Koji Murao

The antidiabetic drug metformin inhibits gastric cancer cell proliferation in vitro and in vivo

Recent studies suggest that metformin, which is commonly used as an oral anti-hyperglycemic agent of the biguanide family, may reduce cancer risk and improve prognosis, but the mechanisms by which metformin affects various cancers, including gastric cancer, remains unknown. The goal of the present study was to evaluate the effects of metformin on human gastric cancer cell proliferation in vitro and in vivo and to study microRNAs (miRNA) associated with antitumor effect of metformin. We used MKN1, MKN45, and MKN74 human gastric cancer cell lines to study the effects of metformin on human gastric cancer cells. Athymic nude mice bearing xenograft tumors were treated with or without metformin. Tumor growth was recorded after 4 weeks, and the expression of cell-cycle-related proteins was determined. In addition, we used miRNA array tips to explore the differences among miRNAs in MKN74 cells bearing xenograft tumors treated with or without metformin in vitro and in vivo. Metformin inhibited the proliferation of MKN1, MKN45, and MKN74 in vitro. Metformin blocked the cell cycle in G0–G1 in vitro and in vivo. This blockade was accompanied by a strong decrease of G1 cyclins, especially in cyclin D1, cyclin-dependent kinase (Cdk) 4, Cdk6 and by a decrease in retinoblastoma protein (Rb) phosphorylation. In addition, metformin reduced the phosphorylation of epidermal growth factor receptor and insulin-like growth factor-1 receptor in vitro and in vivo. The miRNA expression was markedly altered with the treatment of metformin in vitro and in vivo. Various miRNAs altered by metformin also may contribute to tumor growth in vitro and in vivo. Mol Cancer Ther; 11(3); 549–60. ©2012 AACR.

Effects of Glucose and Insulin on Rat Apolipoprotein A-I Gene Expression

We have examined the regulation of apolipoprotein A-I (apoA-I) gene expression in response to glucose and insulin. In Hep G2 cells, endogenous apoA-I mRNA was suppressed by one-half or induced 2-fold following 48 h of exposure to high concentrations of glucose (22.4 mm) or insulin (100 microunits/ml), respectively, compared with control. Transcriptional activity of the rat apoA-I promoter (−474 to −7) in Hep G2 cells paralleled endogenous mRNA expression, and this activity was dependent on the dose of glucose or insulin. Deletional analysis showed that a 50-base pair fragment spanning −425 to −376 of the promoter mediated the effects of both insulin and glucose. Within this DNA fragment there is a motif (−411 to −404) that is homologous to a previously identified insulin response core element (IRCE). Mutation of this motif abolished not only the induction of the promoter by insulin but also abrogated its suppression by glucose. Electrophoretic mobility shift assay analysis of nuclear extracts from Hep G2 cells revealed IRCE binding activity that formed a duplex with radiolabeled probe. The IRCE binding activity correlated with insulin induction of apoA-I expression. In summary, our data show that glucose decreases and insulin increases apoA-I promoter activity. This effect appears to be mediated by a single cis-acting element.

Thiazolidinedione inhibits the production of monocyte chemoattractant protein-1 in cytokine-treated human vascular endothelial cells

The chemokine monocyte chemoattractant protein‐1 is a potent chemoattractant for monocytes. Monocyte chemoattractant protein‐1 is produced by vascular endothelial cells during inflammatory diseases such as atherosclerosis. In this study, we examined the effects of a thiazolidinedione on monocyte chemoattractant protein‐1 expression in human vascular endothelial cells. In human vascular endothelial cells, interleukin‐1β and tumor necrosis factor‐α induced endogenous monocyte chemoattractant protein‐1 protein secretion, mRNA expression and promoter activity. The thiazolidinedione inhibited these effects. In summary, our results indicated that the suppression of the expression of monocyte chemoattractant protein‐1 can be accomplished by thiazolidinedione treatment, raising the possibility that thiazolidinedione may be of therapeutic value in the treatment of diseases such as atherosclerosis.

A Mutant High-Density Lipoprotein Receptor Inhibits Proliferation of Human Breast Cancer Cells

High-density lipoprotein (HDL) stimulates the growth of many types of cells, including those of breast cancer. High levels of HDL are associated with an increased risk of breast cancer development. A scavenger receptor of the B class (SR-BI)/human homolog of SR-BI, CD36, and LIMPII analogous-1 (CLA-1) facilitates the cellular uptake of cholesterol from HDL and thus augments cell growth. Furthermore, HDL is also believed to have antiapoptotic effects on various cell types, and this feature adds to its ability to promote cell growth. These collaborative roles of HDL and CLA-1 prompted us to assess the function of these components on human breast cancer cells. In this study, we created a mutant CLA-1 (mCLA) that lacked the COOH-terminal tail to determine its potential role in breast cancer cell growth. Expression of mCLA inhibited the proliferation of breast cancer cell line MCF-7. This inhibitory action of mCLA required the transcriptional factor activator protein-1 (AP-1), and the mutant receptor also affected the antiapoptotic features of HDL. The effect of HDL on AP-1 activation and [3H]thymidine incorporation was abrogated by wortmannin, a specific inhibitor of phosphoinositide 3-kinase. Furthermore, the dominant negative mutant of Akt abolished the ability of HDL to activate AP-1. These findings raise the possibility that the inhibitors of the effects of HDL may be of therapeutic value for breast cancer.

Molecular cloning and gene expression of growth hormone-releasing peptide receptor in rat tissues.

We cloned a fragment of the rat GH-releasing peptide (GHRP) receptor homologue and examined the tissue distribution of GHRP receptor mRNA in rats. Sequence analysis showed that the open reading frame is well conserved between rat and human with 96% identity in a 364-amino acid overlap. By reverse transcription-polymerase chain reaction we detected GHRP receptor mRNAs in the rat brain including the hypothalamus, anterior pituitary, and renal pelvis in twenty-eight tissues tested. Microdissection revealed that GHRP receptor mRNAs were localized predominantly in the arcuate nucleus and ventromedial hypothalamus.

Expression of Human Scavenger Receptor B1 on and in Human Platelets

Objective—The abundance of HDL particles correlates inversely with the incidence of coronary heart disease. The human scavenger receptor B1 (hSR-B1/CLA-1) is a receptor for HDL. Expression of hSR-B1/CLA-1 mRNA and protein in human platelets was determined using reverse transcriptase–polymerase chain reaction and Western blot, respectively. Presence of the protein on the surface of platelets was shown using flow cytometry. Methods and Results—Immunochemical staining for hSR-B1/CLA-1 showed that it was expressed in megakaryocytes, the platelet precursors of human bone marrow. These findings prompted us to ask whether hSR-B1/CLA-1 was differentially expressed on platelets obtained from patients with atherosclerotic disease compared with those in control subjects. Our findings showed that abundance of hSR-B1/CLA-1 was significantly reduced on the surface of platelets from patients with atherosclerotic disease. The reduced levels of hSR-B1/CLA-1 were associated with increased cholesterol ester content in platelets from patients with atherosclerotic disease compared with control subjects. A negative correlation existed between hSR-B1/CLA-1 expression and platelet aggregation. In summary, our studies show that the HDL receptor hSR-B1/CLA-1 is expressed in platelets and their precursor, the megakaryocyte. The levels of hSR-B1/CLA-1 expression correlate inversely with cholesterol ester content and platelet aggregation. Conclusion—These findings suggest that determining the level of hSR-B1/CLA-1 expression on the platelets may be a useful clinical marker for atherosclerotic diseases.

Rare sugar D-psicose improves insulin sensitivity and glucose tolerance in type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats

A rare sugar, D-psicose has progressively been evaluated as a unique metabolic regulator of glucose and lipid metabolism, and thus represents a promising compound for the treatment of type 2 diabetes mellitus (T2DM). The present study was undertaken to examine the underlying effector organs of D-psicose in lowering blood glucose and abdominal fat by exploiting a T2DM rat model, Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Rats were fed 5% D-psicose or 5% D-glucose supplemented in drinking water, and only water in the control for 13 weeks and the protective effects were compared. A non-diabetic Long-Evans Tokushima Otsuka (LETO), fed with water served as a counter control of OLETF. After 13 weeks feeding, D-psicose treatment significantly reduced the increase in body weight and abdominal fat mass. Oral glucose tolerance test (OGTT) showed the reduced blood glucose and insulin levels suggesting the improvement of insulin resistance in OLETF rats. Oil-red-O staining elucidated that D-psicose significantly reduced lipid accumulation in the liver. Immunohistochemical analysis showed D-psicose induced glucokinase translocation from nucleus to cytoplasm of the liver which enhances glucokinase activity and subsequent synthesis of glycogen in the liver. D-psicose also protected the pathological change of the β-cells of pancreatic islets. These data demonstrate that D-psicose controls blood glucose levels by reducing lipotoxicity in liver and by preserving pancreatic β-cell function.

Human Scavenger Receptor B1 Is Involved in Recognition of Apoptotic Thymocytes by Thymic Nurse Cells

Recognition and uptake of apoptotic cells by neighboring phagocytes is essential for the clearance of dying cells without accompanying inflammation or tissue damage. In the thymus, many apoptotic cells are generated in the process of negative selection, and both thymic macrophages (professional phagocytes) and nursing thymic epithelial cells (nursing TEC; nonprofessional phagocytes) recognize and ingest them. However the receptors responsible for this recognition and uptake have not been identified. In the present study, we have established a human nursing TEC line and examined the expression of several genes of the scavenger receptor family considered to be potential receptors for apoptotic cells. Human scavenger receptor-B1 (hSR-B1)/CLA-1, previously shown to recognize apoptotic cells, was strongly expressed in nursing TEC, whereas there was little or no expression of the other scavenger receptors tested: scavenger receptor class A, CD36, or CD68. Suppression of hSR-B1/CLA-1 expression using antisense oligonucleotides decreased the binding of apoptotic thymocytes to nursing TEC by more than 40%. These results indicate that hSR-B1/CLA-1 may play a major role in the clearance of apoptotic cells in the thymus, mediating the recognition and ingestion of apoptotic thymocytes by nursing TEC.

Gene expression of somatostatin receptor subtypes, SSTR1 and SSTR2, in human lung cancer cell lines.

Somatostatin (SS) acts as a universal endocrine off-swich, and also inhibits the growth of neuroendocrine tumors through its specific receptors. Small cell lung cancer (SCLC) demonstrates some neuroendocrine characteristics and has been proposed as a candidate for treatment with SS and its analogues. In the present study, we investigated the expression of somatostatin receptor (SSTR) subtype (SSTR1 and SSTR2) mRNA in various human lung cancer cell lines by the sensitive reverse-transcription-PCR method and Southern blotting. The levels of expression of SSTR1 mRNA were higher in both SCLC and squamous cell carcinoma than in adenocarcinoma cell lines. Interestingly, SSTR1 gene expression was independent of that of SSTR2 in each SCLC cell line, although the expression of both genes showed a positive correlation in non-SCLC cells. Membranes from a cell line exhibiting highest expression of SSTR2 gene bound SS and its analogue, octreotide, with moderate affinity. These findings may provide useful information for the future clinical application of SS and its analogues for the treatment of lung cancer.

Effect of the anti-diabetic drug metformin in hepatocellular carcinoma in vitro and in vivo.

Metformin is a commonly used oral anti-hyperglycemic agent of the biguanide family. Recent studies suggest that metformin may reduce cancer risk and improve prognosis. However, the antitumor mechanism of metformin in several types of cancers, including hepatocellular carcinoma (HCC), has not been elucidated. The goal of the present study was to evaluate the effects of metformin on HCC cell proliferation in vitro and in vivo, and to study microRNAs (miRNAs) associated with the antitumor effect of metformin in vitro. We used the cell lines Alex, HLE and Huh7, and normal hepatocytes to study the effects of metformin on human HCC cells. In an in vivo study, athymic nude mice bearing xenograft tumors were treated with metformin or left untreated. Tumor growth was recorded after 4 weeks, and the expression of cell cycle-related proteins was determined. Metformin inhibited the proliferation of Alex, HLE and Huh7 cells in vitro and in vivo. Metformin blocked the cell cycle in G0/G1 in vitro and in vivo. This blockade was accompanied by a strong decrease of G1 cyclins, especially cyclin D1, cyclin E and cyclin-dependent kinase 4 (Cdk4). In addition, microRNA (miRNA) expression was markedly altered by the treatment with metformin in vitro and in vivo. In addition, various miRNAs induced by metformin also may contribute to the suppression of tumor growth. Our results demonstrate that metformin inhibits the growth of HCC, possibly by inducing G1 cell cycle arrest through the alteration of microRNAs.