Human Gastric Carcinoma Cell Research Articles

Antitumorigenic effect of plumbagin by induction of SHP1 in human gastric carcinoma cell lines.

74 Background: Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a plant-drived natural agent extracted from the root of Plumbago zeylanic. A recent study reported that plumbagin down-regulated the activity of Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway to show various anti-tumor effects. We aimed in this in vitrostudy to demonstrate the inhibition of JAK2-STAT3 pathway by plumbagin through inducing SH2-containing protein tyrosine phosphatase 1 (SHP1) expression in gastric cancer cell line. Methods: We performed Wetern blot to measure SHP1, phospho-JAK2/STAT3 level, and reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate target gene expression of STAT3. Several functional studies such as water soluble tetrazolium-1 (WST-1) assay, wound closure assay and matrigel invasion assay were also performed. Results: Plumbagin induced SHP1 expression and simultaneously down-regulated phospho-JAK2/STAT3 level via dose-and time-dependant manner in MKN28 cell, a gastric carcinoma cell line which has negative SHP1 expression. This effect was consistent when JAK2-STAT3 signaling was activated by interleukin-6, and ameliorated when cells were treated with prevanadate, a protein tyrosin phosphatase inhibitor. Furthermore, plumbagin significantly reduced gene expression of cyclin D1, VEGF1, survivin, MMP9, known target products of STAT3 activation in gastric cancinogenesis. The functional effect of plumbagin could be validated as inhibition of cell proliferation, migration and invasion, which are the results of activation of JAK2-STAT3 pathway in gastric cancer cells. Conclusions: Plumbagin is a potential negative regulator of cellular growth, migration and invasion by inhibiting both constitutive and inducible STAT3 activity through induction of SHP1 in gastric cancer cells.

Melatonin Induces Cell Apoptosis in AGS Cells Through the Activation of JNK and P38 MAPK and the Suppression of Nuclear Factor-Kappa B: a Novel Therapeutic Implication for Gastric Cancer

Background/Aims: Melatonin, synthesized by the pineal gland and released into the blood, appears to have antitumour properties; however, the mechanisms of its anti-cancer effects are largely unknown, especially in stomach cancer. Here, we explore the antitumour activity of melatonin in a gastric cancer cell line (AGS) and analyse its molecular mechanisms. Methods: AGS cells were treated with melatonin, and cell viability was assessed using a CCK-8 assay. Flow cytometry was performed to evaluate apoptosis, and protein expression was examined by Western blotting. Results: Melatonin significantly inhibited cell viability, clone formation, and cell migration and invasion and induced apoptosis in AGS cells. Moreover, MAPK pathways (p38, JNK and ERK) were activated by melatonin treatment, which also significantly increased caspase-3 cleavage and Bax protein expression and decreased Bcl-2 protein expression in a time-dependent manner. Our results demonstrate that p38 and JNK inhibitors (SB203580 and SP600125, respectively) prevented melatonin-induced apoptosis; thus, the propensity of p38 MAPK and JNK to promote apoptosis could be at least partly due to the inhibition of NF-κB p65 activation by p38 and JNK. Finally, melatonin was able to strengthen cisplatin-mediated antitumour effects in human gastric carcinoma cells by up-regulating the expression of Bax, down-regulating the expression of Bcl-2 and activating the caspase-dependent apoptotic pathway. Conclusion: Melatonin induced apoptosis in AGS cells by activating the caspase-dependent apoptotic pathway and by inhibiting the nuclear translocation of NF-κB p65, two processes that are regulated by p38 and JNK. Furthermore, melatonin significantly enhanced the anti-tumour effects of cisplatin, with low systemic toxicity. These new findings suggest that melatonin may act as a potent anti-tumour agent and may have great potential as an adjuvant therapy in the future.

Colon Cancer Associated Transcript-1 (CCAT1) Expression in Adenocarcinoma of the Stomach.

Background: Long non-coding RNAs (lncRNAs) have been shown to have functional roles in cancer biology and are dys-regulated in many tumors. Colon Cancer Associated Transcript -1 (CCAT1) is a lncRNA, previously shown to be significantly up-regulated in colon cancer. The aim of this study is to determine expression levels of CCAT1 in gastric carcinoma (GC).Methods: Tissue samples were obtained from patients undergoing resection for gastric carcinoma (n=19). For each patient, tumor tissue and normal appearing gastric mucosa were taken. Normal gastric tissues obtained from morbidly obese patients, undergoing laparoscopic sleeve gastrectomy served as normal controls (n=19). A human gastric carcinoma cell line (AGS) served as positive control. RNA was extracted from all tissue samples and CCAT1 expression was analyzed using quantitative real time-PCR (qRT-PCR).Results: Low expression of CCAT1 was identified in normal gastric mucosa samples obtained from morbidly obese patients [mean Relative Quantity (RQ) = 1.95±0.4]. AGS human gastric carcinoma cell line showed an elevated level of CCAT1 expression (RQ=8.02). Expression levels of CCAT1 were approximately 10.8 fold higher in GC samples than in samples taken from the negative control group (RQ=21.1±5 vs. RQ=1.95±0.4, respectively, p<0.001). Interestingly, CCAT1 expression was significantly overexpressed in adjacent normal tissues when compared to the negative control group (RQ = 15.25±2 vs. RQ=1.95±0.4, respectively, p<0.001). Tissues obtained from recurrent GC cases showed the highest expression levels (RQ = 88.8±31; p<0.001). Expression levels increased with tumor stage (T4- 36.4±15, T3- 16.1±6, T2- 4.7±1), however this did not reach statistical significance (p=0.2). There was no difference in CCAT1 expression between intestinal and diffuse type GC (RQ=22.4±7 vs. 22.4±16, respectively, p=0.9). Within the normal gastric tissue samples, no significant difference in CCAT1 expression was observed in helicobacter pylori negative and positive patients (RQ= 2.4±0.9 vs. 0.93±0.2, respectively, p=0.13).Conclusion: CCAT1 is up-regulated in gastric cancer, and may serve as a potential bio-marker for early detection and surveillance.

Screening for genes that are differentially-expressed between gastric cancer cells and gastric tumor sphere cells using the gene chip technique.

The purpose of this study was to screen for genes that were differentially expressed between a human gastric carcinoma cell line (HGC-27) and their tumor spheres, using the gene chip technique. The HGC-27 cells and tumor sphere cells were cultured in vitro in a sterile environment. Total RNA was extracted from both samples and purified using a standard TRIzol reagent. Total RNA was then hybridized onto a GeneChip, according to the standard protocols provided by the manufacturers of the GeneChip IVT Express Kit. The resulting fluorescence signals were analyzed and displayed using the Cluster and Treeview software programs. Under the criteria for significant differential expression (≥2-fold difference), 610 up- and 1135 down-regulated genes were identified in tumor sphere cells, compared to HCG-27 cells. These genes were involved in cell growth, signal transduction, tumorigenesis, and many other functional aspects of tumor cells. In conclusion, a number of genes were differentially expressed in tumor sphere cells compared to HCG-27 cells. In addition, we identified a close correlation between tumor sphere cells and tumorigenesis.

Aberrant EphB/ephrin-B expression in experimental gastric lesions and tumor cells.

To determine whether the expression profiles of EphB receptor and ephrin-B ligand can be used as markers for dysplastic/oncogenic transformation in gastric mucosa. The protein expression and localization of EphB and ephrin-B in normal, ulcerated regenerating, and dysplastic gastric mucosa were examined in a rat experimental model by immunolabeling, and mRNA expression was assessed in four human gastric carcinoma cell lines by reverse transcription-polymerase chain reaction. Ephrin-B- and EphB-expressing regions were divided along the pit-gland axis in normal gastric units. EphB2 was transiently upregulated in the experimental ulcer, and its expression domain extended to gastric pits and/or the luminal surface where ephrin-B-expressing pit cells reside. EphB2, B3, and B4 and ephrin-B1 were coexpressed in the experimental gastric dysplasia, and more than one ligand-receptor pair was highly expressed in each of the gastric carcinoma cell lines. Robust and stable coexpression of EphB and ephrin-B is a feature common to experimentally induced gastric dysplasia and human gastric carcinoma cell lines as compared to normal gastric and ulcerated regenerating epithelia. Thus, EphB/ephrin-B may be a useful marker combination for dysplastic/oncogenic transformation in gastric cancer.

Primary analysis and screening of microRNAs in gastric cancer side population cells.

To explore the microRNA (miRNA) profiles and to determine the key miRNAs within the side population (SP) cells of the gastric cancer cell line MKN-45. We used fluorescence-activated cell sorting and Hoechst 33342 labeling to obtain SP cells from the human gastric carcinoma cell line MKN-45. The miRNA expression profiles of the SP and major population (MP) cells were examined using a miRNA gene chip, and key miRNAs were obtained according to aberrant expression and the miRNAs' possible targets as predicted by bioinformatics. Using a significance criterion of a 1.5-fold or greater difference in expression level, we observed an increase in the expression of 34 miRNAs and a decrease in the expression of 34 miRNAs when comparing SP to MP cells. Using quantitative real-time reverse transcription-polymerase chain reaction to test for differentially expressed miRNAs combined with bioinformatics results, we found that the downregulated miRNAs, such as hsa-miR-3175 and hsa-miR-203, and the upregulated miRNAs, including hsa-miR-130a, hsa-miR-324-5p, hsa-miR-34a, and hsa-miR-25-star, may be important in maintaining and regulating the characteristics of SP cells. There are key miRNAs expressed within the SP cells of the gastric cancer cell line MKN-45, and include hsa-miR-3175, hsa-miR-203, hsa-miR-130a, hsa-miR-324-5p, hsa-miR-34a, and hsa-miR-25-star.

1P1-L05 オープンチップの気液界面制御による希少細胞の自動分離・回収

For molecular analysis of the rare cells, such as Circulating Tumor Cells (CTCs), the single rare cell isolation from the suspension which contains large population of other cells is required. Here, we propose a platform which utilizes surface tension and suction force to array single cells on the microfluidic chip with open channels. Cells flowed to the meniscus of air/liquid interface due to the suction force caused by a suction pump. Then, through surface tension which was generated by the air-liquid interface, the cells were deposited and trapped in the specified pockets surrounded with micropillars. Previously we already succeeded in isolating the rare cell with high speed. We developed a system for cell isolation and pick-up. We purpose to control the position of the air-liquid interface balancing supply and suction pumps. The experiment was performed with human gastric carcinoma cell line (GCIY) cells experiments. By using our isolation methods, the trap rates of cells were nearly 90 %. Also, we utilized mouse CTC model to demonstrate clinical utility of our method. Our platform will be useful for single rare cell analysis because the collected cells could be picked up by glass micro pipette rapidly and easily.

Allicin induces apoptosis of the MGC-803 human gastric carcinoma cell line through the p38 mitogen-activated protein kinase/caspase-3 signaling pathway.

Gastric cancer is one of the most common forms of malignant tumor, and the development of anti‑gastric cancer drugs with minimal toxicity is of clinical importance. Allicin is extracted from Allium sativum (garlic). Recent research, including clinical experiments, has shown that garlic has anticancer and tumor suppressive effects. The present study aimed to investigate the effects of allicin on the MGC‑803 human gastric carcinoma cell line, and to further explore the possible mechanisms of its tumor suppressor effects. The effects of allicin on the MGC‑803 cells were initially examined using an 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. Hoechst staining was also used, in order to demonstrate the impact of allicin on MGC‑803 cell apoptosis. In addition, western blot analysis was performed to determine the abnormal expression levels of apoptosis‑associated proteins, following the treatment of MGC‑803 cells with allicin. Western blotting was also used to investigate the specific mechanisms underlying allicin‑induced apoptosis of MGC‑803 cells. The rate of MGC‑803 apoptosis was significantly increased, when the concentration and treatment time of allicin were increased. Hoechst staining detected an enhanced rate of apoptosis, and enhanced expression levels of cleaved caspase 3 were determined by western blotting. Notably, the protein expression levels of p38 were increased when the MGC‑803 cells were treated with allicin. The results of the present study suggest that allicin may inhibit the proliferation and induce the apoptosis of MGC‑803 human gastric carcinoma cells, and this may partially be achieved through the enhanced expression of p38 and cleaved caspase 3.

CpG island promoter hypermethylation of Ras association domain family 1A gene contributes to gastric carcinogenesis

Methylation rates of the Ras association domain family 1A gene (RASSF1A) have been variously reported as between 7.5 and 66.7% in gastric carcinoma tissues. The role of this gene in gastric cancer also remains to be fully elucidated. The present study aimed to investigate whether promoter hypermethylation of RASSF1A occurs in gastric adenocarcinoma tissues and gastric cancer cell lines, and to determine the effects of RASSF1A in gastric carcinoma cell lines. The results showed a methylation‑specific band only in SNU‑719, MKN28 and AGS human gastric cancer cells (indicating full methylation), none of which exhibited RASSF1A expression. By contrast, SNU‑16, MKN‑45 and KATO‑III human gastric carcinoma cells exhibited methylation as well as unmethylation‑specific bands (indicating partial methylation), and all displayed positive or weakly positive expression of RASSF1A. Bisulfite sequencing in AGS and SNU‑719 cells revealed that virtually all CpG sites were densely methylated. When SNU‑719, MKN‑28 and AGS cells were treated with the demethylating agent 5‑aza‑2'‑deoxycytidine, RASSF1A gene expression was restored and the methylation‑specific polymerase chain reaction pattern was altered in all three cell lines. Transfection of a plasmid expressing RASSF1A into AGS and SNU‑719 cells significantly inhibited cell proliferation. Exogenous RASSF1A also reduced the expression of cyclin D1 and phospho‑retinoblastoma protein, and increased that of p27 as demonstrated by western blot analysis. Furthermore, RASSF1A expression was significantly reduced (P=0.048) and the methylation rate was elevated in gastric adenocarcinoma tissues, compared with those in adjacent healthy intestinal metaplasia (34.6 vs. 66.7%, P=0.029). The present study indicated that epigenetic silencing of RASSF1A is frequently caused by promoter hypermethylation in gastric cancer cell lines as well as in gastric adenocarcinoma tissues, which may contribute to gastric carcinogenesis.

Deoxycholic acid inhibits the growth of BGC-823 gastric carcinoma cells via a p53-mediated pathway

The aim of the present study was to investigate the effects of deoxycholic acid (DCA) on BGC‑823 human gastric carcinoma cells and to explore the possible mechanisms underlying any such effects. Cell proliferation was detected using a 3‑(4,5‑Dimethylthiazol‑2‑yl)‑2,5‑diphenyl tetrazolium bromide assay, cell morphology was observed by inverted microscopy, and cell cycle progression and the mitochondrial membrane potential were analyzed using flow cytometry. The expression of Bcl‑2, Bax, p53, Cyclin D1 and cyclin‑dependent kinase (CDK)2 proteins in BGC‑823 cells was analyzed with western blotting. The results demonstrated that DCA significantly inhibited cell growth, and that the cell cycle was arrested at the G1 phase. DCA was also shown to induce BGC‑823 cell apoptosis, which was associated with the collapse of the mitochondrial membrane potential. The mitochondria‑dependent pathway was activated via an increase in the ratio of Bax:Bcl‑2 in BGC‑823 cells. In addition, the expression of p53, cyclin D1 and CDK2 was altered following DCA treatment. These results suggest that DCA induces apoptosis in gastric carcinoma cells through activation of an intrinsic mitochondrial‑dependent pathway, in which p53 is involved.

Interplay between the prostaglandin transporter OATP2A1 and prostaglandin E2-mediated cellular effects

Prostaglandins such as prostaglandin E2 (PGE2) play a pivotal role in physiological and pathophysiological pathways in gastric mucosa. Little is known about the interrelation of the prostaglandin E (EP) receptors with the prostaglandin transporter OATP2A1 in the gastric mucosa and gastric carcinoma. Therefore, we first investigated the expression of OATP2A1 and EP4 in normal and carcinoma gastric mucosa. Different PGE2-mediated cellular pathways and mechanisms were investigated using human embryonic kidney cells (HEK293) and the human gastric carcinoma cell line AGS stably transfected with OATP2A1. Colocalization and expression of OATP2A1 and EP4 were detected in mucosa of normal gastric tissue and of gastric carcinomas. OATP2A1 reduced the PGE2-mediated cAMP production in HEK293 and AGS cells overexpressing EP4 and OATP2A1. The expression of OATP2A1 in AGS cells resulted in a reduction of [3H]-thymidine incorporation which was in line with a higher accumulation of AGS-OATP2A1 cells in S-phase of the cell cycle compared to control cells. In contrast, the expression of OATP2A1 in HEK293 cells had no influence on the distribution in the S-phase compared to control cells. OATP2A1 also diminished the PGE2-mediated expression of interleukin-8 mRNA (IL-8) and hypoxia-inducible-factor 1α (HIF1α) protein in AGS-OATP2A1 cells. The expression of OATP2A1 increased the sensitivity of AGS cells against irinotecan which led to reduced cell viability. Taken together, these data show that OATP2A1 influences PGE2-mediated cellular pathways. Therefore, OATP2A1 needs to be considered as a key determinant for the understanding of the physiology and pathophysiology of prostaglandins in healthy and tumorous gastric mucosa.

Microwave-assisted solvothermal synthesis and upconversion luminescence of CaF2:Yb3+/Er3+ nanocrystals

Water-dispersible CaF2 and Yb3+/Er3+ codoped CaF2 (CaF2:Yb3+/Er3+) nanocrystals with different sizes and different Yb3+ and Er3+ dopant concentrations were synthesized using ionic liquid 1-n-butyl-3-methyl imidazolium tetrafluoroborate as a fluorine source by the rapid microwave-assisted solvothermal method. It was found that the morphology, size and crystallinity of CaF2:Yb3+/Er3+ nanocrystals could be adjusted by using adenosine 5′-triphosphate disodium salt (ATP). Yb3+ and Er3+ ions were doped into CaF2 nanocrystals to enable upconversion luminescence emission, and the as-prepared CaF2:Yb3+/Er3+ samples exhibited upconversion luminescence upon excitation at 980nm. Confocal laser scanning microscopy images showed that the CaF2:Yb3+/Er3+ nanocrystals could be used for efficient labeling of human gastric carcinoma cells. Moreover, in vitro cytotoxicity experiments indicated that the as-prepared CaF2:Yb3+/Er3+ nanocrystals had essentially little cytotoxicity. These results indicate that the as-prepared CaF2:Yb3+/Er3+ nanocrystals are promising for the application as a luminescent label material in biological imaging.

13-acetoxysarcocrassolide induces apoptosis on human gastric carcinoma cells through mitochondria-related apoptotic pathways: p38/JNK activation and PI3K/AKT suppression.

13-acetoxysarcocrassolide (13-AC), an active compound isolated from cultured Formosa soft coral Sarcophyton crassocaule, was found to possess anti-proliferative and apoptosis-inducing activities against AGS (human gastric adenocarcinoma cells) gastric carcinoma cells. The anti-tumor effects of 13-AC were determined by MTT assay, colony formation assessment, cell wound-healing assay, TUNEL/4,6-Diamidino-2-phenylindole (DAPI) staining, Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining and flow cytometry. 13-AC inhibited the growth and migration of gastric carcinoma cells in a dose-dependent manner and induced both early and late apoptosis as assessed by flow cytometer analysis. 13-AC-induced apoptosis was confirmed through observation of a change in ΔΨm, up-regulated expression levels of Bax and Bad proteins, down-regulated expression levels of Bcl-2, Bcl-xl and Mcl-1 proteins, and the activation of caspase-3, caspase-9, p38 and JNK. Furthermore, inhibition of p38 and JNK activity by pretreatment with SB03580 (a p38-specific inhibitor) and SP600125 (a JNK-specific inhibitor) led to rescue of the cell cytotoxicity of 13-AC-treated AGS cells, indicating that the p38 and the JNK pathways are also involved in the 13-AC-induced cell apoptosis. Together, these results suggest that 13-AC induces cell apoptosis against gastric cancer cells through triggering of the mitochondrial-dependent apoptotic pathway as well as activation of the p38 and JNK pathways.

Geldanamycin mediates the apoptosis of gastric carcinoma cells through inhibition of EphA2 protein expression.

The aim of the present study was to investigate the role of EphA2 in the carcinogenesis and progression of gastric carcinoma. Moreover, we aimed to determine the effect of geldanamycin (GA), an inhibitor of Hsp90, on the proliferation and apoptosis of human gastric carcinoma cells. Gastric carcinoma tissues, paired adjacent mucosa and paired normal mucosa were obtained from resected surgical specimens of gastric carcinoma, and EphA2 mRNA and protein levels were assessed by RT-PCR, immunohistochemistry and western blot analysis. FCM was used to detect cell cycle distribution and apoptosis. MGC803 cell proliferation and apoptosis were assessed by MTT and FCM, respectively. We found that EphA2 protein was increased in the carcinogenesis of gastric epithelial cells. Proliferation index (PI) was significantly upregulated following an increase in EphA2 expression in gastric carcinoma compared with dysplasia and normal samples, and was notably correlated with grade and lymph node metastasis. Knockdown of EphA2 increased the apoptosis rate and decreased the PI of MGC803 cells, which overexpressed the EphA2 protein. GA inhibited the cell proliferation of MGC803 cells in a dose- and time-dependent manner and induced cell apoptosis. In addition, GA decreased the EphA2 protein expression in MGC803 cells. Overexpression of EphA2 inhibited cell growth, blocked cells in the G0/G1 stage and increased cell apoptosis induced by GA in MGC803 cells. However, knockdown of EphA2 in MGC803 cells increased the apoptosis ratio induced by GA. In conclusion, EphA2 overexpression is an important characteristic in the carcinogenesis of gastric epithelial cells, followed by an increase in apoptosis and cell cycle arrest. Knockdown of EphA2 blocked MGC803 cell proliferation and induced cell apoptosis. In conclusion GA inhibits MGC803 cell proliferation and induces cell apoptosis by upregulating expression of EphA2.

Synergistic anti-tumor effect of KLF4 and curcumin in human gastric carcinoma cell line.

Kruppel-like factor 4 is a transcription factor which plays an important role in development and progression of various carcinomas. Curcumin characterized by excellent anti-cancer properties is regarded as a serviceable natural compound used in carcinoma therapy. This study aimed at exploring the impact of KLF4 overexpression in cooperation with curcumin on the proliferation, apoptosis and invasion of human gastric carcinoma BGC- 823 cells. Flow cytometry analysis, CCK-8 assays, transwell assays and Western blot results showed that KLF4 overexpression combined with curcumin had significant anti-proliferation, pro-apoptosis and anti-invasion effects on BGC-823 cells. We also found that KLF4 had synergistic effects with curcumin, better promoting apoptosis and inhibiting proliferation and invasion of gastric carcinona cells. These results indicate that KLF4 could be used as a potential therapeutic target; curcumin could act as an auxiliary and provide a promising therapeutic strategy in stomach cancer.

Abstract 719: Development of systemically-deliverable telomerase-specific oncolytic adenovirus

Abstract Background: Oncolytic virotherapy is an emerging treatment strategy that allows to destroy cancer tissues only but not normal tissues. We previously developed a telomerase-specific oncolytic adenovirus (Telomelysin) whose replication is controlled by the human telomerase reverse tran¬scriptase (hTERT) promoter, and the clinical safety and pharmacokinetics of Telomelysin has been already evaluated in the phase I clinical trial conducted in the US. While oncolytic adenoviruses can produce promising treatment efficacy, their application is basically limited to locoregional administration because of nonspecific trapping in the liver and neutralization by immune response after systemic delivery. Nano-sized materials have been identified as effective and attractive drug carriers in the fields of cancer treatment and diagnosis. Liposome is one of the most popular and widely-used drug carriers. In this study, we investigated the possibility of systemic delivery of telomerase-specific oncolytic adenoviral plasmid DNA via lipid encapsulation as an alternative approach to overcome the shortcomings of oncolytic adenovirotherapy. Methods: TelomeScan, a modified Telomelysin expressing the green fluorescence protein (GFP), is used in this study. Cytotoxic activity of TelomeScan plasmid DNAs encapsulated by liposomes (Lipo-TelomeScan-pDNA) against various types of human cancer cell lines including HCT 116, a human colorectal carcinoma cell line, and MKN1, a human gastric carcinoma cell line, were evaluated by XTT assay and compared with liposome complexes of GFP plasmid DNA controlled by the cytomegalovirus (CMV) promoter (Lipo-GFP-pDNA) as a control agent. Results: Freeze-thaw procedure proved that viable oncolytic adenoviruses were successfully produced in H1299, a human lung cancer cell line, after Lipo-TelomeScan-pDNA transfection. Lipo-TelomeScan-pDNA showed cytotoxic activity on various types of cancer cell lines. Lipo-TelomeScan-pDNA killed HCT116 and MKN1 cells in a dose-dependent manner and produced more effective cytotoxic activity than Lipo-GFP-pDNA. Conclusions: Lipo-TelomeScan-pDNA proved potent cytotoxic effects on cancer cells by producing viable oncolytic adenoviruses. In vivo examination is needed for the next step. Citation Format: Katsuyuki Aoyama, Shinji Kuroda, Hiroshi Tazawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. Development of systemically-deliverable telomerase-specific oncolytic adenovirus. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 719. doi:10.1158/1538-7445.AM2014-719

Silencing of Yap Gene Inhibits Metastasis in a Severe Combined Immune Deficiency Mice Orthotopic Implantation Gastric Cancer Model

YAP signaling has been proved to be involved in cell growth, invasion and metastasis in multiple malignancies. But little has been reported about the effects of YAP transduction on gastric cancer metastases. The present study was aimed to explore the effects of shRNA-mediated targeting of YAP gene on human gastric cancer metastases in severe combined immune deficiency (SCID) mice. Human gastric carcinoma cell line SGC7901 was implanted into the stomach of SCID mice, which were randomly divided into 3 groups (n=10). Via intraperitoneal injection, mice received YAP-shRNA, negative control, or normal saline respectively. The results of TUNEL and FQ-PCR showed an obvious induction of apoptosis in the YAP-shRNA group. Intratumoral vascular density and lymphatic density were suppressed in the YAP-shRNA group. In conclusion, silencing of YAP gene effectively induces the cell apoptosis and inhibits microvascular angiogenesis and lymphagiogenesis of gastric cancer cells in vivo, suggesting that YAP may serve as a promising therapeutic target for treatment of cancer.

묵은지 스타터 Lactobacillus curvatus ML17의 생균제적 특성 및 기능성 조사

This study investigated the probiotic and functional characteristics of Mukeunji starter, Lactobacillus curvatus ML17, isolated from Mukeunji. Lb. curvatus ML17 was confirmed as a safe microorganism due to its non-hemolytic activity and non-production of harmful β-glucuronidase and β-glucosidase. Tolerance to artificial gastric and bile juice of Lb. curvatus ML17 was investigated. After incubation in artificial gastric and bile juice, the number of surviving cells was 1.38×10<SUP>8</SUP> CFU/mL. According to the results of adhesion assay, this strain also exhibited good adhesion to Caco-2 cells. Lb. curvatus ML17 showed good antimicrobial activity against food borne pathogens, especially Micrococcus luteus, Bacillus cereus, Salmonella enterica subsp. enterica, and Pseudomonas aeruginosa. Cell-free extract of Lb. curvatus ML17 exhibited high levels of DPPH scavenging capacity and inhibitory effects on growth of AGS human gastric adenocarcinoma cells and HT-29 human colon carcinoma cells. These results suggest that Lb.curvatus ML17 has potential for application in functional foods.

Cudarflavone B Provides Neuroprotection against Glutamate-Induced Mouse Hippocampal HT22 Cell Damage through the Nrf2 and PI3K/Akt Signaling Pathways

Oxidative cell damage contributes to neuronal degeneration in many central nervous system (CNS) diseases such as Alzheimer’s disease, Parkinson’s disease, and ischemia. Nrf2 signaling-mediated heme oxygenase (HO)-1 expression acts against oxidants that are thought to play a key role in the pathogenesis of neuronal diseases. Cudraflavone B is a prenylated flavone isolated from C. tricuspidata which has shown anti-proliferative activity, mouse brain monoamine oxidase (MAO) inhibitory effects, apoptotic actions in human gastric carcinoma cells and mouse melanoma cells, and hepatoprotective activity. In this study, cudraflavone B showed neuroprotective effects and reactive oxygen species (ROS) inhibition against glutamate-induced neurotoxicity by inducing the expression of HO-1 in mouse hippocampal HT22 cells. Furthermore, cudraflavone B caused the nuclear accumulation of nuclear factor-E2-related factor 2 (Nrf2) and increased the promoter activity of antioxidant response elements (ARE) in mouse hippocampal HT22 cells. In addition, we found that the Nrf2-midiated HO-1 expression by cudraflavone B is involved in the cell protective response and ROS reductions, and cudraflavone B-induced expression of HO-1 was mediated through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in HT22 cells. Our results demonstrated the potential application of naturally occurring cudraflavone B as a therapeutic agent from neurodegenerative disease.

Synergistic inhibitory effect of berberine and d-limonene on human gastric carcinoma cell line MGC803.

This study aims at evaluating the anticancer effects of berberine hydrochloride (berberine) and d-limonene, alone and in combination, on human gastric carcinoma cell line MGC803 to determine whether berberine and d-limonene work synergistically and elucidate their mechanisms. MGC803 cells were treated with berberine and d-limonene, alone and in combination, for 24-48 h. The inhibitory effects of these drugs on growth were determined by MTT assay. The combination index and drug reduction index were calculated with the Chou-Talalay method based on the median-effect principle. Flow cytometry and laser scanning confocal microscopy were employed to evaluate the effects of both drugs on cell-cycle perturbation and apoptosis, generation of reactive oxygen species (ROS), mitochondrial membrane potential, and expression of Bcl-2 and caspase-3 in MGC803 cells. Berberine or d-limonene alone can inhibit the growth of MGC803 cells in a dose- and time-dependent manner. Berberine and d-limonene at a combination ratio of 1:4 exhibited a synergistic effect on anti-MGC803 cells. The two drugs distinctly induced intracellular ROS generation, reduced the mitochondrial transmembrane potential (ΔΨm), enhanced the expression of caspase-3, and decreased the expression of Bcl-2. The combination of berberine and d-limonene showed more remarkable effects compared with drugs used singly in MGC803 cells. The combination of berberine and d-limonene exerted synergistic anticancer effects on MGC803 cells by cell-cycle arrest, ROS production, and apoptosis induction through the mitochondria-mediated intrinsic pathway.