Tissues
Tissue specimens were obtained from patients who were surgically treated at Nagoya University Hospital. All tissue samples were fixed in 10% formalin, embedded in paraffin, and routinely stained with hematoxylin and eosin for histological examination. Endometrial adenocarcinomas were graded according to the criteria of the World Health Organization and classified as grade 1 (well-differentiated), grade 2 (moderately- differentiated), or grade 3 (poorly-differentiated). The relevant institutional review boards approved the study and informed consent from which all patients signed.
Cell lines and culture conditions
We used 3 human endometrial endometrioid adenocarcinoma cell lines (A-MEC, HEC1A, and Ishikawa). A-MEC was a kind gift from Aichi Medical University, Ishikawa from Dr. M. Nishida (Kasumigaura Hospital, Ibaragi, Japan), and HEC1A from Professor H. Kuramoto (Kitazato University, Kanagawa, Japan). Cells were maintained in RPMI 1640 medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal calf serum (FCS) and penicillin-streptomycin. These cells were incubated at 37 C in a humidified atmosphere of 5% CO2.
Immunohistochemistry
Immunohistochemical staining was performed using the avidin-biotin immunoperoxidase technique (Histofine SAB-PO kit, Nichirei, Tokyo, Japan). Sections were cut at a thickness of 4 μm, and immunostained by the streptavidin/biotin/peroxidase method. Deparaffinized sections in 0.01 M citrate buffer were treated three times for 5 min each at 90 C and 750 W using an H2500 microwave oven. Sections were incubated in 0.3 % hydrogen peroxide for 20 min and then further incubated with 10 % normal goat serum for 10 min to block the endogenous peroxidase activity and non-specific immunoglobulin binding, respectively. Rabbit polyclonal antibody (sc-710, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:100 dilution was used for insulin receptor (IR) a, rabbit polyclonal antibody (Upstate, Inc., Lake Placid, NY) at 1:200 dilution was used for insulin receptor substrate-1 (IRS-1), rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:50 dilution was used for GLUT4, and rabbit polyclonal antibody at 1: 100 was used for P-LAP/IRAP. They were added to the tissue sections and incubated for 1 h in a moist chamber at room temperature. Binding of the antibodies was followed with biotinylated goat anti-rabbit IgG and horse-radish peroxidase-conjugated streptavidin (Histofine SAB-PO, Nichirei). Chromogenic development was performed by immersion of the sections in 3-amino-9-ethylcarbazole (AEC, Nichirei). The slides were counterstained with Mayer's hematoxylin.
Plasmid construction and transfection
The eukariotic expression vector pcDNA3.1(-) (Invitrogen Japan K.K., Tokyo, Japan) was used to drive the expression of inserted P-LAP/IRAP cDNA. Transfections were carried out using Lipofectamine according to the manufacturer's instructions (Invitrogen Japan K.K., Tokyo, Japan). A-MEC cells were transfected with pcDNA3.1(-) (A-MEC-pc) or pcDNA3.1(-) inserted with P-LAP/IRAP cDNA (A-MEC-LAP). Stable transfectants were selected by growth in medium supplemented with 400 mg/ml of G418 (Sigma Chemical Co., St. Louis, MO). Several hundred clones resistant to G418 were obtained and polyclonal cells from these transfectants were used in the following experiments to eliminate any effects that could be attributed to clonal variation.
Western blotting
Cell lysates were electrophoresed on 7.5% sodium dodecyl sulfate polyacrylamide gel under reducing conditions. After electrophoresis, the proteins were transferred electrophoretically to an Immobilon membrane (Millipore, Bedford, MA, USA). After blocking, the membrane was incubated for 1 h with rabbit polyclonal antibody against human P-LAP/IRAP, GLUT4, insulin receptor, IRS-1, p-Ser307-IRS1 (Upstate Biotechnology, Lake Placid, NY), p-Tyr1158-IR(Abcam, Cambrige, UK), and mouse monoclonal antibody for phospho-AKT (587F11, Cell Signaling Technology, Inc.) and p85 PI-3 kinase (sc-1637, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) The membrane was washed with TBS-T for 15 min three times, and then incubated with peroxidase-conjugated goat anti-rabbit IgG for 1 h. After washing with TBS-T, the membrane was subjected to ECL-Western blotting detecting reagent (Amersham Biosciences K.K., Tokyo, Japan).
Cellular glucose uptake in P-LAP/IRAP transfection cells
A-MEC-pc and A-MEC-LAP cells were cultured in 12-well plates. Twenty-four h after plating, the cells were serum-starved for 2 h, then incubated in either the presence or absence of 10 nM insulin for 20 min, followed by the addition of 2-deoxy-D-2,6-3H-glucose to a final concentration of 1 μCi/ml. Insulin was purchased from Sigma (Japan).
Cell growth analysis
To evaluate the effect of insulin on cell proliferation, cells were seeded in triplicate in 96-well plates at a density of 5000 cells in a volume of 200 ml. Twenty-four h after plating, insulin was added to the culture medium at concentrations ranging from 10-8 to 10-7 M in the presence 5% FCS. Cell viability after 48 h was assayed using a modified tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay with the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay kit (Promega Corp., Tokyo, Japan) according to the manufacturer's instructions. Absorbance was measured at 490 nm by a microplate reader (Multiskan Bichromatic, Labsystems, Helsinki, Finland).
Statistical analysis
Statistical comparisons among groups were performed using Student's t-test and ANOVA with Bonferroni corrections. Differences between groups were considered significant at p < 0.05.