jkbjts The Journal of the Korean Bone and Joint Tumor Society J Korean Bone Joint Tumor Soc 1226-4962 2233-9841 The Korean Bone and Joint Tumor Society 10.5292/jkbjts.2011.17.1.23 jkbjts-17-23 Original Article Over-expression of PTEN Involved in Troglitazone-induced Apoptosis in Human Osteosarcoma Cells YoonSun JungM.D., Ph.D. ZhouLuM.D. KimJung RyulM.D., Ph.D. Department of Orthopaedic Surgery, Medical School and Research Institute for Endocrine Science, Chonbuk National University, Jeonju, Korea Correspondence to: Jung Ryul Kim, M.D., Ph.D. Department of Orthopedic Surgery, Chonbuk National University Hospital, 634-18, Geumam-dong, Dugjin-gu, Jeonju 561-712, Korea TEL: +82-63-250-1767 FAX: +82-63-271-6538 E-mail: jrkeem@jbnu.ac.kr 12011 712011 171 2329 28022011 08052011 30052011 Copyright © 2011 The Korean Bone and Joint Tumor Society 2011 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Purpose

We investigated the effects of phosphatase and tensin homologue deleted on chromosome 10 gene phosphatase and tensin homologue deleted on chromosome 10 gene (PTEN) expression on the cell proliferation and on the responsiveness of troglitazone in osteosarcoma cells.

 Materials and Methods

Western blotting alnalysis was performed to detect the expression of PTEN in U-2OS cells treated with troglitazone. WST (water-soluble tetrazolium) assay was used to evaluate cell proliferation. Flow cytometry was used to determine cell apoptosis. Further, transfection of wild-type PTEN plasmid DNA was used to upregulate PTEN expression.

 Results

Troglitazone treatment induced growth inhibition of U2-OS cells in a dose- and time-dependent manner. Troglitazone increased the expression of PTEN in a dose-dependent manner. PTEN upregulation induced by troglitazone treatment resulted in cell growth inhibition and apoptosis in U-2OS cells. PTEN over-expression by plasmid transfection enhanced these effects of troglitazone. Moreover, no changes were observed in the mutant type-PTEN group.

 Conclusion

Upregulation of PTEN is involved in the inhibition of cell growth and induction of cell apoptosis by troglitazone. Further, PTEN over-expression can cause cell growth inhibition in osteosarcoma cells and these cell growth inhibitions could be enhance by troglitazone treatment.

 osteosarcoma apoptosis PTEN (phosphatase and tensin homologue deleted on chromosome 10 gene) PPARγ (peroxisome proliferator-activated receptor γ) agonist troglitazone REFERENCES Bramwell VH Osteosarcomas and other cancers of bone Curr Opin Oncol. 2000123306 Koeffler HP Peroxisome proliferator-activated receptor gamma and cancers Clin Cancer Res. 2003919 Young PW Buckle DR Cantello BC Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma J Pharmacol Exp Ther. 19982847519 Kim KY Kim SS Cheon HG Differential anti-proliferative actions of peroxisome proliferator-activated receptor-gamma agonists in MCF-7 breast cancer cells Biochem Pharmacol. 20067253040 Leslie NR Bennett D Lindsay YE Stewart H Gray A Downes CP Redox regulation of PI 3-kinase signalling via inactivation of PTEN EMBO J. 200322550110 Kim JY Kim TK Park JY Kim HJ Lee JW Effects of the peroxisome proliferator-activated receptor ligand troglitazone in osteosarcoma cell lines J Korean Orthop Assoc. 2005405917 Ishiyama M Miyazono Y Sasamoto K Ohkura Y Ueno K A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability Talanta 1997441299305 Ali IU Schriml LM Dean M Mutational spectra of PTEN/MMAC1 gene: a tumor suppressor with lipid phosphatase activity J Natl Cancer Inst. 199991192232 Zhou XP Gimm O Hampel H Niemann T Walker MJ Eng C Epigenetic PTEN silencing in malignant melanomas without PTEN mutation Am J Pathol. 200015711238 Moon SH Lee SH Kim HS Kim CH Chung TW Phosphatase and Tensin Homologue Deleted on Chromosome 10) in Osteosarcoma J Korean Orthop Assoc. 200338 3946 Maehama T Dixon JE The tumor suppressor, PTEN/MMAC1 dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-triphosphate J Biol Chem. 1998273133758 Stoll V Calleja V Vassaux G Downward J Lemoine NR Dominant negative inhibitors of signalling through the phos-phoinositol 3-kinase pathway for gene therapy of pancreatic cancer Gut. 20055410916 Pedrero JM Carracedo DG Pinto CM Frequent genetic and biochemical alterations of the PI 3-K/AKT/PTEN pathway in head and neck squamous cell carcinoma Int J Cancer. 20051142428 Kreisberg JI Malik SN Prihoda TJ Phosphorylation of Akt (Ser473) is an excellent predictor of poor clinical outcome in prostate cancer Cancer Res. 20046452326 Cao LQ Chen XL Wang Q Upregulation of PTEN involved in rosiglitazone-induced apoptosis in human hepatocellular carcinoma cells Acta Pharmacol Sin. 20072887987 Yim HW Jong HS Kim TY Cyclooxygenase-2 inhibits novel ginseng metabolite-mediated apoptosis Cancer Res. 200565195260 Okano H Shiraki K Inoue H 15-deoxy-delta-12-14-PGJ2 regulates apoptosis induction and nuclear factor-kappaB activation via a peroxisome proliferator-activated receptor-gamma-independent mechanism in hepatocellular carcinoma Lab Invest. 200383152939 Lemberger T Desvergne B Wahli W Peroxisome proliferator-activated receptors: a nuclear receptor signaling pathway in lipid physiology Annu Rev Cell Dev Biol. 19961233563 Jow L Mukherjee R The human peroxisome proliferator-activated receptor (PPAR) subtype NUC1 represses the activation of hPPAR alpha and thyroid hormone receptors J Biol Chem. 1995270383640 Caplan AI Bruder SP Mesenchymal stem cells: building blocks for molecular medicine in the 21st century Trends Mol Med. 2001725964 Haydon RC Zhou L Feng T Breyer B Nuclear receptor agonists as potential differentiation therapy agents for human osteosarcoma Clin Cancer Res. 20028128894 Haydon RC Luu HH He TC Osteosarcoma and osteoblastic differentiation: a new perspective on oncogenesis Clin Orthop Relat Res. 200745423746 Figures

Growth inhibition effects of troglitazone in the human osteosarcoma U-2OS cell line in a dose (A) and time (B) -dependent manner. Cells (1×105) were treated with various concentrations of troglitazone. Cell viability was determined by the WST assay and was presented as a calculated percentage of viable cells between troglitazone-treated and -untreated control cells. Each points represents the mean SEM of three determinations. †p<0.01 vs. untreated control.

Effect of troglitazone on the cell survival of osteosarcoma cells by flow cytometry. Histogram patterns of U-2OS cells treated with various concentrations (0, 10, 20, 30, 40, and 50 μM) of troglitazone for 48 hrs by FACS. Cell cycle distribution was analyzed by flow cytometry after coupled staining with Annexin V conjugated to fluorescein isothiocyanate (FITC) and Propidium iodide (PI) as described in Materials and Methods.

Dose-dependent effect of troglitazone on the PTEN expression. U2-OS cells (5×106) were treated with 5, 10, 20, and 30 μM of troglitazone for the indicated concentrations. A representative result was presented from among at least three separate experiments yielding similar results. Each points represents the mean SEM of three determinations. ∗p<0.05 vs. untreated control. †p<0.01 vs. untreated control.

Establishment of PTEN over-expression osteosarcoma cells using transfection. The PTEN expression in the U-2OS cells declined gradually after being treated with troglitazone for a longer time period (eg 72 hrs), since the activity of plasmid trasfection was decreased with time.

Transfection of active PTEN in U2-OS cells enhanced the inhibitory effect of troglitazone on cell growth. The effect of troglitazone-decreased cell viability was enhanced in the wild type-PTEN group, whereas no changes were observed in the mutant type-PTEN group. Each points represents the mean SEM of three determinations. ∗p<0.05 vs. untreated control. †p<0.01 vs. untreated control.