[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 study the possibility and mechanism of construction of tissue engineered bone with human bone marrow mesenchymal stem cells (hBMSCs) as seeding cells and partially demineralized bone matrix (pDBM) as scaffold.

METHODShBMSCs are cultured and mutiplified. The 4th grade hBMSCs are seeded on the pDBM, the growth and adhesion of hBMSCs on pDBM are observed under scanning electro microscope. The adhesion efficiency is assessed. The complexes are implanted in the nude mice subcutaneously, the pDBM without cells as control. The grafts are taken out on the 8th and 12th week.

RESULTSThere is new bone formation on the 8th and 12th week in complex group. There is a layer of osteoblast like cells adhered on the surface of most of the new bone, which suggest the possibility of intramembranous ossification. There is no bone formation in control group.

CONCLUSIONSTissue engineered bone can be constructed with hBMScs and pDBM in vivo, and the mechanism of which could be intramembranous ossification.

Animals ; Bone Marrow Cells ; cytology ; Bone and Bones ; Cell Adhesion ; Cell Differentiation ; Humans ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Nude ; Tissue Engineering ; methods

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

OBJECTIVETo better understand the molecular mechanism of tumorigenesis and progression, the monoclonal antibody against G3BP (Ras-GAP SH3 binding protein), which serves as an important downstream effector of Ras signaling, was prepared, characterized and utilized in analysis of various human tumors.

METHODSBy using the prokaryotic expression vector pGEX-5X1, GST-G3BP fusion protein was expressed in E. coli BL21 under induction of IPTG. Purified GST-G3BP fusion protein was used to immunize BALB/c mice. The monoclonal antibody against G3BP was produced through conventional hybridoma method and characterized by ELISA, Western blot and immunohistochemical staining.

RESULTSA hybridoma cell line secreting anti-G3BP IgG1 subtype antibody was obtained. Western blot and competitive inhibition assay showed that the antibody was G3BP-specific. Immunohistochemical staining demonstrated that G3BP was over-expressed in formalin-fixed and paraffin-embedded tissues of some human tumors, such as lung cancer, colon cancer, gastric cancer and breast cancer. In breast cancer specimens, the degree of G3BP expression correlated positively with the presence of lymph node metastasis and c-erbB2 expression.

CONCLUSIONSThe G3BP-specific monoclonal antibody derived from recombination protein can be used in ELISA, Western blot and immunohistochemical assay. It may provide an important tool in analysis of G3BP in in vitro and in vivo experiments. Besides, G3BP may serve as another prognostic marker for breast cancer.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Biomarkers, Tumor ; Breast Neoplasms ; metabolism ; pathology ; Carrier Proteins ; genetics ; immunology ; metabolism ; DNA Helicases ; Female ; Genetic Vectors ; Humans ; Hybridomas ; secretion ; Lymphatic Metastasis ; Male ; Mice ; Mice, Inbred BALB C ; Poly-ADP-Ribose Binding Proteins ; RNA Helicases ; RNA Recognition Motif Proteins ; Receptor, ErbB-2 ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Tumor Cells, Cultured

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 investigate the mechanism of influence of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behavior of human prostatic cancer cell line PC-3M-1E8.

METHODSThe expression of GST-Raf1-RBD recombinant protein, a specific binding domain of active Ras (GTP-Ras), was induced by IPTG in JM109 bacterium. SDS-PAGE and coomassie brilliant blue staining were performed using cleaved product of the bacterium to determine the expression of the fusion protein. GST-pull down essay was designed to detect the level of active Ras in PGA1 and vector transfected, respectively and in the native PC-3M-1E8 cells. Western blotting was used to detect the expression levels of downstream proteins of Ras signal pathway which may be related to the function of PAG1. Phalloidine labeled by tetramethylrhodamine-5-(and-6) isothiocyanate (TRITC) was used for the staining of intracellular F-actin, Laser passing confocal microscopy was adopted for observing change of the cell morphology and the arrangement of F-actin.

RESULTSAfter IPTG induction, the GST-Raf1-RBD recombinant protein, with a molecular weight of 33 000, was noticed to be highly expressed in JM109 bacterium. GST-pull down assay revealed that the expression level of active Ras markedly decreased after PAG1-transfection while the total Ras remained unchanged. The expression of p-ERK and cyclin D1 in the PAG1-transfected cells decreased in accordance with the level of active Ras. However, the expression of p21(WAF1/CIP1) and p-Akt didn't show any variation. Additionally, the structure of F-actin was turbulent and the pseudopodia of cells diminished conspicuously after PAG1-transfection. There was a high expression of PAG1 in normal human prostate tissue, however, the positive rate of PAG1 immuno-staining decreased in cases of prostatic adenocarcinoma, correspondent with increasing of the grading index of cell differentiation established in the Gleason grading system for the diagnosis of prostate adenocarcinoma.

CONCLUSIONSAn over-expression of PAG1 in PC-3M-1E8 cells effectively suppresses the activation of Ras and ERK, as well as the cyclin D1 expression, leading to an inhibition of the proliferation ability of tumor cells. The turbulence of F-actin and reduction of pseudopodia of cells result in an impairment of cell mobility, invasiveness and metastatic capability. In human prostate and prostatic adenocarcinoma tissues, the expression of PAG1 is related to the cellular differentiation and malignancy.

Actins ; metabolism ; Adaptor Proteins, Signal Transducing ; metabolism ; Adenocarcinoma ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Male ; Membrane Proteins ; metabolism ; Neoplasm Invasiveness ; Prostate ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Transfection ; ras Proteins ; metabolism