Phelligridin D maintains the function of periodontal ligament cells through autophagy in glucose-induced oxidative stress

Purpose@#The objective of this study was to investigate whether phelligridin D could reduce glucose-induced oxidative stress, attenuate the resulting inflammatory response, and restore the function of human periodontal ligament cells (HPDLCs). @*Methods@#Primary HPDLCs were isolated from healthy human teeth and cultured. To investigate the effect of phelligridin D on glucose-induced oxidative stress, HPDLCs were treated with phelligridin D, various concentrations of glucose, and glucose oxidase.Glucose-induced oxidative stress, inflammatory molecules, osteoblast differentiation, and mineralization of the HPDLCs were measured by hydrogen peroxide (H2O2 ) generation, cellular viability, alkaline phosphatase (ALP) activity, alizarin red staining, and western blot analyses. @*Results@#Glucose-induced oxidative stress led to increased production of H2O2 , with negative impacts on cellular viability, ALP activity, and calcium deposition in HPDLCs. Furthermore, HPDLCs under glucose-induced oxidative stress showed induction of inflammatory molecules (intercellular adhesion molecule-1, vascular cell adhesion protein-1, tumor necrosis factor-alpha, interleukin-1-beta) and disturbances of osteogenic differentiation (bone morphogenetic protein-2, and -7, runt-related transcription factor-2), cementogenesis (cementum protein-1), and autophagy-related molecules (autophagy related 5, light chain 3 I/II, beclin-1). Phelligridin D restored all these molecules and maintained the function of HPDLCs even under glucose-induced oxidative stress. @*Conclusions@#This study suggests that phelligridin D reduces the inflammation that results from glucose-induced oxidative stress and restores the function of HPDLCs (e.g., osteoblast differentiation) by upregulating autophagy.

Phelligridin D maintains the function of periodontal ligament cells through autophagy in glucose-induced oxidative stress

Purpose@#The objective of this study was to investigate whether phelligridin D could reduce glucose-induced oxidative stress, attenuate the resulting inflammatory response, and restore the function of human periodontal ligament cells (HPDLCs). @*Methods@#Primary HPDLCs were isolated from healthy human teeth and cultured. To investigate the effect of phelligridin D on glucose-induced oxidative stress, HPDLCs were treated with phelligridin D, various concentrations of glucose, and glucose oxidase.Glucose-induced oxidative stress, inflammatory molecules, osteoblast differentiation, and mineralization of the HPDLCs were measured by hydrogen peroxide (H2O2 ) generation, cellular viability, alkaline phosphatase (ALP) activity, alizarin red staining, and western blot analyses. @*Results@#Glucose-induced oxidative stress led to increased production of H2O2 , with negative impacts on cellular viability, ALP activity, and calcium deposition in HPDLCs. Furthermore, HPDLCs under glucose-induced oxidative stress showed induction of inflammatory molecules (intercellular adhesion molecule-1, vascular cell adhesion protein-1, tumor necrosis factor-alpha, interleukin-1-beta) and disturbances of osteogenic differentiation (bone morphogenetic protein-2, and -7, runt-related transcription factor-2), cementogenesis (cementum protein-1), and autophagy-related molecules (autophagy related 5, light chain 3 I/II, beclin-1). Phelligridin D restored all these molecules and maintained the function of HPDLCs even under glucose-induced oxidative stress. @*Conclusions@#This study suggests that phelligridin D reduces the inflammation that results from glucose-induced oxidative stress and restores the function of HPDLCs (e.g., osteoblast differentiation) by upregulating autophagy.

PPARgamma Inhibits Inflammation through the Suppression of ERK1/2 Kinase Activity in Human Gingival Fibroblasts

Periodontal disease is a major oral disorder and comprises a group of infections that lead to inflammation of the gingiva and the destruction of periodontal tissues. PPARgamma plays an important role in the regulation of several metabolic pathways and has recently been implicated in inflammatory response pathways. However, its effects on periodontal inflammation have yet to be clarified. In our current study, we evaluated the anti-inflammatory effects of PPARgamma on periodontal disease. Human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) showed high levels of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinase-2 (MMP-2), and -9 (MMP-9). Moreover, these cells also showed upregulated activities for extracellular signal regulated kinase (ERK1/2), inducible nitric oxide synthase (iNOS) and cyclooxygnase-2. However, cells treated with Ad/PPARgamma and rosiglitazone in same culture system showed reduced ICAM-1, VCAM-1, MMP-2, -9 and COX-2. Finally, the anti-inflammatory effects of PPARgamma appear to be mediated via the suppression of the ERK1/2 pathway and consequent inhibition of NF-kB translocation. Our present findings thus suggest that PPARgamma indeed has a pivotal role in gingival inflammation and may be a putative molecular target for future therapeutic strategies to control chronic periodontal disease.

Changes in the expression of cytokines and apoptosis-related genes in children with infectious mononucleosis / 소아과

PURPOSE: The aims of this study were to identify the clinical characteristics and determine the changes in the expression of cytokines and apoptosis-related genes in children with infectious mononucleosis. METHODS: Serological examinations of 15 pediatric patients diagnosed with infectious mononucleosis were performed prospectively. Peripheral blood from the patients was used to compare the composition of T cell subsets, cytokines, Epstein-Barr virus (EBV) DNA, and the expression of apoptosis-related genes with those in 10 healthy children. RESULTS: Mean age of the patient group was 5.7+/-3.4 (range, 3-9) years, and the male-to-female ratio was 1.5:1. Fever, sore throat, pharyngitis/tonsillitis, and cervical lymph node enlargement were the most common symptoms and signs. The proportions of CD3+ T cells, CD8+ suppressor cells, and CD56+ natural killer (NK) cells were higher in the patient group than in the control group (P<0.01). The IL-2, IL-6, and interferon (IFN)-gamma levels were higher in the early symptomatic period (P<0.01). Mean amount of EBV DNA in the patients was 10(2.38) copies/microg, and the amount was the highest at the beginning of the symptomatic period and normalized during the convalescent phase. Bcl-2 expression increased during the initial phase, while Bax expression increased during the convalescent phase. Further, FasL expression increased 1 week after symptom presentation and decreased during the convalescent phase. There was no significant change in Fas expression. CONCLUSION: We analyzed the clinical characteristics and changes in the expression ofcytokines and apoptosis-related genes in the patients with infectious mononucleosis.

Iron chelating agent, deferoxamine, induced apoptosis in Saos-2 osteosarcoma cancer cells / 소아과

PURPOSE: Iron is a critical nutritional element that is essential for a variety of important biological processes, including cell growth and differentiation, electron transfer reactions, and oxygen transport, activation, and detoxification. Iron is also required for neoplastic cell growth due to its catalytic effects on the formation of hydroxyl radicals, suppression of host defense cell activities, and promotion of cancer cell multiplication. Chronic transfusion-dependent patients receiving chemotherapy may have iron overload, which requires iron-chelating therapy. We performed this study to demonstrate whether the iron chelating agent deferoxamine induces apoptosis in Saos-2 osteosarcoma cells, and to investigate the underlying apoptotic mechanism. METHODS: To analyze the apoptotic effects of an iron chelator, cultured Saos-2 cells were treated with deferoxamine. We analyzed cell survival by trypan blue and crystal violet analysis, apoptosis by nuclear condensation, DNA fragmentation, and cell cycle analysis, and the expression of apoptotic related proteins by Western immunoblot analysis. RESULTS: Deferoxamine inhibited the growth of Saos-2 cell in a time- and dose-dependent manner. The major mechanism for growth inhibition with the deferoxamine treatment was by the induction of apoptosis, which was supported by nuclear staining, DNA fragmentation analysis, and flow cytometric analysis. Furthermore, bcl-2 expression decreased, while bax, caspase-3, caspase-9, and PARP expression increased in Saos-2 cells treated with deferoxamine. CONCLUSION: These results demonstrated that the iron chelating agent deferoxamine induced growth inhibition and mitochondrial-dependent apoptosis in osteosarcoma Saos-2 cells, suggesting that iron chelating agents used in controlling neoplastic cell fate can be potentially developed as an adjuvant agent enhancing the anti-tumor effect for the treatment of osteosarcoma.

Capsaicin induced apoptosis and the enhanced anticancer effect of anticancer drugs in cancer cells / 소아과

PURPOSE: Capsaicin, the major pungent ingredient in red pepper, has long been used in spices and food additives. It has been recently shown to induce apoptosis in several cell lines through a not well known mechanism. The aim of this study was to investigate the apoptosis-inducing effect of capsaicin on gastric cancer cells, and to provide valuable information concerning the application of capsaicin for therapeutic purposes. METHODS: Cultured SNU-668 cells were treated with capsaicin. We analyzed cell survival by trypan blue and crystal violet analysis, cell cytotoxicity by MTT assay, apoptosis by nuclear condensation and DNA fragmentation, bcl-2 and bax mRNA expression by RT-PCR, and the expression of apoptosis related proteins by Western immunoblot analysis. In order to assess whether the growth inhibitory effect of anticancer drugs is enhanced by capsaicin, we investigated the effects of cell cytotoxicity and the expression of apoptosis related proteins of etoposide and adriamycin treated with capsaicin in cells. RESULTS: Capsaicin inhibited growth of SNU-668 cells in a dose-dependent manner. This inhibitory effect of capsaicin on cell growth was mainly due to the induction of apoptosis as evidenced by DNA fragmentation, nuclear condensation and the expression of apoptosis related proteins. Furthermore, capsaicin prominently reduced the ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax and consequently increased caspase-3 activity. The cells treated with capsaicin were more sensitive to death induced by etoposide and adriamycin than the cells without capsaicin. CONCLUSION: These results demonstrate that capsaicin efficiently induced apoptosis in SNU-668 cells through a caspase-3-dependent mechanism and sensitizes cancer cells to anticancer drugs toward apoptotic cell death, which may contribute to its anticancer effect and chemosensitizer function against gastric cancer.

The anti-tumor mechanisms of p53 through the regulation of expression and glycosylation of insulin-like growth factor binding protein-3 / 소아과

PURPOSE: Insulin-like growth factor binding protein(IGFBP)-3 has been known as a tumor suppressor gene, and its anti-tumor function was divided into insulin-like growth factor(IGF)-dependent and IGF-independent mechanism. In IGF-independent mechanism, IGFBP-3 directly interacts with a cell without binding of IGFs, becoming an interesting object in oncology. Several studies demonstrate that one of the well-known tumor suppressor genes, p53, induces directly IGFBP-3 transcription, and the increment of IGFBP-3 expression induces apoptosis of many cancer cells. Recently, the anti-tumor mechanisms of IGFBP-3 have been reported, but post-translational modification of IGFBP-3 and its anti-tumor mechanism are not well known. In this study, we examined whether p53 regulated the glycosylation of IGFBP-3, and analysed the meaning of IGFBP-3 glycosylation related to the apoptosis of cancer cell. METHODS: The p53-mutated status of MDA-MB-231 human breast cancer cells was used in this experiment. The expression and glycosylation of IGFBP-3 were tested by Western blot analysis after infection of adenovirus mediated Ad/p53 and/or Ad/IGFBP-3. RESULTS: Ad/p53 infected cells resulted in growth retardation and the induced apoptosis. p53 induced direct expression and glycosylation of IGFBP-3. The increase of glcosylated IGFBP-3 was able to promote cellular apoptosis, and the glycosylation of IGFBP-3 was more activated by the double treatment of Ad/p53 and Ad/IGFBP-3. CONCLUSION: From this study, the anti-tumor activity of IGFBP-3 was shown to improve the stabilization of IGFBP-3 through the increment of glycosylation of IGFBP-3 by p53. This result suggests that the combined gene therapy of p53 and IGFBP-3 may appropriate treatment of cancer.

PTEN/MMAC1 enhances the growth inhibition by anticancer drugs with downregulation of IGF-II expression in gastric cancer cells

PTEN/MMAC1 is a tumor suppressor gene that is mutated in a variety of advanced and metastatic cancers. Its major function is likely to be the phosphatase activity that regulates the phosphotidylinositol (PI)3-kinase/ Akt pathway. On the other hand, IGF system plays an important role in cell proliferation and cell survival via PI3-kinase/Akt and mitogen-activated protein kinase pathways in many cancer cells. To evaluate effect of PTEN on cell growth and IGF system in gastric cancer, human gastric adenocarcinoma cells (SNU-5 & -216) were transfected with human PTEN cDNA. Those PTEN- transfected gastric cancer cells had a lower proliferation rate than the pcDNA3-transfected cells. PTEN overexpression induced a profound decrease in the IGF-II and IGF-IR expression levels, and downregulation of IGF-II expression by PTEN was mediated through the regulation of the IGF-II promoter. In addition, a PI3-kinase inhibitor, LY294002, induced the downregulation of IGF-II expression. The PTEN-overexpressing SUN-5 and -216 cells were more sensitive to death induced by etoposide and adriamycin that induce DNA damage than the pcDNA3-transfected cells. These findings suggest that PTEN suppresses the cell growth through modulation of IGF system and sensitizing cancer cells to cell death by anticancer drugs.

A Case of Wiskott-Aldrich Syndrome with Novel Mutation in Exon 2 of the WASP Gene / 소아과

Wiskott-Aldrich syndrome(WAS) is an X-linked recessive immunodeficiency characterized by thrombocytopenia with small platelet volume, eczema, and recurrent infections, and is also characterized by increased incidence of auto immune diseases and malignancies. The phenotype observed in this syndrome is caused by mutation in the Wiskott-Aldrich syndrome protein(WASP) gene localized to the proximal short arm of the X chromosome and recently isolated through positional cloning. The gene encodes a 502 amino acid protein, which contains 12 exons and spans 9 kb of genomic DNA. The function of the encoded protein is not well understood. The clinical diagnosis of WAS can be difficult and is usually confirmed by the detection of WASP gene mutations and the expression of WSAP in patient blood sample using genetic analysis. We reported a case of a 13-month old boy with WAS who was identified with the novel mutation in exon 2 of WASP gene by direct sequencing and the complete absence of WASP expression by immunoblotting.

The Tumor Suppressor Function of PTEN/MMAC1 through the Regulation of IGFs and IGFBPs / 소아과

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.

Diagnostic Approach of Wiskott-Aldrich Syndrome / 소아과

OBJECTIVE: A probable diagnosis of Wiskott-Aldrich syndrome(WAS) should be considered in any boy presenting with unusual bleeding, congenital thrombocytopenia and small platelets. The definitive diagnosis of WAS is usually made by the detection of WASP gene mutation or a decrease or absence of the WAS protein(WASP) in blood cells using molecular genetic analysis. However, these methods are too time-consuming and difficult. In this study, therefore, we tried to compare various diagnostic methods and establish the molecular screening steps for the definitive diagnosis of WAS. METHODS: Peripheral blood was drawn from WAS patient with clinical characteristic symptoms, and analyzed with automated complete blood cell count analysis, immunological analysis, and molecular analysis. The morphologic change of WAS patient's blood cell membrane was examined by scanning electron microscopy(SEM). Protein analysis for the expression of WASP protein in PBMC cells was evaluated by FACS and Western immunoblot. Genetic analysis for the detection of a mutation of the WASP gene was performed by polymerase chain reaction-single strand conformational polymorphism analysis(PCR-SSCP) and direct sequencing of PCR products. RESULTS: In addition to microthrombocytopenia, our investigation revealed morphologic defects in WAS lymphocytes by SEM and abnormal mobility shifting in WAS patients by PCR-SSCP. Sequencing the WASP gene detected a specific single base mutation in exon 2, resulting in missense substitution of adenine for guanine 208(G208A, Gly70Arg). FACS and Western immunoblot demonstrated absent expressions of WASP in WAS patients and reduced expression of WASP in carrier when compared with normal controls. CONCLUSION: From these results, we suggested the following diagnostic approaches in patients suspected of having WAS. Protein-based analysis such as FACS and Western immunoblot should be employed as the first line of investigation. The second line genetic analyses should employ second- step approaches with localization of mutation by screening exons, typically by PCR-SSCP, followed by direct sequencing.

Novel Gap Junction Molecules, Connexin 37, Enhances the Bystander Effect in HSVtk/GCV Gene Therapy

PURPOSE: Gap junction intercellular communication(GJIC) is an important mechanism of the bystander effect in herpes simplex thymidine kinase/ganciclovir(HSVtk/GCV) gene therapy Therefore, we attempted to enhance the bystander effect in vitro by exogenous overexpressing connexin 37(Cx37) in cells to increase GJIC. METHODS: NIH3T3 cells were transfected with the Cx37 and HSVtk gene or the HSVtk gene alone by the calcium phosphate method, and we detected their expression from these cells by RT-PCR. GCV-mediated cytotoxicity and the bystander effect of each transfectant was then assessed and compared. RESULTS: Cells transfected with HSVtk became sensitive to low concentration of GCV. We found significantly increased cytotoxicity in HSVtk/GCV gene therapy after introduction of the HSVtk and Cx37 genes together compared with the cytotoxicity seen after introduction of the HSVtk gene in vitro. Co-expression of the HSVtk and Cx37 genes potentiates HSVtk/GCV gene therapy through the bystander effect. CONCLUSION: These results indicated that the increase of GJIC using Cx37 have potentiated the by stander effect of HSVtk/GCV therapy, and may be a new approach to improve response in suicidal cancer gene therapy.

Induction of Apoptosis in Head and Neck Cancer Cells by Dominant Negative C-myb Gene Therapy / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Although the role of c-myb in head and neck tumor has not been studied, aberrant expression of c-myb in various cancer has been demonstrated recently, suggesting that c-myb may play a role in tumorigenesis. Consequently, disrupting c-myb function might prove a possible stratergy for controlling cancer cell growth. The purpose of this study is to investigate the possibility of clinical application of adenovirus-mediated dominant negative c-myb (DN-myb) gene therapy in head and neck cancer. MATERIALS AND METHOD: All tissues were obtained from patients undergoing therapeutic operation for head and neck tumors and were assayed the expression of c-myb and bcl-2 in tumor and normal tissue by RT-PCR. We have generated a replication-deficient recombinant adenovirus encoding the dominant negative c-myb (Ad/DN-myb) or control adenovirus encoding no transgene (Ad/GFP) and infected adenovirus to SNU-1076, head and neck tumor cell line. We examined cell proliferation by 3H-thymidine assay, apoptosis by DNA fragmentation, bcl-2 expression and Akt/PKB phosphorylation by Western immunoblot, and IGF-II, VEGF mRNA expression by RT-PCR. RESULTS: c-myb expression of head and neck tumor tissues was significantly higher than that of normal tissue, indicating that these genes may play an important role in carcinogenesis of head and neck tumor. Ad/DN-myb infected SNU-1076 cells decreased cell proliferation, IGF-1I and VEGF expression, and remarkably increased their apoptosis through the down-regulation of bcl-2 expression and the inhibition of Akt/PKB pathway activation. CONCLUSION: Results obtains from these study suggest that DN-myb induced apoptosis of head and neck tumor cells and the adenovirusmediated DN-myb gene therapy may be a potential molecular therapy for the treatment of head and neck tumor.

Apoptotic Pathway Induced by Dominant Negative ATM Gene in CT-26 Colon Cancer Cells

PURPOSE: Ataxia telangiectasia mutated(ATM) is involved in DNA damage responses at different cell cycle checkpoints, and signalling pathways associated with regulation of apoptosis in response to ionizing radiation(IR). However, the signaling pathway that underlies IR-induced apoptosis in ATM cells has remained unknown. The purpose of this study was, therefore, to investigate the apoptotic pathway that underlies IR-induced apoptosis in a CT-26 cells expressing dominant negative ATM (DN-ATM). METHODS: We generated a replication-deficient recombinant adenovirus encoding the DN-ATM(Ad/DN-ATM) or control adenovirus encoding no transgene(Ad/GFP) and infected adenovirus to CT-26 cells. After infection, we examined apoptosis and apoptotic pathway by [3H]-thymidine assay, DNA fragmentation, and Western immunoblot analysis. RESULTS: DN-ATM gene served as the creation of AT phenotype in a CT-26 cells as revealed by decreased cell proliferations following IR. In addition, IR-induced apoptosis was regulated through the reduced levels of the anti-apoptotic protein Bcl-2, the increased levels of the apoptotic protein Bax, and the activation of caspase-9, caspase-3, and PARP. CONCLUSION: These results indicate that the pathway of IR-induced apoptosis in CT-26 cells expressing DN-ATM is mediated by mitochondrial signaling pathway involving the activation of caspase 9, caspase 3, and PARP.

Role of the Nuclear Transcription Factor NF-kappa B Caused by Acute Hypoxia in the Heart

PURPOSE: Nuclear factor-kappa B(NF-kappa B) is now recognized as playing a potential role in programmed cell death and the adaptive response to various stress. Cellular hypoxia is a primary manifestation of many cardiovascular diseases. It seems that vascular endothelial growth factor (VEGF) and insulin like growth factor-I(IGF-I) have a function as a protective molecule in the heart against several stress including hypoxia. In this study, the role of NF-kappa B to the cellular response and regulation of protective molecules against the acute hypoxia in the heart was studied. METHODS: To cause acute hypoxic stress to the heart, Sprague Dawley rats were exposed to hypoxic chamer(N2 92% and O2 8%). After the hypoxic exposure, nuclear proteins, total proteins and mRNA were isolated from heart. Translocation of the transcription factors NF-kappa B, NF-ATc, AP-1 and NKX-2.5 were evaluated by electrophoretic mobility shift assay(EMSA). The expression of IGF-I and VEGF were studied before and after the hypoxic stress by competitive-PCR, Northern hybridization and Western hybridization. To confirm the role of the NF-kappa B in the heart, the rats also were pretreated with diethyl-dithiocarbamic acid(DDTC) into peritoneal cavity to block NF-kappa B translocation into nucleus. RESULTS: The expression of NF-kappa B, AP-1 and NF-ATc were increased by the hypoxic stress. Increased expression of the VEGF and IGF-I were also observed by the hypoxic stress. However, the blocking of the NF-kappa B translocation reduced those expressions of VEGF and IGF-I. CONCLUSION: These results suggest that NF-kappa B has a protective role against the acute hypoxia through several gene expression, especially VEGF and IGF-I in heart muscle.

Studies on Functional Differentiation of Small Intestinal Epithelial Cells

PURPOSE: We investigated compounds from food sources given to children that may induce the differentiation of small intestinal epithelial cells in order to signal pathways that induce the prolif eration and differentiation of small intestinal epithelial cells. METHODS: We analyzed small intestinal epithelial cell differentiation using in vitro IEC-6 cells model. The growth curve of IEC-6 cells was obtained by standard MTT assay. Alkaline phospha tase(ALP) activities were determined using the paranitrophenol colorimetric assay for the differ entiation of IEC-6 cells. We did ALP and Brdu double-staining of cultured IEC-6 cells to distin guish between differentiation and proliferation, and investigated compounds' potential for inducing differentiation of small intestinal epithelial cells and protein kinase signal pathway. RESULTS: The calcium ion was essential for the differentiation of IEC-6 cells. Retinol and retinoic acid induced the differentiation of IEC-6 cells. beta-LG stabilized and increased cell permeation of retinoic acid. IEC-6 cells showed 3 or 4 times more ALP activity with co-treatment of retinoic acid and beta-LG. BSA and OVA accelerated differentiation of IEC-6 cells in a similiar fashion to beta -LG. But, pepton and casein didn't. Heat destruction of beta-LG, BSA and OVA lead to loss in the ability of these compounds to induce cellular differentiation. The PKA signal pathway involved differentiation of IEC-6 cells. IEC-6 cells proliferation increased by the activation of PKC signal pathway and decreased differentiation by PKC signal pathway. CONCLUSION: Our results confirm that signal pathways are related to the proliferation and differ entiation of small intestinal epithelial cells and various compounds from food sources of childhood, such as beta-LG, BSA, OVA, and retinoic acid. These compounds appear to induce differentiation of small intestinal epithelial cells and may play a role in stimulating regeneration of epithelial cells after small intestinal mucosal injury.

The Change of Serum Insulin-Like Growth Factor Binding Protein 3 (IGFBP-3) in the Diagnosis and Management of Prostate Cancer / 대한비뇨기과학회지

PURPOSE: Insulin-like growth factor binding protein 3 (IGFBP-3) binds to IGF and acid labile subunit in blood, that regulates the action of IGF by modulating the interaction of IGF with the IGF receptor. Recent studies have shown that IGFBP-3 levels are decreased in prostate cancer patients. We examined IGFBP-3 profiles in prostate cancer patients and determined the effect of treatment on serum IGFBP-3 level in those patients. MATERIALS AND METHODS: Control groups were composed of age-matched healthy subjects and patients with benign prostatic hyperplasia (BPH). Patients with prostate cancer were divided into localized, locally advanced, metastatic and hormone-refractory. Serum samples were collected and stored at 70oC until use. The IGFBP-3 profile was measured by Western ligand blots. RESULTS: The serum IGFBP-3 level in patients with prostate cancer was significantly lower than healthy subjects or patients with BPH. Following the treatment of prostate cancer, IGFBP-3 was increased compared to that seen in pretreated prostate cancer. In hormone-refractory prostate cancer, IGFBP-3 was decreased again. However, there was no correlation between IGFBP-3 and tumor stage or Gleason score. CONCLUSIONS: These data show that IGFBP-3 levels are decreased in pretreated and hormone-refractory prostate cancer. Our results demonstrate that serum IGFBP-3 may be a useful marker in the detection and management of prostate cancer.

The Expression of Insulin-like Growth Factor (IGF), IGF- Binding Protein (IGFBP) and the Role of IGFBP-3 in the Korean Gastric Cancer Cell Lines / 대한암학회지

PURPOSE: Insulin-like growth factor (IGF)-I and II are potent mitogens, postulated to exert autocrine and paracrine effects on growth regulation in human gastric cancer. In this study, we evaluated the expression of IGF-I, -II and IGFBPs in a panel of human gastric cancer cell lines. We also evaluated whether high expression of IGFBP-3 in human gastric cancer cells may increase the sensitivity to the anti-proliferative agents. MATERIALS AND METHODS: 10 human korean gastric cancer ceIl lines and 1 Caucacian gastric adenocarcinoma cell line were used for this study. IGF and IGFBP expressions were evaluated by RT-PCR. IGFBP proteins in conditioned media were detected by Western Ligand Blot. Cell survival after treatment of anti-proliferative agents was assessed by MTT assay. RESULTS: IGF-I and II were expressed in all gastric cancer cell lines. In addition, IGF-I and II stimulated the proliferation of gastric cancer cells. The expression of IGFBP-2 was found in all gastric cancer cell lines. IGFBP-4 was expressed in the most of cell lines. IGFBP-3, -4 and -6 were expressed in about 50% of cell lines. The growth inhibition of IGFBP-3 expressing cells by anti- proliferative agents was more significant than that of IGFBP-3 nonexpressing cells. Cell growth inhibition with treatment of these agents was accompanied by increased IGFBP-3 mRNA level. CONCLUSION: These data confirm that IGF-I, -II, and certain IGFBPs were expressed in gastric cancer cells, and gastric cancer cells show the differential growth inhibition by anti-proliferative agents. The differential growth inhibitory effect of anti-proliferative agents is, at least in part, mediated through up-regulation of IGFBP-3 expression.

A Case of Neonatal Diabetes Mellitus: Transient or Permanent? / 대한소아내분비학회지

A case of neonatal diabetes mellitus is described. The child presented with low birth weight but was normal in appearance. She was acidotic and ketonuria was observed. The HLA typing was DR1 and 3, and insulin autoantibodies were negative. Genetic analysis with polymorphic DNA markers for chromosome 6 indicated biparental inheritance. She required insulin therapy for the control of hyperglycemia, and insulin dependence continues after 8 months of age.

Induction of Apoptosis of Ewing's Sarcoma Cells by Regulating Fusion Protein Expression

PURPOSE: Fusion genes(EWS-Fli-1 and EW.S-erg) function as transcription activators and are essential for maintaining tumorigenic properties in Ewing's sarcoma cells. Several reports have noted that Ets family transcription factors bind with CBP(CREB binding protein) in vitro. To understand the interaction of fusion proteins and CBP, we studied the CBP protein in TC135 cells expressing the EWS-Fli-1 gene. We also studied the hypothesis that downregulation of fusion gene expression may induce susceptibility to apoptosis in Ewing's sarcoma cells. METHODS: For targeting fusion proteins, we reconstructed the antisense EWS-fli-l, EWS-erg and CBP genes in pcDNA3, and transfected these genes to Ewing's sarcoma cells showing high levels of expression for Ve3 and 5838 genes. These vectors were transfected to cells by the calcium phosphate method, and transformed cells were selected using G418. We measured DNA fragments for apoptosis using FACScan. We used crystal violet staining and MTT assay to evaluate cell viability, and Western blot analysis was used to assess CBP gene expression. RESULTS: Cells transfected with antisense fusion genes Ve3 and 5838 showed inhibition of fusion protein expression. These cells also showed decreased cell viability. Susceptibility to apoptosis was induced by treatment with chemotherapeutic agents at low concentrations. Antisense CBP- transfected cells showed loss of cell viability in O.l% and 0.5% serum. This loss of cell viability was similar to the response by antisense fusion protein-transfected cells treated with chemotherapeutic agents at low concentrations. CONCLUSION: Our results suggest that fusion proteins and CBP co-regulate apoptosis in Ewing's sarcoma cells. Antisense fusion gene therapy may be an useful adjunct in combining with chemotherapeutic regimens to downregulate the expression of fusion proteins in Ewing's sarcoma.