Inhibition of tumor development in CCR5^−/− mice.

<p><b>A–C,</b> Tumor images, volumes, and weights. B16 melanoma cells (5×10⁵ cells/mouse) were inoculated s.c. into CCR5^+/+ mice and CCR5^−/− mice (n = 20). Tumor volumes were measured every day and tumor weights were measured at study termination (Day 31). The CCR5^−/− mice had a significant reduction in tumor growth and volume as compared to CCR5^+/+ mice. The results are expressed as mean ± SD. * indicates significant difference from CCR5^+/+ mice (P<0.05). <b>D</b>, The CCR5^−/− mice demonstrated significantly higher survival rates compared to CCR5^+/+ mice (by Log-rank Test). <b>E</b>. Tumor sections were analyzed by H&E stain and expression of proliferating cell nuclear antigen (PCNA) by immunohistochemistry. Data are means ± S.D. of four experimental animals. * indicates significant difference from CCR5^+/+ mice (P<0.05). Scale bar indicates 50 m.</p

Induction of the infiltration of CD8⁺ T cells and NK cells into tumor, and increase in spleen of CCR5^−/− mice.

<p><b>A and B</b>, Immunolfluorescence analysis was used to determine the expression levels of CD8 (CD8⁺ cytotoxic T-cell surface marker) and CD57 (NK cell marker) in tumor and spleen sections. The images shown are representative of three separate experiments performed in triplicate. Scale bars indicate 50 µm (A) and 100 µm (B). <b>C and D</b>, Analysis of lymphocyte phenotypes. The tumor and spleen tissues were separated at study termination (Day 31). Flow cytometry analysis was performed using FACSAria flow cytometry, and represent data were shown. Data are means ± S.D. of four experimental animals.</p

Decrease of NF-κB activity in tumor tissue of CCR5^−/− mice.

<p><b>A</b>, The DNA binding activity of NF-κB was determined in the nuclear extracts of the CCR5^−/− mice and CCR5^+/+ mice tumor tissues by EMSA described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033747#s4" target="_blank">material and methods</a>. <b>B</b>, Expression of p50 and p65 phosphorylation in nuclear extracts (NE), and IκB and IκB phosphorylation in the cytosol (CE) determined by Western blotting. <b>C</b>, Immunolfluorescence analysis of p50 confirmed that the intensities of nuclear staining for p50 were decreased in the tumor tissues of CCR5^−/− mice. Data are means ± S.D. of four experimental animals. * indicates significant difference from CCR5^+/+ mice (p<0.05). Scale bar indicates 50 µm.</p

Induction of apoptotic cell death in tumor tissues of CCR5 ^−/− mice.

<p><b>A</b>, Apoptotic cells in tumor sections were examined by fluorescence microscopy after DAPI and TUNEL staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033747#s4" target="_blank">material and methods</a>. The apoptotic index was determined as the number of DAPI-stained, TUNEL-positive cells that were counted. Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5^+/+ mice. Scale bar indicates 50 µm. <b>B and C</b>, Apoptotic protein (cleaved caspase-3 and Bax) in tumor sections were detected by immunofluorescence assay. The reactive cell number was determined as the number of DAPI-stained, Specific antibody (cleaved caspase-3 and Bax)-positive cells that were counted. Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5^+/+ mice. Scale bar indicates 50 µm. <b>D</b>, The expression of apoptotic proteins was detected by Western blotting using specific antibodies; cleaved caspase-3, cleaved caspase-9, cleaved PARP, Bax, Bcl-2 and c-IAP1 in the tumor tissues. The β-actin protein was used as an internal control. Each band is representative of three independent experimental results. Data are means ± S.D. of three experimental animals. * indicates significant difference from CCR5^+/+ mice (p<0.05).</p

Upregulation of IL-1Ra in the tumor and spleen of CCR5^−/− mice.

<p><b>A</b>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033747#pone-0033747-g004" target="_blank">Figure 4A</a> indicates mouse cytokine array panel coordinates. Nitrocellulose membranes contain 40 different anti-cytokine antibodies printed in duplicate. <b>B</b>, Mouse cytokine array panel indicate the cytokine expression difference in tumor tissues of CCR5^+/+ mice and CCR5^−/− mice, especially IL-1Ra. Representative blot from three independent experiments is shown. Positive controls show the manufacturer’s internal positive control samples on the membrane. <b>C</b>, Protein immune-arrays were performed using Mouse cytokine array in spleen tissues. There were differences in cytokines between CCR5^+/+ mice and CCR5^−/− mice, especially IL-1Ra. Representative blot from three independent experiments is shown. Positive controls show the manufacturer’s internal positive control samples on the membrane. <b>D and E</b>, Immunolfluorescence analysis was used to determine the expression levels of IL-1Ra in tumor and spleen tissues. The reactive cell number was determined as the number of DAPI-stained, IL-1Ra antibody-positive cells that were counted. Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5^+/+ mice. Scale bar indicates 50 µm. <b>F and G</b>, Expression of IL-1Ra was analyzed by western blotting in tumor and spleen tissues. Each band is representative of three independent experimental results. Data are means ± S.D. of three experimental animals. * indicates significant difference from CCR5^+/+ mice (p<0.05).</p

Diterpenoids from the Roots of <i>Euphorbia fischeriana</i> with Inhibitory Effects on Nitric Oxide Production

Bioactivity-guided isolation of a methanolic extract of <i>Euphorbia fischeriana</i> led to the isolation of four new abietane-type diterpenoids, fischeriolides A–D (<b>1</b>–<b>4</b>), together with 11 known diterpenoids. Their structures were elucidated based on the interpretation of 1D and 2D NMR spectroscopic and HRESIMS data. The absolute configuration of compound <b>3</b> was determined by single-crystal X-ray diffraction analysis and electronic circular dichroism methods. Compounds <b>5</b>–<b>9</b> exhibited inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages with IC₅₀ values in the range 4.9–12.6 μM

Effect of thiacremonone on apoptotic cell death and the expression of apoptosis regulatory proteins.

<p>(A) The lung cancer cells were treated in the absence (<i>left panels</i>) and presence of thiacremonone (50 μg/ml, <i>right pannels</i>) for 72 hrs, and then labeled with DAPI and TUNEL solution. Total number of cells in a given area was determined by using DAPI nuclear staining (fluorescent microscope). The green color in the fixed cells marks TUNEL-labeled cells. For quantification, three randomly selected areas were assessed. The apoptotic index (%) was determined as the (TUNEL-positive cell number/total DAPI stained cell number) x 100 (magnification, 200×). Values are mean ±S.D. * P<0.05 compared with significantly different from untreated control cells. (B) The lung cancer cells were treated with different concentrations of thiacremonone (10, 20, and 50 μg/ml) for 72 hrs. Expression of apoptosis regulatory proteins was determined using Western blot analysis. Each image and band is representative of three independent experiments.</p

Anti-Cancer Effect of Thiacremonone through Down Regulation of Peroxiredoxin 6

<div><p>Thiacremonone (2, 4-dihydroxy-2, 5-dimethyl-thiophene-3-one) is an antioxidant substance as a novel sulfur compound generated from High-Temperature-High-Pressure-treated garlic. Peroxiredoxin 6 (PRDX6) is a member of peroxidases, and has glutathione peroxidase and calcium-independent phospholipase A2 (iPLA2) activities. Several studies have demonstrated that PRDX6 stimulates lung cancer cell growth via an increase of glutathione peroxidase activity. A docking model study and pull down assay showed that thiacremonone completely fits on the active site (cys-47) of glutathione peroxidase of PRDX6 and interacts with PRDX6. Thus, we investigated whether thiacremonone inhibits cell growth by blocking glutathione peroxidase of PRDX6 in the human lung cancer cells, A549 and NCI-H460. Thiacremonone (0–50 μg/ml) inhibited lung cancer cell growth in a concentration dependent manner through induction of apoptotic cell death accompanied by induction of cleaved caspase-3, -8, -9, Bax, p21 and p53, but decrease of xIAP, cIAP and Bcl2 expression. Thiacremonone further inhibited glutathione peroxidase activity in lung cancer cells. However, the cell growth inhibitory effect of thiacremonone was not observed in the lung cancer cells transfected with mutant PRDX6 (C47S) and in the presence of dithiothreitol and glutathione. In an allograft in vivo model, thiacremonone (30 mg/kg) also inhibited tumor growth accompanied with the reduction of PRDX6 expression and glutathione peroxidase activity, but increased expression of cleaved caspase-3, -8, -9, Bax, p21 and p53. These data indicate that thiacremonone inhibits tumor growth via inhibition of glutathione peroxidase activity of PRDX6 through interaction. These data suggest that thiacremonone may have potentially beneficial effects in lung cancer.</p></div

Reverse of inhibitory effect of thiacremonone by transfection of mutant prdx6 (C47S).

<p>(A) Lung cancer cells were transfected with pcDNA-prdx6 or pcDNA-prdx6 C47S, and then, thiacremonone was treated (50 μg/ml) for another 72 hrs. The cells were harvested by trypsinization and stained with 0.2% trypan blue. (B) Cell extracts were analyzed by western blotting. Each image and band is representative of three independent experiments. (C) The levels of glutathione peroxidase activity in lung cancer cells were measured using assay kits, as described in Materials and methods. Values are mean ±S.D. #, p<0.05 significantly different from untreated control cells. *, p<0.05, significantly different from untreated control cells transfected with pcDNA-prdx6 C47S. ^&, p<0.05, significantly different between pcDNA-prdx6 and pcDNA-prdx6 C47S treated with thiacremonone.</p

Effect of thiacremonone on tumor growth in allograft model.

<p>(A) Tumor volumes, weights, and images of normal mice. (B) Tumor volumes, weights, and images of PRDX6 overexpressed mice. Values (A and B) are mean ±S.D. * P<0.05 significantly different from untreated mice. (C) Tumor sections of normal mice were analyzed by H&E stain and expression of proteins by immunohistochemistry. The resultant tissues were developed with DAB, and counterstained with hematoxylin. (D) Tumor sections of PRDX6 overexpressed mice were analyzed by H&E stain and expression of proteins by immunohistochemistry. The resultant tissues were developed with DAB, and counterstained with hematoxylin. For quantification, 200 cells at three randomly selected areas were assessed, and the specific protein positively stained cells were counted. Scale bar indicates 50 μm.</p