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A Novel Transforming Growth Factor β Receptor Kinase Inhibitor, A-77, Prevents the Peritoneal Dissemination of Scirrhous Gastric Carcinoma

Authors' Affiliations: ¹ Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka, Japan and ² Department of Pharmaceutical Manufacturing Chemistry, Kyoto Pharmaceutical University, Kyoto, Japan

Requests for reprints: Masakazu Yashiro, Department of Surgical Oncology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. Phone: 81-6-6645-3838; Fax: 81-6-6646-6450; E-mail: m9312510{at}med.osaka-cu.ac.jp.

	Abstract

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Purpose: Transforming growth factor β receptor (TGFβ-R) is reported to correlate with the malignant potential of scirrhous gastric carcinoma. The aim of the current study is to clarify the possibility of molecular target therapy with a TGFβ-R inhibitor, A-77, for the treatment of peritoneal dissemination of scirrhous gastric cancer.

Experimental Design: Three scirrhous gastric cancer cell lines and two fibroblasts were used. For in vivo experiments, the A-77 was administered i.p. to mouse models of peritoneal dissemination. The influences of A-77 on the adhesion ability, invasion ability, and the expression of adhesion molecules were examined in vitro.

Results: The A-77 administration resulted in a significantly (P < 0.01) better prognosis for the mice with peritoneal dissemination (median survival time, 51 days), compared with the control (median survival time, 25 days). A-77 therefore significantly (P < 0.01) decreased the weight and number of metastatic nodes. The adhesive ability and invasion ability of cancer cells were significantly decreased by A-77. A-77 decreased the expression of ₂, ₃, and ₅ integrins in gastric cancer cells. The histologic findings showed the degree of fibrosis to be less in the tumors treated by A-77. A-77 decreased the growth of fibroblast and invasion-stimulating activity of fibroblasts on cancer cells.

Conclusion: The TGFβ-R inhibitor, A-77, decreased the expression of integrins in cancer cells and the proliferation of fibroblasts, which resulted in the decreased adhesive and invasive abilities of scirrhous gastric cancer cells to peritoneum. A-77 is thus considered to be useful for the inhibition of peritoneal dissemination of scirrhous gastric carcinoma.

Transforming growth factor β (TGFβ) expression is reported to be clinically related to the malignant potential of scirrhous gastric carcinoma (8, 10). We also reported that TGFβproduced from stromal fibroblasts or gastric cancer cells stimulated both the invasion and adhesion of scirrhous gastric cancer cells to the peritoneum, thus resulting in an increase of the potential for peritoneal dissemination (11, 12). Various molecules characteristic for scirrhous gastric cancer cells or the cellular environment surrounding them have thus far been elucidated (3, 7, 11). The elucidation of tumor-stroma molecular interactions could profoundly influence targeted cancer therapy and may provide the basis for developing new prevention strategies.

The reported effects of TGFβ include stimulation of invasion, angiogenesis, immunosuppression, fibrosis, and deposition of ECM components (13–16). TGFβ inhibitors are being developed for the treatment of fibrotic disorders and tumors (17, 18). A human monoclonal antibody that targets TGFβ, Metelimumab (Cambridge Antibody Technology), is currently undergoing phase I/II trials of scleroderma (17). The TGFβ antisense oligonucleotide, AP-12009 (Antisense Pharma), is also in the development stage for the treatment of high-grade glioma, and it is currently undergoing phase I/II trials (18). In contrast, there have been few reports of a TGFβ signaling inhibitor for scirrhous gastric carcinoma (19), whereas TGFβ signaling plays an important role in this type of cancer (11, 12).

TGFβ binds to two different serine/threonine kinase receptors (TGFβ-R), termed type II and type I. Both receptors are necessary in the activation of type I receptor kinase signaling. The activated TGFβ-R type I kinase phosphorylates Smad2/3 and activates the Smad signaling pathway (20, 21). Seven type I receptors are present as activin receptor-like kinase (ALK) 1-7 in mammals; ALK-5 serves as a specific receptor for TGFβ (22, 23). An ALK kinase inhibitor, A-77, blocks the signaling of TGFβ in a dose-dependent fashion by the competition with ATP for the activation of Smad2, but does not inhibit extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase (24, 25). Because TGFβ plays an important role for the progression of scirrhous gastric cancer cells (8, 10–12), the TGFβ-R inhibitor, A-77, might inhibit the dissemination of scirrhous gastric cancer. In the present study, we investigated the effect of the TGFβ-R inhibitor on the peritoneal dissemination of scirrhous gastric carcinoma both in vivo and in vitro.

	Materials and Methods

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Compounds. A-77 (Fig. 1A ), a small synthetic molecule that interrupts the phosphorylation of R-Smad by TGFβ-receptor type I, was synthesized as previously reported (24, 25). A-77 was dissolved in PBS (Nikken Bio.), stored at 4°C, and used within 5 d. TGFβ1 was purchased from R&D Systems.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. A-77, a small-molecule inhibitor of the TGFβ-R type-1 kinase domain. A, chemical structure of A-77. Molecular weight is 359. B, the inhibition of Smad2 phosphorylation by A-77 in OCUM-2MD3, OCUM-2D, and NUGC3 cells. Phosphorylation of Smad2 was decreased dose dependently, beginning at 0.3 µmol/L of A-77.

Animal models. Female BALB/c nude mice (Nihon CLEA) were used for the in vivo studies. All experiments were done according to the standard guidelines for animal experiments of Osaka City University Medical School. We examined the effect of A-77 on peritoneal dissemination as follows: OCUM-2MD3 cells (1 x 10⁷) were injected via the right flank of a mouse at day 0 into the abdominal cavity. The i.p. inoculation of OCUM-2MD3 cells into nude mice led to the development of peritoneal metastasis in all mice. In contrast, the i.p. inoculation of OCUM-2D cells (1 x 10⁷) or NUGC3 cells (1 x 10⁷) into nude mice developed peritoneal metastasis in none of 7 mice or in only 1 of 7 mice, respectively. Therefore, OCUM-2D and NUGC3 cells were not used for in vivo study because of their poor tumorigenicity (8, 26). We administered A-77 (150 µg/body/d) i.p. into the mice thrice per week beginning 1 wk after the inoculation to death. Instead of A-77, PBS was administered i.p. as the control. The mice were sacrificed 14 d after tumor inoculation, when the number and weight of peritoneal metastatic nodules were determined between the control groups (n = 6) and the A-77 administration groups (n = 6). Also, we compared the survival between the control groups (n = 7) and the A-77 administration groups (n = 6). For the histopathologic examinations, the tissue blocks were embedded in paraffin, cut in 4-µm-thick sections, and stained with H&E. Immunohistochemical staining for ₂, ₃, and ₅ integrin proteins was carried out using the avidin-biotin-peroxidase complex method. After an autoclave in Target Retrieval Solution (Dako Co.), the slides of 4-µm-thick sections were incubated with a 1:200 dilution of anti–₂ integrin antibody, MAB1998 (Chemicon, Korea); with an anti–₃ integrin antibody, AB1920 (Chemicon); or with anti–₅ integrin antibody, CBL497 (Chemicon).

Western blot analysis. The inhibition by A-77 of the phosphorylation of Smad2 in gastric cancer cells was examined as follows. The cell lines were cultured in DMEM with 2% FBS. The culture was rinsed with PBS and incubated in medium with 10 ng/mL TGFβ1 for 30 min, and then added with A-77 at various concentrations for 60 min. The cells were lysed in a lysis buffer containing 20 mmol/L Tris (pH 8.0), 137 mmol/L EDTA, 100 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 0.25 trypsin inhibitory units/mL aprotinin, and 10 mg/mL leupeptin. Aliquots containing 30 µg of total protein were subjected to SDS-PAGE, and the protein bands were transferred to a polyvinylidene difluoride membrane (Amersham). The membrane was kept in PBS-T (10 mmol/L PBS and 0.05% Tween 20) supplemented with 5% nonfat milk or 5% bovine albumin (Sigma) at room temperature for 1 h. Next, the membrane was placed in a PBS-T solution containing the primary antibody, p-Smad2 (Ser^465/467; 1:1,000) or Smad2 (1:1,000; Cell Signaling Technology), and allowed to react at 4°C overnight. Next, each antibody was washed thrice with PBS-T for 10 min, and a peroxidase-labeled secondary antibody (Amersham) reactive with the primary antibody was added. The bands were detected using an enhanced chemiluminescence system (Amersham). An immunoblot analysis was done twice.

Adhesion assay. According to the method of Nishimura et al. (7), the effects of A-77 on the adhesion ability of cancer cells to peritoneal components were examined using the adhesion assay. The binding of cancer cells to ECM components, Matrigel (Collaborative Research Co.), fibronectin (Mallinckrodt Specialty Chemicals Co.), laminin (Mallinckrodt Specialty Chemicals Co.), and mesothelial cells was also investigated. A 96-well microtiter plate was coated with Matrigel (8 µg/well), fibronectin (4 µg/well), laminin (4 µg/well), or mesothelial cells (1 x 10⁵ cells). Gastric cancer cells (4 x 10⁵) were seeded onto these components in 96-well microtiter plate (Falcon). TGFβ1 was diluted at the concentration of 1.0 or 10 ng/mL with PBS. A-77 was diluted at the concentration of 0.1, 1.0, or 10 µmol/L. The cancer cells were treated overnight. They were allowed to adhere to each well for 30 min at 37°C and then gently washed twice in PBS. The adhesion cancer cells were quantified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (Sigma) colorimetric assay. The percentage of cells adhering was calculated as follows: % bindings = (absorbance of treated surface – only mesothelium or ECM component) / absorbance of total surface x 100. "Total surface" means total cancer cells, 4 x 10⁵, seeded on the microtiter plates with mesothelial components, Matrigel, fibronectin, laminin, and mesothelial cells.

Reverse transcription-PCR. The effect of A-77 on the mRNA expression of the integrin family in gastric cancer cells was examined as follows. The cells were plated in six-well microtiter plates at a density of 2 x 10⁵ per well with or without 10 ng/mL TGFβ1 and 1 µmol/L A-77, and each plate was incubated for 24 h. After incubation, total cellular RNA was extracted from gastric cancer cells with Trizol (Life Technologies) according to the manufacturer's protocol. Next, cDNAs were synthesized from 1 µg of the RNA with a Moloney murine leukemia virus-reverse transcription kit (Life Technologies) using random hexamers. The cDNAs were amplified by PCR for 30 cycles with AmpliTaq Gold DNA polymerase (Perkin-Elmer Cetus) on a thermal cycler. The primer sequences and PCR conditions for integrin subunits, including ₁, ₂, ₃, ₅, ₆, β₁, β₃, and β₅ integrin, have been previously described by Morozevich et al. (27). The following Smad2 primers were used (20): sense, 5'-TCAAGCTTGAGTGTAAACCCTTACCACTATC-3'; antisense, 5'-TAGCGGCCGCGAAAGCTATGATTAACAGGGG-3'. The following glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used as the control: sense, 5'-ACCTGACCTGCCGTCTAGAA-3' and antisense, 5'-TCCACCACCCTGTTGCTGTA-3'. The PCR conditions were as follows: predenaturation, 94°C for 10 min; denaturation, 94°C for 60 s; annealing, 65°C for 60 s; extension, 72°C for 60 s; and final incubation, 72°C for 10 min.

Invasion assay. The in vitro invasiveness was measured by the method of Albini et al. (28) with some modifications. We used the chemotaxicell chambers (Kubota) with a 12-µm membrane filter, and the upper surface of each filter was coated with 50 µg of Matrigel in cold DMEM to form a matrix barrier. The chamber (upper component) was placed in a 24-well culture plate (lower component). Gastric cancer cells were resuspended to a final concentration of 1 x 10⁴/mL in DMEM with 10% FBS. Two hundred microliters of cancer cell suspension and 800 µL of DMEM with 10% FBS or fibroblast cell suspension (1 x 10⁴ cells/mL) were added to the upper and lower components, respectively. The reagents (TGFβ, a final concentration of 0 or 10 ng/mL; A-77, a final concentration of 0, 1, or 10 µmol/L) were diluted into the upper component. After incubation for 72 h at 37°C, cancer cells on the upper surface of the membrane were removed by wiping. The membrane was stained with hematoxylin. Cancer cells that invaded through a filter coated with Matrigel to the lower membrane were manually counted under a microscope at x200 magnification. Six randomly chosen fields were counted for each assay. The mean of six fields was calculated as the sample value. For each group, the culture was done in triplicate.

Proliferation assay of gastric cancer cells and fibroblast cells. According to the method of Inoue et al. (11), 1 x 10⁴ HS-29 or NF-30 cells were seeded on a 24-well plastic plate (Falcon) and incubated with reagents in 1 mL of DMEM containing 10% FBS for 72 h. Next, the cells were dispersed by trypsin/EDTA (Life Technologies) treatment and counted with a Coulter Counter Z2 (Beckman Coulter). TGFβ1 of 10 ng/mL and A-77 of 0.3, 1, 3, or 10 µmol/L were diluted.

Small interfering RNA design. The sequences for Smad2 small interfering RNA (siRNA) are designed as previously reported (29): Smad2 siRNA sense, 5'-GUCCCAUGAAAAGACUUAAtt-3'; antisense, 5'-UUAAGUCUUUUCAUGGGACtt-3'. Control nontargeting siRNA was purchased from Ambion. OCUM-2MD3 cells were prepared at 50% to 60% confluence in six-well dishes. The transfection mixture was prepared by incubating 5 µL of siPORT Neo-Fx (Ambion) and 295 µL of Opti-MEM I for 10 min at room temperature. siRNA was then added to the above mixture and incubated for further 10 min. Finally, the above transfection mixture was added to six-well dish containing 2 mL of DMEM with 10% FBS (final siRNA concentration was 30 nmol/L). Twenty-four hours after transfection, reverse transcription-PCR, a proliferation assay, an adhesion assay, and an invasion assay were done.

Statistical analysis. Comparisons among the data sets were made with an ANOVA, followed by Student's t test. The survival evaluation was carried out using the Kaplan-Meier analysis and the log-rank test. Differences were considered to be statistically significant when the P value was 0.05 or less.

	Results

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Effects of A-77 on Smad2 phosphorylation of gastric cancer cells. Smad2 phosphorylation was increased by 10 ng/mL TGFβ1 and decreased dose-dependently by A-77 from 0.3 to 10 µmol/L in the OCUM-2MD3, NUGC-3, and OCUM-2D cells (Fig. 1B) as previously reported by Tojo et al. (24).

Effects of A-77 administration on the survival of peritoneal dissemination mice. Bloody ascites were observed in the mice in the control group (Fig. 2A ) on day 28 after i.p. inoculation of the OCUM-2MD3 cells, but were not found in the mice in the A-77–administered group (Fig. 2B). OCUM-2MD3 cell inoculation produced many tumor nodules (Fig. 2C) in the peritoneal cavity in the mice in the control group and resulted in death within 28 days in the control group mice. The mice treated with A-77 had fewer tumor nodules (Fig. 2D) in the mesentery than the untreated controls. Microscopically, the diffuse infiltration of cancer cells with abundant fibrous stroma was shown on the disseminated nodule of a control mouse (Fig. 2E). Fibrosis was reduced with A-77 administration, which thus resulted in the medullary growth of the metastatic tumors (Fig. 2F). At 14 days after the inoculation of the OCUM-2MD3 cells, the mice were killed for macroscopic examinations to determine distribution of the peritoneal dissemination. The weight and number of peritoneal metastasis in mice receiving A-77 were significantly (P < 0.01) lower than those of the control. In addition, the volume of the abdominal fluid in mice receiving A-77 was significantly (P < 0.001) lower than that of the control mice (Fig. 2G). The median survival time of the mice in the A-77 administered group was 51 days, whereas that of the control group was 25 days. The survival rate of the mice with peritoneal dissemination was significantly (P < 0.01) extended by A-77 compared with the control group (Fig. 2H).

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. The effect of TGFβ-R inhibitor, A-77, on the peritoneal dissemination in vivo. At 31 d after inoculation of the OCUM-2MD3 cells, some mice were killed for macroscopic examination to determine distribution of the disseminated dissemination. A, bloody ascites were observed in mice of the control group. B, the amount of ascites decreased after the administration of A-77. C, many metastatic nodules were found in the mesentery of control mice. D, in contrast, obvious peritoneal dissemination was not macroscopically observed in a mouse with A-77 administration. E, the nodule on a mouse in the control group shows diffuse infiltration of cancer cells with fibrous stroma in the control group (original magnification, x200). F, compared with the control group, this fibrosis was reduced by A-77 administration (original magnification, x200). G, A-77 significantly (P < 0.01) decreased both the mean weight and the mean number of involved nodes. The mean weights of the metastatic nodules in the control (n = 6) and A-77–treated mice (n = 6) were 223 and 73 mg, respectively. The mean numbers of metastatic nodules in the control and A-77–treated mice were 16 and 4, respectively. The volume of the abdominal fluid in mice receiving A-77 (867 µL) was significantly (P < 0.001) lower than those in the control mice (217 µL). H, survival curves of the mice given peritoneal inoculations of OCUM-2MD3 cells treated with or without A-77. Solid line, control mice (n = 7). Dotted line, mice treated with A-77 (n = 6). The mice were housed under identical conditions, and the date of death of each mouse was recorded. The difference in the survival times between the two groups of mice was statistically significant (P < 0.01, log-rank test).

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. The adhesion ability of scirrhous gastric cancer cells treated with TGFβ-R inhibitor. A, phase contrast photograph of OCUM-2MD3 cancer cells adhered to the confluent mesothelial cells. TGFβ1 stimulation increased the number of adherent OCUM-2MD3 cells to mesothelial cells, compared with the control. A-77 decreased the number of cancer cells under TGFβ1 stimulation that adhered to the mesothelial cells. Arrows, OCUM-2MD3 cells; *, mesothelial cells (original magnification, x100). B, A-77 at 10 µmol/L significantly inhibited the adhesion ability of OCUM-2MD3 to Matrigel, fibronectin, laminin, and mesothelial cells by 23%, 29%, 34%, and 73% in the absence of TGFβ1, respectively. The percentage of OCUM-2MD3 cells treated with 1 or 10 ng/mL TGFβ1 adherent to the various ECM components and mesothelial cells was significantly greater than that without TGFβ1 treatment: 19% (P = 0.034) or 27% (P = 0.007) greater adhesion to Matrigel, 21% (P = 0.044) or 25% (P = 0.011) greater adhesion to fibronectin, 20% (P = 0.040) or 27% (P = 0.004) greater adhesion to laminin, 82% (P = 0.047) or 191% (P = 0.0001) greater adhesion to mesothelial cells, respectively. In the presence of 10 ng/mL TGFβ1,A-77 at 10 µmol/L significantly inhibited the adhesion ability of OCUM-2MD3 to Matrigel, fibronectin, laminin, and mesothelial cells by 60%, 38%, 61%, and 75%, respectively. C, A-77 at 10 µmol/L significantly inhibited the adhesion ability of NUGC3 cells without TGFβ1 treatment to Matrigel, fibronectin, laminin, and mesothelial cells by 31%, 33%, 37%, and 59%, respectively. The percentage of OCUM-2MD3 cells treated with 1 or 10 ng/mL TGFβ1 adherent to the various ECM components and mesothelial cells was significantly greater than those not treated with TGFβ1: 23% (P = 0.008) or 34% (P = 0.001) greater adhesion to Matrigel, 20% (P = 0.022) or 41% (P = 0.001) greater adhesion to fibronectin, 18% (P = 0.004) or 21% (P = 0.006) greater adhesion to laminin, 71% (P = 0.035) or 85% (P = 0.014) greater adhesion to mesothelial cells, respectively. A-77 at 10 µmol/L significantly inhibited the adhesion ability of NUGC3 treated with 10 ng/mL TGFβ1 to Matrigel, fibronectin, laminin, and mesothelial cells by 45%, 39%, 50%, and 39%, respectively. D, A-77 at 10 µmol/L significantly inhibited the adhesion ability of OCUM-2D cells without TGFβ1 treatment to Matrigel, fibronectin, laminin, and mesothelial cells by 33%, 15%, 20%, and 61%, respectively. The percentage of OCUM-2D cells treated with 1 or 10 ng/mL TGFβ1 adherent to the various ECM components and mesothelial cells was significantly greater than those not treated with TGFβ1: 33% (P = 0.005) or 66% (P = 0.001) greater adhesion to Matrigel, 25% (P = 0.012) or 77% (P = 0.001) greater adhesion to fibronectin, 18% (P = 0.013) or 45% (P = 0.001) greater adhesion to laminin, and 52% (P = 0.013) or 178% (P = 0.001) greater adhesion to mesothelial cells, respectively. A-77 at 10 µmol/L significantly inhibited the adhesion ability of OCUM-2D treated with 10 ng/mL TGFβ1 to Matrigel, fibronectin, laminin, and mesothelial cells by 42%, 33%, 50% and 77%, respectively. Columns, mean of six experiments; bars, SD. *, P < 0.05; **, P < 0.01, versus control (a group of same density of TGFβ1 without A-77).

₁, ₂, ₃, ₅, ₆, β₁, β₃

2, ₃

₅

₂, ₃

₅

₂, ₃

₅

View larger version (84K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. The effect of TGFβ receptor inhibitor on the integrin expression of scirrhous gastric cancer cells. A, the 309-, 541-, 306-, 324-, 253-, 452-, 544-, 327-, and 251-bp products correspond to 1, 2, 3, 5, 6, β1, β3, and β5 integrin, and GAPDH, respectively. OCUM-2MD3, NUGC3, and OCUM-2D cells expressed 1, 2, 3, 5, 6, β1, and β5 integrin mRNA bands (lane 1). A-77 (1 µmol/L) down-regulated the 2, 3, and 5 integrin expression (arrows) of the OCUM-2MD3, NUGC3, and OCUM-2D cells (lane 2), compared with the control (lane 1). TGFβ1 (10 ng/mL) up-regulated 2, 3, and 5 integrin expression (arrows) of OCUM-2MD3, NUGC3, and OCUM-2D (lane 3), compared with the control (lane 1). A-77 (1 µmol/L) down-regulated 2, 3, and 5 integrin expression (arrows) of OCUM-2MD3, NUGC3, and OCUM-2D cells (lane 4), compared with lane 3. The intensities of the 251-bp products (GAPDH) were similar for all samples. B, an immunohistochemical study for 2, 3, and 5 integrin expression of metastatic tumors in vivo. The number of 2, 3, and 5 integrin–positive cancer cells in the peritoneal metastatic nodules decreased by A-77 administration, compared with the control group administration (original magnification, x200).

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. The effect of TGFβ-R inhibitor on the invasion ability of scirrhous gastric cancer cells. A, a phase-contrast photograph showing that the migratory capacity of the cancer cells that invaded the pore membrane filter was increased in the presence of TGFβ1, compared with the control. The migratory capacity of the cancer cells by TGFβ1 was significantly decreased in the presence of A-77. Arrowheads, micropore; arrows, invading cancer cells (original magnification, x200). B, the invasiveness of OCUM-2MD3, NUGC3, and OCUM-2D cells treated with 10 µmol/L A-77 significantly decreased in the absence of TGFβ1 compared with the control. The invasiveness of the OCUM-2MD3, NUGC3, and OCUM-2D cells significantly increased by 10 ng/mL TGFβ1. The migration-stimulating activity of TGFβ1 decreased significantly by A-77 in a dose-dependent manner. C, the invasiveness of the OCUM-2MD3, NUGC3, and OCUM-2D cells significantly (P < 0.01) increased by coculture with fibroblast NF30, and the invasion-stimulating effect of fibroblasts significantly decreased by 1 or 10 µmol/L A-77. D, the proliferation ability of HS-29 or NF-30 cells diluted with 10 ng/mL of TGFβ1 (black column) significantly increased compared with the control (open column). The number of HS-29 or NF-30 cells significantly decreased by A-77 at the concentrations in excess of 1 to 3 µmol/L. *, P < 0.05; **, P < 0.01, versus control (a group of same density of TGFβ1 without A-77). Columns, mean and of three independent experiments; bars, SD.

Effect of Smad2 siRNA on the expression of integrins, proliferation, adhesion ability, and invasion ability. We investigated the effect of Smad2 siRNA on expression of Smad2 and integrin mRNA in the OCUM-2MD3 cells (Fig. 6A ). Smad2 siRNA (30 nmol/L) down-regulated the Smad2 mRNA expression of OCUM-2MD3 cells compared with the control, whereas treatment of negative control siRNA (30 nmol/L) had no effect on the expression. No difference in the expression of integrins was found in the cancer cells between the control (lane 1) and negative siRNA (lane 2). In contrast, the expression level of ₂, ₃, and ₅ integrin mRNA in cancer cells treated with Smad2 siRNA (lane 3) was lower than those in the control. The proliferation of OCUM-2MD3 was not affected by 30 nmol/L of Smad2 siRNA treatment or negative control siRNA (Fig. 6B). Smad2 siRNA (30 nmol/L) treatment significantly decreased the adhesive ability of OCUM-2MD3 cells to Matrigel, fibronectin, laminin, and mesothelial cells, compared with either the control or negative control siRNA (Fig. 6C). In the absence of TGFβ1, the invasiveness of OCUM-2MD3 cells treated with Smad2 siRNA decreased, but the difference was not significant. The invasiveness of OCUM-2MD3 cells treated with TGFβ1 significantly increased in the control and negative control siRNA, whereas that of OCUM-2MD3 under TGFβ1 stimulation significantly decreased in the presence of Smad2 siRNA (Fig. 6D).

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. The effect of Smad2 siRNA on the integrin expression, proliferation, adhesion ability, and invasion ability of OCUM-2MD3 cells. A, the 329-bp products correspond to Smad2. OCUM-2MD3 cells expressed Smad2 and 1, 2, 3, 5, 6, β1, and β5 integrin mRNA bands (lane 1). Treatment of the control negative siRNA (30 nmol/L) had no effect on expression of all genes (lane 2), whereas treatment of Smad2 siRNA (30 nmol/L) down-regulated the Smad2 and 2, 3, and 5 integrin expression of the OCUM-2MD3 (lane 3) compared with the control (lane 1). B, Smad2 siRNA or negative control siRNA did not affect the proliferation ability of the OCUM-2MD3 cells. C, the adhesion ability of OCUM-2MD3 cells to Matrigel, fibronectin, laminin, and mesothelial cells was significantly (P < 0.01) inhibited by 66%, 51%, 55%, and 55%, respectively, in the presence of Smad2 siRNA. In contrast, no difference on the adhesion ability of OCUM-2MD3 cells was found between the control and negative siRNA. **, P < 0.01, versus control. D, the invasiveness of OCUM-2MD3 cells treated with 10 ng/mL TGFβ1 was significantly (P < 0.01) increased in the control and negative control siRNA, whereas the invasion-stimulating ability of TGFβ1 significantly (P < 0.05) decreased in the presence of Smad2 siRNA compared with the control.

	Discussion

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A-77 significantly decreased the adhesive ability of scirrhous gastric cancer cells to the peritoneum components of Matrigel, fibronectin, laminin, and mesothelial cells, in the absence and presence of TGFβ1. A-77 down-regulated ₂, ₃, and ₅ integrin expression at the mRNA level both with and without TGFβ1 stimulation, whereas TGFβ1 up-regulated ₂, ₃, ₅, and β₁ integrin expression. An immunohistochemical study also showed that ₂, ₃, and ₅ integrin–positive cancer cells were few in the peritoneal metastatic nodules of mice treated with A-77. We previously reported that ₂β₁ and ₃β₁ integrins might play an important role for the peritoneal dissemination in scirrhous gastric cancer (7, 9). These findings suggested that the down-regulation of ₂ and ₃ integrin expression on scirrhous cancer cells by A-77 might result in the decrease of adhesive ability to peritoneum; consequently, it suppressed peritoneal dissemination of scirrhous gastric cancer. The adhesion ability of cancer cells to laminin was more strongly inhibited by A-77 in all three cell lines, compared with fibronectin. ₂β₁, ₃β₁, and ₆β₁ Integrins bind laminin, whereas ₃β₁, ₄β₁, and ₅β₁ integrins bind fibronectin (9, 30, 31). These findings suggested that ₂β₁ integrins might thus be an important target molecule for A-77 in the adhesion to the peritoneum among integrin families.

The invasion of cancer cells is one important step for peritoneal dissemination (7). TGFβ has been reported to increase the invasiveness of various cancer cells (11, 15, 16, 32). In this study, TGFβ1 increased the invasion ability of scirrhous gastric cancer cells. A-77 significantly inhibited the invasiveness of cancer cells with or without TGFβ. Because gastric cancer cells produce TGFβ1 (33, 34), A-77 might decrease the autocrine manner of invasion ability for gastric cancer cells. The pathologic findings of the peritoneal nodules showed diffuse infiltration of cancer cells with the fibrous stroma on a control mouse. A-77 administration reduced the fibrosis and caused the medullary formation of cancer cells in vivo. In vitro, the proliferation ability of fibroblast cells was stimulated by TGFβ1 and inhibited by A-77 administration. The above findings suggested that A-77 decreased the proliferation of fibroblasts in vivo and in vitro. In a coculture with fibroblasts, the invasiveness of scirrhous gastric cancer cells was significantly increased, and A-77 (1 or 10 µmol/L) significantly decreased the invasion ability of scirrhous gastric cancer cells. We previously reported that TGFβ1 produced by surrounding fibroblasts stimulated the invasion ability of scirrhous gastric cancer cells and were closely associated with the progression of scirrhous gastric carcinoma (3, 7, 11). A-77 was therefore suggested to inhibit the invasion ability of cancer cells by suppressing the intercellular interaction between the scirrhous gastric cancer cells and surrounding fibroblasts. These findings suggested that A-77 inhibited the invasion-stimulating effect of TGFβ not only in an autocrine manner but also in a paracrine manner in scirrhous gastric carcinoma.

To determine whether the above effects are Smad2 dependent, RNA interference was used to achieve selective and specific knockdown of Smad2. The expression level of ₂, ₃, and ₅ integrin mRNA in cancer cells treated with Smad2 siRNA was lower than those of control. Smad2 siRNA treatment significantly decreased the adhesive ability and the invasion ability of OCUM-2MD3 cells. These effects of Smad2 knockdown in adhesion ability, invasion ability, and integrin expression were similar to those of A-77. These findings suggest that the effects of A-77 are related to inhibition of Smad2 phosphorylation.

In conclusion, the TGFβ-R inhibitor, A-77, decreased the expression of integrin mRNA and the growth of fibroblasts, which was associated with the decreased adhesive abilities and the invasion abilities of scirrhous gastric cancer cells. A-77 is therefore considered to be useful for the inhibition of peritoneal dissemination of scirrhous gastric carcinoma.

	Acknowledgments

 
We thank Professor Kohei Miyazono (Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan) for helpful advice.

	Footnotes

 
Grant support: Grants-in-aid for Scientific Research (18591475 and 18390369) from the Ministry of Education, Science, Sports, Culture and Technology of Japan; a Japanese Society of Gastroenterology grant-in aid for scientific research; a grant-in-aid for Kobayashi Foundation for Innovative Cancer Chemotherapy; a grant-in-aid for the Sagawa Foundation for Cancer Research; and a grant-in-aid for the Osaka Medical Research Foundation for Incurable Diseases.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/ 3/07; revised 1/10/08; accepted 1/24/08.

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