Effect of genistein on MAPK signal pathway in the collagen-induced arthritis fibroblast-like synoviocytes / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of genistein (Gen) on MAPK signal pathway in the CIA rat fibroblast-like synoviocytes (FLS).</p><p><b>METHODS</b>The rat model of collagen-induced arthritis (CIA) was established. The cultured FLS of CIA rats were divided using randomized method. The effects of Gen (at the concentration of 50, 100, and 200 micromol/L, respectively) on the proliferation of FLS in CIA rats using methyl thiazolyl tetrazolium (MTT) assay. Effects of Gen (at the concentration of 50, 100, and 200 pmol/L, respectively) on the expressions of extracellular signal-regulated kinase (ERK) and phosphorylated extracellular signal-regulated kinase (p-ERK) in the FLS of CIA rats were detected.</p><p><b>RESULTS</b>Gen could inhibit the proliferation of FLS in CIA rats. The FLS proliferation in the high dose Gen group at 72 h was only 1.10+/-0.04, significantly lower than that in the model group (2.12+/-0.03, P<0.01). Besides, after Gen's action on FLS, the expression of p-ERK was down-regulated. It was only 0.34+/-0.02 in the high dose Gen group, significantly lower than that in the model group (2.68+/-0.14, P<0.01). There was no change in the expression of ERK (P>0.05).</p><p><b>CONCLUSIONS</b>Gen could inhibit the proliferation of FLS in CIA rats. Its mechanism of action was mainly correlated to down-regulating the tyrosine kinase of MAPK signal transduction pathway and inhibiting phosphorylation of ERK.</p>

Synergistic antitumor effects of in vivo production of human endostatin and tissue inhibitor of metalloproteinase-1 in mice after subcutaneous implantation of primary fibroblasts transfected by adenovirus-mediated gene delivery / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tissue inhibitor of metalloproteinase (TIMP)-1 is a multifunctional protein. The aim of the study was to examine the feasibility of using a combination of adenovirus-mediated gene delivery of TIMP-1 plus endostatin and cell transplantation techniques to treat tumor growth and metastasis in mouse melanoma.</p><p><b>METHODS</b>A enzyme-linked immunosorbent assay (ELISA) was used to detect the level of TIMP-1 and endostatin in vitro and in vivo. A tumor bearing mouse model and an experimental lung metastasis model in animal experiments were used to explore the therapeutic effect of in vivo production of human TIMP-1 and endostatin after the implantation of primary fibroblasts infected with the indicated adenovirus into tumor-bearing mice and a cytochemical method was used to observe histopathological changes of the tumor. An experimental lung metastasis model was established by injecting B16BL6 cells into the tail vein of mice and adenovirus-infected primary fibroblasts were subcutaneously implanted into the mice 24 hours later. Twenty-one days after tumor cell injection, mice were sacrificed to examine the effect on nodules visible as black forms on the surface of the lungs in B16BL6 cells.</p><p><b>RESULTS</b>TIMP-1 and endostatin were secreted into the supernatants of cultures of Ad-TIMP-1 and Ad-End-infected mouse primary fibroblasts. We also observed that implantation of fibroblasts infected with Ad-TIMP-1 alone, Ad-End alone, or Ad-TIMP-1 plus Ad-End resulted in detectable blood levels which may clearly inhibit the tumor growth and metastasis in a murine melanoma model.</p><p><b>CONCLUSION</b>These results suggest the high capacity of transfection for the delivery of TIMP-1 or endostatin gene constructs into primary fibroblasts, and demonstrate that the implantation of TIMP-1 and endostatin producing fibroblasts at a site in vivo where direct secretion of TIMP-1 and endostatin into the blood is possible represented a promising approach for the development of cancer therapy.</p>

Effect of hypoxia on migration and adhesion of human pulmonary adenocarcinoma A549 cells / 肿瘤

Objective: To evaluate the effect of hypoxia on migration, invasion and adhesion to endothelial cells of human pulmonary adenocarcinoma A549 cells. Methods: Wound-healing and Transwell invasion assays were performed to study the effect of hypoxia on migration and invasion of A549 cells. Cell adhesion test was used to detect the adhesion of A549 cells to a monolayer of human umbilical vein endothelial cells (HUVECs). Immunofluorescence assay was used to evaluate the effect of hypoxia on distribution of E-cadherin, β-catenin, and F-actin. The luciferase reporter gene assay was performed to detect the transcription of hypoxia-inducible factor-1 (HIF-1) alpha. Results: Hypoxia promotes A549 cell migration, invasion, and adhesion to endothelial cells, and modulated the distribution of E-cadherin and β-catenin and rearrangement of actin cytoskeletal protein, and up-regulated HIF-1-dependent reporter gene expression in A549 cells. Conclusion: Hypoxia promoted A549 cell migration, invasion, and adhesion to endothelial cells by upregulating HIF-1-dependent gene expression, subsequently affecting the redistribution of E-cadherin and β-catenin and rearrangement of F-actin cytoskeletal protein.

Inhibitory effect of genistein on hypoxia-inducible factor-1alpha expression induced by cobalt chloride in leukemia cell line K562 / 中国实验血液学杂志

This study was aimed to investigate the effect of genistein (gen) on the expression of hypoxia inducible factor-1alpha (HIF-1alpha) induced by cobalt chloride (CoCl(2)) in human leukemia cell line K562. The hypoxia condition was simulated by CoCl(2); the dose- and time-effect groups were prepared as follows: the former were exposed to 0, 50, 100 and 150 micromol/L of CoCl(2) for 72 hours, the latter were detected at 0, 24, 48 and 72 hours while treated with CoCl(2) 100 micromol/L. The gen-treated samples were divided into five groups: (1) normal control; (2) CoCl(2) 150 micromol/L; (3) CoCl(2) 150 micromol/L + gen 50 micromol/L; (4) CoCl(2) 150 micromol/L + gen 100 micromol/L; (5) CoCl(2) 150 micromol/L + gen 200 micromol/L. The HIF-1alpha mRNA and protein were detected by RT-PCR and Western blot respectively. The results indicated that the expression of HIF-1alpha protein in K562 cells induced by CoCl(2) increased in dose-and time-dependent manner (p<0.01), while the expression of HIF-1alpha mRNA in K562 cell remained the similar level (p>0.05). Gen significantly inhibited the expression of HIF-1alpha protein induced by CoCl(2) in dose-dependent manner (p<0.01) while the HIF-1alpha mRNA expression was not affected by treatment of gen (p>0.05). It is concluded that CoCl(2) dose- and time-dependently induced the HIF-1alpha protein expression; HIF-1alpha mRNA was constantly expressed regardless of normoxic conditions or in the presence of cobalt ion under normoxic conditions. Gen can inhibit HIF-1alpha expression in K562 cell induced by CoCl(2) at level of protein, but not mRNA.

Effect of arsenic trioxide on VEGF/R and MMP-2, 9 expressed in K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of arsenic trioxide (As2 O3) on the level of VEGF, VEGFR and the activity of MMP-2, 9 in K562 cells.</p><p><b>METHODS</b>The inhibition ratio of K562 cell was detected by MTT assay, the level of VEGF by Enzyme-linked immunosorbent assay (ELISA), the expression ratio of VEGFR by flow cytometry (FCM), and the activity of MMP-2, 9 by gelatin zymography assay.</p><p><b>RESULTS</b>(1) The IC50 of K562 cells was (2.12 +/- 0.11) micromol/L. Proliferation of K562 cells was significantly inhibited at the concentration of 0.4 - 6.4 micromol/L As2 O3 (P < 0.05). (2) The expression of VEGF was slightly up-regulated by 0.05 micromol/L As2 O3 (P > 0.05) and prominently inhibited by 0.4 micromol/L and 3.2 micromol/L As2 O3 (P < 0.05). As2 O3 had no influence on VEGFR. (3) The activity of MMP-2 and 9 was partly inhibited by 0.05 micromol/L As2 O3 incubated 72 hours and by 0.4, 3.2 micromol/L, As2 O3. With the increase of As2 O3 concentration and the incubation time, the inhibited effect on MMP-2 and 9 was enhanced.</p><p><b>CONCLUSIONS</b>As2 O3 may down-regulate the expression of VEGF and inhibit the activity of MMP-2 and 9.</p>

RNA interference and its current application in mammals / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>The aim of this review was to assess RNA interference (RNAi) and its possibility as a potential and powerful tool to develop highly specific double-stranded RNA (dsRNA) or small interfering RNA (siRNA) based gene-silencing therapeutics.</p><p><b>DATA SOURCES</b>The data used in this review were obtained from the current RNAi-related research reports.</p><p><b>STUDY SELECTION</b>dsRNA-mediated RNAi has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms. The discovery that synthetic duplexes of 21 nucleotides siRNAs trigger gene-specific silencing in mammalian cells has further expanded the utility of RNAi in to the mammalian system.</p><p><b>DATA EXTRACTION</b>The currently published papers reporting the discovery and mechanism of RNAi phenomena and application of RNAi on gene function in mammalian cells were included.</p><p><b>DATA SYNTHESIS</b>Since the recent development of RNAi technology in the mammalian system, investigators have used RNAi to elucidate gene function, and to develop gene-based therapeutics by delivery exogenous siRNA or siRNA expressing vector. The general and sequence-specific inhibitory effects of RNAi that will be selective, long-term, and systemic to modulate gene targets mentioned in similar reports have caused much concern about its effectiveness in mammals and its eventual use as a therapeutic mordality.</p><p><b>CONCLUSIONS</b>It is certain that the ability of RNAi in mammals to silence specific genes, either when transfected directly as siRNAs or when generated from DNA vectors, will undoubtedly accelerate the study of gene function and might also be used as a potentially useful method to develop highly gene-specific therapeutic methods. It is also expected that RNAi might one day be used to treat human diseases.</p>

The effect of endostatin mediated by adenovirus on the inflammation and cytokines of arthritis rats / 中华风湿病学杂志

Objective To investigate the effect of recombinant adenovirus mediatied human endostatin (rAD-GFP-ES)on rats with collagen typeⅡinduced arthritis(CIA),and explore the mechanism of inflamma- tion and cytokines inhibition on rats CIA.Methods The rAD-GFP-ES was amplified and purified.The model of rat CIA was induced by intradermal injection of typeⅡcollagen combined with complete Freund's adjuvant(CFA). On the second day after the injection,the therapeutic administration of rAD-GFP-ES(1?10~(11)pfu?kg~(-1)?week~(-1)?4 weeks)were performed to the rats.The mean arthritis index(AI)was scored every week since then.The relative concentrations of ES,IL-I?,TNF-?in sera collected at the fourth week were evaluated by western blotting. Results①The titer of the purified rAD-GFP-ES and rAD-GFP was 6.6?10~(12)pfu/ml and 4.8?10~(12)pfu/ml,re- spectively(A_(260nm)/A_(280nm)＞1.3).②The concentration of ES in sera of the group treated with rAD-GFP-ES was 2.4-lold higher compared to the normal group.③The mean arthritis index of the group treated with rAD-GFP- ES was much lower than that of the model group.The administration of rAD-GFP-ES could significantly de- creas the production of IL-1?and TNF-?in sera.Conclusions①The rAD-GFP-ES is efficiently expressed in vivo.②The rAD-GFP-ES has an inhibitory effect on the arthritis index of rat CIA.③IL-1?and TNF-?are involved in the pathogenesis of RA.The rAD-GFP-ES has an inhibitory effect on the expression of IL-1?and TNF-?in rat CIA.