ANTICANCER RESEARCH 28 : 3593-3600 (2008)

Pioglitazone, a Ligand for Peroxisome Proliferator-activated Receptor-γ Acts as an Inhibitor of Colon Cancer Liver Metastasis SHOTA TAKANO, TETSURO KUBOTA, HIDEKI NISHIBORI, HIROTOSHI HASEGAWA, YOSHIYUKI ISHII, NOBUHIRO NITORI, HIROKI OCHIAI, KOJI OKABAYASHI, YUKO KITAGAWA, MASAHIKO WATANABE and MASAKI KITAJIMA

Department of Surgery, Keio University School of Medicine, Shinanomachi 35, Shinjuku-ku, Tokyo 160-8582, Japan

Abstract. Background: Peroxisome proliferator-activated proliferator-activated receptor (PPAR) is a member of the receptor-γ (PPARγ) is a member of the steroid receptor nuclear hormone receptor family, the largest family of superfamily. Liganded PPARγ can inhibit cancer cell transcription factors (1). PPARγ is highly expressed in proliferation. The in vitro and in vivo inhibitory effect of the multiple cell types, including adipose tissue, various synthetic ligands, ciglitazone (CGZ) and pioglitazone (PGZ), carcinomas and immune cells. PPARγ has an influence on on human colon cancer was investigated. Materials and adipocyte differentiation and its role has been suggested in Methods: Cell proliferation and the expression of PPARγ, cell proliferation, immunoregulation and inflammation (2). cyclooxygenase (COX)-2 and cyclin D1 were assessed in Its expression in colon cancer cells is well established and colon cancer cells treated with CGZ or PGZ . After ligand activation can inhibit the proliferation of cancer cells subcutaneous or splenic inoculation of severe combined and induce cell differentiation and apoptosis (3), although immunodeficient (SCID) mice using colon cancer HT-29 and several reports have suggested that PPARγ agonists may SW480 cells, PGZ was administered orally and tumor growth promote colon cancer tumorigenesis under certain inhibition was assessed by xenograft volume. The COX-2, circumstances (4, 5). PPARγ heterodimerizes with the cyclin D1 and PPARγ expression in the HT-29 cells was retinoid X receptor and binds to specific DNA-binding sites, evaluated. Results: Cultured HT-29 and SW480 cells expressed known as the peroxisome proliferater response element PPARγ and proliferation was inhibited by CGZ and PGZ. Oral (PPRE), to regulate transcription of numerous target genes. PGZ inhibited xenograft tumor growth and liver metastases in Forman et al. and Kliewer et al. identified 15-deoxy- the SCID mouse and suppressed expression of COX-2 and prostaglandin J2 (15d-PGJ2) as a PPARγ endogenous ligand cyclin D1 in HT-29 cells. Conclusion: PGZ down-regulates (6, 7). Thiazolidinediones (TZD) and non-steroidal anti- COX-2 and cyclin D1 and inhibits colon cancer proliferation inflammatory drugs (NSAIDs) are known synthetic PPARγ and liver metastasis, making PPARγ a candidate target for the ligands, as are troglitazone (TGZ), ciglitazone (CGZ), treatment/prevention of colon cancer metastasis. rosiglitazone (RGZ) and pioglitazone (PGZ). Since induction of differentiation is a non-toxic therapeutic The prevention and control of hepatic metastasis remain approach, PPARγ ligands, such as PGZ, a clinically used problematic in the treatment of colonic cancer. In spite of anti-diabetic drug, may be candidates for a novel, non-toxic recent remarkable advances in colon cancer chemotherapy, approach to the prophylactic or chemopreventive treatment the toxicity of the cytotoxic agents used has a significantly of colon cancer (8-11). It has been reported that TZD s, negative impact on quality of life. The peroxisome mainly TGZ, inhibit the growth of colon cancer cells in vitro and in vivo (12). It has been shown by luciferase assay, that TGZ transactivates the transcription of a PPRE (13). While high dose (>100 μM) PPARγ ligand-induced apoptosis is Correspondence to: Tetsuro Kubota, MD, Center for Comprehensive known, cancer cell growth is inhibited by G1 cell cycle arrest and Advanced Medicine, Keio University Hospital, Shinanomachi rather than apoptosis at a dose of about 10 μΜ (14). The 35, Shinjuku-ku, Tokyo 160-8582, Japan. Tel: +81 353633966, Fax: +81 333530246, e-mail: [email protected] purpose of the present study was to determine the inhibitory effect and mechanism of PPARγ ligands on the growth and Key Words: Pioglitazone, colon cancer, liver metastasis. liver metastasis of human colon cancer.

0250-7005/2008 $2.00+.40 3593 ANTICANCER RESEARCH 28 : 3593-3600 (2008)

Figure 1. Expression of PPARγ and inhibitory effect of pioglitazone (PGZ) on colon cancer cell lines in vitro. Western blotting shows PPARγ expression in all colon cancer cell lines tested (lower panel). The cells were incubated with 20 or 50 μM PGZ for 6 days, and the number of cells was counted (upper panel). The experiments were triplicated.

Materials and Methods were then probed with anti-PPARγ mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by Colon cancer cell lines. The colon cancer cell lines used were incubation with secondary antibody (anti-mouse IgG). Specific HT-29, WiDr, SW480, LoVo and HCT116, which were obtained bands were visualized by electrochemiluminescence (ECL) Western from the American Type Culture Collection, Manassas, VA, USA. Blotting Detection Reagents (Amersham Biosciences, They were cultured in RPMI-1640 with 10% fetal bovine serum Buckinghamshire, England). (FBS) at 37°C using 5% CO 2. Cell proliferation assay. The colon cancer cells were incubated with Chemicals. PGZ was kindly provided by Takeda Chemical 20 or 50 μM PGZ or DMSO. After incubation for 6 days, the cells Industries (Osaka, Japan). CGZ was purchased from Wako Pure were counted, and the tumor inhibition rate (IR) was calculated Chemical Industries (Tokyo, Japan). The agents were dissolved in using the following formula: IR=100 – (treated-cell count)/(control- dimethyl sulfoxide (DMSO). cell count) ×100 (%). HT-29 and SW480 cells were also cultured with 10-100 μM PGZ Animals. Four-week-old male severe combined immunodeficient or CGZ, for 2, 4 or 6 days and the inhibiting curves were drawn by (SCID) mice with a genetic background of CB-17/lcr-scid/Jcl were plotting IR against the concentration. purchased from CLEA Japan Inc. (Tokyo, Japan). The mice were used in this study after they were kept in our facility for 1 week. SCID mouse xenograft model of colon cancer. HT-29 or SW480 The mice were maintained according to the rules of our Institutional cells at 1×10 7 cells/mouse were injected subcutaneously into the Review Board. flank of SCID mice on day 0. From day 7, 200 mg of PGZ per kg suspended in 0.2 ml of DMSO per mouse or DMSO alone (control Expression of PPARγ in colon cancer cell lines. Western blot vehicle) was orally administered to the mice for 5 consecutive days analysis was performed according to the method of Yamauchi et al. followed by 2 days of rest, for 5 weeks. The inhibitory effect was (15). The colon cancer cells were incubated on 6 cm plates for 2 assessed according to the volume of the tumor xenograft, which was days. Whole cell lysates were extracted from the cultured cells with estimated using the following formula: TV=a × b 2/2 (TV: estimated radioimmunoprecipitation assay buffer. Ten μg of each cell lysate tumor volume [mm 3], a: length [mm], b: width [mm]). were assayed using the Bio-Rad DC Protein Assay Kit (Bio-Rad The growth curve was drawn by plotting the mean TV with Laboratories, Hercules, CA, USA), then revolved in a 10% SDS standard deviation against time. Upon completion of the experiments, PAGE gel and transferred to Immobillon transfer membranes the IR was calculated as 1 – (TV of treated tumor/TV of control (Millipore, Billerica MA, USA) via electroblotting. The membranes tumor) in %, and IRs were compared statistically by Student’s t-test.

3594 Takano et al : Pioglitazone Inhibits Colon Cancer Liver Metastasis

Figure 2. The in vitro inhibitory effect of pioglitazone (PGZ) and ciglitazone (CGZ) on HT-29 and SW480 cells. HT-29 and SW480 cells were incubated with 10-100 μM PGZ, CGZ, or vehicle alone and the number of cells was counted, and the inhibition rate calculated. The experiments were triplicated. * p< 0.05.

3595 ANTICANCER RESEARCH 28 : 3593-3600 (2008)

Figure 3. Effect of PGZ on tumor growth after HT-29 and SW480 cells were subcutaneously implanted in SCID mice. After injection, 200 mg of PGZ per kg or vehicle alone was orally administered for 5 consecutive days followed by 2 days rest, for 5 weeks. The inhibitory effect was assessed according to the xenograft volume. * p=0.031, HT-29; and p=0.028, SW480.

The mice were weighed twice weekly to assess for toxicity of PGZ. The primary antibodies used were anti-PPARγ rabbit polyclonal Two days after the last administration, the mice were sacrificed by antibody (Santa Cruz Biotechnology), anti-COX-2 mouse cervical dislocation under ether-anesthesia, the liver was weighed monoclonal antibody (Cayman Chemical), and anti-cyclin D1 and the xenografts were removed for immunohistochemical analysis. monoclonal antibody (BD Biosciences-Pharmingen).

Murine models of colon cancer liver metastasis. HT-29 or SW480 at Statistical analysis. The differences in number of tumor cells, tumor 3×10 5 cells/mouse were injected into the spleen of SCID mice on volume, liver volume and the number of liver metastases were day 0 under ether-anesthesia. From day 1, either 200 mg of PGZ analyzed by Student’s t- test. Differences with p-values of <0.05 per kg suspended in 0.2 ml of DMSO per mouse or DMSO control were statistically significant. vehicle was orally administered to the mice for 5 consecutive days followed by 2 days of rest, for 3 weeks. The mice were sacrificed Results two days after the last administration of PGZ by cervical dislocation under ether-anesthesia. The inhibitory effect of PGZ on liver metastasis was assessed according to the number of metastatic By Western blotting assessment, PPARγ was expressed in all nodules on the liver surface and the total liver weight at the end of the cell lines tested (Figure 1, lower panel). After incubation experiments on day 21. The IR of metastasis was calculated using with PGZ, tumor cell growth was inhibited in a the following formula: IR of metastasis = 100 – (number of concentration-dependent manner (Figure 1, upper panel). metastatic nodules on the liver surface of treated mice)/(number of The proliferation of the HT-29 and SW480 cells was also metastatic nodules on the liver surface of control mice) ×100 (%). inhibited by CGZ as well as PGZ in a concentration- and Immunohistochemistry. The xenografts used for the subcutaneous time-dependent manner (Figure 2a and b). There was a treatment by PGZ in the SCID mouse model were used for the statistically significant ( p< 0.05) difference between the immunohistochemical assay according to the method described by incubation times of 2 and 6 days, and the concentrations of Yamauchi et al. (16). The subcutaneous xenografts of HT-29 were 10 and 100 μM in each incubation time. The antitumor effect fixed in 20% formalin. Paraffin-embedded sections were incubated of PGZ on the HT-29 and SW480 cells in the SCID mouse is with Mouse on Mouse (MOM) IgG blocking reagent (Vector shown in Figure 3, where the subcutaneous tumor growth Laboratories, Burlingame, CA, USA) to reduce the non-specific staining of mouse tissue by mouse antibodies. After blocking with was inhibited by PGZ. The inhibition rates after 5 weeks of biotinylated anti-mouse IgG blocking reagent (Vector Laboratories), PGZ were 60.0% for HT-29 and 60.6% for SW480 with a the sections were stained using the Dako EnVision System (Dako statistically significant difference of p= 0.031 and p= 0.028, Corporation, Carpinteria, CA, USA). respectively. No significant differences in the body weights

3596 Takano et al : Pioglitazone Inhibits Colon Cancer Liver Metastasis

Figure 4. Effect of PGZ on colon cancer liver metastasis in SCID mice. HT-29 or SW480 cells were injected into the spleen of SCID mice. After injection, 200 mg of PGZ per kg or vehicle alone was orally administered for 5 consecutive days followed by 2 days of rest, for 3 weeks. Liver metastasis was assessed by the number of metastatic nodules on the liver surface and the total liver weight. * p<0.05.

of the mice or the liver weights were observed (data not shown), suggesting that these PGZ dosages and incubation time periods had no adverse effect on the tumor-bearing SCID mice. The oral administration of PGZ significantly reduced the number of metastatic nodules and the total liver weight of the SCID mice inoculated with HT-29 or SW480 cells ( p< 0.05) (Figure 4). When the expression of COX-2, cyclin D1 and PPARγ in the HT-29 cells was assessed by Western blotting in vitro (Figure 5), PGZ was shown to suppress the expression of COX-2 and cyclin D1 but not PPARγ in a dose-dependent manner. The effect on the expression of COX-2, cyclin D1 and PPARγ was also assessed in the xenograft of the SCID mice by immunohistochemical staining (Figure 6). PPARγ expression was localized in the nucleus and cytoplasm of the tumor cells and no variation was observed regarding the expression levels and localization in the PGZ-treated and control mice. COX-2 expression was localized in the cytoplasm, while cyclin D1 expression was localized in the nucleus of the tumor cells. The expression of COX-2 and Figure 5. Effect of PGZ on the expression of PPARγ, COX-2 and cyclin cyclin D1 was decreased in the PGZ-treated tumors D1 in HT-29 cells. HT-29 cells were incubated with 20 or 50 μM PGZ or vehicle alone for 2 days. Expression of COX-2, cyclin D1 and PPARγ compared to the controls. was assessed by Western blotting. The experiments were triplicated. Discussion

In the present study, the synthetic PPARγ ligands, CGZ and Furthermore, PGZ suppressed the expression of COX-2 and PGZ, inhibited the growth of colon cancer cells in both a time- cyclin D1 both in vitro and in vivo . These data suggest that the and dose-dependent manners as does TGZ and had an suppression of these molecules was related to the inhibitory inhibitory effect on the proliferation and liver metastasis of effect of PGZ on the colon cancer cells. It is thought that TZD colon cancer cell lines HT-29 and SW480, in vivo . transactivation of PPRE influences or controls gene expression

3597 ANTICANCER RESEARCH 28 : 3593-3600 (2008)

Figure 6. Effect of PGZ on the expression of PPARγ, COX-2 and cyclin D1 in HT-29 xenografts. After 5 weeks of treatment with PGZ or vehicle, the subcutaneous tumors were assessed by immunohistochemistry. COX-2 and cyclin D1 expression showed greater decrease in PGZ-treated mice, compared to untreated mice, but PPARγ expression did not differ (Original magnification, ×400).

in a manner resulting in G1 cell cycle arrest. Furthermore, SW480 xenografts, suggesting that PPARγ inhibited the cyclin D1, c-myc, β-catenin and COX-2 have been reported as tumor growth by down-regulating cyclin D1 and COX-2. target genes of PPARγ (17-20). The COX-2 and cyclin D1 The precise role of TZD in liver metastasis of cancer is not suppression by PGZ suggested that PGZ inhibited the growth yet fully understood. Speculation about potential mechanisms of the colon cancer cells by controlling the expression of these includes reports that TGZ inhibits the production of matrix molecules. Cyclin D1 plays a pivotal role in the development metalloproteinase-7 and interferes with adhesion of colon of a subset of human carcinomas, including parathyroid cancer cells (25). Additionally, PPARγ inhibits cell adenoma, breast cancer, and colon cancer and promotes invasion/motility and alters cell morphology in human progression through the G1-S-phase of the cell cycle (21). On pancreatic cancer (26). These reports suggest that TZD the other hand, it is known that COX-2 is overexpressed in inhibits liver metastasis by acting on cancer cell movement, cancer of the colon, stomach, esophagus and lung, and is on inflow and outflow into blood vessels, and on cell related to growth, infiltration and metastasis of these cancer adhesion. Further experimental studies to elucidate the cells (22). Furthermore, Inoue et al. proposed the idea that the mechanism of TDZ in cancer metastasis are required and expression of COX-2 is regulated by a negative feedback loop should include more in-depth investigation of these pathways. mediated through PPARγ (23). Recent investigation has provided amazing advances in the Coi et al. reported that low-dose PPARγ ligand promoted area of chemotherapy for colon cancer, including cancer that the growth of adenomatous polyposis coli (APC)-mutated has metastasized to the liver. While surgical resection is the HT-29 in vitro and in vivo (4). The AP C gene mutation is the first choice for localized liver metastasis, conventional most common cause of colon cancer related to the cytotoxic regimens with monoclonal antibodies, bevacizumab wingless/int (Wnt)/β-catenin pathway (24). The present and cetuximab, show promise in terms of tumor reduction and administration of a high dose (200 mg/kg) of PGZ to the prolonged survival periods. However, a multitude of adverse mice may have suppressed the expression of cyclin D1, as a effects such as bone marrow suppression, gastrointestinal result of inhibitory effects on the β-catenin pathway. In spite toxicity and neurotoxicity remain factors that severely of the high dosage of PGZ, the body and liver weights of the compromise the patient’s quality of life. In view of this, PGZ SCID mice did not indicate toxic effects. A representative would be an ideal candidate drug for cancer treatment as it is PPARγ ligand was reported to inhibit both lung and lymph already used clinically for diabetes with minimal toxic side- node metastases of human colon cancer cells, involving effects. Since the inhibitory effect of PGZ alone against liver modulation of the E-cadherin/β-catenin system and the metastasis of colon cancer in the SCID mouse model is differentiation-promoting system (11). In the present study, limited, potential combined therapy with other existing PGZ significantly inhibited the liver metastasis of HT-29 and chemotherapy regimens is promising.

3598 Takano et al : Pioglitazone Inhibits Colon Cancer Liver Metastasis

In conclusion, PPARγ may provide a target for the 15 Yamauchi T, Watanabe M, Hasegawa H, Nishibori H, Ishii Y, prevention and treatment of primary and metastatic colon Tatematsu H, Yamamoto K, Kubota T and Kitajima M: The cancer. potential for a selective cyclooxygenase-2 inhibitor in the prevention of liver metastasis in human colon cancer. Anticancer Res 23 : 245-250, 2003. References 16 Yamauchi T, Watanabe M, Kubota T, Hasegawa H, Ishii Y, Endo T, Kabeshima Y, Yorozuya K, Yamamoto K, Mukai M and Kitajima 1 Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, M: Cyclooxygenase-2 expression as a new marker for patients with Umesono K, Blumberg B, Kastner P, Mark E, Chambon P and colorectal cancer. Dis Colon Rectum 45 : 98-103, 2002. Evans RM: The nuclear receptor superfamily: the second decade. 17 Wang C, Fu M, D’Amico M, Albanese C, Zhou JN, Brownlee M, Cell 83 : 835-839, 1995. Lisanti MP, Chatterjee VKK, Lazar MA and Pestell RG: Inhibition 2 Fajas L, Debrill MB and Auwerx J: Peroxisome proliferator- of cellular proliferation through I κB kinase-independent and activated receptor-gamma: from adipogenesis to carcinogenesis. peroxisome proliferator-activated receptor γ-dependent repression J Mol Endocrinol 27 : 1-9, 2001. of cyclin D1. Mol Cell Biol 21 : 3057-3070, 2001. 3 Bull AW: The role of peroxisome proliferator-activated receptor- 18 Yamakawa-Karakida N, Sugita K, Inukai T, Goi K, Nakamura gamma in colon cancer and inflammatory bowel disease. Arch M, Uno K, Sato H, Kagami K, Barker N and Nakazawa S: Pathol Lab Med 127 : 1121-1123, 2003. Ligand activation of peroxisome proliferator-activated receptor 4 Coi IK, Kim YH, Kim JS and Seo JH: PPAR-gamma ligand γ induces apoptosis of leukemia cells by down-regulating the c- promotes the growth of APC-mutated HT-29 human colon myc gene expression via blockade of the Tcf-4 activity. Cell cancer cells in vitro and in vivo . Invest New Drugs 26(3) : 283- Death Differ 9: 513-526, 2002. 288, 2008. 19 Gerhold DL, Liu F, Jiang G, Li Z, Xu J, Lu M, Sachs JR, Babchi 5 Thompson EA: PPAR-gamma physiology and pathology in A, Fridman A, Holder DJ, Doebber TW, Berger J, Elbrecht A, gastrointestinal epithelial cells. Moll Cells 24(2) : 167-176, 2007. Moller DE and Zhang BB: Gene expression profile of adipocyte 6 Forman BM, Tontonz P, Chen J, Brun RP, Spiegelman BM and differentiation and its regulation by peroxisome proliferator- Evans RM: 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand activated receptor γ agonists. Endocrinology 143 : 2106-2118, for the adipocyte determination factor PPAR gamma. Cell 83(5) : 2002. 803-812, 1995. 20 Subbaramaiah K, Lin DT, Hart JC and Dannenberg AJ: 7 Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC and Peroxisome proliferator-activated receptor γ ligands suppress the Lehmann JM: A prostaglandin J2 metabolite binds peroxisome transcriptional activation of cyclooxygenase-2. J Biol Chem 276 : proliferator-activated receptor gamma and promotes adipocyte 12440-12448, 2001. differentiation. Cell 83(5) : 813-819, 1995. 21 Wang C, Fu M, Sakamaki T and Pestell RG: Minireview: Cyclin 8 Lowell BB: PPARγ: An essential regulator of adipogenesis and D1: Normal and abnormal functions. Endocrinology 145 : 5439- modulator of fat cell function. Cell 99 : 239-242, 1999. 5447, 2004. 9 Willson TM, Lehmann JM and Kliewer SA: Discovery of 22 Wendum D, Masliah J, Trugnan G and Flejou JF: ligands for the nuclear peroxisome proliferator-activated Cyclooxygenase-2 and its role in colorectal cancer development. receptor. Ann NY Acad Sci 804 : 276-283, 1996. Virchows Arch 445 : 327-333, 2004. 10 Tontonz P, Singer S, Forman BM, Sarraf P, Fletcher JA, Fletcher 23 Inoue H, Tanabe T and Umesono K: Feedback control of CD, Brun RP, Mueller E, Altiok S, Oppenheim H, Evans RM cyclooxygenase-2 expression through PPAR γ. J Biol Chem and Spiegelman BM: Terminal differentiation of human 275(36) : 28028-28032, 2000. liposarcoma cells induced by ligands for peroxisome 24 Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, proliferator-activated receptor-gamma and the retinoid X Vogelestein B and Kinzler KW: Activation of beta-catenin-Tcf receptor. Proc Natl Acad Sci USA 94 : 237-241, 1997. signaling in colon cancer by mutation in beta-catenin or APC. 11 Yoshizumi T, Ohta T, Ninomiya I, Terada I, Fushida S, Fujimura Science 275 : 1787-1790, 1997. T, Nishimura G, Shimizu K, Yi S and Miwa K: Thiazolodinedion, 25 Sunami E, Tsuno NH, Kitayama J, Saito S, Osada T, Yamaguchi a peroxisome proliferator-activated receptor-gamma ligand, H, Tomozawa S, Shibata Y and Nagawa H: Decreased synthesis inhibits growth and metastasis of HT-29 human colon cancer cells of matrix metalloproteinase-7 and adhesion to the extracellular through differentiation-promoting effects. Int J Oncol 25 : 631-639, matrix proteins of human colon cancer cells treated with 2004. troglitazone. Surg Today 32 : 343-350, 2002. 12 Sarraf P, Mueller E, Jones D, King FJ, DeAngelo DJ, Partrige 26 Motomura W, Nagamine M, T anno S, Sawamukai M, Takahashi JB, Holden SA, Chen LB, Singer S, Fletcher C and N, Kohgo Y and Okumura T: Inhibition of cell invasion and Spiegelman BM: Differentiation and reversal of malignant morphological change by troglitazone in human pancreatic changes in colon cancer through PPAR gamma. Nature Med cancer cells. J Gastroenterol 39 : 461-468, 2004. 4: 1046-1052, 1998. 13 Kitamura S, Miyazaki Y, Shinomura Y, Kondo S, Kanayama S and Matsuzawa Y: Peroxisome proliferator-activated receptor- gamma induces growth arrest and differentiation markers of human colon cancer cells. Jpn J Cancer Res 90 : 75-80, 1990. 14 Takahashi N, Okumura T, Motomura W, Fujimoto Y, Kawabata I and Kohgo Y: Activation of PPARγ inhibits cell growth and Received June 26, 2008 induces apoptosis in human gastric cancer cells. FEBS Lett 455 : Revised September 8, 2008 135-139, 1999. Accepted September 29, 2008

3599