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 activates calpain activity.

<p>Calpain activity was measured with the fluorescent calpain substrate Suc-LLVY-AMC in N87, AGS, MKN45 and SCM-1 cells. (A) Time course responses to honokiol (20 µM) treatment. Data are expressed in terms of fold of control conditions. (B) Honokiol (20 and 40 µM) increases calpain activity at 60 min treatment. (C) Calpain inhibitors ALLN, ALLM, and Z-Leu-Leu-CHO; 25 and 50 µM) significantly inhibited honokiol-increased calpain activity. Data are presented as mean±SEM (n = 4).</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

Silencing of GRP94 by siRNA induces cell apoptosis.

<p>(A) Apoptosis in GRP94-siRNA-transfected MKN45 cells was analyzed 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>, which was compared with cells treated with honokiol (40 µM) or etoposide (40 µM). (B-a) GRP94 protein levels were detected by Western blot analysis in control and GRP94-siRNA-transfected MKN45 cells. (B-b) Honokiol and etoposide induce GRP94 cleavage in MKN45 cells. Cells were treated with 20 µM honokiol and 40 µM etoposide (Etopo.) as indicated for 4 h. 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

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

Effect of honokiol on metastasis of gastric tumor cells <i>in vivo</i>.

<p>[¹⁸F]-FDG-PET served as a surrogate measure of therapeutic efficacy. Tumors in nude mice were established for 7 days after the intraperitoneal inoculation of gastric cancer cells (MKN45, A; SCM-1, B). Mice were then injected intraperitoneally with honokiol (5 mg/kg/twice per week). Twenty-eight days after honokiol treatment, FDG-PET/CT images of mice were taken, and then mice were sacrificed for macroscopic examination of the distribution of disseminated metastasis. Representative FDG-PET/CT images of animals inoculated with MKN45 (A-a) or SCM-1 (B-a) gastric cancer cells with or without honokiol (HK) treatment are shown. HK (5 mg/kg) was administered by intraperitoneal injection. The maximum intensity projection of typical representative nude mice (left, tumor control; right, HK treatment) is shown. Moreover, many metastatic nodules were found in the mesentery of the control mice inoculated with MKN45 (A-b) or SCM-1 (B-b) cells. In contrast, peritoneal metastasis was observed sporadically in honokiol-treated mice.</p

Schematic representation of peritoneal metastasis mouse model with or without honokiol treatment.

<p>(A) Four groups of mice were investigated and listed. (B) The metastatic conditions in cancer cell inoculation by PET/CT surveillance and further then for honokiol treatment. The human gastric cancer cells (4∼5×10⁶ cells) MKN45 (a) and SCM-1 (b) were inoculated to mice at day 7.</p