Human Gastric Cancer AGS Cells Research Articles

AMBRA1 drives gastric cancer progression through regulation of tumor plasticity

BackgroundStomach adenocarcinoma (STAD) is an aggressive malignancy characterized by high tumor plasticity and heterogeneity. This study investigates the role of Autophagy and Beclin 1 Regulator 1 (AMBRA1) in regulating tumor plasticity in STAD progression.MethodsCombined with clinical data, the pan-cancer analysis of AMBRA1 was performed to analyze the role of AMBRA1 in STAD. Western blot, Flow Cytometry (FCM) assay, trans-well assay, wound healing assay, MTT, Reactive Oxygen Species (ROS) assay, Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and staining were performed to study the effects of AMBRA1 in AGS human gastric cancer cells. An AGS gastric cancer xenograft model was constructed to further verify the role of AMBRA1 in the development of STAD.ResultsAMBRA1 overexpression correlated with poor overall survival in STAD and was positively associated with T cell CD4+ infiltration. High AMBRA1 expression also indicated worse prognosis in patients with high cancer-associated fibroblast infiltration. AMBRA1 depletion suppressed STAD cell proliferation, migration, and invasion in vitro. Mechanistically, AMBRA1 knockdown induced G1/S cell cycle arrest and triggered cellular senescence through epigenetic alterations, including changes in H3K9me3 levels. AMBRA1 inhibition also sensitized STAD cells to chemotherapeutic agents. In vivo studies confirmed the tumor-suppressive effects of AMBRA1 loss, resulting in reduced tumor growth and increased cellular senescence.ConclusionsOur findings uncover an oncogenic role for AMBRA1 in STAD. Targeting AMBRA1 may induce tumor cell senescence, apoptosis, and potentiate anti-tumor immunity, providing a rationale for developing AMBRA1-targeted precision therapies to improve clinical outcomes in STAD patients.

Cytotoxic effects of the standardized extract from Curcuma aromatica Salisb. rhizomes via induction of mitochondria-mediated caspase-dependent apoptotic pathway and p21-mediated G0/G1 cell cycle arrest on human gastric cancer AGS cells

ABSTRACT Curcuma aromatica Salisb. (C. aromatica) is one of the traditional herbs used to treat microbial infection, skin eruption, coronary heart disease, and other diseases, including cancer. However, the inhibitory effects and underlying mechanisms of action of C. aromatica on gastric cancer cells have not yet been fully elucidated. Our study aimed to examine the possible molecular mechanisms underlying the cytotoxic effects attributed to C. aromatica rhizome standardized extract against gastric cancer cells. The components of two major active compounds in C. aromatica rhizome extract were quantitatively analyzed using a simple and validated HPLC method. Cytotoxicity was determined in different gastric cancer and non-cancer cell lines. The biological activities of the extract targeting apoptosis and cell cycle-related genes on gastric cancer AGS cells were also investigated to elucidate the mechanisms relating to the anti-proliferative effect of C. aromatica rhizomes. The two major active compounds curdione and germacrone, in the C. aromatica extract were standardized to 0.64% and 1.12% w/w, respectively. The standardized extract (CAE) exerted cytotoxic effects on various cancer cells, whereas minimal effects at equivalent doses were noted for normal cells. CAE concentration-dependently suppressed growth of gastric cancer AGS cells via induction of apoptosis. Further studies revealed that CAE treatment disrupted mitochondrial membrane potential (ΔΨm), increased Bax/Bcl-2 ratio, and cytochrome c release, resulting in activation of caspase-9/-3 and subsequent cleavage of PARP. Further, the inhibitory effects of caspase-9/-3 expression by a synthetic pan-caspase inhibitor partially protected cells against apoptosis following CAE treatment. In addition, CAE significantly promoted cell death in AGS cells via an accumulation of cells in the G0/G1 phase. This effect was associated with upregulation of the CDK inhibitor p21 and downregulation of cyclin D1, cyclin E, CDK4, and CDK2 expression. Our data indicated that CAE exerted anti-proliferative activity by activating the mitochondria-mediated caspase-dependent apoptotic pathway and arresting the p21-mediated G0/G1 cell cycle on human gastric cancer AGS cells.

The selective proapoptotic impact of the myricetin-loaded alginate-cellulose hybrid nanocrystals (MAC-NCs) on the human AGS gastric cancer cells.

Myricetin, a flavanol present in fruits, tea, and vegetables, has the potential to reduce chronic diseases like gastric cancer by promoting cell death and stopping cell growth. However, its limited bioactivity due to its short lifespan and poor solubility in water has been a challenge. The current research focuses on incorporating myricetin into alginate-cellulose hybrid nanocrystals to enhance its selective proapoptotic effects on human AGS gastric cancer cells. MAC-NCs, myricetin-loaded alginate-cellulose hybrid nanocrystals, were synthesized using a combined co-precipitation/ultrasonic homogenization method and characterized through Dynamic Light Scattering (DLS), Fourier Transform Infrared Spectroscopy (FTIR), Field Emission Scanning Electron Microscope (FESEM), and Zeta-potential analyses. Their cytotoxic activity was tested on cancerous (AGS) and normal (Huvec) cells, revealing selective toxicity. Apoptotic markers, Caspase 8 and Caspase 9, gene expression was measured, and cell death type was confirmed using DAPI staining and flow cytometry on AGS cells. Synthesized MAC-NCs, measuring 40nm, showed significant selective toxicity on human gastric cells (IC50 of 31.05µg/mL) compared to normal endothelial cells (IC50 of 214.26µg/mL). DAPI and annexin flow cytometry revealed increased apoptotic bodies in gastric cells, indicating apoptosis. However, the apoptosis was found to be independent of Caspase-8 and Caspase-9. The current study provides critical insights into the therapeutic potential of MAC-NCs for gastric cancer treatment. Based on the notable induction of apoptosis in the AGS cancer cell line, the synthesized MAC-NCs exhibit promising potential as a selective anti-gastric cancer agent. However, further in-vivo studies are necessary to confirm and quantify the nanoparticle's selective toxicity and pharmaceutical properties in future investigations.

GPR137 inactivates Hippo signaling to promote gastric cancer cell malignancy

As the fifth most common cancer in the world, gastric cancer (GC) ranks as the third major cause of cancer-related death globally. Although surgical resection and chemotherapy still remains the mainstay of potentially curative treatment for GC, chemotherapy resistance and adverse side effects limit their clinical applications. Thus, further investigation of the mechanisms of carcinogenesis in GC and discovery of novel biomarkers is of great concern. We herein report that the elevated expression of GPR137 is correlated with GC. Overexpression of GPR137 potentiates human gastric cancer AGS cell malignancy, including proliferation, migration, invasion, colony formation and xenograft growth in nude mice in vivo, whereas knockout of GPR137 by CRISPR/Cas9 gene editing exerts the opposite effects. Mechanistically, GPR137 could bind to MST, the upstream kinases in Hippo pathway, which disrupts the association of MST with LATS, subsequently activating the transcriptional co-activators, YAP and TAZ, and thereby triggering the target transcription and the alterations in GC cell biological actions consequently. Therefore, our findings may provide with the evidence of developing a potentially novel treatment method with specific target for GC.

Efficacy of Hyperthermic Pressurized Intraperitoneal Aerosol Chemotherapy in an In Vitro Model Using a Human Gastric Cancer AGS Cell Line and an Abdominal Cavity Model.

Peritoneal carcinomatosis (PC) presents a major challenge in the treatment of late-stage, solid tumors, with traditional therapies limited by poor drug penetration. We evaluated a novel hyperthermic pressurized intraperitoneal aerosol chemotherapy (HPIPAC) system using a human abdominal cavity model for its efficacy against AGS gastric cancer cells. A model simulating the human abdominal cavity and AGS gastric cancer cell line cultured dishes were used to assess the efficacy of the HPIPAC system. Cell viability was measured to evaluate the impact of HPIPAC under 6 different conditions: heat alone, PIPAC with paclitaxel (PTX), PTX alone, normal saline (NS) alone, heat with NS, and HPIPAC with PTX. Results showed a significant reduction in cell viability with HPIPAC combined with PTX, indicating enhanced cytotoxic effects. Immediately after treatment, the average cell viability was 66.6%, which decreased to 49.2% after 48 hours and to a further 19.6% after 120 hours of incubation, demonstrating the sustained efficacy of the treatment. In contrast, control groups exhibited a recovery in cell viability; heat alone showed cell viability increasing from 90.8% to 94.4%, PIPAC with PTX from 82.7% to 89.7%, PTX only from 73.3% to 74.8%, NS only from 90.9% to 98.3%, and heat with NS from 74.4% to 84.7%. The HPIPAC system with PTX exhibits a promising approach in the treatment of PC in gastric cancer, significantly reducing cell viability. Despite certain limitations, this study highlights the system's potential to enhance treatment outcomes. Future efforts should focus on refining HPIPAC and validating its effectiveness in clinical settings.

Traditional Banxia Xiexin decoction inhibits invasion, metastasis, and epithelial mesenchymal transition in gastric cancer by reducing lncRNA TUC338 expression

BackgroundBanxia Xiexin decoction (BXD) is a classic traditional Chinese medicine (TCM) formula clinically used to treat chronic gastritis, gastric ulcers, gastric cancer, and many other gastrointestinal diseases. Long noncoding RNAs (lncRNAs) have been shown to play an important role in maintaining the malignant phenotype of tumors. However, no relevant studies have shown whether Banxia Xiexin decoction regulates and controls lncRNA TUC338, and the effect of TUC338 on the regulation of gastric cancer invasion and metastasis remains unclear. PurposeTo investigate the ability of the traditional Chinese medicine (TCM) Banxia Xiexin decoction (BXD) to inhibit the migration and invasion of human gastric cancer AGS cells by regulating the long noncoding RNA (lncRNA) TUC338. MethodsUHPLC‒MS/MS was used to analyze the chemical components of BXD. MTT was performed to determine the effects of BXD on the proliferation of AGS cells. qRT‒PCR was used to determine the expression of lncRNA TUC338 in gastric cancer tissues, paracarcinoma tissues, AGS human gastric cancer cells and GES-1 normal gastric mucosa cells and to evaluate the effects of BXD on the expression of lncRNA TUC338 in AGS cells. Lentiviral transfection was used to establish human gastric cancer AGS cells with knocked down lncRNA TUC338 expression. The effects of lncRNA TUC338 knockdown on the migration and invasion of AGS cells were observed by a scratch assay and Transwell migration assay, respectively. Western blotting was performed to analyze the effects of lncRNA TUC338 knockdown on epithelial-to-mesenchymal transition (EMT) in AGS cells. We performed quality control on three batches of BXD. We used UHPLC‒MS/MS to control the quality of three random batches of BXD used throughout the study. ResultsNinety-five chemical components were identified from the water extract of BXD, some of which have anticancer effects. The expression of TUC.338 in gastric cancer tissues was higher than that in para-carcinoma tissues. BXD inhibited the invasion and migration of gastric cancer cells by inhibiting the expression of lncRNA TUC338, which reduced EMT. After knockdown of lncRNA TUC338, the migration and invasion of AGS cells were reduced; the expression of the EMT-related protein E-cadherin was increased, and the expression of N-cadherin and vimentin was reduced. ConclusionsThe present results suggest that BXD has potential as an effective treatment for gastric cancer through the inhibition of lncRNA TUC338 expression.

Production of the exopolysaccharide from Lactiplantibacillus plantarum YT013 under different growth conditions: optimum parameters and mathematical analysis

ABSTRACT Exopolysaccharides (EPSs) are highly heterogeneous natural polymers, can exhibit some unique bio-logical activities, such as anti-oxidation, inflammation regulation, anticancer and anti-viral activities, etc. A new type of EPS was obtained from the fermentation supernatant of Lactiplantibacillus plantarum YT013. However, the low yield of EPS posed a challenge for further research and application. Therefore, in this study, the production process of EPS was optimized, and anti-cancer cells proliferation activity was investigated. Identified the best single-factor conditions for EPS production, including initial pH 6, inoculation amount 5% (v/v), temperature 37°C, cultivation time 36 h, glucose 3% (w/v), soy peptone 1.5% (w/v), KH2PO4 0.3% (w/v). Under these conditions, the maximum production of EPS reached 0.630 mg/mL, which is 1.31 times higher than that before optimization. Concurrently, the nonlinear model was used for fitting, and it was found that the Voigt nonlinear model was in better agreement with the optimization results, which can be used as an effective tool for optimizing EPS production conditions. Furthermore, the anti-proliferative activity of EPS was evaluated using the human gastric cancer AGS cell line in vitro. MTT assay revealed a strong inhibitory effect of EPS on AGS cell proliferation. Additionally, flow cytometry analysis indicated that EPS promoted apoptosis in AGS cells. Lactiplantibacillus plantarum YT013 EPS, as a biodegradable and nontoxic natural active substance, shows potential as a natural antitumor substance.

Activity Changes of the Peptic Lactoferrin Hydrolysate in Human Gastric Cancer AGS Cells in Response to Cu(II) or Mn(II) Addition.

Lactoferrin is an interesting bioactive protein in milk and can interact with various metal ions of trace elements such as copper, iron, manganese, and others. In this study, a lactoferrin hydrolysate (LFH) was generated from commercial bovine lactoferrin by protease pepsin, fortified with Cu2+ (or Mn2+) at two levels of 0.64 and 1.28 (or 0.28 and 0.56) mg/g protein, respectively, and then measured for the resultant bioactivity changes in the well-differentiated human gastric cancer AGS cells. The assaying results indicated that the LFH and Cu/Mn-fortified products had long-term anti-proliferation on the cells, while the treated cells showed DNA fragmentation and increased apoptotic cell proportions. Regarding the control cells, the cells treated with the LFH and especially Cu/Mn-fortified LFH had remarkably up-regulated mRNA expression of caspase-3 and Bax by respective 1.21-3.23 and 2.23-2.83 folds, together with down-regulated mRNA expression Bcl-2 by 0.88-0.96 folds. Moreover, Western-blot assaying results also indicated that the cells exposed to the LFH and Cu/Mn-fortified LFH (especially Mn at higher level) for 24 h had an enhanced caspase-3 expression and increased ratio of Bax/Bcl-2. It can thus be concluded that the used Cu/Mn-addition to the LFH may lead to increased bioactivity in the AGS cells; to be more specific, the two metal ions at the used addition levels could endow LFH with a higher ability to cause cell apoptosis by activating caspase-3 and increasing the Bax/Bcl-2 ratio.

Allyl isothiocyanate inhibits cell migration and invasion in human gastric cancer AGS cells via affecting PI3K/AKT and MAPK signaling pathway in vitro.

Metastasis is commonly occurred in gastric cancer, and it is caused and responsible for one of the major cancer-related mortality in gastric cancer patients. Allyl isothiocyanate (AITC), a natural product, exhibits anticancer activities in human many cancer cells, including gastric cancer. However, no available report shows AITC inhibits gastric cancer cell metastasis. Herein, we evaluated the impact of AITC on cell migration and invasion of human gastric cancer AGS cells in vitro. AITC at 5-20 μM did not induce significant cell morphological damages observed by contrast-phase microscopy but decreased cell viability assayed by flow cytometry. After AGS cells were further examined by atomic force microscopy (AFM), which indicated AITC affected cell membrane and morphology in AGS cells. AITC significantly suppressed cell motility examined by scratch wound healing assay. The results of the gelatin zymography assay revealed that AITC significantly suppressed the MMP-2 and MMP-9 activities. In addition, AITC suppressed cell migration and invasion were performed by transwell chamber assays at 24 h in AGS cells. Furthermore, AITC inhibited cell migration and invasion by affecting PI3K/AKT and MAPK signaling pathways in AGS cells. The decreased expressions of p-AKTThr308 , GRB2, and Vimentin in AGS cells also were confirmed by confocal laser microscopy. Our findings suggest that AITC may be an anti-metastasis candidate for human gastric cancer treatment.

Effects of Probiotic Saccharomyces boulardii Supernatant on Viability, Nano-Mechanical Properties of Cytoplasmic Membrane and Pro-Inflammatory Gene Expression in Human Gastric Cancer AGS Cells.

Gastric cancer has been recognized as the second most probable cause of death in humans from cancer diseases around the world. Postbiotics, supernatant, and metabolites from probiotic microorganisms have recently been used widely to prevent and treat cancer diseases in humans, without any undesirable side effects. This study explores the antiproliferative and antitumor activities of the probiotic Saccharomyces cerevisiae var. boulardii supernatant (SBS) against AGS cancer cells, a human gastric adenocarcinoma cell line. We evaluated cell growth inhibitory and mechanical properties of the cytoplasmic membrane and the downregulation of survivin and proinflammatory genes in AGS cells treated with SBS after 24 and 48 h. SBS significantly inhibits the AGS cell growth, and the concentrations with IC50 values after 24 and 48 h treatments are measured as 2266 and 1956 µg/mL, respectively. Regarding the AFM images and Young`s modulus analysis, SBS significantly induces morphological changes in the cytoplasmic membrane of the treated AGS cells. Expression of survivin, NFƙB, and IL-8 genes is significantly suppressed in AGS cells treated with SBS. Considering the antitumor activities of SBS on AGS cell line, it can be regarded as a prospective therapeutic and preventive strategy against human stomach cancer disease.

Co-delivery of doxorubicin and paclitaxel via noisome nanocarriers attenuates cancerous phenotypes in gastric cancer cells

Gastric cancer (GC) is known as a deadly malignancy all over the world, yet none of the current therapeutic regimens have achieved efficacy. this current study has aimed to optimize and reduce treatment doses and overcome multidrug resistance in GC by developing optimum niosomal formulation for the delivery of doxorubicin (DXR), paclitaxel (PTX), and their co-delivery. The particles' size, polydispersity index (PDI), and entrapment efficacy (EE%) were optimized using statistical techniques, i.e., Box-Behnken and Central Composite Design. In contrast to soluble drug formulations, the release rate of medicines from nanoparticles were higher in physiological and acidic pH. Niosomes were more stable at 4 °C, compared to 25 °C. The MTT assay revealed that the IC50 of drug-loaded niosomes was the lowest among all developed formulations. The apoptosis-related genes (CASPASE-3, CASPASE-8, and CASPASE-9) and tumor suppressor genes (BAX, BCL2) were evaluated in cancer cells before and after treatment. In comparison to control cells and cells treated with soluble forms of DXR and PTX, while the expression of BCL2 decreased, the expression of BAX, CASPASE-3, CASPASE-8, and CASPASE-9 was enhanced in cells treated with drug-loaded niosomes. Drug-loaded niosomes inhibited colony formation capacity and increased apoptosis in human AGS gastric cancer cells. Our results indicate that co-delivery of DXR and PTX-loaded niosomes may be an effective and innovative therapeutic approach to gastric cancer.

Multi-Targeting Anticancer Activity of a New 4-Thiazolidinone Derivative with Anti-HER2 Antibodies in Human AGS Gastric Cancer Cells

Combining chemotherapy with immunotherapy still remains a regimen in anticancer therapy. Novel 4-thiazolidinone-bearing hybrid molecules possess well-documented anticancer activity, and together with anti-HER2 antibodies, may represent a promising strategy in treating patients with gastric cancer with confirmed human epidermal growth factor receptor 2 (HER2) expression. The aim of the study was to synthesize a new 4-thiazolidinone derivative (Les-4367) and investigate its molecular mechanism of action in combination with trastuzumab or pertuzumab in human AGS gastric cancer cells. AGS cell viability and antiproliferative potential were examined. The effect of the tested combinations as well as monotherapy on apoptosis and autophagy was also determined. Metalloproteinase-2 (MMP-2), intercellular adhesion molecule 1 (ICAM-1), pro-inflammatory and anti-inflammatory cytokine concentrations were also demonstrated by the ELISA technique. We proved that pertuzumab and trastuzumab were very effective in increasing the sensitivity of AGS gastric cancer cells to novel Les-4367. The molecular mechanism of action of the tested combination is connected with the induction of apoptosis. Additionally, the anticancer activity is not associated with the autophagy process. Decreased concentrations of pro-inflammatory cytokines, MMP-2 and ICAM-1-were observed. The novel combination of drugs based on anti-HER2 antibodies with Les-4367 is a promising strategy against AGS gastric cancer cells.

In vitro and in vivo phototoxicity on gastric mucosa induced by methylene blue.

Methylene blue (MB) is used endoscopically to demarcate tumors and as a photosensitizer in photodynamic therapy (PDT). However, there are few in vivo studies about its toxicity in healthy stomach tissue. We performed sequential in vitro and in vivo analyses of MB-induced phototoxicity. We performed in vitro experiments using the AGS human gastric cancer cell line treated with light-emitting diode (LED) irradiation (3.6 J/cm2) and MB. Cytotoxicity was evaluated using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. In vivo toxicity was evaluated in the stomach of beagles using the same dose of fiber-optic LED via gastroscopy, after spraying 0.1% and 0.5% MB solutions. Stomach tissue was also evaluated using the TUNEL assay. In vitro, increased concentrations of MB led to higher TUNEL scores. However, cell viability was significantly lower after MB plus LED irradiation than after treatment with MB alone (P < 0.001). In vivo, the TUNEL score was highest immediately after treatment with 0.1% or 0.5% MB plus light irradiation, and the score was significantly higher in the LED illumination plus MB group than in the control group (P < 0.05). The elevated TUNEL score was maintained for 3 days in the MB plus light irradiation group but returned to normal levels on day 10. : Endoscopic light application with MB 0.5% concentration to the stomach may be regarded as a safe procedure despite some DNA injuries in the early period.

ISLR interacts with MGAT5 to promote the malignant progression of human gastric cancer AGS cells.

Gastric cancer is a common malignant tumor with high morbidity and mortality. The present study aimed to investigate the role of the immunoglobulin superfamily containing leucine-rich repeat (ISLR) gene in gastric cancer and examine whether ISLR could interact with N-acetylglucosaminyltransferase V (MGAT5) to affect the malignant progression of gastric cancer. The expression of ISLR and MGAT5 in human normal gastric epithelial cells and human gastric cancer cells, and the transfection efficiency of ISLR interference plasmids and MGAT5 overexpression plasmids were all detected by reverse transcription-quantitative PCR (RT-qPCR) and western blot. The viability, proliferation, migration and invasion, and epithelial-mesenchymal transition (EMT) of gastric cancer cells after indicated transfection were detected by Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, wound healing assay, and transwell assay. The interaction between ISLR and MGAT5 was confirmed by co-immunoprecipitation. The expression of proteins related to migration, invasion, and EMT was detected by immunofluorescence and western blot. As a result, ISLR was highly expressed in gastric cancer and was associated with poor prognosis. Interference with ISLR inhibited the viability, proliferation, migration, invasion, and EMT of gastric cancer cells. ISLR interacted with MGAT5 in gastric cancer cells. MGAT5 overexpression weakened the effects of ISLR knockdown on suppressing the viability, proliferation, migration, invasion, and EMT of gastric cancer cells. ISLR interacted with MGAT5 to promote the malignant progression of gastric cancer.

Ginsenoside compound K-loaded gold nanoparticles synthesized from Curtobacterium proimmune K3 exerts anti-gastric cancer effect via promoting PI3K/Akt-mediated apoptosis

BackgroundCompound K (CK) is the minor ginsenoside present in fermented Panax ginseng extract. Despite the pharmacological efficacy of CK, its industrial use has been restricted due to its low water solubility and poor permeability. To overcome this defect, our study was to synthesize gold nanoparticles from CK (CK-AuNPs) to investigate their potential as anticancer candidates.MethodsTo biologically synthesize CK-AuNPs, a novel strain, Curtobacterium proimmune K3, was isolated from fermented ginseng beverage, then combined with CK and gold salts to biosynthesize gold nanoparticles (CurtoCK-AuNPs). Their physicochemical characteristics were evaluated using UV–Vis spectrometry, FE-TEM, EDX, elemental mapping, XRD, SAED, DLS and TGA.ResultsCurtoCK-AuNPs exerted significant selective cytotoxic effects on AGS human gastric cancer cells. Fluorescence staining with Hoechst, propidium iodide, and MitoTracker demonstrated that CurtoCK-AuNPs induce apoptosis and mitochondrial damage, respectively. Quantitative real-time PCR and western blotting analyses showed that cytotoxic effect of CurtoCK-AuNPs were involved in apoptosis, based on their activation of Bax/Bcl-2, cytochrome c, caspase 9, and caspase 3, as well as their suppression of PI3K–Akt signaling.ConclusionOur findings provide data for understanding the molecular mechanisms of nanoparticles; thus, providing insight into the development of alternative medications based on gold nanoparticles of ginseng-derived CK.

Flavonoid-Rich Extract of Oldenlandia diffusa (Willd.) Roxb. Inhibits Gastric Cancer by Activation of Caspase-Dependent Mitochondrial Apoptosis.

To evaluate the apoptosis and cycle arrest effects of Oldenlandia diffusa flavonoids on human gastric cancer cells, determine the action mechanisms in association with the mitochondrial dependent signal transduction pathway that controls production of reactive oxygen species (ROS), and evaluate the pharmacodynamics of a mouse xenotransplantation model to provide a reference for the use of flavonoids in prevention and treatment of gastric cancer. Flavonoids were extracted by an enzymatic-ultrasonic assisted method and purified with D-101 resin. Bioactive components were characterized by high-performance liquid chromatography. Cell lines MKN-45, AGS, and GES-1 were treated with different concentrations of flavonoids (64, 96, 128, 160 µg/mL). The effect of flavonoids on cell viability was evaluated by MTT method, and cell nuclear morphology was observed by Hoechst staining. The apoptosis rate and cell cycle phases were measured by flow cytometry, the production of ROS was detected by laser confocal microscope, the mitochondrial membrane potential (MMP) were observed by fluorescence microscope, and the expression of apoptotic proteins related to activation of mitochondrial pathway were measured by immunoblotting. MKN-45 cells were transplanted into BALB/c nude mice to establish a xenograft tumor model. Hematoxylin and eosin staining was used to reveal the subcutaneous tumor tissue. The tumor volume and tumor weight were measured, the expression levels of proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67 were detected by immunohistochemistry, and the expression levels of CA72-4 were measured by enzyme linked immunosorbent assay. Oldenlandia diffusa flavonoids inhibited proliferation of MKN-45 and AGS human gastric cancer cells, arrested the cell cycle in G1/S phase, induced accumulation of ROS in the process of apoptosis, and altered MMP. In addition, flavonoids increased Apaf-1, Cleaved-Caspase-3, and Bax, and decreased Cyclin A, Cdk2, Bcl-2, Pro-Caspase-9, and Mitochondrial Cytochrome C (P<0.05). The MKN-45 cell mouse xenotransplantation model further clarified the growth inhibitory effect of flavonoids towards tumors. The expression levels of PCNA and Ki-67 decreased in each flavonoid dose group, the expression level of CA72-4 decreased (P<0.05). Flavonoids derived from Oldenlandia diffusa can inhibit proliferation and induce apoptosis of human gastric cancer cells by activating the mitochondrial controlled signal transduction pathway.

Anti-Cancer Activity of Gedunin by Induction of Apoptosis in Human Gastric Cancer AGS Cells.

Currently, gastric cancer is considered one of the major causes of high mortality and morbidity worldwide. Recent advances in therapeutics, clinical treatment, staging procedures, and imaging techniques are high, yet the prevalence of gastric cancer has not been reduced. Usage of the synthetic drug has many side effects that can lead to other ailments. Gedunin, a phytochemical derived from Azadirachta indica (neem tree), exhibits several pharmacological activities including antitumor, anti-inflammatory, antiulcer, antipyretics, antibacterial, antifungal, anti-diabetic, and antimalarial properties. In the current investigation, the effect of gedunin on the cell viability; reactive oxygen species (ROS) generation by DCFH-DA staining; mitochondrial membrane potential (MMP) by Rh-123 staining; apoptosis by AO/EtBr staining; cell migration and wound healing ability by wound scratch assay; and Bcl-2, Bax, caspase-3, and caspase-9 by ELISA techniques were analyzed in the AGS cells. The treatment with gedunin effectively inhibited the cell viability with IC50 = 20µM, increased the ROS generation, and triggered the apoptosis in AGS cells. The gedunin-treated AGS cells also demonstrated a decreased MMP status. The increment in the ROS generation leads to oxidative stress which in turn induce the apoptosis. The activity of Bax gene was upregulated and the activity of Bcl-2 gene was down-regulated in the AGS cells after the treatment with gedunin. In the AGS cells treated with gedunin, the caspase-3 and caspase-9 activities were increased. In overall, these findings suggested that gedunin can be used as a potent chemotherapeutic agent in the future to treat gastric cancer.

Endoplasmic Reticulum Stress and Impairment of Ribosome Biogenesis Mediate the Apoptosis Induced by Ocimum x africanum Essential Oil in a Human Gastric Cancer Cell Line.

Background and Objectives: Gastric cancer remains a major unmet clinical problem worldwide. Although conventional medical treatments are available, their curative effects are generally unsatisfactory. Consequently, it remains necessary to search natural products for potential alternatives in treating gastric cancer patients. Ocimum x africanum Lour. is a culinary herb that has been used in folk medicine for various diseases, but little is known regarding its anti-cancer activity against gastric cancer cells. In the current study, we focus on the anti-cancer mechanisms of O. x africanum essential oil (OAEO) in the AGS human gastric cancer cell line. Materials and Methods: After OAEO treatment, AGS cell viability was evaluated by MTT assay. Cell migration and apoptotic nuclear morphology were determined by wound-healing assay and DAPI staining, respectively. Gene expression levels of apoptosis-related genes were quantified by qRT–PCR. Differential protein expression was determined with an LC–MS/MS-based proteomics approach to identify the key proteins that may be important in the anti-cancer mechanisms of OAEO on AGS cells. The chemical constituents of OAEO were identified by GC–MS analysis. Results: We found OAEO to exhibit a potent growth-inhibiting effect on AGS cells, with an IC50 value of 42.73 µg/mL. After OAEO treatment for 24 h, AGS cell migration was significantly decreased relative to the untreated control. OAEO-treated AGS cells exhibited common features of apoptotic cell death, including cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation. Apoptotic cell death was confirmed by qRT–PCR for apoptosis-related genes, revealing that OAEO decreased the expression of anti-apoptotic genes (BCL2 and BCL-xL) and activated pro-apoptotic genes and apoptotic caspase genes (TP53, BAX, CASP9, CASP12, and CASP3). Moreover, expression of CASP8 was not changed after treatment. Proteomic analysis revealed that OAEO may produce a signature effect on protein clusters relating to unfolded protein accumulation, thereby inducing severe ER stress and also impairing ribosome synthesis. STRING analysis revealed seven up-regulated and 11 down-regulated proteins, which were significantly associated with protein folding and ribosome biogenesis, respectively. Using GC–MS analysis, 6-methyl-5-hepten-2-one, citral, neral, and linalool were found to be the major chemical constituents in OAEO. Conclusions: Taken together, these results indicate that OAEO has a potential anti-proliferative effect on AGS cells. Our molecular findings show evidence supporting an important role of ER stress and ribosome biogenesis impairment in mediating the induction of cell death by OAEO through the mitochondrial-apoptotic pathway. This study, therefore, provides fundamental knowledge for future applications using OAEO as an alternative therapy in gastric cancer management.

Sulforaphane Suppresses the Nicotine-Induced Expression of the Matrix Metalloproteinase-9 via Inhibiting ROS-Mediated AP-1 and NF-κB Signaling in Human Gastric Cancer Cells.

Sulforaphane, a natural phytochemical compound found in various cruciferous vegetables, has been discovered to present anti-cancer properties. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in gastric cancer metastasis. However, the role of sulforaphane in MMP-9 expression in gastric cancer is not yet defined. Nicotine, a psychoactive alkaloid found in tobacco, is associated with the development of gastric cancer. Here, we found that sulforaphane suppresses the nicotine-mediated induction of MMP-9 in human gastric cancer cells. We discovered that reactive oxygen species (ROS) and MAPKs (p38 MAPK, Erk1/2) are involved in nicotine-induced MMP-9 expression. AP-1 and NF-κB are the critical transcription factors in MMP-9 expression. ROS/MAPK (p38 MAPK, Erk1/2) and ROS functioned as upstream signaling of AP-1 and NF-κB, respectively. Sulforaphane suppresses the nicotine-induced MMP-9 by inhibiting ROS-mediated MAPK (p38 MAPK, Erk1/2)/AP-1 and ROS-mediated NF-κB signaling axes, which in turn inhibit cell invasion in human gastric cancer AGS cells. Therefore, the current study provides valuable evidence for developing sulforaphane as a new anti-invasion strategy for human gastric cancer therapy.

Synthesis of 8-Fluoroneocryptolepine and Evaluation for Cytotoxic Activity against AGS Cancer Cells.

Neocryptolepine derivatives have attracted great interest because of their unique cytotoxic activity. 8-Fluoroneocryptolepine (8FNC) was synthesized, and its cytotoxicity was evaluated by MTT assay in AGS gastric cancer cells and gastric mucosa GES-1 cells. 8-Fluoroneocryptolepine showed greater selectivity and cytotoxicity to AGS cells than the cisplatin (CIS) and fluorouracil (5-Fu) commonly used in clinical treatment of gastric cancer. Most importantly, we significantly improved the cytotoxic effect of 8FNC against AGS cells by structural modification and reduced the cytotoxicity against GES-1 cells compared with neocryptolepine. We further evaluated the activity of 8FNC against AGS cells in vitro. Our results indicate that 8FNC arrests the AGS cell cycle in the G2/M phase, reduces the mitochondrial membrane potential of AGS cells, and drives the initiation of apoptotic body formation in 8FNC-induced apoptosis. Moreover, 8FNC exhibits strong inhibitory effects on AGS cell migration. Studies on the molecular mechanisms of the cytotoxic activities of 8FNC revealed that it may play a significant role in the inhibitory effect on AGS human gastric cancer cells through the PI3K/AKT signaling pathway. In conclusion, 8FNC may become a promising lead compound in the development of potential clinical drug candidates for the treatment of gastric cancer.