AKT INHIBITOR INHIBITS CELL PROLIFERATION IN MALIGNANT FIBROUS HISTIOCYTOMA CELLS

• Published in: Anticancer research Vol. 28; no. 5C
• Main Authors: Hitora, T, Kanji, S, Nakamura, O, Youji, K, Tetsuji, Y
• Format: Journal Article
• Language: English
• Published: 01-10-2008
• ISSN: 0250-7005

Introduction: The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays an important role in various cellular processes including cell growth, survival, and motility. Recently, accumulating evidence indicated that PI3K/Akt pathway plays a crucial role in tumorigenesis and tumor progression by promoting cell proliferation and inhibiting apoptosis. In addition, abnormal function of the PI3K/Akt pathway has been reported in many human tumors and this signaling pathway has been suggested to be a potential target for cancer chemotherapy. We examined the expression of Akt and the existence of PI3K/Akt signaling pathway in human malignant fibrous histiocytoma (MFH) cell lines, and the inhibitory effect of Akt inhibitor on the cell proliferation. Materials and Methods: Cell lines and reagent. Three human MFH cell lines (Nara-H, GBS-1, TNMY1) were used in this study. TNMY1 was previously established in our laboratory. All cell lines were grown in culture medium consisting of Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich). The cell lines were routinely maintained at 37 degree C in a humidified 5% CO sub(2) atmosphere. Akt inhibitor X, a specific Akt kinase inhibitor was purchased from Calbiochem (San Diego, CA, USA). The inhibitory effect of Akt inhibitor. The cell proliferation was assayed using the MTS assay (CellTiter 96 registered Aqueous One Solution Cell Proliferation Assay; Promega, Madison, WI, USA). Cells were seeded in 96-well cell culture plates in culture medium with 10% FBS. After 24 hours (h), the medium was refreshed with 1% FBS containing AKT inhibitor in the indicated concentrations. After 24 and 48 h, the medium was removed and washed with phosphate-buffered saline, then refreshed with fresh medium containing MTS reagent. The optical density was measured at 490 nm using an automatic microplate reader after 2 h of further incubation. The percent age viability of each well was calculated. At least three independent cultures were performed for each study. The data were analyzed statistically using ANOVA with Fisher's PLSD post hoc test. Western blotting. Cells were pretreated for 60 min with 1% FBS containing AKT inhibitor at different concentrations. Whole cell lysates were collected for protein content, and cell lysates were separated by SDS polyacrylamide gel electrophoresis under reducing conditions. Then gels were electrophoretically transferred to PVDF membrane, and immunoblotted with anti-AKT1/2/3 antibody (Santa Cruz Biotechnology, CA, USA) and anti-phospho-AKT1/2/3 antibody (Ser 473, Thr 308; Santa Cruz Biotechnology). Bound antibodies were detected using the ECL plus Western blotting detection system (GE Healthcare Bio-Sciences, Piscataway, NJ). Results: The effect of the Akt inhibitor: Akt inhibitor inhibited the cell proliferation of all 3 cell lines in a dose- and time-dependent manner; 10 mu M AKT inhibitor inhibited the cell proliferation of Nara-H and GBS-1 at the percent viability of 50% or less; 25 uM AKT inhibitor inhibited the cell proliferation of TNMY1 at viability of 50% or less. Expression of Akt and phospho-Akt: Western blotting analysis revealed that not only Akt but phospho-Akt were expressed in all cell lines under the normal condition, so it was suggested that Akt signaling pathway was always activated in all 3 cell lines under the normal condition. Phosphorylation of Akt was reduced by 25 mu M Akt inhibitor in all cell lines. Discussion: The PI3K/Akt pathway is very important as a target of the molecular targeting therapy. In our study, Akt inhibitor showed a dose-dependent inhibitory effect on the cell proliferation of human MFH cells. Akt inhibitor reduced the phosphorylation of Akt. These results suggest that the PI3K/Akt signaling pathway exists and plays an important role in cell proliferation in MFH cells. Although further studies are needed to explore the mechanisms of cell proliferation, Akt inhibitor will be a potent chemotherapeutic agent for human MFH.