[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

To report a case of cutaneous and subcutaneous coinfection caused by Lichtheimia corymbifera and Candida parapsilosis.A 67-year-old female peasant consulted about proliferative granuloma developing on her left forearm after topical application of a Chinese herbal drug and splint fixation for the treatment of suspected fracture of the wrist.Direct microscopic examination showed gram positive budding yeast cells in lesion secretions.Pathological study with periodic acid-Schiff (PAS) and gormori methenamine silver (GMS) staining revealed broad non-separate hyphae in the corneum and dermis.Fungal culture of lesional tissue at 35℃ grew both mould and yeast.The mould was identified as Lichtheimia corymbifera based on morphological findings and sequences of the internal transcribed space (ITS) 1-4 regions.Thermal tolerance study revealed that the isolate grew fast at 37℃ but slowly at 40℃.Under a scanning electron microscope,the acrogenous sporangia were pear-shaped with conical sporangiophores originating from the top of stolon,which were among but not opposite to the rhizoids.The yeast was identified as Candida parapsilosis by Chromagar test and D1/D2 region sequencing.As antimicrobial susceptibility test indicated,the Lichtheimia corymbifera isolate was most sensitive to terbinafine and itraconazole.The proteolytic activity of Lichtheimia corymbifera was higher than that of Candida parapsilosis.The granuloma completely subsided after surgical resection and 6-week treatment with oral itraconazole 200 mg twice a day.No recurrence was observed during a 4-year follow-up.

The interspecific association of companion species in Artemisia annua community in 48 region of southwest China was analyzed by variance analysis, chi2-test and association indices. The total related variance ratio among species in A. annua community was 2.05. Among 45 species pairs of 10 main species in the community, only 4 pairs showed significant negative correlations. Chi2 test, PC, OI, DI and AC values indicated pairs 1-8 (A. annua- A. lactiflora), 1-9 (A. annua- Setaria viridis) and 1-10 (A. annua- Bidens pilosa) showed a high correlations, and common utilization to non-restrictive resources. The results indicated that there was a significant positive correlation among species,and the community was at a stable stage, showed strong ability to human interference.
Analysis of Variance ; Artemisia annua ; China ; Conservation of Natural Resources ; Drugs, Chinese Herbal ; Symbiosis

OBJECTIVETo investigate the effects of tumor metastasis suppressor gene 1 (TMSG-1) overexpression on the proliferation, invasion and apoptosis of breast cancer cells and to determine possible correlations of TMSG-1 and metastasis of breast cancer.

METHODSFull-length human TMSG-1 coding sequences were cloned into plasmid pcDNA3.0-FLAG. The recombinant plasmids constructs were transfeced into MDA-MB-231, a highly malignant breast cancer cell line. Parental, vector-only stable transfectant and TMSG-1 stable transfectant clones were tested by MTT, soft agar colony formation and Boyden chamber assays. At twenty-four hours and forty-eight hours post transient transfection, double staining with Annexin-V-FITC and PI were employed to distinguish apoptotic cells from living cells by flow cytometry analysis.

RESULTSThree TMSG-1 overexpression clones were selected. Compared with the control cells, TMSG-1 overexpression MDA-MB-231 cells showed strong inhibition of proliferation and decreased clonogenicity in soft agar (P<0.05). Transfection of TMSG-1 into MDA-MB-231 cells significantly suppressed the cell invasion ability in vitro (decreased numbers of cells trespassing the matrigel in three experiments: 72.3+/-8.1, 85.0+/-4.2, and 73.5+/-7.8) in comparison with nave cells without transfection (187.5+/-2.1) and cells transfected with the control vector (162.3+/-6.8) (P<0.01). Transient transfection of TMSG-1 into MDA-MB-231 cells could promote cell apoptosis at 24 and 48 hours after transfection (P<0.05).

CONCLUSIONSTMSG-1 protein may have multiple functions in the regulation of proliferation, invasion and apoptosis of metastatic breast cancer cells, likely as a metastasis suppressor gene.

Apoptosis ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Membrane Proteins ; genetics ; metabolism ; physiology ; Neoplasm Invasiveness ; Plasmids ; Recombinant Proteins ; metabolism ; Sphingosine N-Acyltransferase ; genetics ; metabolism ; physiology ; Transfection ; Tumor Suppressor Proteins ; genetics ; metabolism ; physiology

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 investigate the expression of RhoC in breast cancer cells with different metastatic potential and its correlation with invasiveness.

METHODSExpression of RhoC mRNA and protein in human breast cancer cells MCF-7 with low metastatic potential and MDA-MB-231 with high metastatic potential was detected by RT-PCR, Western blot, immunohistochemistry and immunofluorescence staining. Eukaryotic expression plasmids of RhoC were constructed and transfected into MCF-7 cells. The biological effects were observed, including in vitro invasion by Boyden charmber assay, motility by would healing assay, alteration of microfilament network by TRTIC-phalloidin staining and expression of p-Akt by Western blot assay.

RESULTSThe expression levels of RhoC mRNA and protein varied in the two different metastatic breast cancer cell lines. RhoC was significantly up-regulated in the highly metastatic cells in comparison to the weakly metastatic counterpart (P < 0.01). As shown by Boyden charmber assay, the invasive capacity of transfected cells overexpressing RhoC was significantly promoted as reflected by more penetrating cells (56.88 +/- 4.18) than that of the antisense transcripts (23.12 +/- 3.22), the negative (23.77 +/- 3.64) and blank controls (28.44 +/- 2.48). Further study by would healing assay indicated that cells overexpressing RhoC were more motile in actin-based active movement. The wound healing ratio after 24 h of the sense transcripts, antisense transcripts, negative controls and blank controls was 58.28% +/- 2.14%, 22.36% +/- 2.73%, 28.23% +/- 2.62%, 30.18% +/- 2.86%, respectively. The TRITC-phalloidin staining revealed less actin filament bundles and a reorganized cytoskeleton within the sense transcripts. In addition, p-Akt expression level was upregulated in the sense transcripts.

CONCLUSIONRhoC overexpression may promote the invasive capacity of human breast cancer cells in vitro and its expression level is positively correlated with the metastatic capacity of those cells. So RhoC may be a potential target in the development of a novel strategy for treating metastasis of breast cancer.

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Movement ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Neoplasm Invasiveness ; Phosphorylation ; Plasmids ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; metabolism ; Transfection ; Up-Regulation ; rho GTP-Binding Proteins ; genetics ; metabolism ; rhoC GTP-Binding Protein