Honokiol Induces Calpain-Mediated Glucose-Regulated Protein-94 Cleavage and Apoptosis in Human Gastric Cancer Cells and Reduces Tumor Growth

Background. Honokiol, a small molecular weight natural product, has been shown to possess potent anti-neoplastic and anti-angiogenic properties. Its molecular mechanisms and the ability of anti-gastric cancer remain unknown. It has been shown that the anti-apoptotic function of the glucose-regulated proteins (GRPs) predicts that their induction in neoplastic cells can lead to cancer progression and drug resistance. We explored the effects of honokiol on the regulation of GRPs and apoptosis in human gastric cancer cells and tumor growth. Methodology and Principal Findings. Treatment of various human gastric cancer cells with honokiol led to the induction of GRP94 cleavage, but did not affect GRP78. Silencing of GRP94 by small interfering RNA (siRNA) could induce cell apoptosis. Treatment of cells with honokiol or chemotherapeutics agent etoposide enhanced the increase in apoptosis and GRP94 degradation. The calpain activity and calpain-II (m-calpain) protein (but not calpain-I (mu-calpain)) level could also be increased by honokiol. Honokiol-induced GRP94 down-regulation and apoptosis in gastric cancer cells could be reversed by siRNA targeting calpain-II and calpain inhibitors. Furthermore, the results of immunofluorescence staining and immunoprecipitation revealed a specific interaction of GRP94 with calpain-II in cells following honokiol treatment. We next observed that tumor GRP94 over-expression and tumor growth in BALB/c nude mice, which were inoculated with human gastric cancer cells MKN45, are markedly decreased by honokiol treatment. Conclusions and Significance. These results provide the first evidence that honokiol-induced calpain-II-mediated GRP94 cleavage causes human gastric cancer cell apoptosis. We further suggest that honokiol may be a possible therapeutic agent to improve clinical outcome of gastric cancer

Association of IS605 and cag-PAI of Helicobacter pylori Isolated from Patients with Gastrointestinal Diseases in Taiwan

Background. The cag pathogenicity island (cag-PAI) is one of the most important virulent determinants of Helicobacter pylori. An insertion sequence (IS) element of cag-PAI (IS605) has been found to generate H. pylori strains with varying virulence. Aim. To evaluate the impact of IS605 and cag-PAI on H. pylori strains isolated from Taiwanese patients with severity of gastric diseases. Methods. H. pylori isolates were cultured from gastric biopsies from 99 patients with peptic ulcer, chronic gastritis, and gastric carcinoma. Six distinct, well-separated colonies were isolated from each patient and analyzed by genotyping. Results. cagA, cagE, cagM, cagT, orf10, and orf13 were found to be present in 90.0%–100.0% of the H. pylori isolates. A total deletion of cagA, cagE, cagM, cagT, orf10, and orf13 was found in 1 isolate (1.0%). The IS605 element was found to be positive in 15.2% of the isolates. The presence of IS605 was higher in H. pylori isolated from patients with gastric carcinoma (25.0%) than in patients with duodenal ulcer (6.5%) or chronic gastritis (6.3%) (P<0.001). Conclusions. The majority of the patients examined had intact cag-PAI. IS605 was present in 15.2% and was higher in H. pylori isolated from patients with gastric carcinoma than in those with peptic ulcer

Targeting proteins to deliver therapeutic or diagnostic reagents

The present invention is directed to compositions comprising an angiogenesis inhibitor coupled to a therapeutic or diagnostic agent. In a specific embodiment, the composition is a fusion gene or fusion gene product encoding the angiogenesis inhibitor coupled to a therapeutic or diagnostic agent. In a particular embodiment, the composition is used for methods to treat angiogenesis-related diseases, such as cancer.Board of Regents, University of Texas Syste

Effects of honokiol on the expressions of GRP94 and GRP78 in human gastric cancer cell lines.

<p>(A) Western blot analyses for GRP94 and GRP78 in MKN45 cells treated with honokiol for 8 h in a dose-response manner. (B) Western blot analyses for GRP94 and GRP78 in MKN45 cells treated with honokiol (a, 20µM; b, 40 µM) in a time-response manner. (C) Comparison of GRP94 protein expression among human gastric cancer cell lines (SCM-1, AGS, N87, and MKN45), tumor isolated from MKN45 cells-inoculated mice (T), normal mouse gastric epithelium tissue (N), and human umbilical vein endothelial cells (HUVEC). All results shown are representative of at least four independent experiments.</p

Suppression of calpain activity by calpain inhibitors or c siRNA-calpain-II decreased honokiol-induced cell apoptosis.

<p>Human gastric cancer cells were analyzed for apoptosis by Annexin V/PI staining as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001096#s2" target="_blank">Materials and Methods</a>. (A) SCM-1 cells were treated with honokiol (HK, 40 µM) for 4 h in the presence or absence of calpain inhibitors (ALLN and ALLM, 50 µM). (B) AGS cells transfected with siRNA-calpain-I- or siRNA-calpain-II were treated with honokiol (HK, 20 µM) for 4 h. Results shown are representative of at least four independent experiments.</p

Kinetics changes of GRP94 and GRP78 protein expression in various human gastric cancer cell lines following honokiol treatments.

<p>Western blot analyses for GRP94 and GRP78 in cells (N87 (A), AGS (B), MKN45 (C) and SCM-1 (D)) treated with 40 µM honokiol for various time courses as indicated. Data are presented as mean±SEM (n = 5).</p

Honokiol induces calpain-mediated GRP94 degradation and apoptosis.

<p>Western blotting for determination of GRP94 degradation and calpain-I and II expression and activation of caspase-7 and caspase-12 in SCM-1 cells 24 h after honokiol (40 µM) treatment in the presence or absence of calpain inhibitors (ALLN and ALLM, 25 and 50 µM) was detected. In some experiments, SCM-1 cells were transfected with calpain-II-siRNA or control-siRNA. Results shown are representative of at least four independent experiments.</p

Effects of honokiol on calpain-I and II protein levels and interaction of calpain and GRP94.

<p>Cells were treated with honokiol (20–60 µM) for various time courses as indicated. (A) Calpain-I and II protein levels were detected by Western blot analysis in honokiol-treated SCM-1 cells. (B) Primary antibodies for calpain-II and GRP94 were applied to the cells (MKN45 and SCM-1) followed by secondary antibodies coupled with FITC-conjugated or TRITC-conjugated, respectively. Co-localization of two labeled antigens was detected as a single image when the images from both channels were overlaid. (C) Interaction of calpain and GRP94 were detected in N87, AGS, MKN45 and SCM-1 cells. Immunoprecipitated proteins were collected and subjected to SDS-PAGE and immunoblotting with anti-calpain-II or anti-GRP94 antibodies. Results shown are representative of at least four independent experiments.</p

Honokiol induces a GRP94 degradation-associated apoptotic response in human gastric cancer cells.

<p>(A) Western blot analyses for PARP and GADD153 in MKN45 cells treated with honokiol for 8 h in a dose-response manner. (B) Time course responses for PARP, GADD153, caspase-7 and caspase-12 in MKN45 cells treated with honokiol (20 µM). Results shown are representative of at least four independent experiments.</p