HPLC chromatogram of AKBA in plasma (left) and PaCa tissue (right) in mice.

<p>HPLC chromatogram of AKBA in plasma (left) and PaCa tissue (right) in mice.</p

AKBA potentiates gemcitabine to inhibit distant organ metastasis in orthotopic PaCa nude mice.

<p>AKBA and gemcitabine combined inhibited metastases to the spleen, liver, and lungs. Mice were killed, and their abdomen was opened surgically, and then the number of metastasis foci in each organ was counted. Columns, mean; bars, SE.</p

AKBA enhances the effect of gemcitabine to inhibit tumor cell proliferation and angiogenesis in PaCa.

<p>A, the results of an immunohistochemical analysis of proliferation marker Ki-67 indicated that PaCa cell proliferation was inhibited in mice treated with AKBA alone and in combination with gemcitabine. B, quantification of Ki-67 positive cells was described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026943#s4" target="_blank"><i>Materials and Methods</i></a>. Columns, mean; bars, SE. C, the results of an immunohistochemical analysis of CD31 for microvessel density in PaCa indicated that angiogenesis was inhibited by AKBA alone and in combination with gemcitabine. D, quantification of CD31 positive cells was described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026943#s4" target="_blank"><i>Materials and Methods</i></a>. Columns, mean; bars, SE.</p

AKBA enhances the effect of gemcitabine against the expression of NF-κB and NF-κB–regulated gene products in pancreatic cancer tissues.

<p>A, detection of NF-κB by DNA binding in orthotopic tumor tissue samples showed the inhibition of NF-κB by AKBA. B, Western blot analysis showed that AKBA alone or combination with gemcitabine inhibited the expression of NF-κB–dependent genes. C, immunohistochemical analysis of NF-κB–regulated genes (COX-2, MMP-9, CXCR4 and VEGF) in pancreatic tumor tissues from mice. Percentages indicate positive staining for the given biomarker. Samples from three animals in each group were analyzed, and representative data are shown.</p

AKBA inhibits proliferation of PaCa cells and enhances the apoptosis of gemcitabine<i>in vitro</i>.

<p>A, MTT assay results showed dose-dependent suppression of cell proliferation in all four pancreatic cancer cell lines tested. Data are representative of three independent experiments. B, PANC-28 (1×10⁶) cells were treated with AKBA (10, 25, and 50 µmol/L) for 12 h; nuclear extracts were prepared and then assayed for NF-κB activation by EMSA. C, Western blot analysis showed that AKBA inhibited constitutive expression of NF-κB–regulated gene products that regulate antiapoptosis, proliferation, and metastasis in pancreatic cancer cells. The MIA PaCa-2 (1×10⁶) cells were treated with AKBA (10, 25 and 50 µmol/L) for 24 h. Whole-cell lysates were prepared and assayed for NF-κB–regulated gene products by Western blotting. Data represent two independent experiments. D, AKBA potentiates the apoptotic effects of gemcitabine in pancreatic cancer cells <i>in vitro</i>. LIVE/DEAD assay results indicated that AKBA potentiates gemcitabine-induced cytotoxicity. Percentages are proportions of apoptotic pancreatic cancer cells. Data are representative of two independent experiments. E, The MIA PaCa-2 (1×10⁶) cells were treated with AKBA (25 µmol/L) for 12 h and then gemcitabine (500 nmol/L) was added for 24 h. Whole-cell lysates were prepared and subjected to Western blotting. Data represent two independent experiments.</p

AKBA potentiates the effect of gemcitabine in blocking the growth of pancreatic cancer in nude mice.

<p>A, schematic of experimental protocol described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026943#s4" target="_blank"><i>Materials and Methods.</i></a> Group I was given corn oil (100 µL, p.o., daily), group II was given AKBA (100 mg/kg, p.o., daily), group III was given gemcitabine (25 mg/kg, i.p., twice weekly), and group IV was given AKBA (100 mg/kg, p.o., daily) and gemcitabine (25 mg/kg, i.p., twice weekly; n = 5). B, bioluminescence IVIS images of orthotopically implanted pancreatic tumors in live, anesthetized mice and measurements of photons per second depicting tumor volume at various time points using live IVIS imaging at the indicated times (n = 5). Points, mean; bars, SEM. C, Necropsy photographs of mice. D, tumor volumes in mice measured on the last day of the experiment at autopsy with Vernier calipers and calculated using the formula V = 2/3πr³ (n = 5). Columns, mean; bars, SE.</p

Cardamonin suppresses RANKL-induced IκBα degradation and phosphorylation through inhibition of IKK activity.

<p>(<b>A</b>) RAW 264.7 cells (1.5×10⁶/well) were incubated with 20 µM of cardamonin for 12 h and then treated with 10 nM RANKL for the indicated times (min). Cytoplasmic extracts were examined for IκBα degradation by western blot using an anti- IκBα antibody. Anti−β-actin was used as a loading control. (<b>B</b>) RAW 264.7 cells (1.5×10⁶/well) were pretreated with cardamonin (20 µM) for 12 h, then incubated with ALLN (50 µg/mL) for 30 min, and then treated with RANKL (10 nM) for 15 min. Cytoplasmic extracts were prepared and analyzed by western blot using phospho-IκBα antibody. The same membrane was reprobed with IκBα and β-actin antibody. (<b>C</b>) RAW 264.7 cells were pretreated with cardamonin (20 µM) for 12 h and then incubated with RANKL (10 nM) for the indicated times (min). Whole-cell extracts were immunoprecipitated using an antibody against IKKα and analyzed by an immune complex kinase assay using recombinant GST-IκBα as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064118#s2" target="_blank">Materials and Methods</a>. To examine the effect of cardamonin on the level of IKK proteins, whole-cell extracts were analyzed by western blot using anti-IKKα and anti-IKKβ antibodies. Values below the blot indicate fold change (GST-IκBα/IKKα) compared with control. Control was set as fold 1. (<b>D</b>) RAW 264.7 cells (1.5×10⁶/well) were pre-incubated with cardamonin for 12 h and then exposed to RANKL (10 nM) for the indicated times. Whole-cell extracts were analyzed by western blotting using relevant antibodies.</p

Cardamonin inhibits RANKL-induced osteoclastogenesis.

<p>(<b>A</b>) RAW 264.7 cells (5×10³/well) were incubated with cardamonin (500 nM) or RANKL (5 nM) alone, or with RANKL plus cardamonin (0, 50, 100, 250, 500 nM) for 5 days, and then stained for TRAP expression. TRAP-positive cells were photographed. Original magnification,×100. (<b>B</b>) Quantification of multinucleated osteoclasts (i.e., those containing three nuclei) after treatment with medium, RANKL (5 nM) alone, or RANKL plus cardamonin (0, 50, 100, 250, 500 nM) for 5 days. Values represent means ± SD. Data are representative of two independent experiments performed in triplicates; *, <i>P</i><0.01, and **, <i>P</i><0.05 <i>vs</i>. RANKL alone. (<b>C</b>) RAW 264.7 cells (5×10³ cells) were incubated with Indicated concentration of cardamonin for 12 h, and then treated with 5 nM RANKL for 5 days. To determine cell viability, cells were trypsinized, and then subjected to trypan blue exclusion assay. Data represent the means ± SD of triplicate samples. (<b>D</b>) RAW 264.7 cells (3×10³/well) were seeded into calcium phosphate apatite-coated plates, treated with cardamonin (500 nM) or RANKL (5 nM) alone, or with RANKL plus cardamonin. After 5 days, remaining cells were imaged under light microscope. Arrows indicate pit formation.</p

Cardamonin suppresses osteoclastogenesis induced by tumor cells.

<p>(<b>A</b>) RAW 264.7 cells (5×10³/well) were incubated in the presence of MDA-MB-231 cells (1×10³/well) and exposed to cardamonin (500 nM) for 5 days, and finally stained for TRAP expression. Multinucleated osteoclasts were counted (right panel). Values represent means ± SD. Data are representative of two independent experiments performed in triplicates; *, <i>P</i><0.05, and ***, <i>P</i><0.001. (<b>B</b>) RAW264.7 cells (5×10³/well) were incubated in the presence of U266 cells (1×10³/well) and exposed to cardamonin (500 nM) for 5 days, and finally stained for TRAP expression. Multinucleated osteoclasts were counted (right panel). Values represent means ± SD. Data are representative of two independent experiments performed in triplicates; <b>*</b>, <i>P</i><0.05, and **, <i>P</i><0.01. (<b>C</b>) U266 cells (1×10⁶/well) were incubated with indicated concentration of cardamonin for 24 h. Whole cell extracts were prepared and analyzed by western blotting using RANKL antibody. Anti−β-actin was used as a loading control.</p

RANKL induces NF-κB activation and cardamonin inhibits it in a dose- and time-dependent manner.

<p>(<b>A</b>) The structure of cardamonin. (<b>B</b>) RAW 264.7 cells (1.5×10⁶/well) were incubated with different concentrations of cardamonin for 12 h, treated with 10 nM RANKL for 30 min, and examined for NF-κB activation by EMSA. Fold value is based on the value for medium (control), arbitrarily set at 1. (<b>C</b>) RAW 264.7 cells (1.5×10⁶/well) were incubated with 20 µM of cardamonin for 12 h, treated with 10 nM RANKL for the indicated times (min), and examined for NF-κB activation by EMSA. Fold value is based on the value for medium (control), arbitrarily set at 1. CV, cell viability.</p