Journal: Journal of the National Cancer Institute

Article Title: Specific peptide ligand for Grb7 signal transduction protein and pancreatic cancer metastasis.

doi: 10.1093/jnci/djj105

Figure Lengend Snippet: Fig. 1. Histologic analysis of Grb7 expression in human pancreatic cancer tissues. A ) Immunohistochemistry. Repre sentative examples of pancreatic cancer tissue and adjacent normal tissue are shown. Similar tissues gave similar results. Immunohistochemistry used an anti-Grb7 monoclonal anti body. Left ) Positive staining for Grb7 protein in pancreatic cancer cells, compared with surrounding noncancer acinar and duct cells (original magnifi cations: upper = ×100, lower = ×200). Positive staining = solid arrowheads ; negative staining = open arrowheads . Right ) Lack of staining for Grb7 protein in pancreatic cancer cells (original magnifi cations: upper = ×100) or noncancer acinar and duct cells (original magnifi cations: lower = ×200). Scale bars = 100 μ m. B ) Immunoblot analysis. Upper ) Expression of total Grb7. Lower ) Expression of phosphorylated Grb7 (Grb7/pY). Expression was detected with an anti-Grb7 protein antibody C-20 and with an anti-phosphotyrosine antibody, respectively, in a lysate from pancreatic tumor tissue ( lanes T ) and a lysate from adjacent noncancerous tissue ( lanes N) . Left ) Case 1, a Grb7-positive tumor. Right ) Case 2, a Grb7- negative tumor. For the analysis of phosphorylated Grb7 protein, the lysate was fi rst immunoprecipitated with the anti-Grb7 antibody and then the immunoprecipitated proteins were separated by electrophoresis and examined by immunoblot analysis. C ) Grb7 expression and metastatic spread to lymph nodes of pancreatic cancer in 36 patients with pancreatic cancer. Bars = percentage of Grb7 overexpression in tumors with no metastasis (stage N0; bar N0), metastasis only to the regional lymph nodes (stage N1; bar N1), or metastasis to the distant lymph nodes (stage N2; bar N2). A two-sided Fisher’s exact test was used to determine that the difference in Grb7 expression between tumors from patients without (N0) and with metastasis to lymph nodes (N1 + N2) was statistically signifi cant ( P = .006).

Article Snippet: The anti-Grb7 polyclonal antibody (product C-20; Santa Cruz Biochemistry, Santa Cruz, CA) was used at 1 : 50 dilution in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (Sigma Chemical, St. Louis, MO), followed by incubation for 6 hours at room temperature.

Techniques: Expressing, Immunohistochemistry, Staining, Negative Staining, Western Blot, Immunoprecipitation, Electrophoresis, Over Expression

Journal: Journal of the National Cancer Institute

Article Title: Specific peptide ligand for Grb7 signal transduction protein and pancreatic cancer metastasis.

doi: 10.1093/jnci/djj105

Figure Lengend Snippet: Fig. 2. RNA interference of Grb7 expression in human pancreatic cancer cells. A ) Transfection of fl uorescent-labeled siRNAs in the Panc-1 living human pancreas cancer cells. Fluorescently labeled siRNAs are shown as green on multichannel confocal laser – scanning microscopy. B ) Expression of Grb family proteins in fi bronectin-stimulated Panc-1 cells transfected with Grb7 siRNA or with control siRNA or nontransfected control Panc-1 cells (none). C ) Fibronectin migration assay. The number of cells migrating into the fi bronectin was determined for Panc-1 cells transfected with Grb7 siRNA or with control siRNA or nontransfected control Panc-1 cells (none) with a modifi ed Boyden chamber method. The mean migration rate, expressed as cell numbers, derived from three independent experi- ments for duplicates per point (control siRNA = 84.8 cells and Grb7 siRNA = 20.7 cells; difference = 64.2 cells, 95% confi dence interval = 47.3 to 81.1 cells; P <.001). All statistical tests were two-sided.

Article Snippet: The anti-Grb7 polyclonal antibody (product C-20; Santa Cruz Biochemistry, Santa Cruz, CA) was used at 1 : 50 dilution in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (Sigma Chemical, St. Louis, MO), followed by incubation for 6 hours at room temperature.

Techniques: Expressing, Transfection, Labeling, Confocal Laser Scanning Microscopy, Control, Migration, Derivative Assay

Journal: Journal of the National Cancer Institute

Article Title: Specific peptide ligand for Grb7 signal transduction protein and pancreatic cancer metastasis.

doi: 10.1093/jnci/djj105

Figure Lengend Snippet: Fig. 5. Grb7 peptide inhibitor and metastasis. BALB/c nu/nu mice were inoculated intraperitoneally with Panc-1 human pancreatic cancer cells ( 22 ) . A ) Representative peritoneal metastases of Panc-1 cells in mice treated with the control peptide ( left ) or with the Grb7 peptide inhibitor G7-18NATE-P ( right ). Upper ) Peritoneal metastases. Arrows = nodules. Lower ) Representative sections of Panc-1 tumor cells invading the parietal peritoneum in mice treated with the control peptide ( arrows , left ) but not in mice treated with G7-18NATE-P ( right ). Similar effects were detected with other human pancreas cancer cell lines examined. B ) Number of nodules and total weight of metastatic nodules per mouse. Upper ) Number of nodules. The number of nodules was statistically signifi cantly lower in mice treated with the Grb7 inhibitor (3.2 nodules, 95% confi dence interval [CI] = 1.6 to 4.8 nodules; P <.001, t test) than in mice treated with control peptide (control = 72.6 nodules, 95% CI = 55.8 to 89.4 nodules) ( P <.001, t test). Lower ) Total weight of nodules. The total weight of nodules was statistically signifi cantly lower in mice treated with the Grb7 peptide inhibitor (Grb7 peptide = 0.19 g, 95% CI = 0.06 to 0.32 g) than in mice treated with control peptide (control = 4.13 g, 95% CI = 3.40 to 4.86 g) ( P <.001, t test). Error bars = 95% CIs. All statistical tests were two-sided.

Article Snippet: The anti-Grb7 polyclonal antibody (product C-20; Santa Cruz Biochemistry, Santa Cruz, CA) was used at 1 : 50 dilution in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (Sigma Chemical, St. Louis, MO), followed by incubation for 6 hours at room temperature.

Techniques: Control