BACKGROUND: Sex steroid hormones are believed to play an important role in the genesis and growth of uterine myoma. Several studies suggest a possible role of insulin-like growth factor(IGF) as a mediator of estradiol in uterine myama. We have recently demonstrated that some IGF binding proteins(IGFRPs) messenger ribonucleic acid(mRNA) expressions in myoma are dependent on the in vivo esttogen status. The purposes of this study are to evaluate the in vitro effects of sex steroid hormones including estrogen on the IGFBPs gene expression in tissues from uterine myoma and adjacent normal myometrium. METHODS: Tissues from myoma and adjacent normal myometrium of patients with uterine myoma during early proliferative phase of menstrual cycle were cultured in the absence(control) and presence of 17b-estradiol(10M/L) or/and progesterone(10M/L) for 3 days. The IGFBPs mRNA expressions in these explants were analyzed by Nothern blot using specific human complementary deoxyribonucleic acid(cDNA) probes. RESULTS: The addition of 17b-estradiol, progesterone alone and in combination to conditioned media of explants from myoma and adjacent normal myornetrium did not result in any changes in the expression of IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-6 mRNA. With progesterone addtion, lGFBP-3 rnRNA expression was significantly reduced in myoma explant but not in adjacent ncemal myometrium explant. There was no significant change in the IGFBP-3 mRNA expression with 17b-estradiol and with the combination of both 17b-estradiol and progesterone. CONCLUSION: 17b-estradiol does not affect IGFBPs gene expression in the myoma and adjacent normal myometrium explant regardless of the presence of progesterone in vitro. However progesterone alone induces a decrease in IGFBP-3 synthesis in myoma explant.
Animals ; Culture Media, Conditioned ; Estradiol ; Estrogens ; Female ; Gene Expression ; Gonadal Steroid Hormones ; Humans* ; Insulin-Like Growth Factor Binding Protein 2 ; Insulin-Like Growth Factor Binding Protein 3 ; Insulin-Like Growth Factor Binding Protein 4 ; Insulin-Like Growth Factor Binding Protein 5 ; Insulin-Like Growth Factor Binding Protein 6 ; Insulin-Like Growth Factor Binding Proteins ; Leiomyoma* ; Menstrual Cycle ; Mice ; Myoma ; Myometrium* ; Progesterone ; RNA, Messenger*

PURPOSE: PTEN/MMAC1, a novel tumor suppressor gene, is mutated in a variety of advanced and metastatic cancers. It acts as a phosphatase, and thereby, regulates the PI-3 kinase/Akt pathway. In this study, we examined to evaluate the new function of anti-tumor effects of PTEN/MMAC1 through the regulation of the IGFs-IGFBPs in gastric cancer cells. METHODS: PTEN/MMAC1 was expressed in an adenovirus-mediated gene delivery system and introduced into gastric cancer cells(SNU-484 & SNU-668) in vitro. The effect of cell growth and the expression of IGFs and IGFBPs after Ad/PTEN infection was analyzed by MTT assay, RT-PCR and Western immunoblot. RESULTS: Ad/PTEN infected cells were inhibited in cell growth compared with moak cells and Ad/ LacZ infected cells. Overexpression of PTEN/MMAC1 induced decrease in expression of IGF-I, -II and IGF-I receptors which are known as growth prompt molecules in a variety of cancers. Of the six IGFBPs, the expressions of IGFBP-4 and IGFBP-6 were decreased in Ad/PTEN infected cells. In contrast, IGFBP-3 expression was markedly increased by up to 3-fold in Ad/PTEN infected cells. Overexpression of PTEN/MMAC1 inhibited the activation of Akt/PKB pathway, but had no effect on the MAPK pathway. CONCLUSION: These findings suggest that the tumor suppressor function of PTEN/MMAC1 is, at least in part, mediated through the down-regulation of IGF-I abd IGF-II, and up-regulation of IGFBP-3 in gastric cancer cells by the inhibition of PI-3 kinase pathway.
Blotting, Western ; Down-Regulation ; Gene Transfer Techniques ; Genes, Tumor Suppressor ; Insulin-Like Growth Factor Binding Protein 3 ; Insulin-Like Growth Factor Binding Protein 4 ; Insulin-Like Growth Factor Binding Protein 6 ; Insulin-Like Growth Factor Binding Proteins* ; Insulin-Like Growth Factor I ; Insulin-Like Growth Factor II ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases ; Receptor, IGF Type 1 ; Stomach Neoplasms ; Up-Regulation

OBJECTIVETo study the nuclear localization of insulin-like growth factor binding protein-6(IGFBP-6) in PC-3M cells.

METHODSThe two fragments of the nuclear localization sequence (NLS)-deleted IGFBP-6 and the NLS-mutated IGFBP-6 were obtained by overlapping PCR, and then the fragment was inserted into a pEGFP-C1 vector. PC-3M cells were transfected with the expression constructs containing wild-type IGFBP-6 or the two mutants (pEGFP-C1-BP6 Delta NLS and pEGFP-C1-BP6-Mut), and the different distribution of the three EGFP-fusion proteins was observed by confocal laser microscope. The statistical analysis of the ratio of the nuclear fluorescence to the cytoplasmic fluorescence (Fn/c) was performed. Results Confocal microscopic images of transfected cells showed that the green fluorescence of EGFP-IGFBP-6 was concentrated mostly in the nuclei, whereas the control cells expressing EGFP showed green fluorescence distributed uniformly. The results of Fn/c from EGFP and EGFP-IGFBP-6 were significant different (P<0.05). The NLS-deleted IGFBP-6 completely eliminated nuclear accumulation of the green fluorescent signal; in contrast, nuclear accumulation was only slightly reduced for the NLS-mutated IGFBP-6; compared with wild-type IGFBP-6, both mutants were significantly different (P<0.05). Conclusions IGFBP-6 can be translocated to the nucleus in PC-3M cell that is mediated by a putative NLS sequence. Our study provides new evidence for further studies on the insulin-like growth factor-independent activity of IGFBP-6.

Cell Line, Tumor ; Cell Nucleus ; metabolism ; Genetic Vectors ; Humans ; Insulin-Like Growth Factor Binding Protein 6 ; genetics ; metabolism ; Plasmids ; genetics ; Protein Transport ; genetics ; Transfection

Fibroblast-like synoviocytes (FLSs) constitute a major cell subset of rheumatoid arthritis (RA) synovia. Dysregulation of microRNAs (miRNAs) has been implicated in activation and proliferation of RA-FLSs. However, the functional association of various miRNAs with their targets that are characteristic of the RA-FLS phenotype has not been globally elucidated. In this study, we performed microarray analyses of miRNAs and mRNAs in RA-FLSs and osteoarthritis FLSs (OA-FLSs), simultaneously, to validate how dysregulated miRNAs may be associated with the RA-FLS phenotype. Global miRNA profiling revealed that miR-143 and miR-145 were differentially upregulated in RA-FLSs compared to OA-FLSs. miR-143 and miR-145 were highly expressed in independent RA-FLSs. The miRNA-target prediction and network model of the predicted targets identified insulin-like growth factor binding protein 5 (IGFBP5) and semaphorin 3A (SEMA3A) as potential target genes downregulated by miR-143 and miR-145, respectively. IGFBP5 level was inversely correlated with miR-143 expression, and its deficiency rendered RA-FLSs more sensitive to TNFα stimulation, promoting IL-6 production and NF-κB activity. Moreover, SEMA3A was a direct target of miR-145, as determined by a luciferase reporter assay, antagonizing VEGF165-induced increases in the survival, migration and invasion of RA-FLSs. Taken together, our data suggest that enhanced expression of miR-143 and miR-145 renders RA-FLSs susceptible to TNFα and VEGF165 stimuli by downregulating IGFBP5 and SEMA3A, respectively, and that these miRNAs could be therapeutic targets.
Arthritis, Rheumatoid* ; Fibroblasts* ; Insulin-Like Growth Factor Binding Protein 5 ; Interleukin-6 ; Luciferases ; MicroRNAs ; Osteoarthritis ; Phenotype* ; RNA, Messenger ; Semaphorin-3A ; Synovial Fluid

PURPOSE: IGFBP-3 leads to the induction of insulin resistance in 3T3-L1 adipocytes. We carried out a series of experiments to elucidate the effects of IGFBP-3 on adipokines and gene expressions. METHODS: We treated fully-differentiated 3T3-L1 adipocytes with IGFBP-3 (0.5, 1, and 2 microg/mL) for one day and measured the mRNA levels of adiponectin, leptin, resistin, and TNF-alpha by RT-PCR, and adiponectin, leptin, resistin, and IL-6 protein levels in the culture supernatant were measured using multiplex adipokine assay ELISA Kits (Linco Research, St. Charles, Missouri). Gene expression in 3T3-L1 adipocyte cells using a microarray method was performed. RESULTS: IGFBP-3 inhibited the expression of adiponectin, leptin, resistin, and TNF-alpha mRNA. IGFBP-3 at 0.5 and 1 micro/mL decreased adiponectin release, but IL-6 release was increased at 2 micro/mL IGFBP-3. A dose-dependent inhibition of leptin was released by IGFBP-3 at 50%. Resistin release was decreased by 40%. The effect of IGFBP-3 on the gene expression in 3T3-L1 adipocyte cells using a microarray assay related to an increase of agouti-realted proteins (Agrp) and Janus kinase 2 (JAK2), and a decrease of the ras homolog gene family (Rhoq), acyl-CoA synthetase long-chain family member 6 (Acsl6), and the interleukin-1 receptor-associated kinase 1 (Irak1). CONCLUSION: IGFBP-3 regulates several adipokines gene expressions that are known to modulate insulin sensitivity, and this regulation may be attributable to the insulin resistance effect of IGFBP-3 on adipocytes.
Adipocytes ; Adipokines ; Adiponectin ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Insulin Resistance ; Insulin-Like Growth Factor Binding Protein 3 ; Interleukin-1 Receptor-Associated Kinases ; Interleukin-6 ; Janus Kinase 2 ; Leptin ; Ligases ; Proteins ; Resistin ; RNA, Messenger ; Tumor Necrosis Factor-alpha

OBJECTIVETo investigate the effect of the environmental carcinogenic factor-TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) on cell apoptosis and gene regulation of insulin-like growth factor binding protein 6 (IGFBP-6) in osteogenic sarcoma (SaOS-2) cells.

METHODSThe SaOS-2 cells were cultured with TCDD (1 x 10(-9), 1 x 10(-8), 1 x 10(-7) mol/L) for 24 hours. The MTT reduction assay and flow cytometry were used to measure the cell proliferation and the cell apoptosis in TCDD-treated SaOS-2 cells. The Nitrophenol phosphate salt method was used to measure activity of alkaline phosphatase (ALP) in SaOS-2 cells. The IGFBP-6 mRNA and protein in SaOS-2 cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and western blotting analysis.

RESULTSSaOS-2 cell proliferation was up-regulated with TCDD (1 x 10(-9), 1 x 10(-8), and 1 x 10(-7) mol/L) about 20%, 47% and 93% (18.4 +/- 4.5, 22.5 +/- 3.6 and 29.4 +/- 4.2), respectively. The synthesis of ALP was up-regulated about 28%, 95%, and 142% (1.12 +/- 0.28, 1.58 +/- 0.14 and 1.96 +/- 0.17), respectively (P < 0.05). The cell apoptosis was down-regulated in dose-dependent biological manner about 5%, 26% and 52%, respectively (P < 0.05). The expression of IGFBP-6 mRNA and protein was decreased in 1 x 10(-7) mol/L TCDD-treated SaOS-2 cells about 76% and 72% (P < 0.05).

CONCLUSIONTCDD at low concentration may have the negative effect on cell apoptosis and down-regulation on gene expression of IGFBP-6 in SaOS-2 cells.

Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Insulin-Like Growth Factor Binding Protein 6 ; genetics ; metabolism ; Osteosarcoma ; metabolism ; pathology ; Polychlorinated Dibenzodioxins ; toxicity ; RNA, Messenger ; genetics

OBJECTIVETo investigate the antiestrogenic effect of environment teratogen on the gene expression of insulin-like growth factors (IGFs) family in osteoblast cells during rat skeleton development.

METHODSThe fetal rat models with congenital skeleton malformation were constructed by treating 20 female Wistar rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on pregnant day 10. The MC-3T3-E1 cells were cultured with estrogen, TCDD, or a combination of the two chemicals for 24 hours. The IGF-II and IGFBP-6 mRNA levels in rat calvaria bone tissue and MC-3T3-E1 cells were detected by reverse transcription-polymerase chain reaction. Flow cytometer was used to determine the cell proliferation.

RESULTSTCDD at the concentration of 5-15 microg/kg induced developmental skeleton defect of fetal rat, and the effect was dose-dependent. The expression of IGF-II mRNA gene was enhanced by estrogen in rat calvaria bone tissue and MC-3T3-E1 cells, whereas IGFBP-6 mRNA was decreased. Estrogen increased the cell proliferation in MC-3T3-E1 cells. TCDD, however, inhibited the effect of estrogen on regulation of IGF-II gene and IGFBP-6 gene as well as MC-3T3-E1 cell proliferation.

CONCLUSIONThese findings provide the evidence that TCDD can induce congenital fetal skeleton malformation under the condition of high estrogen level in pregnant Wistar rats. TCDD has antiestrogenic effect and hence exerts negative influence on the osteoblast cells through target IGF-II and IGFBP-6 of IGFs family.

Abnormalities, Drug-Induced ; etiology ; Animals ; Bone and Bones ; abnormalities ; Dose-Response Relationship, Drug ; Estrogen Receptor Modulators ; toxicity ; Female ; Insulin-Like Growth Factor Binding Protein 6 ; genetics ; Insulin-Like Growth Factor II ; genetics ; Osteoblasts ; drug effects ; metabolism ; Polychlorinated Dibenzodioxins ; toxicity ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar

PURPOSE: Laparoscopic colectomy has clinical benefits such as short hospital stay, less postoperative pain, and early return of bowel function. However, objective evidence of its immunologic and oncologic benefits is scarce. We compared functional recovery after open versus laparoscopic sigmoidectomy and investigated the effect of open versus laparoscopic surgery on acute inflammation as well as tumor stimulation. MATERIALS AND METHODS: A total of 57 patients who were diagnosed with sigmoid colon cancer were randomized for elective conventional or laparoscopically assisted sigmoidectomy. Serum samples were obtained preoperatively and on postoperative day 1. C-reactive protein (CRP) and interleukin-6 (IL-6) were measured as inflammation markers, and vascular endothelial growth factor (VEGF) and insulin-like growth factor binding protein-3 (IGFBP-3) were used as tumor stimulation factors. Clinical parameters and serum markers were compared. RESULTS: Postoperative hospital stay (p=0.031), the first day of gas out (p=0.016), and the first day of soft diet (p<0.001) were significantly shorter for the laparoscopic surgery group than the open surgery group. The levels of CRP, IL-6, and VEGF rose significantly, and the concentration of IGFBP-3 fell significantly after both open and laparoscopic surgery. However, there were no significant differences in the preoperative and postoperative levels of CRP, IL-6, VEGF, and IGFBP-3 between the two groups. CONCLUSION: Our data suggest that both open and laparoscopic surgeries are accompanied by significant changes in IL-6, CRP, IGFBP-3, and VEGF levels. Acute inflammation markers and tumor stimulating factors may not reflect clinical benefits of laparoscopic surgery.
Aged ; Biological Markers/blood ; C-Reactive Protein/metabolism ; Colectomy/*adverse effects/methods ; Female ; Humans ; Inflammation/etiology/metabolism ; Insulin-Like Growth Factor Binding Protein 3/blood ; Interleukin-6/blood ; Laparoscopy/adverse effects ; Male ; Middle Aged ; Postoperative Period ; Sigmoid Neoplasms/*surgery ; Treatment Outcome ; Vascular Endothelial Growth Factor A/blood

OBJECTIVETo explore the relationship between the methylation status of CpG islands in the promoter region of 10 genes in breast cancer cells and their sensitivity to 5-fluouracil (5-Fu), and to identify the genes responsible for the 5-Fu resistance in breast cancer.

METHODSThree cell lines (differently resistant to chemotherapy) were used in this study: Bcap-37 (IC(50): 289.77 microg/ml), T47D (IC(50): 134.16 microg/ml) and ZR-75-30 (IC(50): 4.20 microg/ml). The methylation profile of 10 genes (BAG1, C11ORF31, CBR1, CBR4, GJA1, FOXL2, IGFBP6, P4HA1, SRI and TYMS) in the 3 breast cancer cell lines was determined by methylation specific PCR. The steady-state mRNAs of ABCC8, CHFR and IGFBP6 genes were quantified by real-time RT PCR analysis.

RESULTSAmong the 10 genes, only genes IGFBP6 and FOXL2 displayed differential DNA methylation pattern between the 5-Fu-resistant and 5-Fu-sensitive cell lines. The mRNA expression level of genes PRSS21, LOX, IGFBP6, ABCC8 and CHFR was quantified by real-time RT-PCR analysis. Except for CHFR, the expression level of the other 4 genes was correlated with the methylation status of CpG islands, namely, a lower expression level with methylation status and a higher level with demethylation status.

CONCLUSIONThe results of the present study have demonstrated that there are 8 genes with differential methylation status in chemosensitive and chemoresistant breast cancer cell lines, i.e. two genes more than the six genes we reported previously. Our findings provide both mechanistic insights for the drug resistance of breast cancer and the basis for further studies on potential application of the DNA methylation in this set of genes for prediction of chemosensitivity of breast cancer.

Antimetabolites, Antineoplastic ; pharmacology ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; CpG Islands ; genetics ; DNA Methylation ; Drug Resistance, Neoplasm ; Fluorouracil ; pharmacology ; Forkhead Box Protein L2 ; Forkhead Transcription Factors ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Insulin-Like Growth Factor Binding Protein 6 ; genetics ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism

PURPOSE: The prevalence of asthma and obesity is increasing concomitantly, but the link between asthma and obesity is unclear. We sought to address possible roles of leptin and adiponectin in the development of asthma, and changes in pulmonary function in overweight children. METHODS: Four study groups of 61 children aged 6 to 18 years (mean age, 9.69+/-2.16) were enrolled: (1) 14 mild-to-moderate asthmatics with overweight, (2) 16 mild-to-moderate asthmatics with normal weight, (3) 16 obese subjects without asthma, and (4) 15 healthy controls. We measured biomarkers in blood, including total and allergen-specific IgE, eosinophil, eosinophilc cationic protein (ECP), leptin, adiponectin, interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha), lipid profiles, insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein 3 (IGF-BP3). Body mass index (BMI), antioxidants and micronutrients in a daily diet were evaluated by the questionnaire. We performed the bronchial challenge test by methacholine inhalation and free running, respectively. RESULTS: The leptin levels was apparently high, and the adiponectin level was low in the over-weight children, as depicting a significant inverse correlation between the 2 variables (R=-0.479; P<0.001). The FEV(1)/FVC ratio was low in the overweight children regardless of the presence of asthma. However, the effect of IL-6, TNF-alpha, nutrients, and other variables on asthma development in the overweight children with asthma was not verified. CONCLUSION: In this study, the levels of leptin, adiponectin or other obesity-related biomarkers were not independently associated with asthma. Therefore, it is concluded that obesity may not be an important factor in pulmonary function impairment.
Adiponectin ; Aged ; Aluminum Hydroxide ; Antioxidants ; Asthma ; Biomarkers ; Body Mass Index ; Bronchial Provocation Tests ; Carbonates ; Child ; Diet ; Eosinophils ; Humans ; Immunoglobulin E ; Inhalation ; Insulin-Like Growth Factor Binding Protein 3 ; Interleukin-6 ; Interleukins ; Leptin ; Methacholine Chloride ; Micronutrients ; Obesity ; Overweight ; Prevalence ; Surveys and Questionnaires ; Running ; Tumor Necrosis Factor-alpha