[Objective] The aim of the present study was to investigate the role of membrane estrogen receptor (mER) mediated pathway in the proliferation and apoptosis of endothelial progenitor cells (EPCs). [Methods] Bone marrow (BM)-derived EPCs were cultured. The cells were divided into different groups, plus or not plus estrogen receptor blocker (ICI 182,780), PI3K inhibitors (LY294002), and NOS inhibitor (L-NAME) to show the effect of E_2-BSA on EPCs. The proliferation of EPCs was determined by MTT and nitric oxide (NO) release was measured by chromatometry. Apoptotic cell death was determined using the Hochest 33258 staining. The expression of phosphorylated eNOS (p-eNOS) were detected by Western blot. [Results] E_2-BSA could increase EPCs proliferation, and this effect was inhibited by estrogen receptor blocker ICI 182,780, thus indicated that mER-initiated membrane signaling pathways were involved in the action of estrogen on EPCs. E_2-BSA increased nitric oxide production and inhibited apoptosis induced by serum withdrawal, and this effect also inhibited by PI3K inhibitor (LY294002), NOS inhibitor (L-NAME)and estrogen receptor blocker(ICI 182,780), thus indicated that PI3K/Akt/NO pathway was involved the effect of estrogen on EPCs apoptosis. Moreover, E_2-BSA treatment increased phosphorylation of eNOS (p-eNOS). PI3K inhibitors (LY294002) also blocked these effects. [Conclusions] The results of present study suggested that mER mediated EPCs proliferation and apoptosis were related to the PI3K/Akt/eNOS pathway.

To investigate the anticancer effects of ring C in 18β-glycyrrhetinic acid (GA), a series of GA derivatives featured with 9(11)-ene moiety in ring C were designed and synthesized. The structures were confirmed by IR, LC-MS and 1H NMR. Their inhibitory effects towards human prostate cancer PC-3 and leukemia HL-60 cell lines were determined. Most of the derivatives displayed stronger antiproliferative activities than GA. Particularly, compound 14 showed promising anticancer activity with the GI50 values of 4.48 µmol · L(-1) and 1.2 µmol · L(-1) against PC-3 and HL-60 cells respectively, which is worth further study.
Antineoplastic Agents ; chemistry ; pharmacology ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; drug effects ; Drug Design ; Glycyrrhetinic Acid ; analogs & derivatives ; chemistry ; HL-60 Cells ; drug effects ; Humans ; Male ; Prostatic Neoplasms ; pathology

AIMTo isolate and determine the structures of chemical constituents from the seeds of Descurainia sophia (L.) Webb ex Prantl.

METHODSThe chemical constituents were extracted from the seeds of Descurainia sophia (L.) Webb ex Prantl with 75% ethanol and purified by polyamide, silica gel, RP-C18 and Sephadex LH-20 on column chromatography. Chemical methods and spectroscopic methods, such as 1H and 13CNMR, HSQC, HMBC and TOCSY spectra were used for the structural identification.

RESULTSFifteen compounds were obtained. Twelve of them were identified as quercetin-3-O-beta-D-glucopyranosyl-7-O-beta-gentiobioside (I), kaempferol-3-O-beta-D-glucopyranosyl-7-O-beta-gentiobioside (II), isorhamnetin-3-O-beta-D-glucopyranosyl-7-O-beta-gentiobioside (III), quercetin-7-O-beta-gentiobioside (IV), kaempferol-7-O-beta-gentiobioside (V), isorhamnetin-7-O-beta-gentiobioside (VI), quercetin-3,7-di-O-beta-D-glucopyranoside (VII), kaempferol-3, 7-di-O-beta-D-glucopyranoside (VIII), isorhamnetin-3, 7-di-O-beta-D-glucopyranoside (IX), kaempferol-3-O-beta-D-glucopyranosyl-7-O-[(2-O-trans-sinnapoyl)-beta-D- glucopyranosyl(1-->6)]-beta-D-glucopyranoside) (X), sinapic acid ethyl ester (XI) and 3, 4, 5-trimethoxyl-cinnamic acid (XII).

CONCLUSIONCompounds X and VI are new compounds. IV, V, VII, VIII and IX were isolated from Cruciferae family for the first time. I, II, III were obtained from Descurania genus and XI, XII from D. sophia for the first time.

Brassicaceae ; chemistry ; Flavonols ; chemistry ; isolation & purification ; Glucosides ; chemistry ; isolation & purification ; Molecular Structure ; Plants, Medicinal ; chemistry ; Seeds ; chemistry

Bacille Calmette-Guerin (BCG) induces potent Th1 responses with the help of interleukin (IL)-10 and IL-12 released from dendritic cells (DCs), and suppresses Th2- associated allergic reactions. However, there are still some controversies on therapeutic effects of BCG in asthmatics. This study investigated whether BCG administration to DCs suppresses IL-5 production from T cells in atopic asthmatics. DCs derived from peripheral blood of subjects were cultured with or without BCG and Dermatophagoides farinae extract. Some DCs were co-cultured with T cells in the presence of BCG or the above culture supernatants. In the atopic asthmatics, BCG significantly increased IL-10 and IL-12 production from DCs. In the presence of D. farinae extract, BCG further increased IL-10 production. BCG-induced IL-10 production was significantly higher in the atopics (n=14) than in the non-atopics (n=9). Both BCG and the BCG-treated DCs culture supernatant significantly increased IFN-gamma production from T cells. Both BCG and the supernatant from DCs+BCG+D. farinae co-cultures significantly decreased IL-5 production (all p<0.05), but the supernatant from DCs+BCG co-cultures did not. In conclusion, administration of BCG together with D. farinae extract effectively decreased IL-5 production from T cells, probably through the action of IL-10 and IL-12 released from DCs in D. farinaesensitive asthmatics.
Adult ; Asthma/*immunology ; BCG Vaccine/*immunology ; Dendritic Cells/*immunology ; Female ; Humans ; Interferon-gamma/biosynthesis ; Interleukin-10/biosynthesis ; Interleukin-12/biosynthesis ; Interleukin-5/*biosynthesis ; Lymphocyte Activation ; Male ; T-Lymphocytes/*immunology

ObjectiveTo evaluate the neurobiological characteristics of human histio-amniotic mesenchymal (hAMCs) and effect of hAMCs transplantation into the brain to treat Parkinson's disease(PD) modle mice.MethodsThe expressions of mesenchymal stem cells, neural stem cells, dopaminergic neurons and markers related to neurogenesis such as Vimentin, STRO-1, nestin, CD133, β-tubulin, TH, DAT, Ngn2 and mash-1 in hAMCs were evaluated through immunocytochemical stain; and the mRNA transcriptions of neural stem cell markers, Vimentin and nestin in hAMCs were detected by RT-PCR. The PD model was induced by MPTP(i.p.) in C57BL/6 mice transplanted with hAMCs into the right striatum. The therapeutical effect of hAMCs on PD mice was evaluated by spontaneous movement, rotating bar test and the immunohistochemistry of anti-human chondrosome and TH antibodies in striatum.ResultshAMCs induced by nerve cells culture medium, expressed mesenchymal stem cells, neural stem cells, dopaminergic neurons and other specific markers related to neurogenesis mentioned above. The frequency of spontaneous movement in PD mice was significantly increased(P<0-05), and the time of rotating bar was obviously prolonged(P<0-05) after transplantation with hAMCs.ConclusionhAMCs possess the characteristics of nerve cells after cultured in vitro and can significantly recover the damage of motor function induced by MPTP after transplantation into striatum in PD model mice.

OBJECTIVETo investigate the effect of blood-activating Chinese medicinal compounds and water-draining Chinese medicinal compounds on tumor necrosis factor alpha (TNF-α) and nuclear factor kappaB (NF-κ B) expressions in rats with intracerebral hemorrhage (ICH) at the acute stage, and to monitor their therapeutic effect and mechanism of action on inflammation and cerebral edema.

METHODSA rat model of cerebral hemorrhage was achieved by injecting autologous arterial blood into the caudate nucleus. A total of 168 rats were randomly divided into 4 groups: blood-activating medicine group (n=42), water-draining medicine group (n=42), sham operated group (n=42), and the model group (n=42). A series of brain samples were obtained at days 1, 3 and 5 after ICH from rats in all groups. Protein expression levels of TNF-α and NF-κ B were measured by immunohistochemical staining and gene expression levels of TNF-α and NF-κ B were measured by real-time fluorescent PCR.

RESULTSCompared to the sham operated group, protein expression levels of TNF-α and NF-κ B in the model group significantly increased (P<0.01). Protein and gene expressions of TNF-α from the blood-activating medicine group and water-draining medicine group significantly decreased when compared to those in the model group P<0.05). Meanwhile, compared to the model group, the expression of NF-κ B in the blood-activating medicine group significantly decreased (P<0.05), while expression of NF-κ B in the water-draining medicine group did not differ (P>0.05).

CONCLUSIONSBlood-activating Chinese medicinal compounds and water-draining Chinese medicinal compounds can alleviate inflammation of peripheral tissue and cerebral edema. However, the blood-activating Chinese medicinal compounds were more effective than the water-draining Chinese medicinal compounds. The possible effective mechanism may be by means of inhibiting the activation of NF-κ B so as to suppress the transcription of target genes including gene expression of TNF-α.

Animals ; Base Sequence ; Blood ; Body Water ; DNA Primers ; Intracranial Hemorrhages ; metabolism ; Male ; Medicine, Chinese Traditional ; NF-kappa B ; metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the effects of tumor metastasis-related gene TMSG-1 overexpression on the proliferation and invasion of a highly metastatic prostate cancer cell line in vitro.

METHODSThe eukaryotic expression plasmids containing full-length TMSG-1 cDNAs were stably transfected into the highly metastatic prostate cancer cell line PC-3M-1E8. Clones highly expressing TMSG-1 were identified by RT-PCR and Western Blot analysis after G418 screening. The cell proliferation was detected by cell growth curve, MTT assay and soft agar colony formation assay. The invasive potential of tumor cells in vitro was tested by Matrigel invasion assay.

RESULTSThree TMSG-1 overexpression clones were selected. Cell growth curve and MTT assay showed that TMSG-1 overexpression clones exhibited a strong inhibition of proliferation compared with that of the parental cells or those transfected with vector alone from the third day of culture (P <0.05). In vitro analysis also showed that the TMSG-1 transfected clones exhibited a decreased clonogenicity in soft agar compared with that of the parental cells or those transfected with vector only (P < 0.05). TMSG-1 expression significantly suppressed cell invasion in vitro of TMSG-1-transfected PC-3M-IE8 cells (P < 0.05).

CONCLUSIONThe TMSG-1 protein may serve as a tumor metastasis suppressor due to inhibiting cell proliferation and invasion of the highly metastatic prostate cancer cell line PC-3M-1E8.

Animals ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; Mice ; NIH 3T3 Cells ; Neoplasm Invasiveness ; Prostatic Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Sphingosine N-Acyltransferase ; Transfection ; Tumor Suppressor Proteins ; genetics ; metabolism

OBJECTIVETo identify the putative specific localization signal sequence of tumor metastasis suppressor gene-1 (TMSG-1) and to explore the mechanism of subcellular localization of TMSG-1 protein.

METHODSVectors expressing green fluorescence protein (GFP) tagged different TMSG-1 fragments were generated and transfected into human embryo kidney 293 (HEK293) cells. The expression of those fusion proteins was detected by Western blotting and their subcellular localizations were observed by laser confocal microscope.

RESULTSGFP was fused with the native TMSG-1(aa1-380) or different fragments including T1 (aa1-70), T2 (aa1-128), T3 (aa129-380), T4 (aa71-128), T5 (aa71-179) and T6 (aa71-380). Anti-GFP Western blotting showed that these fusion proteins were successfully expressed. Under laser confocal microscope, GFP fused with fragment T4 (aa71-128) localized mainly in the nucleolus; GFP fused with fragment T6 (aa71-380) localized diffusely in the nucleus; while other fusion proteins with TMSG-1 (aa1-380) or fragment T1 (aa1-70), T2 (aa1-128), T3 (aa129-380) and T5 (aa71-179) localized in the cytoplasm. Fragment T4(Δ119-128) was generated from T4 with deletion of 10 amino acid of the C terminal. GFP fused with fragment T4(Δ119-128) remained in the nucleus, but no longer in the nucleolus.

CONCLUSIONSThere is a nucleolar localization signal (aa119-128 RRRRNQDRPS) within TMSG-1. This finding may have laid the foundation for further investigations into subcellular localization and function of TMSG-1.

Amino Acid Sequence ; Blotting, Western ; Cell Nucleolus ; metabolism ; Cell Nucleus ; metabolism ; Cytoplasm ; metabolism ; Green Fluorescent Proteins ; metabolism ; HEK293 Cells ; Humans ; Membrane Proteins ; genetics ; metabolism ; Microscopy, Confocal ; Nuclear Localization Signals ; Plasmids ; Recombinant Fusion Proteins ; metabolism ; Sphingosine N-Acyltransferase ; genetics ; metabolism ; Transfection ; Tumor Suppressor Proteins ; genetics ; metabolism

OBJECTIVETo investigate the regulatory mechanism of the transcription of tumor metastasis suppressor gene TMSG-1.

METHODSLuciferase reporter assay and site-directed mutagenesis were used to analyze the regulatory region of TMSG-1. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were carried out to verify the interaction of KLF6 and Sp1 with the regulatory region of TMSG-1. Co-immunoprecipitation (CoIP) was performed to analyze the interaction between KLF6 and Sp1. TMSG-1 and wt-KLF6 mRNA expressions in cells with different metastatic capacities were quantitated by real-time PCR. Cell invasive capability was determined by Matrigel invasion assay.

RESULTSA 63 bp inducible regulatory region (+59 bp - +123 bp) in exon 1 was identified by luciferase assay using reporter plasmids with a series of TMSG-1 regulatory region deletions. Mutations in KLF6/Sp1 binding sites of this region resulted in a decrease of luciferase activity, while cotransfection with KLF6 or Sp1 expressing plasmids led to a remarkable increase of luciferase activity. EMSA and ChIP demonstrated that KLF6 as well as Sp1 interacted with this region. CoIP also indicated a possible interaction between KLF6 and Sp1 proteins. In the highly metastatic cell sublines, a low level of wild type KLF6 was associated synchronously with a low TMSG-1 level. Prostate carcinoma cells overexpressing KLF6 exhibited a higher TMSG-1 level and a lower invasive capability.

CONCLUSIONSTranscription factor complex of KLF6 and Sp1 may participate in the inducible transcriptional regulation of TMSG-1, and a decreased wild type KLF6 expression is likely associated with a low TMSG-1 level in the highly metastatic cell sublines.

Binding Sites ; genetics ; Cell Line, Tumor ; Electrophoretic Mobility Shift Assay ; Humans ; Immunoprecipitation ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Male ; Membrane Proteins ; genetics ; metabolism ; Mutagenesis, Site-Directed ; Mutation ; Neoplasm Invasiveness ; Prostatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Sp1 Transcription Factor ; genetics ; metabolism ; Sphingosine N-Acyltransferase ; genetics ; metabolism ; Transcriptional Activation ; Transfection ; Tumor Suppressor Proteins ; genetics ; metabolism

Aged ; Cholangiopancreatography, Endoscopic Retrograde ; Fibrosis ; drug therapy ; pathology ; surgery ; therapy ; Gallstones ; complications ; Humans ; Middle Aged ; Somatostatin ; therapeutic use ; Varicose Veins ; drug therapy ; pathology ; surgery ; therapy