Human Fibroblast Cell Line MRC-5 Research Articles

Copper(II) complexes with different diamines as inhibitors of bacterial quorum sensing activity

Three copper(II) complexes, trans-[Cu(1,3-pd)2Cl2]?H2O (Cu1; 1,3-pd is 1,3-propanediamine), trans-[Cu(2,2-diMe-1,3-pd)2Cl2] (Cu2; 2,2-diMe- -1,3-pd is 2,2-dimethyl-1,3-propanediamine) and trans-[Cu(1,3-pnd)2Cl2]?H2O (Cu3; 1,3-pnd is (?)-1,3-pentanediamine), were synthesized and structurally characterized by elemental microanalyses, IR, electronic absorption and reflectance spectroscopy and molar conductivity measurements. The antimicrobial efficiency of the complexes against four clinically relevant microorganisms and their antiproliferative effect on the normal human lung fibroblast cell line MRC-5 were evaluated. Since in many bacteria, pathogenicity is regulated by an intercellular communication process called quorum sensing (QS), the effect of the copper(II) complexes Cu1?3 on bacterial QS was examined. The obtained results showed that these complexes inhibited violacein production in Chromobacterium violaceum CV026, indicating their anti-QS activity via the homoserine lactone (HSL) pathway. Two biosensor strains were used to determine which pathway, C4-HSL (N-butanoylhomoserine lactone) or 3OC12-HSL (N-(3-oxododecanoyl)homoserine lactone), was affected by the copper(II) complexes. The biological activities of the copper(II) complexes were compared with those for the nickel(II) complexes of the general formula trans- -[Ni(L)2(H2O)2]Cl2 (L = 1,3-pd, 2,2-diMe-1,3-pd and 1,3-pnd).

In vitro antimicrobial activity and cytotoxicity of nickel(II) complexes with different diamine ligands

Three diamines, 1,3-propanediamine (1,3-pd), 2,2-dimethyl-1,3-propanediamine (2,2-diMe-1,3-pd) and (?)-1,3-pentanediamine (1,3-pnd), were used for the synthesis of nickel(II) complexes 1?3, respectively, of the general formula [Ni(L)2(H2O)2]Cl2. The stoichiometries of the complexes were confirmed by elemental microanalysis, and their structures were elucidated by spectroscopic (UV?Vis and IR) and molar conductivity measurements. The complexes 1?3, along with NiCl2?6H2O and the diamine ligands, were evaluated against a panel of microbial strains that are associated with skin, wound, urinary tract and nosocomial infections. The obtained results revealed no significant activity of 1?3 against the investigated bacterial strains. On the other hand, they showed good antifungal activity against pathogenic Candida strains, with minimum inhibitory concentration (MIC) values in the range from 15.6 to 62.5 ?g mL-1. The best anti-Candida activity was observed for complex 2 against C. parapsilosis, while the least susceptible to the effect of the complexes was C. krusei. The antiproliferative effect on normal human lung fibroblast cell line MRC-5 was also evaluated in order to determine the therapeutic potential of nickel(II) complexes 1?3. These complexes showed lower negative effects on the viability of the MRC-5 cell line than the clinically used nystatin and comparable selectivity indexes to that of this antifungal drug.

The potential of aqueous extracts of Bellucia dichotoma Cogn. (Melastomataceae) to inhibit the biological activities of Bothrops atrox venom: A comparison of specimens collected in the states of Pará and Amazonas, Brazil

Ethnopharmacological importanceThe effectiveness of aqueous extract of Bellucia dichotoma Cogn. (Melastomataceae) specimems collected in Santarém, PA, against some biological activities of Bothrops atrox venom (BaV) has been scientifically proven. Here, we analyzed the components and assessed the anti-snakebite potential of aqueous extracts of bark of B. dichotoma collected in Manaus, AM, (AEBd-MAO) and Santarém, PA, (AEBd-STM), both in Brazil. Materials and methodsThe phytochemical profiles of the aqueous extracts were identified using thin layer chromatography (TLC), and the concentrations of phenolics were determined by colorimetric assay. The inhibitory potential of the extracts was tested against the phospholipase A2, coagulant and gelatinolytic activities of BaV in vitro and its defibrinating and edema-inducing activities in vivo. Interaction between BaV and the extracts was investigated using SDS-Page electrophoresis and Western blotting. Extract cytotoxicity and antioxidant potential were assessed using the human fibroblast cell line MRC-5 and the DPPH assay in cell culture, respectively. ResultsWhile there was no difference between the phytochemical profiles of the extracts, AEBd-MAO had higher concentrations of total phenolics, total tannins and hydrolysable tannins. The extracts inhibited 100% of the phospholipase and coagulant activity of BaV when pre-incubated. Without pre-incubation, however, there was no reduction in phospholipase activity, although significant inhibition of coagulant activity was observed. In the doses used in folk medicine, without pre-incubation, both extracts inhibited 100% of the coagulant activity of BaV. In vivo, the extracts were unable to inhibit the defibrinating activity of the venom but were effective in inhibiting its edema-inducing activity. In the profiles of the extracts pre-incubated with BaV, not all the protein bands revealed by SDS-PAGE and Western blot were observed. Both extracts had a high antioxidant potential and neither had a cytotoxic effect. ConclusionAlthough the concentrations of phenolics in each extract were different, the anti-snakebite potential was similar for the concentrations of extract tested. Our findings are of importance for the quality control of this raw material, which, once tested in accordance with Brazilian National Health Surveillance Agency recommendations, may be suitable for use as a phytomedicine to complement treatment of the local effects induced by Bothrops venoms.

Rheum emodin inhibits enterovirus 71 viral replication and affects the host cell cycle environment.

Human enterovirus 71 (EV71) is the primary causative agent of recent large-scale outbreaks of hand, foot, and mouth disease (HFMD) in Asia. Currently, there are no drugs available for the prevention and treatment of HFMD. In this study, we compared the anti-EV71 activities of three natural compounds, rheum emodin, artemisinin and astragaloside extracted from Chinese herbs Chinese rhubarb, Artemisia carvifolia and Astragalus, respectively, which have been traditionally used for the treatment and prevention of epidemic diseases. Human lung fibroblast cell line MRC5 was mock-infected or infected with EV71, and treated with drugs. The cytotoxicity of the drugs was detected with MTT assay. The cytopathic effects such as cell death and condensed nuclei were morphologically observed. The VP1-coding sequence required for EV71 genome replication was assayed with qRT-PCR. Viral protein expression was analyzed with Western blotting. Viral TCID50 was determined to evaluate EV71 virulence. Flow cytometry analysis of propidium iodide staining was performed to analyze the cell cycle distribution of MRC5 cells. Rheum emodin (29.6 μmol/L) effectively protected MRC5 cells from EV71-induced cytopathic effects, which resulted from the inhibiting viral replication: rheum emodin treatment decreased viral genomic levels by 5.34-fold, viral protein expression by less than 30-fold and EV71 virulence by 0.33107-fold. The fact that inhibition of rheum emodin on viral virulence was much stronger than its effects on genomic levels and viral protein expression suggested that rheum emodin inhibited viral maturation. Furthermore, rheum emodin treatment markedly diminished cell cycle arrest at S phase in MRC5 cells, which was induced by EV71 infection and favored the viral replication. In contrast, neither astragaloside (50 μmol/L) nor artemisinin (50 μmol/L) showed similar anti-EV71 activities. Among the three natural compounds tested, rheum emodin effectively suppressed EV71 viral replication, thus is a candidate anti-HFMD drug.

Stereocontrolled synthesis of the four 16-hydroxymethyl-19-nortestosterone isomers and their antiproliferative activities

Novel 16-hydroxymethyl-19-nortestosterone diastereomers were prepared by Birch reduction from the corresponding 3-methoxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol isomers with known configurations. The synthesized compounds are 16α- and 16β-hydroxymethyl-substituted 19-nortestosterone and their 17α-epimers. To prepare 17α-19-nortestosterone, the Mitsunobu inversion reaction of 19-nortestosterone with different alkyl and aryl carboxylic acids was chosen. Deacylation of the new compounds by the Zemplén method yielded the required 17α-19-nortestosterone.The antiproliferative activities of the structurally related compounds were determined in vitro through microculture tetrazolium assays on a panel of human adherent cervical (HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231, MDA-MB-361 and T47D) and ovarian (A2780) cell lines. The 17α epimer of 19-nortestosterone demonstrated considerable activity, selectively for HeLa cells, with a calculated IC50 of 0.65μM. The reference compound, cisplatin, displayed an order of magnitude higher IC50 (12.4μM). The cancer selectivity of 17α-19-nortestosterone was tested by MTT assay performed with noncancerous human fibroblast cell line MRC-5. The results indicated that 17α-19-nortestosterone selectively disturbs the viability of HeLa cells without greatly affecting other cancer cell types and intact fibroblasts.

Synergistic effects by combination of ganciclovir and tricin on human cytomegalovirus replication in vitro

It has been demonstrated as the first report that combination treatment with ganciclovir (GCV) and tricin (4′,5,7-trihydroxy-3′,5′ -dimethoxyflavone), a derivative of Sasa albo-marginata, after human cytomegalovirus (HCMV) infection has synergistic effects on both infectious virus production and HCMV DNA synthesis in the human embryonic fibroblast cell line MRC-5. In this paper, we examined the anti-HCMV effects of GCV plus various concentrations of tricin, and tricin plus various concentrations of GCV in MRC-5 cells. We found that expression of the HCMV UL54 gene was significantly inhibited by combination of GCV with tricin when compared with GCV mono-treatment. These results suggest that tricin is a novel compound for combination therapy with GCV against HCMV replication. In addition, reduced-dose combination therapy may provide a direction for treatment in patients with HCMV infection while reducing drug toxicity.

In vitro anticancer activity of gold(III) complexes with some esters of (S,S)-ethylenediamine-N,N'-di-2-propanoic acid.

Five novel gold(III) complexes of general formulas [AuCl2{(S,S)-R2eddip}]PF6, ((S,S)-eddip = (S,S)-ethylenediamine-N,N'-di-2-propanoate, R = n-Bu, n-Pe, i-Bu, i-Am, cPe; 1-5, respectively) were synthesized and characterized by UV/Vis, IR and NMR spectroscopy and mass spectrometry. DFT calculations indicated that (R,R)-N,N'-configuration diastereoisomers were the most stable for 1-5. 3 is stable in DMSO for at least 24 h, but immediate hydrolysis in PBS occurs. 3 is readily reduced with ascorbic acid and forms adducts with bovine serum albumin (BSA). In vitro anticancer activity of the gold(III) complexes against human cervix adenocarcinoma HeLa, human myelogenous leukemia K562, human melanoma Fem-x tumor cell lines, as well as against non-cancerous human embryonic lung fibroblast cell line MRC-5 was determined using MTT assay. Complex 4 showed highest activity and selectivity (IC50(Fem-x) = 1.3 ± 0.2; IC50(MRC-5)/IC50(Fem-x) = 72.5 ± 12.4), 4 times more active and 28 times more selective than cisplatin. Complexes induced apoptotic mode of death in a time-dependent manner in HeLa cells.

Abstract 3369: Differential biotherapeutic advantages of honey in targeting the Warburg effect and survival of MRC-5 and A549 cell lines

Abstract Lung cancer is a one of the most prevalent and deadly cancers in United States. Experimental evidence support that cancer cells do exhibit higher glycolytic rates than normal cells (Warburg effect). To exploit this unique cancer-dependent ATP generation phenomenon, we hypothesize that exposure of cancer cells to organic inhibitors of glycolysis would negatively impact their survival and alter their growth and viability resulting from the vast decrease in their essential glycolytic ATP production; no negative consequences will be seen on normal lung cells. The human lung fibroblast cell line MRC-5 and the human alveolar epithelial cell line A549 were used as models for normal lung and lung cancer in vitro in this study. Using standard methods, both cell lines were maintained and exposed to honey and D-glucose reagents at concentration levels ranging from 31.3-2,000 µg/ml in 96 well plates in quadruplets and experiments repeated at least three times using MTT, and cell counting (T4 Cellometer) assays as well as phase-contrast photo-imaging. Our results indicate that exposure of both cell lines to these natural nutraceutic organics resulted in concentration dependent cell destruction/cell survival depending on the cell line exposed. Honey and D-glucose showed statistically significant (p<0.05) differential negative effects on the A549 line in comparison to its unexposed control as well as to their effects on the MRC-5 cell line. Results show a promising role of honey and D-glucose as metabolites of interest for selective management of cancerous cells. Citation Format: Ibrahim O. Farah, Veshell L. Lewis, Wellington K. Ayensu, Joseph A. Cameron. Differential biotherapeutic advantages of honey in targeting the Warburg effect and survival of MRC-5 and A549 cell lines. [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 3369. doi:10.1158/1538-7445.AM2014-3369

Anti-Inflammatory and Pro-apoptotic Effects of Curcumin and Resveratrol on the Human Lung Fibroblast Cell Line MRC-5

Background: The naturally occuring polyphenols curcumin and resveratrol are considered to be powerful antioxidants and anti-inflammatory compounds and both inhibit the proliferation of different types of cancer cells. In the present study, we investigated possible anti-inflammatory and pro-apoptotic effects of curcumin and resveratrol on the human lung fibroblast cell line MRC-5. Methods: MRC-5 cells were stimulated for 6 h with interleukin (IL)-1β or phorbol 12- myristate 13-acetate (PMA) in the absence or presence of different concentrations of curcumin or resveratrol. The release of interleukin (IL)-6 was quantified by enzyme-linked immunosorbent assay (ELISA). The modulation in phosphorylation of the transcription factor nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) such as p38 and ERK1/2 were analyzed by Western blot. Cytotoxic and pro-apoptotic effects of curcumin and resveratrol were monitored by the measurement of lactate dehydrogenase (LDH) activity and by Annexin-V/7-AAD staining. Results: Both curcumin and resveratrol effectively attenuated IL-1β and PMA-induced IL-6 expression in MRC-5 cells. Furthermore, curcumin treatment induced apoptosis via caspase-3 signaling and caused endoplasmic reticulum (ER) stress. Salubrinal, an inhibitor of serine/threonine phosphatase PP1, and antioxidants such as N-acetyl-cysteine (NAC), reduced glutathione (GSH) and sodium hydrogen sulfide (NaHS) diminished the cytotoxic effects of curcumin on MRC-5 cells. In contrast to curcumin, resveratrol had no negative effects on cell viability

Synthesis and biological evaluation of the pirfenidone derivatives as antifibrotic agents

A total of 24 pirfenidone derivatives were designed, synthesized and evaluated for their inhibitory activity against the human lung fibroblast cell line MRC-5. These compounds showed the remarkable proliferation inhibition against MRC-5 compared to pirfenidone as the positive control. The possible mechanism of this kind of derivatives as antifibrotic agents was explored. The molecular docking and p38 binding affinity assays demonstrated that the antifibrotic potential of the pirfenidone derivatives was possibly through the inhibition of p38 MAPK signaling pathway. The data from this study suggested that p38 might be a potential therapeutic target for the new generation antifibrotics. All the pirfenidone derivatives are reported here for the first time.

Induction of an EMT-like transformation and MET in vitro

BackgroundThe epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) play pivotal roles in metastasis of epithelial cancers. The distinction between them has shed new light on the molecular mechanisms of tumor metastasis. Recently, tumor microenvironment (TM) has been identified as one of the most potent inducers of EMT and MET. TM is characterized by its complexity and flexibility. The purpose of this study was to ascertain the exact effect of each distinct TM component on the evolution hepatocellular carcinoma (HCC) metastasis.MethodsTwo different cell culture models were used. The HCC cell line Bel-7402 was co-cultured with the normal liver cell line HL-7702 or with the retinal vascular endothelial cell line RF/6A in double-layer six-well plates, imitating the direct interaction between tumor-host cells and tumor cells. Bel-7402 was also cultured in the conditioned medium (CM) of the human lung fibroblast cell line MRC-5, HL-7702 or RF/6A, imitating an indirect interaction. Integrin β1, β3, β4, β7, laminin β3, E-cadherin and Snail levels were measured by quantitative RT-PCR in tumor sepecimens from 42 resected HCC.ResultsWe found that Bel-7402 cells co-cultured with HL-7702 or RF/6A cells were induced to undergo MET. The expression of E-cadherin, α-catenin and β-catenin was up-regulated, accompanied with a strengthened E-cadherin/catenin complex on the membrane of co-cultured Bel-7402 cells. Consequently, the invasion and migration ability of cells was declined. Conversely, Bel-7402 cells cultured in conditioned medium from MRC-5 cells underwent an EMT-like transformation as the cells became elongated with increased invasion and migration ability. Furthermore, we demonstrated that HL-7702 cells could generally inhibit the tumorigenicity and viability of Bel-7402 cells. We also found that integrin β1 expression was negatively associated with capsular formation, and that integrin β4 expression was negatively associated with CK19 expression.ConclusionOur findings highlight the strong influences exerted by TM on tumor progression through EMT and MET by impacting the expression of adhesion molecules, including the E-cadherin/catenin complex, laminins and integrins.

Experimental study on A549 cell death mediated by xenoantigen α-gal in human serum

背景与目的人体α-1, 3半乳糖基转移酶（α-1, 3Galactosyltransferase, α-1, 3GT）基因功能失活而不表达α-半乳糖基（α-galactosyl, α-gal）表位，但天然存在着大量抗α-gal抗体，异种器官移植研究结果提示，在人肿瘤细胞上重新表达异种移植抗原α-gal，可能诱发类似于宿主抗移植物超急性排斥反应的抗肿瘤效应。本研究通过基因导入手段，建立稳定表达异种移植抗原α-gal的转基因人肺癌细胞系，探讨α-gal介导的人血清抗肿瘤的可能性及其机制。方法将前期成功构建的α-1, 3GT基因真核表达质粒pEGFP-N1-GT瞬时转染人肺腺癌细胞A549，筛选并建立稳定的转基因细胞系A549-GT。MTT增殖实验和显微镜观察转基因细胞生物学特性变化；RT-PCR检测A549-GT中α-1, 3GT基因mRNA表达；荧光素标记的凝集素（FITC-BS-IB4 lectin）染色检测α-1, 3GT在肿瘤细胞表面合成α-gal的能力；A549-GT细胞及其培养基分别与正常人细胞共培养，检验α-1, 3GT基因的稳定性和酶活性的稳定性；人血清结合实验检测A549-GT与IgM和补体C3结合情况。结果RT-PCR检测到转基因细胞系A549-GT中有α-1, 3GT mRNA表达。荧光显微镜和流式细胞术检测结果显示：A549-GT能够长期稳定表达异种移植抗原α-gal表位；A549-GT与其亲本细胞在生长形态及增殖速度上无明显差异；A549-GT细胞及其培养基分别与正常人胚肺成纤维细胞MRC-5共培养均不能使MRC-5获得α-gal合成能力；经人血清处理后，荧光免疫实验观察到转基因细胞系A549-GT能与血清抗体IgM结合并诱导补体C3结合。结论异种移植抗原α-gal在肿瘤细胞上的重新表达，可能通过补体依赖的细胞毒机制，介导类似于异种器官移植排斥反应的抗肿瘤效应。

Synthesis and antibacterial evaluation of a new series of N-Alkyl-2-alkynyl/(E)-alkenyl-4-(1H)-quinolones.

To gain further insight into the structural requirements of the aliphatic group at position 2 for their antimycobacterial activity, some N-alkyl-4-(1H)-quinolones bearing position 2 alkynyls with various chain length and triple bond positions were prepared and tested for in vitro antibacterial activity against rapidly-growing strains of mycobacteria, the vaccine strain Mycobacterium bovis BCG, and methicillin-resistant Staphylococcus aureus strains, EMRSA-15 and -16. The compounds were also evaluated for inhibition of ATP-dependent MurE ligase of Mycobacterium tuberculosis. The lowest MIC value of 0.5 mg/L (1.2–1.5 µM) was found against M. fortuitum and M. smegmatis. These compounds displayed no or only weak toxicity to the human lung fibroblast cell line MRC-5 at 100 µM concentration. The quinolone derivatives exhibited pronounced activity against the epidemic MRSA strains (EMRSA-15 and -16) with MIC values of 2–128 mg/L (5.3–364.7 µM), and M. bovis BCG with an MIC value of 25 mg/L (66.0–77.4 µM). In addition, the compounds inhibited the MurE ligase of M. tuberculosis with moderate to weak activity showing IC50 values of 200–774 µM. The increased selectivity towards mycobacterial bacilli with reference to MRC-5 cells observed for 2-alkynyl quinolones compared to their corresponding 2-alkenyl analogues serves to highlight the mycobacterial specific effect of the triple bond. Exploration of a terminal bromine atom at the side chain of N-alkyl-2-(E)-alkenyl-4-(1H)-quinolones showed improved antimycobacterial activity whereas a cyclopropyl residue at N-1 was suggested to be detrimental to antibacterial activity.

Anti-cytomegalovirus effects of tricin are dependent on CXCL11

It has been reported that treatment with tricin (4′,5,7-trihydroxy-3′,5′-dimethoxyflavone), a derivative of Sasa albo-marginata, after human cytomegalovirus (HCMV) infection significantly suppressed both infectious virus production and HCMV replication in the human embryonic fibroblast cell line MRC-5. In this paper, we examined the mechanisms for the anti-HCMV effects of tricin in MRC-5 cells. Exposure of fibroblasts to tricin inhibited infectious HCMV production, with concomitant decreases in levels of transcripts of the CXC chemokine IFN-inducible T cell alpha chemoattractant (I-TAC or CXCL11) gene. We also found that the transcripts of the HCMV immediate early (IE) gene and replication of HCMV were lower in CXCL11 gene-knockdown cells. These results suggest that tricin is a novel compound with potential anti-HCMV activity and that CXCL11 is one of the chemokines involved in HCMV replication. In addition, it is possible that CXCL11 is the one of the targets of tricin.

Expression of multiple tumor suppressor gene p16 and its relationship with prognosis of gastric cancer patients

To investigate the relationship between p16 expression and cell proliferation and prognosis in gastric cancer patients. Gastric cancer cell lines SGC-7901, MKN45, MKN28, human embryonic kidney cell line HEK293, human fibroblast cell line MRC-5, and surgical specimens of gastric carcinoma and adjacent normal gastric mucosa from 65 patients were included in this study. RT-PCR, MTT and FCM assays were used to detect p16 expression in gastric cancer cell lines and surgical specimens of gastric cancer. MTT assay was used to determine cancer cell viability and FCM to detect cell cycle. Kaplan-Meier survival curve and Log-Rank statistics were used to analyze the relationship between p16 expression and survival of petients with gastric cancer. Gastric cancer cell lines were mostly negative for p16 expression, and p16 was re-expressed after the cells transfected with p16 gene by adenovirus AdCMV-p16. p16 re-expression resulted in the decrease of cancer cell viability and cancer cell cycle arrest with increased G(1) phase and decreased S phase. p16 expression in cancer specimens was 32.3% (21/65), significantly lower than the 81.5% (53/65) in normal mucosa (χ(2) = 32.124, P < 0.001). The disease-free survival was significantly shorter in p16-negative patients than that in p16-positive patients (P < 0.01), but not the overall survival (P > 0.05). p16 expression was significantly correlated with differentiation and lymph node metastasis, but not significantly correlated with sex, age, tumor size or invasion depth of the gastric cancer. p16 gene is important for cancer cell proliferation. The inactivation gives cancer cells a high activity for proliferation and metastasis, and then influences the disease-free survival of gastric cancer patients.

Resistin and insulin/insulin-like growth factor signaling in rheumatoid arthritis

Human resistin has proinflammatory properties that activate NF-κB-dependent pathways, whereas its murine counterpart is associated with insulin resistance. The aim of this study was to examine potential cross-talk between resistin and insulin/insulin-like growth factor (IGF) signaling in rheumatoid arthritis (RA). Levels of IGF-1, IGF binding protein 3, and resistin were measured in the blood and synovial fluid of 60 patients with RA and 39 healthy control subjects. Human RA synovium was implanted subcutaneously into SCID mice, and the mice were treated with resistin-targeting small interfering RNA. Primary synovial fibroblasts from patients with RA, as well as those from patients with osteoarthritis, and the human fibroblast cell line MRC-5 were stimulated with resistin. Changes in the IGF-1 receptor (IGF-1R) signaling pathway were evaluated using histologic analysis, immunohistochemistry, and reverse transcription-polymerase chain reaction. Resistin and IGF-1R showed different expression profiles in RA synovia. Low levels of IGF-1 in RA synovial fluid were associated with systemic inflammation and inversely related to the levels of resistin. Stimulation of synovial fibroblasts with resistin induced phosphorylation of IGF-1R to a degree similar to that with insulin, and also induced phosphorylation of transcription factor Akt. This was followed by gene expression of GLUT1, IRS1, GSK3B, and the Akt inhibitors PTPN and PTEN. Abrogation of resistin expression in vivo reduced the expression of IGF-1R, the phosphorylation of Akt, and the expression of PTPN and PTEN messenger RNA in RA synovium implanted into SCID mice. Resistin utilizes the IGF-1R pathway in RA synovia. Abrogation of resistin synthesis in the RA synovium in vivo leads to reductions in the expression of IGF-1R and level of phosphorylation of Akt.

Inhibitory effects of tricin derivative from Sasa albo-marginata on replication of human cytomegalovirus

The anti-human cytomegalovirus (HCMV) activity of tricin (4′,5,7-trihydroxy-3′,5′-dimethoxyflavone), a derivative from Sasa albo-marginata, was studied in the human embryonic fibroblast cell line MRC-5. In a plaque assay, tricin and ganciclovir (GCV) showed concentration-dependent inhibitory properties from 0.05 to 3.6 μM and 0.01 to 1.0 μM, respectively. Tricin had no virucidal effects on cell-free HCMV. Treatment with tricin 1 h before, or 1 h or 3 h after viral infection significantly suppressed HCMV replication. Moreover, tricin inhibited the expression of immediate early (IE) 2 mRNA and DNA polymerase (UL54) mRNA in HCMV-infected cells. Western blot analysis also demonstrated that tricin decreased the expression of IE antigen (especially IE2) and cyclooxygenase 2 (COX-2) expression in HCMV-infected cells. In the presence of tricin, prostaglandin E2 (PGE 2) accumulation by HCMV infection was completely inhibited. These results suggest that tricin is a novel compound with potential COX inhibitor-dependent anti-HCMV activity.

Cytotoxic Furanogermacranolides from the Flowers ofHelianthus angustifolius

The dichloromethane extract of the flowers of Helianthus angustifolius L. (Asteraceae, Heliantheae) was investigated in vitro for its cytotoxic activity using human cancer cell lines: CCRF-CEM leukemia, MDA-MB-231 breast cancer, U251 glioblastoma, HCT 116 colon cancer cells, and the human lung fibroblast cell line MRC-5. Cytotoxicity-guided fractionation led to the isolation of four related heliangolide-type sesquiterpene lactones. The structures were elucidated by means of NMR spectroscopy and high-resolution mass spectrometry. Of the investigated compounds, 8-methacrylyl-4,15-iso-atriplicolide (1) showed the highest activity against all tested cancer cell lines with IC₅₀ values ranging from 0.26 ± 0.01 µM for CCRF-CEM cells to 4.22 ± 0.26 µM for MRC-5 cells.

Prognostic significance of mortalin expresion in gastric adenocarcinoma with wild-type p53.

e14662 Background: Mortalin is a heat non-inducible member of the heat shock protein 70 family that is expressed in various stress responses such as glucose deprivation, low-dose ionizing radiation, and caloric restriction. Mortalin and p53 form a complex in the cytoplasm or in the centrosome, which compromises the p53 activity, and thus prevents apoptosis under stress conditions. Methods: This study included 182 unselected Japanese patients with primary gastric cancer. The expression of mortalin and aberrant p53 was investigated by immunohistochemical staining. In addition, the human fibroblast cell line MRC-5 and the gastric cancer cell line MKN-74, which has a mutation in the TP53 gene, were grown under normal or hypoxic conditions, and changes in localization of mortalin and p53 were examined. Results: Mortalin-positive and aberrant-p53-positive samples were found in 75.2% and 62.6% of cases, respectively. The Mortalin and aberrant p53 expression was associated with tumor invasion and lymph node and l...

Analysis of the Biological Response of Endothelial and Fibroblast Cells Cultured on Synthetic Scaffolds with Various Hydrophilic/Hydrophobic Ratios: Influence of Fibronectin Adsorption and Conformation

In this study we developed polymer scaffolds intended as anchorage rings for cornea prostheses among other applications, and examined their cell compatibility. In particular, a series of interconnected porous polymer scaffolds with pore sizes from 80 to 110 microns were manufactured varying the ratio of hydrophobic to hydrophilic monomeric units along the polymer chains. Further, the effects of fibronectin precoating, a physiological adhesion molecule, were tested. The interactions between the normal human fibroblast cell line MRC-5 and primary human umbilical vein endothelial cells (HUVECs) with the scaffold surfaces were evaluated. Adhesion and growth of the cells was examined by confocal laser scanning microscopy. Whereas MRC-5 fibroblasts showed adhesion and spreading to the scaffolds without any precoating, HUVECs required a fibronectin precoating for adhesion and spreading. Although both cell types attached and spread on scaffold surfaces with a content of up to a 20% hydrophilic monomers, cell adhesion, spreading, and proliferation increased with increasing hydrophobicity of the substrate. This effect is likely due to better adsorption of serum proteins to hydrophobic substrates, which then facilitate cell adhesion. In fact, atomic force microscopy measurements of fibronectin on surfaces representative of our scaffolds revealed that the amount of fibronectin adsorption correlated directly with the hydrophobicity of the surface. Besides cell adhesion we also examined the inflammatory state of HUVECs in contact with the scaffolds. Typical patterns of platelet/endothelial cell adhesion molecule-1 expression were observed at intercellular boarders. HUVECs adhering on the scaffolds retained their proinflammatory response potential as shown by E-selectin mRNA expression after stimulation with lipopolyssacharide (LPS). The proinflammatory activation occurred in most of the cells, thus confirming the presence of a functionally intact endothelium. Little or no expression of the proinflammatory activation markers in the absence of LPS stimulation was observed for HUVECs growing on scaffolds with up to a 20% of hydrophilic component, whereas activation of these markers was observed after stimulation. In conclusion, scaffolds containing up to 20% hydrophilic monomers exhibited excellent cell compatibility toward human fibroblast cell line MRC-5 and human endothelial cells. Atomic force microscopy confirmed that adsorbed serum proteins such as fibronectin probably accounted for the positive correlation of HUVEC adhesion and surface hydrophobicity.