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Biomedical subjects

Fang Tian

Publications and source records attributed to Fang Tian.

36 records · Page 2Linked to original sources

PE, a new sulfated saponin from sea cucumber, exhibits anti-angiogenic and anti-tumor activities in vitro and in vivo.

Here, we examined the in vitro and in vivo anti-angiogenesis and anti-tumor activities of PE, a new marine-derived compound. Inhibition of angiogenesis was assessed in vitro using proliferation, migration, adhesion, tube-formation and apoptosis assays in PE-treated HMECs and HUVECs. In vivo, CAM assays were used to assess inhibition effect of PE on physiological angiogenesis, and immunofluorescent microscopy was used to examine tumor microvessel density and apoptosis in PE-treated mouse tumor models. Finally, Western blotting analyses were performed to examine the effect of PE on VEGF signaling in HMECs. The results showed that PE inhibited proliferation of HMECs and HUVECs with IC50 values of 2.22 +/- 0.31 microM and 1.98 +/- 0.32 microM, induced endothelial cell apoptosis at concentrations <2 microM, induced dose-dependent suppression of cell migration, cell adhesion and tube formation in HMECs and HUVECs, and showed anti-proliferative activities against several tumor cell lines (IC50 values of approximately 4 microM). In vivo, PE (5 nM/egg) suppressed spontaneous angiogenesis in our CAM assay, and induced marked growth inhibition in mouse sarcoma 180 and hepatoma 22 models. Specifically, PE treatment reduced mouse sarcoma 180 tumor volume by triggering apoptosis of both tumor and tumor-associated endothelial cells, preferentially targeting on endothelial cells comparable with tumor cells. Finally, PE treatment suppressed the active (phosphorylated) forms of VEGFR2, Akt, ERK, FAK and paxillin, which are involved in endothelial cell survival, proliferation, adhesion and migration. Our results indicate that PE exerts an anti-angiogenic activity associated with inhibition of VEGFR2 signaling, and an anti-tumor activity associated with decreased proliferation of tumor cells and increased apoptosis of both endothelial cells and tumor cells.

Angiogenesis Inhibitors↗

Improving P-gp expression in human mononuclear cells in vitro transfected by multidrug resistance-1 mRNA.

OBJECTIVE: To evaluate the expression and function activity of P-glycoprotein (P-gp) in human mononuclear cells (MNCs) in vitro transfected by multidrug resistance-1 (MDR1) mRNA. METHODS: Two MDR1 cDNA vectors, pT7TS_MDR1 and pGEM5Zf(+)_MDR1, were constructed and transcripted in vitro. Vector pGEM5Zf(+)_MDR1 only contained the coding region of mdr1 cDNA, and pT7TS_MDR1 also included Xeponus beta-globin 5' and 3' untranslated region. MNCs were prepared from peripheral blood of parvicellular lung cancer patient. The two human mdrl mRNAs were then transferred into human MNCs in vitro by DOTAP. And the expression efficiency and pump function of P-gp were measured with flow cytometry. RESULTS: Expression of P-gp significantly elevated in both transferred cells compared with untransferred cells (P < 0.01). And pT7TS_MDR1 showed higher capability in elevating the expression of P-gp than pGEM5Zf(+)_MDR1 (P < 0.01). The P-gp function was elevated in both pT7TS_MDR1 and pGEM5Zf(+) MDR1 groups. The survival ratio of MNCs in erythrocyte-lysis-solution (ELS, 86.07%) and lymphocyte-isolation-solution (LIS, 83.67%) had no significant difference. The CD34+ cells content of the MNCs used for transfection was 2.65% and 1.01% in ELS and LIS group, respectively (P < 0.01). CONCLUSIONS: It is a feasible approach to improve P-gp expression in human MNCs by transfection of MDR-1 mRNA. And the ELS may be more suitable for purifing MNCs for mRNA transfection than LIS.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Smad-binding defective mutant of transforming growth factor beta type I receptor enhances tumorigenesis but suppresses metastasis of breast cancer cell lines.

The role of transforming growth factor beta (TGF-beta) in carcinogenesis is complex, with tumor suppressor and pro-oncogenic activities depending on the particular tumor cell and its stage in malignant progression. We previously have demonstrated in breast cancer cell lines that Smad2/3 signaling played a dominant role in mediating tumor suppressor effects on well-differentiated breast cancer cell lines grown as xenografts and prometastatic effects on a more invasive, metastatic cell line. Our present data based on selective interference with activation of endogenous Smad2 and Smad3 by stable expression of a mutant form of the TGF-beta type I receptor (RImL45) unable to bind Smad2/3 but with a functional kinase again show that reduction in Smad2/3 signaling by expression of RImL45 enhanced the malignancy of xenografted tumors of the well-differentiated MCF10A-derived tumor cell line MCF10CA1h, resulting in formation of larger tumors with a higher proliferative index and more malignant histologic features. In contrast, expression of RImL45 in the more aggressive MCF10CA1a cell line strongly suppressed formation of lung metastases following tail vein injection. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in these cells. Using an in vitro assay, we further show that non-Smad signaling pathways, including p38 and c-Jun NH(2)-terminal kinase, cooperate with TGF-beta/Smads in enhancing migration of metastatic MCF10CA1a cells, but that, although necessary for migration, these other pathways are not sufficient for metastasis.

Animals↗

Glucose biosensor based on the microcantilever.

Diagnosis and management of diabetes require quantitative and selective detection of blood glucose levels. We report a technique for micromechanical detection of biologically relevant glucose concentrations by immobilization of glucose oxidase (GOx) onto a microcantilever surface. Microfabricated cantilevers have traditionally found utility in atomic force microscope imaging. During the past decade, however, microcantilevers have been increasingly used as transducers in chemical-sensing systems. This paper describes the combination of this technology with enzyme specificity to construct a highly selective glucose biosensor. The enzyme-functionalized microcantilever undergoes bending due to a change in surface stress induced by the reaction between glucose in solution and the GOx immobilized on the cantilever surface. Experiments were carried out under flow conditions. The common interferences for glucose detection in other detection schemes have been tested and have shown to have no effect on the measurement of blood glucose level by this technique.

Biosensing Techniques↗

Recombinant mouse canstatin inhibits chicken embryo chorioallantoic membrane angiogenesis and endothelial cell proliferation.

Human canstatin, a 24 kD fragment of the alpha2 chain of type IV collagen, has been proved to be one of the most effective inhibitors of angiogenesis and tumor growth. To investigate in vivo antiangiogenesis activity and in vitro effects on endothelial cell proliferation of recombinant mouse canstatin, the cDNA of mouse canstatin was introduced into an expression vector pQE40 to construct a prokaryotic expression vector pQE-mCan. The recombinant mouse canstatin efficiently expressed in E. coli M15 after IPTG induction was monitored by SDS-PAGE and by Western blotting with an anti-hexahistidine tag antibody. The expressed mouse canstatin, mainly as inclusion bodies, accounted for approximately 35% of the total bacterial proteins. The inclusion bodies were washed, lysed and purified by the nickel affinity chromatography to a purity of approximately 93%. The refolded mouse canstatin was tested on the chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. In addition, recombinant mouse canstatin potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrate that the recombinant mouse canstatin effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells.

Angiogenesis Inhibitors↗

[Identification of acute leukemia-specific genes from leukemia recipient/sibling donor pairs by distinguishing study with oligonucleotide microarrays].

To explore differentially expressed genes in leukemia gene expression profile and identify main related genes in acute leukemia, gene expression profiles were analyzed in bone marrow/leucopheresis peripheral blood stem cells samples from 9 acute leukemia patients and their sibling donors with the use of oligonucleotide microarrays. 163 reported leukemia-related genes were involved in the study. The oligonucleotide primers were designed, synthesized and spotted on the chemical-material-coated-glass plates in array. The total RNAs were isolated from nine patients' bone marrow or leucopheresis peripheral blood cells and from nine their sibling donors peripheral blood stem cells treated by G-CSF, then collected by CS-3000 cell selection machine, and were reversely transcribed to cDNAs with the incorporations of fluorescent dUTP. The mixed probes were then hybridized to the oligonucleotide microarray. The results showed that in four patient/donor pairs with B-ALL, 5 up-regulated (RIZ, STK-1, T-cell leukemia/lymphoma 1A, Cbp/p300, Op18) and 1 down-regulated genes (hematopoietic proteoglycan core protein) were identified; In five patient/donor pairs with AML-M(4) and AML-M(5), 6 up-regulated (STAT5B, ligand p62 for the Lck SH2, CST3, LTC4S, myeloid leukemia factor 2 and epb72) and 1 down-regulated genes (CCR5) were identified. In conclusion, on the basis of distinguishing study of specific genetic related recipient/sibling donor pairs, screening leukemia-related genes with oligonucleotide microarrays, a set of 13 up-regulated or down-regulated genes among 163 leukemia-related genes has been identified. The result has further confirmed that above genes play critical role in the molecular mechanism of acute leukemia.

Blood Donors↗

[Molecular cloning and functional analysis of STGC3 - a novel gene on chromosome 3p21].

BACKGROUND & OBJECTIVE: A locus of loss of heterozygosity (LOH) with high frequency has been found on chromosome 3p21 in nasopharyngeal carcinoma (NPC). On the basis of our former research, this study was designed to clone and analyze a novel NPC-associated gene at this locus. METHODS: The full-length cDNA sequence of this gene was obtained by plasmid cDNA sequencing and RACE,and analyzed by bioinformatics. The pEGFP-C2/STGC3 fusion mammalian expression vector was constructed, and transfected into COS7 and CNE2 cell lines mediated by lipofectin to analyze the subcellular localization of gene expressing proteins. The expression of STGC3 was detected in normal tissues and tumor cell lines by Northern blot. RESULTS: An 1 271 bp full-length cDNA sequence of gene,which had no obvious homology with other known genes in bioinformatic databases,was obtained. This gene,named STGC3 (GenBank accession number:AY078383), localized on chromosome 3p21, and encodes a protein consisting of 146 amino acids. Protein localization analysis under fluorescent microscope indicated that STGC3 fusion protein was distributed in nucleus and cytoplasm 24-48 hours after transfection. MTE(TM)Array2 Northern blot analysis showed STGC3 expressed in both normal tissues and tumor cells,while its expression down-regulated in many tumor cell lines, such as Burkitt's lymphoma cell line Daudi. CONCLUSIONS: STGC3 is a novel gene, and down-regulated in NPC and many other tumor cell lines. STGC3 fusion protein distributes in cytoplasm and nucleus.

Amino Acid Sequence↗

Reduction in Smad2/3 signaling enhances tumorigenesis but suppresses metastasis of breast cancer cell lines.

The role of transforming growth factor beta in breast cancer is controversial with tumor suppressor and pro-oncogenic activities having been demonstrated. To address whether the same or different signal transduction pathways mediate these opposing activities, we manipulated the Smad2/3 signaling pathway in cells of common origin but differing degrees of malignancy derived from MCF10A human breast cells. We show that interference with endogenous Smad2/3 signaling enhances the malignancy of xenografted tumors of premalignant and well-differentiated tumor cells but strongly suppresses lung metastases of more aggressive carcinoma cells after tail vein injection. Overexpression of Smad3 in the same cells has opposite effects. The data demonstrate that the Smad2/3 signaling pathway mediates tumor suppressor and prometastatic signals, depending on the cellular context.

Animals↗

Human ADAM33: protein maturation and localization.

ADAM33 (a disintegrin and metalloprotease) was recently found to be a novel asthma susceptibility gene. Domain-specific antibodies were used to study its expression and processing. When the pro-domain and catalytic domain were expressed by a stable-transfected cell line, the pro-domain was removed by cleavage within a putative furin cleavage site. The catalytic domain was active in an alpha(2)-macroglobulin complex formation assay and mutation of the catalytic site glutamic acid (E346A) eliminated activity. In transient transfections using the full-length protein, a pro-form and mature form were detectable and alternate glycosylation was demonstrated at sites within the catalytic domain. ADAM33 was detected on the cell surface, with the majority of protein detected intracellularly. The E346A mutation had no significant effect on protein processing. Endogenous ADAM33 was detected in bronchus tissue, bronchial smooth muscle cells, and MRC-5 fibroblasts, consistent with a role in the pathophysiology of asthma.

ADAM Proteins↗

[Identification of down-regulated expressed sequence tag at chromosome 3p21 in nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: It was reported that a locus of high frequency loss of heterozygosity was found at 3p14-25 of nasopharyngeal carcinoma (NPC) cells. This study was designed to identify the expressed sequence tags (ESTs) associated with NPC at chromosome 3p21 in order to clone new candidate NPC-associated genes at the locus. METHODS: The expression of relative ESTs at 3p21 was determined in nasopharyngeal carcinoma tissues and normal nasopharyngeal epithelia using ESTs homology analysis with bioinformatics in computer network combined with reverse transcription-polymerase chain reaction (RT-PCR). The expression of EST was detected in other normal tissues and tumor cell lines using Northern blot analysis. RESULTS: Comparing with expression of normal nasopharyngeal epithelia, EST (N31985) was down-regulated in 60.00% (3/5) nasopharyngeal carcinoma cell lines and 47.06% (16/34) nasopharyngeal carcinoma tissues (P < 0.05). CONCLUSION: N31985 is down-regulated in NPC, suggesting it may play a role in NPC carcinogenesis.

Carcinoma, Squamous Cell↗

Aberrant expression and deletion of FHIT gene in leukemias.

FHIT (fragile histidine triad) gene at chromosome 3p14.2 usually expresses at a very low level in human tissue and cells. A high frequency of abnormalities in FHIT gene has been demonstrated in various cancers. FHIT is proposed as a putative tumor-suppressor gene. To evaluate the expression of the FHIT gene in various leukemias, bone marrow or peripheral blood samples from 98 leukemia patients were tested by RT-PCR: 38 from patients with AML-[M(2)(9), M(3)(12), M(4)(8), M(5)(9)], 16 with ALL, and 34 with CML-[CP(20), AP(4), BC(10)] of various FAB types, as well as 10 patients with other hematological malignancies. To detect a deletion in sequencing the FHIT gene, the representative aberrant PCR products were cloned and then sequenced. The results showed that 22/38 (58%) patients with AML, 9/16 (56%) patients with ALL and 19/34 (56%) patients with CML were detectable of aberrant FHIT mRNA transcripts or deletion of FHIT. In 6 (16%) AML patients, 3 (19%) ALL patients, and 5 (15%) CML patients, the wild-type product was absent. Some patient's samples - 6 (42%) AML, 6 (38%) ALL, and 14 (41%) CML revealed aberrant FHIT transcripts in addition to a normal-sized band. Samples from healthy donors (PB, n = 12; BM, n = 5) did not indicate any abnormal expression. Eleven isolated fragments from various patterns of FHIT gene expression were investigated using cDNA sequencing. Sequence analysis revealed deletion of exon 4-8, exon 5-8, and exon 5-6 in various leukemias, as well as the deletion of the full FHIT gene sequence. The fused transcripts included: exon 3 and exon 9, exon 3 and exon 7, exon 4 and exon 9, exon 5 and exon 7. Sequence analysis of aberrant fragments present in samples from an AML and a CML patients was detected for point mutations and insert mutations located in exons 2, 8 and 10, plus a variety of aberrant transcripts. Deletion or aberrant FHIT mRNA transcripts in 50/98 (51%) leukemia patients were found. All samples with aberrant FHIT lacked gene product. A Kaplan-Meier plot of survival in patients with AML in relation to FHIT expression revealed that aberrance or loss of FHIT gene significantly correlated with a low clinical remission rate and poor overall survival.

Acid Anhydride Hydrolases↗

Identification of full, partial and inverse CC chemokine receptor 3 agonists using [35S]GTPgammaS binding.

Study of the CC chemokine receptor 3 (CCR3) has been limited to using radiolabeled agonist chemokines. A small molecule CCR3 antagonist, 2-[(6-amino-2-benzothiazolyl)thio]-N-[1-[(3,4-dichlorylphenyl)methyl]-4-piperidinyl]acetamide, Banyu (I), was tritiated and used for pharmacological studies. Banyu (I) has a K(d) of 5.0+/-0.4 and 4.3+/-1.8 nM on human CCR3 transfectants and eosinophils, and noncompetitively inhibits [125I]eotaxin binding and eotaxin-induced [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding. The proportion of [125I]eotaxin: [3H]Banyu (I) binding sites in eosinophils or transfectants was 35% or 13%, although both binding sites were overexpressed in transfectants. CCR3 spontaneously couples to G-proteins in CCR3 transfectants, demonstrated by changes in basal and eotaxin-induced [35S]GTPgammaS binding under reduced NaCl and GDP concentrations. Consequently, Banyu (I) was identified as an inverse agonist. In contrast, CCL18 and I-TAC (interferon-inducible T cell alpha-chemoattractant) were neutral antagonists, inhibiting eotaxin-induced [35S]GTPgammaS binding, with minimal effect on basal coupling of CCR3 to G proteins. Eotaxin, eotaxin-2 and monocyte chemoattractant protein (MCP)-4 are full agonists inducing [35S]GTPgammaS binding; eotaxin-3, MCP-3, RANTES (regulated on activation normal T cell expressed and secreted), vMIP-I (Kaposi's sarcoma-associated herpesvirus macrophage inflammatory protein-) and vMIP-II are partial agonists, indicating that this is a sensitive method to quantitate agonist efficacy.

Animals↗

[Transfection of MDR1-mRNA into human mononuclear cells to improve their resistance to anticancer agents, an in vitro study].

OBJECTIVE: To investigate the expression and function of P-glycoprotein (P-gp), an efflux pump encoded by multidrug resistance complementary DNA (MDR1), in human mononuclear cells (MNCs) transfected with MDR1 mRNA. METHOD: Two kinds of human MDR1 mRNA, pT7TS-MDR1 and pGEM5Zf (+)-MDR1 with difference in the 5' and 3' untranslated regions only, were constructed and modified and then were transfected into human MNCs rich in hematopoietic progenitor cells from a patient with small cell lung cancer. The expression efficiency and pump function of P-gp were measured with FACS. Rhodomine 123 efflux test was used to examine the function of P-gp. Un- transfected mononuclear cells from the same person were used as controls. RESULT: The expression of P-gp 12 hours after transfection was 2.16 % in control group, 8.94% in pGEM5Zf (+)-MDR1 group, and 19.14% in pT7TS-MDR1 group. The expression of P-gp 72 hours after transfection was 2.12% in control group, 6.12% in pGEM5Zf(+)-MDR1 group, and 10.71% in pT7TS-MDR1 group, significantly higher in the third group. Rhodamine-123 efflux test showed that the efflux-pump function of P-gp 12 hours after the trransfection was 19.20% in control group, 25.59% in GEM5Zf (+)- MDR1 group, and 35.02% in pT7TS-MDR1 group (all P < 0.01). The expression and presence of P-gp by transfected cells lasted at least 72 hours. CONCLUSION: Transfection of human mononuclear cells with MDR1 mRNA significantly increases their resistance to anticancer agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

[Adhesion induces matrix metalloproteinase-9 gene expression in ovarian cancer cells].

OBJECTIVE: This work was conducted to investigate the expression of matrix metalloproteinase 9 (MMP 9) gene in cancer cells by fibronectin adhesion and the underlying mechanism of cell invasion. METHODS: Following adhesion of ovarian cancer cells A2780 to fibronectin, mRNA expression of MMP cells were assayed by reverse transcription-polymerase chain reaction (RT-PCR). MMP9 promoter was cloned from genomic DNA of HT1080 cells with PCR. The MMP-9-pGL2 reporter gene vector was constructed and then transiently transfected into A2780 cells. RESULTS: Adhesion induced the increase of cellular MMP9 mRNA content in A2780 cells, not affecting the expression of MMP2 or TIMP-1 gene. The stimulation was enhanced with the increase adhesion time. When the transfected cells were allowed to adhere and spread on FN-coated surface, the promoter activity of MMP9 gene was also enhanced dramatically. CONCLUSION: Cell-ECM adhesion may stimulate the expression of MMP9 gene through stimulating the promoter activity, thereby enhancing cancer cell invasion and metastasis.

Cell Adhesion↗

Isolation and Identification of cDNA Sequences Differentially Expressed in Laryngeal Carcinoma.

The isolation of the genes related to laryngeal carcinoma(LC) is necessary for revealing mechanisms of carcinogenesis and genetic predisposition to LC. The mRNA differential display method was used to compare and analyze mRNAs prepared from two adult laryngeal carcinoma tissues and paired tumor-adjacent normal tissues. A total of twenty-two differential display experiments was performed and thirty-five cDNA fragments differentially expressed in normal or malignant laryngeal epithelial tissues were identified. Differential expression of six of these thirty-five cDNA fragmens was confirmed by reverse northern dot blot. Subsequent cloning of six differentially expressed cDNA fragments and sequencing and BLASTn analysis resulted in the identification of twelve distinct cDNA sequences. Four of these were shown to be novel gene sequences that have not been reported. Eight of the remaining cDNA sequences showed sequence homology to those previously reported. The differential expression of these twelve cDNA sequences in the carcinoma or normal tissue of the larynx were confirmed by fixing the twelve cDNA sequences on the membrane, followed by the hybridization with the total cDNA probes from laryngeal carcinoma or normal tumor-adjacent tissues and by differential RT-PCR. These results suggest that these cDNA sequences might be involved in carcinogenesis of laryngeal carcinoma.

Journal Article↗

The tumor suppressor gene RIZ in cancer gene therapy (review).

In recent years, significant progress has been made in understanding the nature of the human genome and the role that genes and their related proteins play in both normal and diseased cells. It has been proven that many cancers are caused by the mutation of certain genes or lack of gene function. The introduction of those genes into cancer cells where gene function is compromised, can work to restore gene function and stop tumor progression. There have been numerous clinical trials, which have shown that gene therapy products are efficacious in humans. The RIZ1 gene is a member of a superfamily of histone/protein methyltransferases. The gene is commonly inactivated in human cancers. Gene knock-out study has established RIZ1 as a tumor susceptibility gene in mice. The gene has potent tumor suppressive activities in causing apoptosis, G2/M arrest, or both. Preclinical animal studies have shown that a recombinant adenovirus expressing the gene, AdRIZ1, can suppress the growth of colon cancer xenografts. Therefore, AdRIZ1 shows promise as a new generation of gene therapy products to enter the clinic.

DNA-Binding Proteins↗