• Title/Summary/Keyword: Cell-based assay

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miR-200a Overexpression in Advanced Ovarian Carcinomas as a Prognostic Indicator

  • Zhu, Cheng-Liang;Gao, Guo-Sheng
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.20
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    • pp.8595-8601
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    • 2014
  • Background: miR-200a expression is frequently altered in numerous cancers. The aim of the present study was to determine the role of microRNA-200a in advanced ovarian carcinomas. Materials and Methods: We measured miR-200a expression in 72 matched normal ovarian tissues and advanced ovarian carcinomas, and also two ovarian carcinoma cell lines (SKOV3 and SKOV3.ip1 - the latter being more invasive and metastatic than the parental SKOV3) by stem-loop real-time RT-PCR based on TaqMan microRNA assay using U6 as a reference. Levels of miR-200a expression were compared by disease stage, tumor grade, histology, and lymph node involvement. To evaluate the role of microRNA-200a, cell proliferation and invasion of SKOV-3 and SKOV-3.ip1 were analyzed with miR-200a inhibitor/mimic transfected cells. Results: Of 72 paired samples, 65 cancer tissues overexpressed microRNA-200a greater than two fold in comparison with matched normal epithelium. Specifically, patients with lymph node metastasis showed significant elevation. The level correlated with clinicopathological features, including high tumor grade, late disease stage, most notably with lymph node metastasis, but not with tumor histology. In addition, SKOV-3.ip1 cells also overexpressed miR-200a compared with SKOV-3, and miR-200a inhibitor transfected SKOV-3.ip1 cells showed significant reduction in cellular proliferation and invasion, while a miR-200a mimic stimulated the opposite behavior. Conclusions: We provide definitive evidence that miR-200a is up-regulated in a significant proportion of advanced ovarian carcinomas, and that elevated miR-200a expression facilitates tumor progression. Our findings support the notion that miR-200a is an onco-microRNA for ovarian cancer, and elevation is a useful potential diagnostic indicator. This study also provides a solid basis for further functional analysis of miR-200a in advanced ovarian cancer.

Inhibitory effect of Scrophulariae Radix extract on $TNF-{\alpha},\;IL-1{\beta}$, IL-6 and Nitric Oxide production in lipopolysaccharide - activated Raw 264.7 cells (현삼메탄올 추출물이 LPS로 유도된 Raw 264.7 cell에서의 $TNF-{\alpha},\;IL-1{\beta}$, IL-6, 및 nitric oxide 생성에 미치는 영향)

  • Byun, Sung-Hui;Yang, Chae-Ha;Kim, Sang-Chan
    • The Korea Journal of Herbology
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    • v.20 no.2
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    • pp.7-16
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    • 2005
  • Objectives : Scrophulariae Radix (SRE) is commonly used in combination with other herbs as a nutrient and health strengthening agent, and to remove 'heat' and replenish vital essence. The water-based extract of this herb can lower blood pressure in both anesthetized and concious animals, and exhibits an anti-inflammatory activity. But, there is lack of studies regarding the effects of SRE on the immunological activities in molecular levels. The present study was conducted to evaluate the effect of SRE on the regulatory mechanism of cytokines and nitric oxide (NO) in Raw 264.7 cells. Method : After the treatment of Scrophulariae Radix methanol extract, cell viability was measured by MTT assay, NO production was monitored by measuring the nitrite content in culture medium. COX-2 and iNOS were determined by Immunoblot analysis, and levels of cytokine were analyzed by sandwich immunoassays. Results : Results provided evidence that SRE inhibited the production of nitrite and nitrate (NO), inducible nitric oxide synthase (iNOS), $interleukin-1{\beta}\;(IL-1{\beta})$ and interleukin-6 (IL-6), and the activation of phospholylation of inhibitor ${\kappa}B{\alpha}\;(p-I{\kappa}B{\alpha})$ in Raw 264.7 cells activated with lipopolysaccharide (LPS). Conclusion : These findings suggest that Scrophulariae Radix can produce anti-inflammatory effect, which may playa role in adjunctive therapy in Gram-negative bacterial infections.

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Cellular-uptake Behavior of Polymer Nanoparticles into Consideration of Biosafety

  • Do, Jeong-Hoe;An, Jeong-Ho;Joun, Yong-Seung;Chung, Dong-June;Kim, Ji-Heung
    • Macromolecular Research
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    • v.16 no.8
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    • pp.695-703
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    • 2008
  • Nanoparticles have tremendous potential in cancer prevention, detection and augmenting existing treatments. They can target tumors, carry imaging capability to document the presence of tumors, sense pathophysiological defects in tumor cells, deliver therapeutic genes or drugs based on the tumor characteristics, respond to external triggers to release an appropriate agent, document the tumor response, and identify the residual tumor cells. Nanoparticles < 30 nanometers in diameter show unexpected and unique properties. Furthermore, particles < 5 nanometers in size can easily penetrate cells as well as living tissues and organs. This study evaluated the safety of nano materials in a living body and the relationship between the living tissue and synthetic nano materials by examining the in-vitro cytotoxicity of poly(lactic-co-glycolic) acid (PLGA) nano-spheres and fluorescein isothiocynate(FITC)-labeled dendrimers as polymer nanoparticles. PLGA was chosen because it has been used extensively for biodegradable nanoparticles on account of its outstanding bio-compatibility and its acceptance as an FDA approved material. The dendrimer was chosen because it can carry a molecule that recognizes cancer cells, a therapeutic agent that can kill those cells, and a molecule that recognizes the signals of cell death. Cytotoxicity in L929 mouse fibroblasts was monitored using MTT assay. Microscopic observations were also carried out to observe cell growth. All assays yielded meaningful results and the PLGA nanoparticles showed less cytotoxicity than the dendrimer. These nano-particles ranged in size from 10 to 100 nm according to microscopy and spectroscopic methods.

Effects of dihydrocubebin, a lignan isolated from Indonesian plant Piper cubeba, on the histamine release from rat mast cells

  • Nugroho, Agung Endro;Wahyono, Wahyono;Wahyuono, Subagus;Maeyama, Kazutaka
    • Advances in Traditional Medicine
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    • v.10 no.3
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    • pp.200-207
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    • 2010
  • The fruits of Piper cubeba L. are used traditionally to treat respiratory disorders in Indonesia. In order to determine the compounds responsible for this activity, the fruits were extracted with nhexane followed by ethanol to give n-hexane and ethanol extracts. Based on tracheospasmolytic assay on these two extracts, the n-hexane extract was more active to inhibit trachea contraction than that of ethanol extract. Upon bioassay guided isolation of the n-hexane extract, a tracheospasmolytic active compound was isolated and identified as dihydrocubebin [(3,4),(3',4')-bis-methylenedioxy-9,9'dihydroxylignan] $(\underline{1})$. Compound $\underline{1}$ was tested further for its ability to inhibit histamine released from mast cells, using rat basophilic leukemia (RBL-2H3) cell line and rat peritoneal mast cells RPMCs) as models; and $DNP_{24}$-BSA, thapsigargin, ionomycin, compound 48/80 and PMA were used as inducers for histamine released from mast cell. The test result showed that $\underline{1}$ inhibited histamine release from RBL-2H3 cells induced by $DNP_{24}$-BSA, thapsigargin and ionomycin. In addition, $\underline{1}$ suppressed histamine release from RPMC induced by either thapsigargin or ionomycin. However, $\underline{1}$ did not inhibit histamine release from RPMC induced by either compound 48/80 or combination PMA-sub optimum dose of ionomycin. Therefore, it was concluded that the inhibitory effects of $\underline{1}$ on the histamine released from mast cells may involve mechanisms related to intracellular $Ca^{2+}$ signaling events or downstream processes of intracellular $Ca^{2+}$ signaling in mast cells.

The Effect of a Community-Based Self-Management Program for Patients at Thyroid Cancer-Diagnosis Stage : a Pilot Study (재가 갑상선암 환자를 위한 지역암센터 자가관리프로그램 중재 효과에 대한 예비연구)

  • Yoo, Hyera;Boo, Sunjoo;Chun, Mison;Jo, Eun Mi
    • Journal of Korean Public Health Nursing
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    • v.29 no.3
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    • pp.582-593
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    • 2015
  • Purpose: This study was conducted to examine the effectiveness of a self-management program on patients with thyroid cancer, particularly during the time of waiting for surgery after cancer diagnosis. Psychological distress, biological responses of immune cell counts, and quality of life were the variables of this study. Methods: One group pre-post test design was used with the nature of a pilot study. Ten newly diagnosed thyroid cancer patients were recruited through physicians' referrals. After drop out of 4 participants, final data were collected from six participants. Small group technique, a one and half hour-session per week for one month (total 4 sessions, 6 hours) was used. Relaxation techniques, meditation training, and strategies to reduce distress were provided by researchers. Standardized questionnaires and an established bio-assay were used for collection of data. Results: Participants showed significant lowering of psychological distress (p<.05) and improvement in global quality of life (p<.05). Biological responses of immune cell counts did not show statistical significance. Conclusion: The self-management program may reduce psychological distress and improve quality of life of patients with thyroid cancer between the time of diagnosis and surgery. The self-management program would be a valuable approach for patients with an unexpected cancer diagnosis to prepare for their disease experience in a community setting.

Experimental Research of ZrO2/BCP/PCL Scaffold with Complex Pore Pattern for Bone Tissue Regeneration (골 조직 재생을 위한 복합 공극 패턴을 가진 ZrO2/BCP/PCL 인공지지체의 실험적 평가)

  • Sa, Min-Woo;Shim, Hae-Ri;Kim, Jong Young
    • Transactions of the Korean Society of Mechanical Engineers A
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    • v.39 no.11
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    • pp.1153-1159
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    • 2015
  • Recently, synthetic biopolymers and bioceramics such as poly (${\varepsilon}$-caprolactone)(PCL), hydroxyapatite, tricalcium phosphate, biphasic calcium phosphate(BCP), and zirconia have been used as substrates to generate various tissues or organs in tissue engineering. Thus, the purpose of this study was the characterization of $ZrO_2$/BCP/PCL(ZBP) scaffold for bone tissue regeneration. Based on the result of single-line test, blended 3D ZBP scaffolds with fully interconnected pores and new complex pore pattern of $45^{\circ}+135^{\circ}$-type and staggered-type were successfully fabricated using a polymer deposition system. Furthermore, the effect of ZBP scaffold on mechanical property was analyzed. In addition, in vitro cell interaction of ZBP scaffold on MG63 cells was evaluated using a cell counting kit-8(CCK-8) assay.

Suppression of the Wnt/${\beta}$-catenin Pathway by Bryostatin-1 (Bryostatin-1에 의한 Wnt/${\beta}$-Catenin 신호전달체계 저해효과)

  • Park, Seoyoung;Oh, Sangtaek
    • Microbiology and Biotechnology Letters
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    • v.42 no.1
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    • pp.89-92
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    • 2014
  • The Wnt/${\beta}$-catenin pathway plays important roles in a variety of biological processes, such as cell proliferation, differentiation, and organ development. Here, we used a cell-based reporter assay to identify bryostatin-1, a natural macrocyclic lactone, as an inhibitor of the Wnt/${\beta}$-catenin pathway. Bryostatin-1 suppressed ${\beta}$-catenin response transcription (CRT), which was activated by a Wnt3a-conditioned medium (Wnt3a-CM), through a decrease in the intracellular ${\beta}$-catenin protein levels, without affecting its mRNA level. In addition, pharmacological inhibition of proteasome abrogated bryostatin-1-mediated down-regulation of the ${\beta}$-catenin protein level. Our findings suggest that bryostatin-1 attenuates the Wnt/${\beta}$-catenin pathway through the promotion of proteasomal degradation of ${\beta}$-catenin.

Anti-Proliferative Effects of β-Cyclodextrin Inclusion Complexes with Coumarinolignans from Acer mono (고로쇠 Coumarinolignan의 β-Cyclodextrin 포접화합물 제조 및 암세포증식 억제활성)

  • Yim, Soon-Ho;Jung, Da-Woon;Williams, Darren R.;Geckeler, Kurt E.;Kim, Kyung Keun;Shin, Boo Ahn;Lee, Ik-Soo;Kim, Huyn Jung
    • Korean Journal of Pharmacognosy
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    • v.46 no.2
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    • pp.133-139
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    • 2015
  • Two coumarinolignans, cleomiscosins C (1) and D (2) were isolated from the heartwood of Acer mono, together with four compounds, 5-O-methyl-(E)-resveratrol-3-O-${\beta}$-D-glucopyranoside (3), 5-O-methyl-(E)-resveratrol-3-O-${\beta}$-D-apiofuranosyl-(1$\rightarrow$6)-${\beta}$-D-glucopyranoside (4), scopoletin (5), and (E)-resveratrol-3-O-${\beta}$-D-glucopyranoside (6). Of them, cleomiscosins C (1) and D (2) were applied to preparing inclusion complex molecules with ${\beta}$-cyclodextrin (${\beta}$-CD) to improve the very poor solubility in cell media. The CD complexes of 1 and 2 exhibited an enhancement of water solubility which is feasible to measure their cytotoxicity using a spectrophotometer in a cell-based assay. Anti-proliferative activity of these complex molecules was successfully estimated on HCT116 human colon cancer cells, and cleomiscosin D (2) showed anti-proliferative effects at the concentration of 1.95~31.2 ${{\mu}g}$/mL in a dose-dependent manner.

Characterization of an Iron- and Manganese-containing Superoxide Dismutase from Methylobacillus Sp. Strain SK1 DSM 8269

  • Seo, Sung Nam;Lee, Jae Ho;Kim, Young Min
    • Molecules and Cells
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    • v.23 no.3
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    • pp.370-378
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    • 2007
  • A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes.

Effect of sweet potato purple acid phosphatase on Pseudomonas aeruginosa flagellin-mediated inflammatory response in A549 cells

  • Heyeon, Baik;Jaiesoon, Cho
    • Animal Bioscience
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    • v.36 no.2
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    • pp.315-321
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    • 2023
  • Objective: The study was conducted to investigate the dephosphorylation of Pseudomonas aeruginosa flagellin (PA FLA) by sweet potato purple acid phosphatase (PAP) and the effect of the enzyme on the flagellin-mediated inflammatory response in the A549 lung epithelial cell line. Methods: The activity of sweet potato PAP on PA FLA was assayed at different pH (4, 5.5, 7, and 7.5) and temperature (25℃, 37℃, and 55℃) conditions. The release of interleukin-8 (IL-8) and the activation of nuclear factor kappa- light-chain-enhancer of activated B cells (NF-κB) in A549 cells exposed to PA FLA treated with or without sweet potato PAP was measured using IL-8 and NF-κB ELISA kits, respectively. The activation of toll-like receptor 5 (TLR5) in TLR5-overexpressing HEK-293 cells exposed to PA FLA treated with or without sweet potato PAP was determined by the secreted alkaline phosphatase-based assay. Results: The dephosphorylation of PA FLA by sweet potato PAP was favorable at pH 4 and 5.5 and highest at 55℃. PA-FLA treated with the enzyme decreased IL-8 release from A549 cells to about 3.5-fold compared to intact PA FLA at 1,000 ng/mL of substrate. Moreover, PA-FLA dephosphorylated by the enzyme repressed the activation of NF-κB in the cells compared to intact PA FLA. The activation of TLR5 by PA-FLA was highest in TLR-overexpressing HEK293 cells at a substrate concentration of 5,000 ng/mL, whereas PA FLA treated with the enzyme strongly repressed the activation of TLR5. Conclusion: Sweet potato PAP has the potential to be a new alternative agent against the increased antibiotic resistance of P. aeruginosa and may be a new conceptual feed additive to control unwanted inflammatory responses caused by bacterial infections in animal husbandry.