Biomedical subjects

Ming Fu

Publications and source records attributed to Ming Fu.

33 records · Page 2Linked to original sources

PubMed2004

Overexpression of Aurora-A contributes to malignant development of human esophageal squamous cell carcinoma.

PURPOSE: Aurora-A/STK15/BTAK, a centrosome-associated oncogenic protein, is implicated in the control of mitosis. Overexpression of Aurora-A has been shown to result in chromosomal aberration and genomic instability. Multiple lines of evidence indicate that Aurora-A induces cell malignant transformation. In the current study, we are interested in investigating the expression of Aurora-A in human esophageal squamous cell carcinoma (ESCC) and characterizing the association of Aurora-A with ESCCmalignant progression. EXPERIMENTAL DESIGN: Aurora-A protein expression was examined in 84 ESCC tissues and 81 paired normal adjacent tissues by either immunohistochemistry or Western blot analysis. In addition, a gene-knockdown small interfering RNA technique was used in ESCC cells to investigate whether Aurora-A contributes to the ability of a tumor to grow invasively. RESULTS: The amount of Aurora-A protein in ESCC was considerably higher than that in normal adjacent tissues. Overexpression of Aurora-A was observed in 57 of 84 (67.5%) ESCC samples. In contrast, <2% of normal adjacent tissue displayed high expression of Aurora-A. Interestingly, overexpression of Aurora-A seemed to correlate with the invasive malignancy of ESCC. Disruption of endogenous Aurora-A using small interfering RNA technique substantially suppressed cell migrating ability. CONCLUSION: The findings presented in this report show that Aurora-A expression is elevated in human esophageal squamous cell carcinoma and is possibly associated with tumor invasion, indicating that overexpression of Aurora-A may contribute to ESCC occurrence and progression.

Aurora Kinase A↗

PubMed2004

Sonic hedgehog regulates the proliferation, differentiation, and migration of enteric neural crest cells in gut.

Enteric neural crest cells (NCCs) migrate and colonize the entire gut and proliferate and differentiate into neurons and glia of the enteric nervous system in vertebrate embryos. We have investigated the mitogenic and morphogenic functions of Sonic hedgehog (Shh) on enteric NCCs in cell and organ culture. Enteric NCCs expressed Shh receptor Patched and transcripts encoding the Shh signal transducer (Gli1). Shh promoted the proliferation and inhibited the differentiation of NCCs. The pro-neurogenic effect of glial cell line--derived neurotrophic factor (GDNF) on NCCs was abolished by Shh. In gut explants, NCCs migrated from the explants onto the adjacent substratum if GDNF was added, whereas addition of Shh abolished this migration. Neuronal differentiation and coalescence of neural crest--derived cells into myenteric plexuses in explants was repressed by the addition of Shh. Our data suggest that Shh controls the proliferation and differentiation of NCCs and modulates the responsiveness of NCCs toward GDNF inductions.

Animals↗

PubMed2004

[Mechanisms of the drug resistance of a 2', 2-difluorodeoxycytide (gemcitabine)-resistant variant of the human lung adenocarcinoma cell line].

OBJECTIVE: To investigate the mechanisms of the drug resistance of gemcitabine resistance variant of the human lung adenocarcinoma cell line A549-Gem. METHOD: Immunohistochemistry and RT-PCR were used to tested the expression of P-glycoprotein and transcription of mRNA of multidrug resistance gene and deoxycytidine kinase. Using a cDNA microarray compared the expression profiles between the resistant A549-Gem and the parent cell line A549. RESULTS: The A549-Gem shown slight positive expression of P-glycoprotein compared with positive control by immunohistochemistry, but A549 was negative for P-gp. RT-PCR amplified mRNA, using specific dCK and multidrug resistant gene 1 (mdr1) primers, demonstrated that A549-Gem express amplicon of mdr1 but no products of mdr1 was detected in A549. In the parent cell line, dCK mRNA amplication product could be detected, but did not found in resistant cells. About 18.8% of the total DNA elements had substantially altered level of expression in resistant A549-Gem compared with A549 by cDNA microarray. The biochemical functions of the different expressed genes are diverse and include oncogene and tumor suppressor gene, cell cycle regulator, heat shock protein, apoptotic and antiapoptotic factors, DNA transcription factors, DNA repair and recombination factors, signal transduction genes, protein translation genes, as well as a large number of metabolic genes. Several cluster genes overexpressed in resistant cell line, including ubiquitin-proteasome system, zinc finger protein, glutaredoxin and heat shock protein, may be related to the mechanisms of gemcitabine resistance. CONCLUSION: The mechanisms of resistance to gemcitabine are multifactorial, may include multidrug resistance and dCK deficiency. Differential expression genes monitored by cDNA microarray between A549-Gem and A549 may be related to the mechanisms of gemcitabine resistance in human lung cancer and potentially suggest the prevalence of expression of these genes in drug resistance. The use of cDNA microarray has provided a global view of the response of lung cancer cells to gemcitabine at the genomic level, it should be suitable for examining the development of drug resistance in cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

[Gadd45 mediated G2/M cell cycle arrest induced by BRCA1].

BACKGROUND & OBJECTIVE: It is considered that tumor suppressor gene BRCA1 is an important factor in the regulation of cell cycle checkpoint, but the molecular mechanism by which BRCA1 regulates cell cycle G(2)/M arrest is less known. The objective of this study was to investigate the effects of Gadd45 on the BRCA1-induced cell growth suppression. METHODS: BRCA1 induction of Gadd45 protein was analyzed using Western-blot assay following cells transfection with BRCA1 expression vector and cell sorting. Activation of the Gadd45 promoter by BRCA1 was determined by CAT assay. Effect of antisense Gadd45 on the BRCA1-induced cell cycle G(2)-M arrest was examined by flow cytometry analysis. And the effects of antisense Gadd45 on BRCA1-mediated growth suppression in HeLa and HCT116 cell lines was determined by colony formation assay. RESULTS: Gadd45 protein was highly induced after expression of BRCA1. BRCA1 strongly activated the Gadd45 promoter. Antisense Gadd45 substantially abrogated BRCA1-activated cell cycle G(2)-M arrest and BRCA1-induced cell growth suppression on HeLa and HCT116 cell lines. CONCLUSION: Gadd45 is a BRCA1-regulated downstream gene and mediates the role of BRCA1 in the control of cell cycle G(2)/M arrest and growth suppression.

BRCA1 Protein↗

PubMed2004

[Development and characterization of a gemcitabine-resistant variant of human lung adenocarcinoma cell line A549].

BACKGROUND & OBJECTIVE: Gemcitabine (2',2-difluorodeo- xycytide) has antitumor activity in both experimental and clinical treatment of solid tumors. Although resistance to gemcitabine in ovarian cancer cell line and erythroleukemic cell line was described, there was no report on the lung cancer resistant variant. In order to elucidate the mechanism by which gemcitabine induce resistance in lung cancer, we have established the resistance to gemcitabine in human lung adenocarcinoma cell line A549 and described the characteristics of its resistant variant. METHODS: Resistance to gemcitabine was established by exposing A549 cells to increasing concentration of gemcitabine, which was designated as A549-Gem. The IC(50) and resistance index(RI) were tested by MTT assay and colony formation test. The growth curve and cell cycle of A549 and A549-Gem were compared. The cross-resistance profile of A549-Gem was also tested. RESULTS: The IC(50) increased from 6.56+/-1.19 micromol/L in A549 to 921.09+/-225.27 micromol/L in A549-Gem as tested by MTT assay at 72h exposure, the RI was 140.52 (P=0.019 5). The RI of colony formation test was 132.95. Double time of A549 and A549-Gem were 29.7 h and 36.4 h, respectively, as evaluated by the growth curve. A549-Gem was cross-resistant to vincristine and etoposide(54.38-fold and 6.18-fold)(P< 0.01), but not to adriamycin, cisplatin, cytarabine, and paclitaxel. CONCLUSION: A549-Gem, the gemcitabine resistant phenotype, is stable and suitable for the study of gemcitabine resistance in lung cancer. A549-Gem is cross-resistant to vincristine and etoposide, but not resistant to adriamycin, cisplatin, cytarabine and paclitaxel.

Adenocarcinoma↗

PubMed2003

[Suppression of HCT116 growth of cells by Gadd45].

OBJECTIVE: To study the mechanism of suppression of growth of HCT116 human colon carcinoma cells by Gadd45 gene. METHODS: HCT116 human colon carcinoma cells were transfected with pTRE-Gadd45 vector so as to establish Gadd45-inducible cell line that was cultured in medium with tetracycline. Then tetracycline was withdrawn. The number of cell clones was counted. Flow cytometry was used to detect the percentages of cells at the G1, S, and G2-M phases. TUNEL technique was used to detect the apoptosis of cells. Western blotting was used to analyze the expression of Gadd45 protein, and apoptosis-related proteins: poly-ADP-ribose polymerase (PARP), and caspase 3 protein. RESULTS: Gadd45 protein was not expressed in the HCT116 cells cultured in the medium with tetracycline, however, it was expressed with a gradually increased level in the cells cultured in the medium from which tetracycline was withdrawn, The clone formation rate of HCT116 cells was 100% in the medium with tetracycline, however, was only 14.2% in the medium with tetracycline withdrawal, with a suppression rate of more than 85%. The percentage of cells in G(2)-M phase was significantly increased in the cells cultured in the medium with tetracycline withdrawal. 24-36 hours after the withdrawal of tetracycline, PARP and caspase 3 protein were activated remarkably. CONCLUSION: High expression of Gadd45 inhibits the growth of HCT116 cells, through inducing G2-M arrest and activating apoptotic pathway.

Blotting, Western↗

PubMed2003

HOXB5 expression is spatially and temporarily regulated in human embryonic gut during neural crest cell colonization and differentiation of enteric neuroblasts.

HOX genes from paralogous groups 4 and 5 are particularly relevant to the gut neuromusculature development because these genes are expressed at the splanchnic mesoderm surrounding the gut diverticulum, and at the level of the neural tube from where the vagal neural crest cells (NCCs) originate. In this study, we examined the migration and differentiation of NCCs, and investigated the expression patterns of HOXB5 in human embryonic guts. Human embryos of gestational week-4 to -8.5 were studied. Vagal NCCs enter the esophagus, migrate, and colonize the entire gut in a rostrocaudal manner between week-4 and week-7. The migrating NCCs in gut express HOXB5. Two separate and discontinuous mesenchymal expression domains of HOXB5 were detected in the gut: the distal domain preceding the migratory NCCs; and the proximal domain overlapping with the NCCs. The two expression domains shift caudally in parallel with the rostrocaudal migration of NCCs between week-4 and week-5. Neuron and glia differentiation of NCCs are concomitant with HOXB5 down-regulation in NCCs and the mesenchyme. By week-7, myenteric plexuses have formed; HOXB5 expression is switched on in the plexuses. We found that (1) the migratory route of NCCs in human embryonic gut was similar to that in mice and chicks; and (2) the expression pattern of HOXB5 correlated with the migration and differentiation of NCCs, suggesting a regulatory role of HOXB5 in the development of NCCs.

Cell Differentiation↗

PubMed2003

[Redox reactions of Sep15 and its relationship with tumor development].

BACKGROUND & OBJECTIVE: Sep15 is a selenium-containing protein identified in 1998. This protein may be involved in cancer etiology and it may have redox function. The objective of this study was to investigate the relationship between the redox function of Sep15 and tumor development. METHODS: The full-length DNA sequence of Sep15 was obtained by RT-PCR and then recombined to eukaryotic expression vector pcDNA3.1(+). The BEL-7402- Sep15 cell line, which expressed the high levels of Sep15 by transfecting the cultured hepatocarcinoma cell line BEL-7402 with pcDNA3.1-Sep15 was generated. From morphologic investigation, cell growth curve, clone formation and nude mice tumor growth curve, the relationship between Sep15 and hepatocarcinoma cell line BEL-7402 was determined. Furthermore, the redox reaction of sep15 was detected by MTT assay. RESULTS: There was no distinct effect of transfection of Sep15 gene on BEL-7402-Sep15 cell. The cell survival rate was drastically different between BEL-7402-Sep15 cell and both BEL-7402- pcDNA cell and BEL-7402-Sep15 cell after foreign H2O2 reactive oxygen stress (P<0.05). CONCLUSION: Transfecting Sep15 gene did not influence the growth characteristics of BEL-7402 cell line and Sep15 may have redox function.

Animals↗

PubMed2003

[Antitumor effect of gene gun-mediated DNA vaccine pWRG-neu immunization in C57BL/6 mice].

OBJECTIVE: To investigate the inhibitory effect of DNA vaccine immunization on neu-overexpressed melanoma growth in prophylactic treatment and anti-lung-metastasis experiments in C57BL/6 mice. METHODS: pcDNA-neu transfected into B16F10 with transfection reagent Fugene 6, neu-overexpressed cell clone B16F10-neu was selected with limited dilution method. The growth curve was drawn to analyse its proliferating character in vitro. With Helios gene gun system, DNA vaccine pWRG-neu was immunized to 8-week-old C57BL/6 mice in the shaved abdominal skin for 3 times at two-weekly interval. After immunization, the life span was analyzed. Using MTT assay, the cytolysis activity of the DNA immunized mice spleen cells was compared. RESULTS: One clone of neu-overexpressed B16F10-neu was selected and its proliferating character was the same as B16F10 and B16F10-pcDNA. In prophylactic, treatment and anti-lung-metastasis experiments, gene gun-mediated pWRG-neu immunization could exhibit antitumor effects. The growth and metastasis of neu-overexpressed melanoma was reduced dramatically. The spleen cells of the immuned mice showed cytotoxic T lymphocyte (CTL) activity. CONCLUSION: Gene gun-mediated gene transfer is effective and practicable. DNA vaccine pWRG-neu is potent in preventing subsequent tumor cells challenge, inhibiting the tumor growth and metastasis.

Animals↗

PubMed2003

Bladder cancer therapy using combined proliferating cell nuclear antigen antisense oligonucleotides and recombinant adenovirus p53.

OBJECTIVE: To evaluate the antitumor efficacy of proliferating cell nuclear antigen antisense oligonucleotide (PCNA-ASO) in combination with recombinant adenovirus p53 (Ad-p53) against bladder cancer EJ and BIU-87 cells in vitro and in vivo. METHODS: Cells were transfected with Ad-p53 (100 MOI), and PCNA-ASO (1.6 micro mol/L) was then introduced into the cells using a cationic lipid (lipofectamine, 20 micro l/ml). In vitro and in vivo antitumor effects of combining PCNA-ASO with Ad-p53 were measured using the MTT assay, flow cytometry, clone formation, and a nude mice model. RESULTS: The combination of PCNA-ASO and Ad-p53 inhibited cell viability in both the EJ (89.3%) and BIU-87 (78.6%) cell lines. The ability of the cells to form foci was also reduced by 74.8% in EJ cells and by 67.5% in BIU-87 cells (P < 0.01). A significant decrease of cells in the S phase (11.4% in EJ cells, 14.6% in BIU-87 cells) and a significant increase of cells in G1 phase (62.2% in EJ, 56.8% in BIU-87) were noted. The mean tumor volume after 7 days of treatment with PCNA-ASO or Ad-p53 in combination decreased to 47.6% or 36.4% of the initial tumor size in the two cell lines respectively. CONCLUSION: These results indicate that combined PCNA-ASO and Ad-p53 in the treatment of bladder cancer with mutant p53 has important therapeutic potential, significantly suppressing the growth of human bladder cancer both in vitro and in vivo.

Adenoviridae↗

PubMed2003

[Construction of eukaryotic expression vector of murine SLC gene and characterization of its chemotactic function].

AIM: To construct the eukaryotic expression vector of murine SLC gene and study the chemotactic function of murine SLC in-vitro and in-vivo. METHODS: Murine SLC gene was cloned by RT-PCR from the thymus tissue of a C57BL/6 mouse. Eukaryotic expression vector of SLC gene-pcDNA3.1 mSLC was constructed and transfected into B16F10 cells by gene gun. Culture supernatant was collected 48 hours after the transfection and chemotactic function of expression product to lymphocytes was detected by a chemotaxis chamber. SLC expression was detected by RT-PCR. Lymphocytic infiltration was observed in SLC gene transfected murine abdominal skin. RESULTS: The gene cloned from the C57BL/6 mouse thymus tissue was SLC gene Scya21b. Transfected cells expressed SLC mRNA, and culture supernatant of those cells had a potent chemotactic function to lymphocytes. Histological examination of transfected skin showed obvious lymphocytic infiltration. CONCLUSION: The SLC gene was cloned from the mouse thymus tissue and could be expressed in B16F10 cells. The expression product in-vitro and in-vivo had a remarkable chemotactic function to lymphocytes.

Animals↗

PubMed2002

[Experimental study on adenovirus-mediated transfer of p53 or adenovirus- mediated transfer of p53 combined with radiotherapy for human cervical cancer cell line HeLa cells].

OBJECTIVE: To evaluate the therapeutic efficiency of adenovirus-mediated transfer of p53(Ad-p53) or Ad-p53 combined with radiotherapy for human cervical cancer cell line HeLa cells. METHODS: HeLa cells were either transfected with Ad-p53 alone or with Ad-p53 combining with radiotherapy. Reverse transcription polymerase chain reaction(RT-PCR), the cell growth, morphological changes, cell cycle, apoptosis and molecular changes were measured using cell counting, microscopy, flow cytometry, DNA ladder, and in vivo therapy experiments to evaluate the therapeutic efficiency of the regimens. RESULTS: Ad-p53 could be transfected into HeLa cells and p53 mRNA could be expressed more in the targeted HeLa cells. Ad-p53 transfer had strong therapeutic effects to HeLa cells in vitro and in vivo (the inhibition rates were 61.8% and 54.8%, respectively), moreover, the combined administration of Ad-p53 and radiotherapy had stronger therapeutic effects in vitro and in vivo (the inhibition rate was 85.5% in vitro, and the inhibition rate was 77.4% in vivo). The inhibition rate by radiotherapy alone was 44.6% in vitro and 58.4% in vivo. Massive apoptosis of HeLa cells was induced by these regimens. Cell cycle analysis demonstrated that all these regimens could arrested HeLa cells in G(2)-M. CONCLUSIONS: After introduced into HeLa cells, Ad-p53 shows therapeutic efficiency for HeLa cells both in vitro and in vivo. Moreover, Ad-p53 could enhance therapeutic efficiency for HeLa cells when combined with radiotherapy.

Adenoviridae↗

PubMed2002

Adenovirus-mediated transfer of p53 and p16 inhibiting proliferating activity of human bladder cancer cell EJ in vitro and in vivo.

To evaluate the effects of adenovirus (Ad)-mediated transfer of p53 and p16 on human bladder cancer cells EJ, EJ were transfected with Ad-p53 and Ad-p16. Cell growth, morphological change, cell cycle, apoptosis were measured using MTT assay, flow cytometry, cloning formation, immunocytochemical assays. Ad-p16 or Ad-p53 alone could inhibit the proliferating activity of EJ cells in vitro. Ad-p53 could induce apoptosis of partial EJ cells. G1 arrest was observed 72 h after infection with Ad-p16, but apoptosis was not obvious. The transfer of Ad-p16 and Ad-p53 could significantly inhibit the growth of EJ cells, decrease the cloning formation rate and induce apoptosis of large number of EJ cells. The occurrence time of subcutaneous tumor was delayed and the tumor volume in 4 weeks was diminished by using Ad-p53 combined with Ad-p16 and the difference was significant compared with using Ad-p53 or Ad-p16 alone. It was suggested that the transfer of wild-type p53 and p16 could significantly inhibit the growth of human bladder cancer in vitro and in vivo.

Adenoviridae↗

PubMed2002

[Research of the anti-tumor effect of human endostatin mediated by recombinant adeno-associated virus].

BACKGROUND & OBJECTIVE: It was reported that endostatin could inhibit tumor angiogenesis, then inhibit the growth and metastasis of tumor. However, there was few report about the treatment usage of endostatin. This study was designed to explore the effect of endostatin mediated with recombinant adeno-associated virus(rAAV) on tumor growth and metastasis. METHODS: To obtain the endostatin gene complete cDNA by RT-PCR, and clone it into the plasmid pSNAV and package the recombinant rAAV-SS-endostatin; to analyze its anti-tumor effect through animal experiments. RESULTS: In the B16F10 melanoma tumor model, C57BL/6 mice were inoculated with 10(11)TU rAAV-SS-endostatin i.m. injection, which the inhibition rate was 57.1%; In the lung metastatic model, the inhibition rate of metastases was 70.7%. CONCLUSIONS: rAAV-SS-endostatin can effectively inhibit tumor growth and metastasis.

Animals↗

PubMed2002

[Determination of arsenic in animal liver by atomic fluorescence spectrometry].

An atomic fluorescence spectrometry for the determination of micro arsenic in animal liver was developed with high sensitivity and fine accuracy. Under the selected experimental conditions, there was a linear relationship between the fluorescence intensity and arsenic concentration in the range of 0-160 ng.mL-1 with correlation coefficient of 0.9996. The limit of detection was 0.4 ng.mL-1, and the recovery was 94.2%-98.5%.

Animals↗