomics-based evaluation on H. pylori infection. AGS human gastric carcinoma cells were infected with H. pylori strain 26695, and then the altered metabolite pathways in the infected AGS cells were analyzed by metabolomics. Metabolites related to the glutathione (GSH) cycle were downregulated by H. pylori infection. Next, we evaluated the effects of H. pylori on the GSH-related pathway in AGS cells infected with H. pylori isolated from patients with atrophic gastritis (AG), duodenal ulcer (DU) and gastric cancer (GC). We found that the declined degree of GSH levels and oxidative stress were greater in AGS cells infected with GC strains than DU and AG-derived strains. There were no significant differences in almost mRNA expressions of GSH-related factors among different clinical strains, but the protein expression of glutathione synthetase was lower in AGS cells infected with GC-derived strains than DU and AG-derived strains. Our data demonstrates that GC-derived H. pylori-induced oxidative stress in a host is stronger and GC-derived strains may have suppressive influences on the host’s GSH-related defense systems.

Characterization and evaluation of cytotoxic and apoptotic effects of green synthesis of silver nanoparticles using Artemisia Ciniformis on human gastric adenocarcinoma

In recent years, using leaf extract for synthesizing silver nanoparticles (AgNPs) has been introduced as an easy, low-cost, and eco-friendly alternative to chemical and physical methods. In this study, the green synthesis of AgNPs was investigated using Artemisia ciniformis (A. ciniformis) leaf extract and their anti-proliferative and apoptotic effects on human gastric adenocarcinoma (AGS) cell line were evaluated. The properties of green synthesized AgNPs were confirmed using the transmission electron microscopy (TEM), scanning electron microscopy (SEM), UV–vis spectroscopy, X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, Energy-dispersive X-ray spectroscopy (EDX), Dynamic light scattering (DLS) and Zeta potential. To assess the anti-proliferative activity and induction of apoptosis by green synthesized AgNPs such methods as MTT assay, annexin FI-TC, cell cycle analysis, and Real-Time PCR were performed.TEM images showed that the AgNPs were spherical in shape with a size ranging from 4–14 nm. Green synthesized AgNPs inhibited the growth of AGS human gastric carcinoma cells in a time- and dose-dependent manner. Flow cytometric results confirmed cell death by apoptosis. Green synthesized AgNPs caused up-regulation of pro-apoptotic genes, caspase-3, caspase-9, and Bax; moreover, they caused down-regulation of Bcl2 gene and inhibited the expression of cyclin D1 and MMP2 genes. Cell cycle analysis revealed the arrest of cancer cells in the G0/G1 phase. We have shown that green synthesized AgNPs inhibit gastric cancer cell proliferation through apoptosis and have the potential to be used as a novel biological anti-cancer agent in the treatment of gastric cancer.

Anti-microbial and anti-inflammatory effects of Cheonwangbosim-dan against Helicobacter pylori-induced gastritis.

BackgroundThere are various Helicobacter species colonizing the stomachs of animals. Although Helicobacter species usually cause asymptomatic infection in the hosts, clinical signs can occur due to gastritis associated with Helicobacter in animals. Among them, Helicobacter pylori is strongly associated with chronic gastritis, gastric ulcers, and gastric cancers. As the standard therapies used to treat H. pylori have proven insufficient, alternative options are needed to prevent and eradicate the diseases associated with this bacterium. Cheonwangbosim-dan (CBD), a traditional herbal formula that is popular in East Asia, has been commonly used for arterial or auricular flutter, neurosis, insomnia, and cardiac malfunction-induced disease.ObjectivesThe present study investigated the antimicrobial effect of CBD on H. pylori-infected human gastric carcinoma AGS cells and model mice.MethodsAGS cells were infected with H. pylori and treated with a variety of concentrations of CBD or antibiotics. Mice were given 3 oral inoculations with H. pylori and then dosed with CBD (100 or 500 mg/kg) for 4 weeks or with standard antibiotics for 1 week. One week after the last treatment, gastric samples were collected and examined by histopathological analysis, real-time quantitative polymerase chain reaction, and immunoblotting.ResultsOur results showed that CBD treatment of AGS cells significantly reduced the H. pylori-induced elevations of interleukin-8, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). In the animal model, CBD treatment inhibited the colonization of H. pylori and the levels of malondialdehyde, inflammation, proinflammatory cytokines, iNOS, and COX-2 in gastric tissues. CBD also decreased the phosphorylation levels of p38 mitogen-activated protein kinase family.ConclusionsThis study suggests that CBD might be a prospective candidate for treating H. pylori-induced gastric injury.

Usnic Acid Inhibits Cell Proliferation Via Downregulation Of PCNA Expression In Gastric Carcinoma AGS Cells

Usnic acid is a secondary metabolite obtained from various species of lichen. Previous studies have shown various biological activities of usnic acid, such as anti-oxidant, anti-microbial, anti-viral, anti-protozoal, anti-inflammatory, and anti-proliferative activities in different models. Its anti-proliferative activities in gastric cancer cells are still unexplored. Herein, we have investigated the effects of usnic acid on cell proliferation and viability and associated molecular alterations in human gastric carcinoma AGS cells. The treatment of usnic acid (2.5-25μM) dose-dependently reduced cell proliferation. The mRNA expression of tumor suppressor gene phosphatase tensin homolog (PTEN)in the usnic acid-treated AGS cells was increased, which may play a role in the inhibition of cell proliferation and induction of cell death. We also observed a decrease in the expression of PCNA that regulates cell proliferation by playing an important role in DNA replication. The expression of cyclin-dependent kinase inhibitor p21, which may play a role in cell cycle and proliferation inhibition was found uninfluenced with usnic acid treatment. Thus, collectively these results revealed that usnic acid inhibits the cell proliferation of AGS cells through downregulating the expression of PCNA and can be further evaluated in vivo models for its therapeutic potentials.

Antioxidant Activity of Ruthenium Complexes Containing Nitro‐imidazole Derivatives

Ruthenium‐based coordination complexes are compounds with great pharmacological potential. Once their toxicity and pharmacokinetics properties can be easily modulated changing the ligands on the six coordination sites of the metal, those species can present a broad spectrum of applications, such as: antineoplastic, bactericide, antiparasitic, vasorelaxant, among others. However, antioxidant properties of that kind of substance is still poorly explored, even after recent works have shown evidence that nitrosyl ruthenium complexes can scavenger free radicals. In addition, nitro‐imidazole derivatives are organic compounds with antioxidant properties and are able to work as ligands in coordination complexes. Considering that, the compounds cis‐[Ru(NO2)(bpy)2(4NIMN)](PF6) (1) and cis‐[RuCl(bpy)2(MTZ)](PF6) (2), where bpy=2,2′‐bipyridine, 4NIMN=4‐nitroimidazole and MTZ=metronidazole, were selected for analysis. Thus, the aim of the present study was to investigate in vitro antioxidant activity of those ruthenium complexes. The complexes were synthesized using microwave‐assisted synthesis, adapting methods previously described. Structural characterization was held through spectroscopic and spectrometric techniques, confirming the proposed structures. Cytotoxicity of the compounds at 3.12 to 100.0 μM was evaluated on A549 human lung carcinoma, RAW 264.7 murine macrophages, AGS human gastric carcinoma cells and human neutrophils at 5×106 cells/mL. No significant cytotoxicity was observed at any concentration of the compounds. Antioxidant properties were evaluated by quantifying malondialdehyde (MDA) and reactive oxygen species (ROS) production, and by evaluating superoxide dismutase activity in LPS‐stimulated RAW 264.7 cells treated with 1 and 2 at 3 – 25 μM. All the concentrations of both complexes were able to significantly reduce the production of superoxide anion in relation to the vehicle group, similarly to the reference treatment (α – tocopherol) All concentrations could also reduce the amount of MDA and ROS produced. Finally, the ruthenium complexes 1 and 2 were not cytotoxic to human neutrophils, lung and gastric carcinomas, and presented potent antioxidant properties. Tests are being carried out in order to elucidate antioxidant mechanisms and to analyze possible anti‐inflammatory effects.Support or Funding InformationCAPES; CNPqThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

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.

Gastroprotective effects of Hwanglyeonhaedok-tang against Helicobacter pylori-induced gastric cell injury

Ethnopharmacological relevanceHelicobacter pylori, which is found in the stomachs of approximately half of the world's population, has been associated with the development of chronic gastritis and gastric cancer. Hwanglyeonhaedok-tang (HHT) is a popular traditional medicine for the therapies of gastric ulcers and gastritis. Aim of the studyThe emerging resistance of H. pylori to antibiotics arouses requirement on alternative nonantibiotic-based therapies. In the present study, we investigated the anti-inflammatory activity and anti-microbial activity of HHT against H. pylori in vitro and in an H. pylori-infected mouse model. Materials and methodsH. pylori were treated with various concentrations of HHT and then incubated with human gastric carcinoma AGS cells. For the in vivo study, mice were orally infected with H. pylori three times over the course of 1 week, and then subjected to daily administration of HHT (120 or 600 mg/kg) for 4 weeks or standard triple therapy for 1 week. At the scheduled termination of the experiment, all mice were killed and their stomachs were collected for histological examination, quantitative real-time PCR, and Western blot analysis. ResultsOur in vitro studies showed that HHT treatment inhibited the adhesion of H. pylori to AGS cells and suppressed the H. pylori-induced increases of inflammatory regulators, such as interleukin (IL)-8, cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS). In the mouse model, HHT treatment significantly reduced H. pylori colonization, inflammation, and the levels of IL-1β, IL-6, C-X-C motif chemokine ligand 1 (CXCL1), tumor necrosis factor alpha (TNF-α), COX-2, and iNOS in gastric mucosa. Further investigation showed that HHT treatment reduced the H. pylori-induced phosphorylations of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK), and nuclear factor-kappa B (NF-κB). ConclusionsOur findings collectively suggest that HHT has anti-inflammatory activity and antibacterial activity against H. pylori and could be an alternative to antibiotics for preventing H. pylori infection.

The Extracts from Allium hookeri induces p53-independent Apoptosis through Mitochondrial Intrinsic Pathways in AGS Human Gastric Carcinoma Cells

Allium hookeri is a traditional plant to treat inflammatory diseases in India and Myanmar. Allium hookeri has anti-cancer and anti-microbial properties. However, till date, apoptotic mechanisms of Allium hookeri extract (AHE) were not investigated yet. In the present study, we aimed to investigate the apoptotic effects of AHE in AGS human gastric carcinoma cells. We confirmed the anti-proliferative activity and apoptotic effects of AHE by MTT assay and Annexin-V staining. In addition, treatment with AHE reduced the expression level of p-Akt. Akt plays an important role in cancer cell survival, growth, and division. Akt down-regulates apoptosis-mediated proteins, such as antiapoptotic proteins. Moreover, AHE increases expression level of tumor suppressor p53 and pro-apoptotic proteins. We treated LY294002 (inhibitor of Akt) and rapamycin (inhibitor of mTOR), pifitrin-α (p53 inhibitor) to determine the relationship between signal transduction of proteins associated with apoptosis. Taken together, our results indicate that AHE could induce apoptosis in AGS human gastric carcinoma cells.

Apoptotic Effects of Linum album Extracts on AGS Human Gastric Adenocarcinoma Cells and ZNF703 Oncogene Expression

Introduction:Linum album is a medicinal plant endemic in Iran that is very important pharmaceutically. The present study concerns the effect of different extracts of L. album on ZNF703 gene expression and apoptosis in human gastric carcinoma AGS cells.Method and material:Hydro alchoholic L. album extracts from various plant sources were produced by Maceration. AGS cells were treated with different concentrations (200, 400, 600, 800 and 1000 µg/ml) and the cytotoxicity potency was assessed after 24 h by MTT assay. Then, quantitative real time PCR was conducted for ZNF703 gene expression in AGS cells. Also, cell apoptosis/necrosis was assessed with the aid of Annexin V/PI staining and quantification by flow cytometry.Results:L. album extracts exerted dose-dependent toxicity in the AGS cell line. The mRNA levels of ZNF703 gene expression were significantly decreased with rhizome, fruit at fruiting, leaf and stem at anthesis (P<0.001), and leaf and stem at fruiting extracts as compared to the controls (P<0.01). Also, the number of apoptotic cells was increased from 2.70% (statistically significant; p<0.05) in untreated AGS cells to 44%, following treatment with the leaf and stem at anthesis example.Discussion:Our findings revealed that the L. album extracts can induce apoptosis and might modulate cytotoxicity by down regulating ZNF703 gene expression in AGS cells. Therefore, this extract could be a good candidate for inhibiting cancer cell growth, especially that of gastric cancer. In addition, ZNF703 may have potential as a therapeutic target.

Diallyl trisulfide induces apoptosis and mitotic arrest in AGS human gastric carcinoma cells through reactive oxygen species-mediated activation of AMP-activated protein kinase

Diallyl trisulfide (DATS), one of the principal constituents of garlic oil, is a kind of organosulfur compound with high anti-cancer activity. Although inhibition of cancer cell proliferation by DATS is known to be associated with the induction of apoptosis and cell cycle arrest related to reactive oxygen species (ROS) production, it is still necessary to study the detailed mechanisms. In this study, we investigated the role of ROS on the activation of AMP-activated protein kinase (AMPK) in DATS-induced apoptosis and cell cycle arrest in AGS human gastric carcinoma cells. The results of the present study indicate that DATS inhibited proliferation of AGS cells by promoting apoptosis, and accumulating cellular portion of G2/M phase via the induction of cyclin B1 and cyclin-dependent kinase p21(WAF1/CIP1). The phosphorylation of histone H3 was also markedly increased following treatment with DATS, revealing that DATS stimulated a mitotic arrest, not the G2 phase. Furthermore, we found that DATS concurrently induced phosphorylation of AMPK; however, chemical inhibition of AMPK by compound C, an AMPK inhibitor, significantly blocked apoptosis induced by DATS, suggesting that DATS induces cytotoxicity of AGS cells through the AMPK-dependent pathway. Moreover, DATS provoked intracellular ROS generation and the loss of mitochondrial membrane potential, and in particular, when ROS production was blocked by antioxidant N-acety-l-cysteine, both AMPK activation and growth inhibition by DATS were completely abolished. Collectively, these findings suggest that DATS inhibited growth of AGS cells, which was mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis, and cell cycle arrest, through a ROS-dependent activation of AMPK pathway.

감마 오리자놀의 위암세포증식억제 및 세포사멸 유도 효능

Gamma (γ)-oryzanol is a substance abundant in rice, which is widely cultivated in Asian countries. In this study, we evaluated the effect of γ-oryzanol treatment on proliferation and apoptosis of AGS human gastric carcinoma cells. AGS cells were treated with γ-oryzanol for 72 h in a dose dependent manner. Treatment of γ-oryzanol (50, 100, and 200 μg/mL) resulted in decreased AGS cell proliferation and increased number of cells in the sub-G1 population. Additionally, apoptotic cells were investigated by annexin V staining and mitochondrial membrane potential assays. Our results indicated that γ-oryzanol treatment increased the number of annexin V-positive cells and depolarized cells. This demonstrated that γ-oryzanol is effective for the induction of apoptosis in AGS cells. We next examined the expression of promising anticancer drug target molecules, including PTEN and HSP90. We found that treatment of γ-oryzanol induced the expression of PTEN in AGS cells. Under the same treatment conditions, γ-oryzanol reduced the expression of HSP90 in AGS cells. These results suggest that γ-oryzanol-induced apoptosis was accompanied by changes in regulation of PTEN and HSP90 in AGS cells. Taken together, γ-oryzanol could be used as a functional substance for the prevention of gastric cancer.

Evaluation of Cell- Morphological Changes by Helicobacter pylori CagA and Pragmin in AGS Human Gastric Carcinoma Cells

Background: Helicobacter pylori CagA oncoprotein was injected into mammalian host cells via type IV secretion system, where it was tyrosine phosphorylated at the Glu-Pro-Ile-Tyr-Ala (EPIYA) sequence. Tyrosine phosphorylation of CagA was shown to be a prerequisite for the induction of actin cytoskeletal rearrangement in AGS gastric epithelial cells. The needle-like projections, known as the hummingbird phenotype, are thought to be involved in gastric disease. Moreover, Pragmin, a mammalian protein, contains a single EPIYA motif, and tyrosine phosphorylation of Pragmin at EPIYA motif in AGS cells induce cell-morphological changes, which are characterized by elongated cell shape with invasive phenotype that contributes to tumor invasion and metastasis. Methods: In this study, AGS cells were transfected by CagA or/and Pragmin using lipofectamine 2000 reagent, then, cell-morphological changes were investigated using light microscope. Finally, elongated cells were counted and the results were compared. Results: Our results revealed that there is a competition between CagA and Pragmin to interact with CSK via EPIYA motif. We also found that although the cell-morphological changes are particularly dependent on tyrosine phosphorylation at EPIYA motifs in both, changes of cell morphology are different in CagA and Pragmin transfected cells. Conclusions: Our findings suggest that the mechanisms inducing the elongated cell morphology in CagA or Pragmin transfected cells may be different.

The expression of HDAC7 in cancerous gastric tissues is positively associated with distant metastasis and poor patient prognosis.

To characterize the expression patterns of HDAC7 in patients with gastric cancer and evaluate the prognostic value of HDAC7 in gastric cancer. The expression of histone deacetylase 7 (HDAC7) was detected in paraffin-embedded gastric cancer samples from 86 patients by immunohistochemistry, and the differences in the expression of HDAC7 between cancerous and corresponding adjacent noncancerous tissues were compared using the Wilcoxon matched-pairs signed rank test. The correlation between HDAC7 expression and Ki-67 expression or clinicopathologic characteristics was evaluated using a Spearman rank correlation test. Prognostic outcomes that correlated with HDAC7 were examined using a Kaplan-Meier analysis and Cox proportional hazards model. Moreover, the effects of HDAC7 on the proliferation, migration and invasion of gastric cancer cells were investigated in vitro using human gastric carcinoma AGS cells. We found that HDAC7 was downregulated in cancerous gastric tissues (P=0.0019). However, the expression of HDAC7 in cancerous gastric tissues positively correlated with Ki-67 expression (P=0.0325) and distant metastasis (P=0.020). Moreover, overall survival was shorter for patients expressing higher levels of HDAC7 in cancerous tissues (P=0.042). Mechanistically, the disruption of the HDAC7 gene attenuated the capacity of cell growth, migration and invasion and induced G0/G1 arrest in AGS cells. Conversely, forced ovperexpression of HDAC7 promoted cell growth, migration and invasion and G1/S transition in AGS cells. These results indicate that high HDAC7 expression in cancerous gastric tissues correlates with distant metastasis and predicts a poor prognosis for patients with gastric cancer.

AGS 인체 위암 세포에서 Akt/mTOR/GSK-3β 신호경로 조절을 통한 개똥쑥 추출물의 Apoptosis 유도 효과

개똥쑥은 예로부터 항암, 항바이러스 및 항균의 효능을 지니는 것으로 알려져 왔지만 작용 기작에 대한 내용이 많이 알려지지 않았다. 본 연구에서는 AGS 인체 위암 세포를 대상으로 개똥쑥 추출물(AAE)에 의한 apoptosis 효과와 신호경로 연구를 시행하였다. AAE의 암세포 성장에 미치는 영향을 확인하기 위하여 AGS cell에 AAE를 처리하고 MTT assay와 LDH assay를 수행한 결과 AAE 농도 의존적으로 나타난 세포 성장 억제가 세포 손상에 의한 것임을 확인하였다. 또한, AAE에 의한 암세포 증식 억제 효과가 apoptosis에 의한 것인지 확인하기 위하여 Hoechst 33342 staining과 Annexin Ⅴ-PI staining을 수행한 결과, Hoechst 33342 staining에서 apoptotic body와 세포질 응축이 농도 의존적으로 증가하는 것을 확인하였고, Annexin Ⅴ-PI staining에서 apoptotic cells의 변화가 농도 의존적으로 증가함을 확인하였다. Western blotting의 결과 AAE가 농도 의존적으로 세포 생장에 관여하는 신호 단백질인 p-Akt, p-TSC2, p-mTOR, p-GSK-3β의 발현이 감소함을 확인하였고, anti-apoptotic 단백질인 Bcl-2의 발현이 억제됨으로써 proapoptotic 단백질인 Bax, Bak의 발현이 증가하는 일련의 신호경로를 조절할 수 있다는 것을 확인하였다. 미토콘드리아 막 전위의 탈분극 유도를 확인하기 위한 JC-1 assay 수행 결과, AAE 농도 의존적으로 미토콘드리아 막 전위의 탈분극이 유도됨을 확인하였다. 탈분극에 의한 caspase 활성을 확인하기 위해 caspase-3/7 activity assay를 수행한 결과, AAE 농도 의존적으로 caspase activity 증가를 확인하였다. 또한, apoptosis가 일어나는 일련의 신호경로를 확인하기 위해 apoptosis 상위 단백질인 Akt, mTOR, GSK-3β의 활성을 억제하는 LY294002, Rapamycin, BIO를 각각 AGS cell에 처리하고 세포증식에 미치는 영향과 신호 단백질의 발현 양상을 알아보기 위해 MTT assay, LDH assay, western blotting을 수행하였다. 그 결과 AAE와 LY294002, Rapamycin 처리군에서 세포증식 억제와 LDH 방출량 증가 뿐만 아니라 세포 생장 신호 단백질인 p-mTOR, p-TSC2, p-Akt, p-GSK-3β의 발현이 감소하는 것을 확인하였고, Bcl-2의 발현이 억제됨으로써 Bax와 Bak의 발현을 증가시키는 신호경로를 조절할 수 있다는 것을 확인하였다. 따라서 AGS cell에 개똥쑥 추출물을 처리하였을 때 유도되는 apoptosis 효과는 Akt/mTOR/GSK-3β 경로 활성 억제를 통해 Bcl-2 발현이 감소함에 따라 Bax, Bak를 활성화해 세포질로의 cytochrome C 유리에 따른 caspase 활성으로 이루어진다는 것을 알 수 있었다.

Coordination-Driven Self-Assembly and Anticancer Potency Studies of Ruthenium-Cobalt-Based Heterometallic Rectangles.

Three new cobalt-ruthenium heterometallic molecular rectangles, 1-3, were synthesized through the coordination-driven self-assembly of a new cobalt sandwich donor, (η5 -Cp)Co[C4 -trans-Ph2 (4-Py)2 ] (L; Cp: cyclopentyl; Py: pyridine), and one of three dinuclear precursors, [(p-cymene)2 Ru2 (OO∩OO)2 Cl2 ] [OO∩OO: oxalato (A1 ), 5,8-dioxido-1,4-naphthoquinone (A2 ), or 6,11-dioxido-5,12-naphthacenedione (A3 )]. All of the self-assembled architectures were isolated in very good yield (92-94 %) and were fully characterized by spectroscopic analysis; the molecular structures of 2 and 3 were determined by single-crystal X-ray diffraction analysis. The anticancer activities of bimetallic rectangles 1-3 were evaluated with a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, an autophagy assay, and Western blotting. Rectangles 1-3 showed higher cytotoxicity than doxorubicin in AGS human gastric carcinoma cells. In addition, the autophagic activities and apoptotic cell death ratios were increased in AGS cells by treatment with 1-3; the rectangles induced autophagosome formation by promoting LC3-I to LC3-II conversion and apoptotic cell death by increasing caspase-3/7 activity. Our results suggest that rectangles 1-3 induce gastric cancer cell death by modulating autophagy and apoptosis and that they have potential use as agents for the treatment of human gastric cancer.

Novel nuclear targeting coiled-coil protein of Helicobacter pylori showing Ca(2+)-independent, Mg(2+)-dependent DNase I activity.

HP0059, an uncharacterized gene of Helicobacter pylori, encodes a 284-aa-long protein containing a nuclear localization sequence (NLS) and multiple leucine-rich heptad repeats. Effects of HP0059 proteins in human stomach cells were assessed by incubation of recombinant HP0059 proteins with the AGS human gastric carcinoma cell line. Wild-type HP0059 proteins showed cytotoxicity in AGS cells in a concentration-dependent manner, whereas NLS mutant protein showed no effect, suggesting that the cytotoxicity is attributed to host nuclear localization. AGS cells transfected with pEGFP-HP0059 plasmid showed strong GFP signal merged to the chromosomal DNA region. The chromosome was fragmented into multiple distinct dots merged with the GFP signal after 12 h of incubation. The chromosome fragmentation was further explored by incubation of AGS chromosomal DNA with recombinant HP0059 proteins, which leaded to complete degradation of the chromosomal DNA. HP0059 protein also degraded circular plasmid DNA without consensus, being an indication of DNase I activity. The DNase was activated by MgCl2, but not by CaCl2. The activity was completely blocked by EDTA. The optimal pH and temperature for DNase activity were 7.0-8.0 and 55°C, respectively. These results indicate that HP0059 possesses a novel DNase I activity along with a role in the genomic instability of human gastric cells, which may result in the transformation of gastric cells.

Phytosynthesis of silver nanoparticles using Artemisia marschalliana Sprengel aerial part extract and assessment of their antioxidant, anticancer, and antibacterial properties.

A rapid phytosynthesis of silver nanoparticles (AgNPs) using an extract from the aerial parts of Artemisia marschalliana Sprengel was investigated in this study. The synthesized AgNPs using A. marschalliana extract was analyzed by UV–visible spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy and further characterized by transmission electron microscopy, scanning electron microscopy, zeta potential, and energy-dispersive spectroscopy. Characteristic absorption bands of AgNPs were found near 430 nm in the UV–vis spectrum. Energy-dispersive spectroscopy analysis of AgNPs in the energy range 2–4 keV confirmed the silver signal due to surface plasmon resonance. Scanning electron microscopy and transmission electron microscopy results revealed that the AgNPs were mostly spherical with an average size ranging from 5 nm to 50 nm. The zeta potential value of −31 mV confirmed the stability of the AgNPs. AgNPs produced using the aqueous A. marschalliana extract might serve as a potent in vitro antioxidant, as revealed by 2,2-diphenyl-1-picryl hydrazyl assay. The present study demonstrates the anticancer properties of phytosynthesized AgNPs against human gastric carcinoma AGS cells. AgNPs exerted a dose-dependent inhibitory effect on the viability of cells. Real-time polymerase chain reaction was used for the investigation of Bax and Bcl-2 gene expression in cancer and normal cell lines. Our findings show that the mRNA levels of pro-apoptotic Bax gene expression were significantly upregulated, while the expression of anti-apoptotic Bcl-2 was declined in cells treated with AgNPs compared to normal cells. In addition, flow cytometric analysis showed that the number of early and late apoptotic AGS cells was significantly enhanced following treatment with AgNPs as compared to untreated cells. In addition, the AgNPs showed strong antibacterial properties against tested pathogenic bacteria such as Staphylococcus aureus, Bacillus cereus, Acinetobacter baumannii, and Pseudomonas aeruginosa. Based on the obtained data, we suggest that phytosynthesized AgNPs are good alternatives in the treatment of diseases because of the presence of bioactive agents.