Volume 131, Issue 5, Pages 1530-1541 (November 2006) A Novel Molecular Targeting Compound as K-samII/FGF-R2 Phosphorylation Inhibitor, Ki23057, for Scirrhous Gastric Cancer  Kazunori Nakamura, Masakazu Yashiro, Tasuku Matsuoka, Masashige Tendo, Toshiyuki Shimizu, Atsushi Miwa, Kosei Hirakawa  Gastroenterology  Volume 131, Issue 5, Pages 1530-1541 (November 2006) DOI: 10.1053/j.gastro.2006.08.030 Copyright © 2006 AGA Institute Terms and Conditions

Figure 1 Chemical structure of Ki23057 (molecular weight, 538.1). Gastroenterology 2006 131, 1530-1541DOI: (10.1053/j.gastro.2006.08.030) Copyright © 2006 AGA Institute Terms and Conditions

Figure 2 Effect of Ki23057 on kinase activities. (A) Kinase inhibitory activities of Ki23057 were evaluated by detecting 32P radioactivity using GST-fused proteins of kinase domein of tyrosine kinase receptors. Ki23057 inhibited the autophosphorylation of FGF-R2, FGF-R1, and VEGF-R but not EGF-R. (B) For cellular assay, cultured cells were incubated with serial concentrations of Ki23057. Each RTK was immunoprecipitated from cell lysates and immunoblotted by antiphosphotyrosine. Ki23057 inhibited the autophosphorylation of K-samII, VEGF-R, and PDGF-R but not EGF-R and IGF-1R-GST; glutathione-S-transferase. Gastroenterology 2006 131, 1530-1541DOI: (10.1053/j.gastro.2006.08.030) Copyright © 2006 AGA Institute Terms and Conditions

Figure 3 Inhibitory effect of various concentrations of Ki23057 on proliferation of gastric cancer cell lines. (A) Ki23057 at concentrations of 30 nmol/L, 100 nmol/L, 300 nmol/L, and 1000 nmol/L suppressed the cell viability of OCUM-2MD3 cells by 9.7%, 91.4%, 94.4%, and 95.1% respectively, compared with untreated controls. In OCUM-8 cells, Ki23057 addition inhibited the growth by 30 nmol/L, 100 nmol/L, 300 nmol/L, and 1000 nmol/L of Ki23057 by 43.4%, 83.7%, 85.9%, and 86.5%, respectively, compared with untreated controls. In contrast, no growth-inhibitory effect of Ki23057 was found in nonscirrhous gastric carcinoma cells (MKN-7, MKN-74, and MKN-45). Percentage of growth inhibition was calculated as 100 × (1 − treated cells/untreated cells). The results are presented as the mean of 4 experiments. (B) Numbers of scirrhous gastric cancer cells, OCUM-2MD3 and OCUM-8, were decreased significantly by addition of Ki23057. Proliferation of scirrhous gastric cancer cells with 30 nmol/L Ki23057 or more Ki23057 was significantly decreased, compared with the control at 48 hours and 72 hours. (C) Proliferation of nonscirrhous gastric carcinoma cells, MKN-7, MKN-45, and MKN-74, was not suppressed by addition of Ki23057 at any concentration. MKN-7 and MKN-74 cells were derived from well-differentiated adenocarcinoma; MKN-45 was derived from poorly differentiated adenocarcinoma without scirrhous characteristics. Ki23057 concentrations: 0 nmol/L as control (○), 10 nmol/L (●), 30 nmol/L (□), 100 nmol/L (■), 300 nmol/L (▴), 750 nmol/L (), and 1000 nmol/L (▴). The results are presented as the mean and standard deviation (bars) of 2 independent experiments. *P < .05 vs control. **P < .01 vs control. Gastroenterology 2006 131, 1530-1541DOI: (10.1053/j.gastro.2006.08.030) Copyright © 2006 AGA Institute Terms and Conditions

Figure 4 Receptor tyrosine kinase expression of gastric cancer cells. (A) The overexpression of K-samII was in scirrhous gastric cancer cell lines, OCUM-2MD3, and OCUM-8 cells. In contrast, K-samII overexpression was not found in nonscirrhous gastric cancer cell lines, MKN-7, MKN-45, and MKN-74 cells. VEGF-R expression was found in 4 of 5 cancer cell lines including OCUM-2MD3 and OCUM-8 cells. The expression of PDGF-Rβ and c-kit was not observed in scirrhous gastric cancer cell lines (A). (B) Expression of K-samII was recognized at the membrane of OCUM-2MD3 cells but not MKN-45 cells. Gastroenterology 2006 131, 1530-1541DOI: (10.1053/j.gastro.2006.08.030) Copyright © 2006 AGA Institute Terms and Conditions

Figure 5 Inhibition of K-samII, VEGF-R, ERK, and Akt phosphorylation by Ki23057 in gastric cancer cells. (A) K-samII phosphorylation (α-pY) was decreased by 100 nmol/L or more of Ki23057 in scirrhous gastric cancer OCUM-2MD3 cells, whereas no inhibition of α-K-samII was found. Ki23057 at 30 nmol/L or more decreased α-pY in scirrhous gastric cancer OCUM-8 cells. In contrast, no inhibition of α-pY was found in non-scirrhous gastric cancer cells, MKN-7, MKN-45, and MKN-74. (B) VEGF-R phosphorylation was not decreased at any Ki23057 concentration compared with total VEGF-R expression in scirrhous gastric cancer cell lines, OCUM-2MD3 and OCUM-8. (C) Gastric cancer cells were exposed to different concentrations of Ki23057 for 30 minutes. Following harvesting, cells were lysed and processed for immunoblot analysis using antibodies directed against p-ERK, ERK1, p-Akt, and Akt. OCUM-2MD3 cells, p-ERK was decreased at concentrations of 100 nmol/L or more Ki23057, and p-Akt was decreased at concentrations of 1000 nmol/L. OCUM-8 cells, p-ERK, and p-Akt were decreased at concentrations of 100 nmol/L or more of Ki23057. In contrast, no inhibition of p-ERK or p-Akt expression was evident in MKN-7 cells, MKN-45 cells, or MKN-74 cells. Gastroenterology 2006 131, 1530-1541DOI: (10.1053/j.gastro.2006.08.030) Copyright © 2006 AGA Institute Terms and Conditions

Figure 6 Apoptosis induction by Ki23057 in vitro. Ki23057 at concentrations of 100 nmol/L and 1000 nmol/L significantly increased the apoptosis (12.1% and 20.0%, respectively) in OCUM-2MD3 cells compared with no treatment (2.3%). Apoptotic cells show condensation and aggregation of nuclear chromatin into dense masses beneath the nuclear membrane. *P < .05 vs control. Gastroenterology 2006 131, 1530-1541DOI: (10.1053/j.gastro.2006.08.030) Copyright © 2006 AGA Institute Terms and Conditions

Figure 7 Inhibitory effect of Ki23057 on growth and phosphorylation of xenografted tumor. (A) Size of the xenografted tumor was decreased compared with size on day 6 by oral administration of Ki23057 at 25 mg/kg/day (■; n = 8). In contrast, xenografted tumor size in controls (n = 8) continued to increase with time (●). (B) To assess phosphorylation in tumor cells, tumor-bearing mice were treated with vehicle control or a single dose of Ki23057 (25 mg/kg/day). K-samII phosphorylation was suppressed in tumors of Ki23057-treated mice (mouse 3 and mouse 4), compared with that in vehicle mice (mouse 1 and mouse 2). Analysis of downstream K-samII signaling proteins, ERK and Akt, showed similarly decreased phosphorylation by an administration of Ki23057. VEGF-R phosphorylation was not suppressed in both vehicle and Ki23057-treated mice. **P < .01 vs control. Gastroenterology 2006 131, 1530-1541DOI: (10.1053/j.gastro.2006.08.030) Copyright © 2006 AGA Institute Terms and Conditions

Figure 8 Effects of Ki23057 on peritoneal metastasis. (A) Bloody ascites was observed 18 days after intraperitoneal inoculation of OCUM-2MD3 cells. (B) Ascites was decreased by oral administration of Ki23057 at 25 mg/kg/day. At 28 days after tumor inoculation, some mice were killed for macroscopic examination to determine distribution of disseminated metastasis (C and D). (C) Many metastatic nodules were found in the mesentery of a control mouse. (D) In contrast, peritoneal metastasis was not observed macroscopically in a mouse with Ki23057 administration. (E) The survival rate in the Ki23057-treated group (n = 11) was significantly higher than in the control group (n = 13). Gastroenterology 2006 131, 1530-1541DOI: (10.1053/j.gastro.2006.08.030) Copyright © 2006 AGA Institute Terms and Conditions

Figure 9 Immunohistochemistry with p-ERK antibody in a tumor xenograft and peritoneal nodules. Ki23057 decreased immunohistochemical staining indicating phosphorylation of MAP kinase, p-ERK, in OCUM-2MD3 tumor xenografts, peritoneal nodules, and ascites (A) compared with the control (B). Gastroenterology 2006 131, 1530-1541DOI: (10.1053/j.gastro.2006.08.030) Copyright © 2006 AGA Institute Terms and Conditions

Figure 10 Apoptotic indices in tumor xenografts in vivo. Apoptotic cells show condensation and aggregation of nuclear chromatin into dense masses beneath the nuclear membrane. Numbers of apoptotic (TUNEL-positive) cells were significantly decreased following (A) Ki23057 administration (B) compared with controls. (C) Apoptotic indices in tumor xenograft and peritoneal nodules were significantly increased following Ki23057 treatment. Data are presented as mean ± SD (4 samples per group). **P < .01 vs control. Gastroenterology 2006 131, 1530-1541DOI: (10.1053/j.gastro.2006.08.030) Copyright © 2006 AGA Institute Terms and Conditions