Effects of MAPK inhibitors on iNOS, COX-II, NF-κB, and IκBα expression in LPS-stimulated RAW 264.7 cells.

<p>Cells were pre-treated with (A) ERK inhibitor (PD98059), (B) p38 inhibitor (SB203580), or (C) JNK inhibitor (SP600125) for 1 h and stimulated with LPS (0.1 μg/mL) for 15 min (for evaluation of expression of phospho-ERK, phospho-p38, phospho-JNK, ERK, p38, and JNK) or 24 h (for expression of iNOS and COX-II). For data in (A), (B), and (C), phospho-JNK, phospho-ERK, phospho-p38, ERK, p38, JNK, iNOS, and COX-II levels were detected by western blot analysis. Cells were pre-treated with 10 μM JNK inhibitor (SP600125) for 1 h before treatment with or without LPS (0.1 μg/mL) for 24 h. (D) IL-6 and (E) TNF-α production were determined by an ELISA assay. (F) Cells were pre-treated with JNK inhibitor (SP600125) (10 μM) and exposed to 0.1 μg/mL LPS for 15 min. The expression of cytosolic NF-κB subunit (p65) and IκBα and nuclear NF-κB subunit (p65) was determined by western blot analysis. All data are expressed as the means ± SEM from three independent experiments. *P < 0.05 and **P < 0.01 are significantly different from the control.</p

Effects of PNAPs on IL-6 and TNF-α expression in LPS-stimulated RAW 264.7 cells.

<p>After pre-treatment with PNAPs at different concentrations for 1 h, cells were stimulated with or without LPS (0.1 μg/mL) for 24 h. The concentration of (A) IL-6 or (B) TNF-α in medium was measured by ELISA. The + symbols on the x-axis indicate pre-treatment with PNAPs. Data are expressed as the means ± SEM from three independent experiments. *p < 0.05 or **p < 0.01 compared to the LPS group. Note: in panel A and B, for cells treated with PNAP only, IL-6 and TNF-α concentrations were low or undetectable using the IL-6 and TNF-α ELISA kit.</p

Effect of PNAPs on NO expression in LPS-stimulated RAW 264.7 cells.

Cells were pre-treated with PNAPs in different concentrations and exposed to 0.1 μg/mL LPS for 24 h. NO production in the supernatant was measured using the Griess reaction. The + symbols on the x-axis indicate pre-treatment with PNAPs. Data represent the mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, indicate significant differences compared to the LPS-treated group.</p

Chemical Structures of 2-Phenylnaphthalene Derivatives PNAP-1−PNAP-8.

<p>Chemical Structures of 2-Phenylnaphthalene Derivatives PNAP-1−PNAP-8.</p

Effect of PNAPs on the viability of RAW 264.7 cells.

<p>Cells were incubated with PNAPs (0–50 μM) for 24 h. Cell viability was examined by MTT assays. (A) <b>PNAP</b>-<b>1</b>, (B) <b>PNAP</b>-<b>2</b>, (C) <b>PNAP</b>-<b>3</b>, (D) <b>PNAP</b>-<b>4</b>, (E) <b>PNAP</b>-<b>5</b>, (F) <b>PNAP</b>-<b>6</b>, (G) <b>PNAP</b>-<b>7</b>, and (H) <b>PNAP</b>-<b>8</b>. Data represent the mean ± SEM from three independent experiments. *P < 0.05 and **P < 0.01 compared to the control.</p

Effects of PNAPs on iNOS and COX-II expression in LPS-stimulated RAW 264.7 cells.

<p>Cells were pre-treated with PNAPs at different concentrations for 1 h and then stimulated with or without 0.1 μg/mL LPS for 24 h. iNOS and COX-II protein expression were detected by western blotting. Results were normalized to β-actin expression. The protein levels of iNOS and COX-II after LPS treatment is expressed as the control, which was arbitrarily assigned a value of 1.0.</p

Effects of PNAPs on MAPK and NF-κB activation in LPS-stimulated RAW 264.7 cells.

<p>Cells were pre-treated with (A) <b>PNAP</b>-<b>6</b> or (B) <b>PNAP</b>-<b>8</b> (2.5, 5, and 10 μM) and exposed to 0.1 μg/mL LPS for 15 min. phospho-ERK, phospho-p38, phospho-JNK, ERK, p38, JNK, IκBα, and NF-κB subunit (p65), were then analyzed by western blotting. The + symbols on the x-axis indicate pre-treatment with PNAPs. The data are expressed as the means ± SEM from three independent experiments. *P < 0.05 and **P < 0.01 are significantly different from the control.</p

Effect of PNAP-1, -3h, and -6h on MCF-7 cell cycle progression.

<p>MCF-7 cells were treated with <b>PNAP</b>-<b>1</b>, -<b>3h</b>, and -<b>6h</b> at three concentrations (5, 10, and 25 μM) for 48 h, and then, (A) the cells were fixed and stained with propidium iodide to analyze the DNA content by flow cytometry. (B, C) Cell cycle-associated proteins (cyclin D1, CDK4, cyclin E, CDK2, p21, p27, cyclin B1, and CDK1) were detected and analyzed by western blot. The expression was quantified with the computerized Gel-Pro Analyzer image analysis system. The data are expressed as the means ± SEM from three independent assays. *P<0.05 and **P<0.01 are significantly different from the control.</p

Proposed model of PNAP-6h-mediated MCF-7 cell cycle arrest and apoptosis.

<p><b>PNAP</b>-<b>6h</b> causes cell cycle arrest at the S and G2/M phases. <b>PNAP</b>-<b>6h</b> modulates MCF-7 apoptosis via activation of the intrinsic/extrinsic pathways and the p38/ERK pathway.</p

Effect of 2-Phenylnaphthalenes PNAP-1−PNAP-8, Their Demethylation Derivatives PNAP-2h−PNAP-7h, and Amino PNAP-8h on the Viability of MCF-7 Cells.

<p>The MCF-7 cell line was treated with <b>PNAP</b>-<b>1</b>−<b>PNAP</b>-<b>8</b>, and <b>PNAP</b>-<b>2h</b>−<b>PNAP</b>-<b>8h</b> at eight concentrations and then incubated at 37°C for 48 h. The cell viability was examined via the MTT assay.</p><p>^a The number of viable cells remaining after PNAP treatment is expressed as a percentage of the vehicle control, which was arbitrarily assigned a value of 100.0%. The results are presented as the means ± SEM from three independent assays.</p><p>*P<0.05 and **P<0.01 compared with the control.</p><p>Effect of 2-Phenylnaphthalenes PNAP-1−PNAP-8, Their Demethylation Derivatives PNAP-2h−PNAP-7h, and Amino PNAP-8h on the Viability of MCF-7 Cells.</p