GRP78 Knockdown Enhances Apoptosis via the Down-Regulation of Oxidative Stress and Akt Pathway after Epirubicin Treatment in Colon Cancer DLD-1 Cells

INTRODUCTION: The 78-kDa glucose-regulated protein (GRP78) is induced in the cancer microenvironment and can be considered as a novel predictor of responsiveness to chemotherapy in many cancers. In this study, we found that intracellular reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation were higher in GRP78 knockdown DLD-1 colon cancer cells compared with scrambled control cells. METHODOLOGY/PRINCIPAL FINDINGS: Treatment with epirubicin in GRP78 knockdown DLD-1 cells enhanced apoptosis and was associated with decreased production of intracellular ROS. In addition, apoptosis was increased by the antioxidants propyl gallate (PG) and dithiothreitol (DTT) in epirubicin-treated scrambled control cells. Epirubicin-treated GRP78 knockdown cells resulted in more inactivated Akt pathway members, such as phosphorylated Akt and GSK-3β, as well as downstream targets of β-catenin expression. Knockdown of Nrf2 with small interfering RNA (siRNA) increased apoptosis in epirubicin-treated GRP78 knockdown cells, which suggested that Nrf2 may be a primary defense mechanism in GRP78 knockdown cells. We also demonstrated that epirubicin-treated GRP78 knockdown cells could decrease survival pathway signaling through the redox activation of protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase that negatively regulates the Akt pathway. CONCLUSIONS: Our results indicate that epirubicin decreased the intracellular ROS in GRP78 knockdown cells, which decreased survival signaling through both the Akt pathway and the activation of PP2A. Together, these mechanisms contributed to the enhanced level of epirubicin-induced apoptosis that was observed in the GRP78 knockdown cells

The effect of the Akt pathway on epirubicin (epi) treatment.

<p>Scrambled control and GRP78 knockdown DLD-1 cells were treated with epirubicin (500 ng/ml) for the indicated times. The phosphorylation of (A) Akt and (C) GSK 3β was evaluated by western blotting. (B) Akt activity was determined as outlined in the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035123#s2" target="_blank">Materials and Methods</a></i>. The protein on the PVDF membrane stained with Coomassie brilliant blue is shown as an internal control. These experiments were performed at least 3 times, and a representative experiment is presented.</p

The effect of Nrf2 on epirubicin (epi) treatment.

<p>(A) Scrambled control and GRP78 knockdown DLD-1 cells were cultured for 48 h, and nuclear Nrf2 expression was evaluated by western blotting. (B) The GRP78 knockdown DLD-1 cells were pretreated with scrambled siRNA (sc) or Nrf2 siRNA for 24 h, and nuclear Nrf2 expression was evaluated by western blotting. (C) The GRP78 knockdown DLD-1 cells were pretreated with scrambled siRNA (scramble) or Nrf2 siRNA for 24 h and treated with 500 ng/ml epirubicin (epi) for another 48 h. After treatment, the percentage of cells that were in the subG₁ phase indicated by DNA damage was determined as outlined in the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035123#s2" target="_blank">Materials and Methods</a></i>. These experiments were performed at least 3 times, and a representative experiment is presented.</p

Expression and activity of protein phosphatase 2A (PP2A) after epirubicin (epi) treatment.

<p>Scrambled control and GRP78 knockdown DLD-1 cells were treated with epirubicin (500 ng/ml) for the indicated times. (A) The expression of PP2A was evaluated by western blotting, and (B) the activity of PP2A was determined as outlined in the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035123#s2" target="_blank">Materials and Methods</a></i>. These experiments were performed at least 3 times, and a representative experiment is presented.</p

The effect of intracellular oxidative stress on epirubicin (epi) treatment.

<p>Scrambled control and GRP78 knockdown DLD-1 cells were (A) cultured for 24 and 48 h or (B) treated with epirubicin (500 ng/ml) for 24 or 48 h. (C) GRP78 knockdown DLD-1 cells were treated with epirubicin (500, 750, 1000 ng/ml) for 48 h. Intracellular ROS were evaluated by DCFH-DA staining and flow cytometry as outlined in the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035123#s2" target="_blank">Materials and Methods</a></i>. (D) Scrambled control DLD-1 cells were treated with 500 ng/ml epirubicin (epi) alone, 25 µM propyl gallate (PG) alone, 1 mM dithiothreitol (DTT) alone or epirubicin combined with PG or DTT for 48 h . The percentages of cells that were in the subG₁ phase (indicated by DNA damage) were determined as outlined in the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035123#s2" target="_blank">Materials and Methods</a></i>. The values are represented as the mean ± standard deviation (n = 5–8) of the individual experiments. A significant difference from the control group was set at <i>P</i><0.05 (*).</p

Matrine Attenuates COX-2 and ICAM-1 Expressions in Human Lung Epithelial Cells and Prevents Acute Lung Injury in LPS-Induced Mice

Matrine is isolated from Sophora flavescens and shows anti-inflammatory effects in macrophages. Here we evaluated matrine’s suppressive effects on cyclooxygenase 2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expressions in lipopolysaccharide- (LPS-) stimulated human lung epithelial A549 cells. Additionally, BALB/c mice were given various matrine doses by intraperitoneal injection, and then lung injury was induced via intratracheal instillation of LPS. In LPS-stimulated A549 cells, matrine inhibited the productions of interleukin-8 (IL-8), monocyte chemotactic protein-1, and IL-6 and decreased COX-2 expression. Matrine treatment also decreased ICAM-1 protein expression and suppressed the adhesion of neutrophil-like cells to inflammatory A549 cells. In vitro results demonstrated that matrine significantly inhibited mitogen-activated protein kinase phosphorylation and decreased nuclear transcription factor kappa-B subunit p65 protein translocation into the nucleus. In vivo data indicated that matrine significantly inhibited neutrophil infiltration and suppressed productions of tumor necrosis factor-α and IL-6 in mouse bronchoalveolar lavage fluid and serum. Analysis of lung tissue showed that matrine decreased the gene expression of proinflammatory cytokines, chemokines, COX-2, and ICAM-1. Our findings suggest that matrine improved lung injury in mice and decreased the inflammatory response in human lung epithelial cells

Analysis of GRP78 expression, cell viability and apoptosis.

<p>(A) Analysis of GRP78 expression in the scrambled and GRP78 knockdown DLD-1 cells by western blotting. Analysis of (B) cell viability and (C) apoptosis after treatment with vehicle (control) and epirubicin (epi). Scrambled control and GRP78 knockdown DLD-1 cells were treated with epirubicin (500 ng/ml) for 48 h. Cell viability was evaluated by the trypan blue exclusion assay. The percentages of cells that were in the subG₁ phase (indicated by DNA damage) were determined as outlined in the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035123#s2" target="_blank">Materials and Methods</a></i>. The values are represented as the mean ± standard deviation (n = 5–8) of the individual experiments. Significant differences for the control group and the scrambled control group are <i>P</i><0.05 (*) and <i>P</i><0.05 (#), respectively. These experiments were performed at least 3 times, and a representative experiment is presented.</p

Evaluation of the cytosolic cytochrome c and mitochondrial membrane potential in shikonin treatment.

<p>(A) U87MG and Hs683 cells were treated with 2 μM shikonin for 3, 6, 12 or 24 h. After treatment, cytosolic protein was extracted to assess cytochrome c expression. Fifty micrograms of protein were loaded onto a 12% SDS-polyacrylamide gel and evaluated by western blotting. These experiments were performed at least 3 times; a representative experiment is presented. (B) U87MG and Hs683 cells were treated with 2 μM shikonin for 6 h. After treatment, cells were stained with 5 μM rhodamine 123 (Rho 123) for 30 min. The rhodamine 123 fluorescence represented the mitochondrial membrane potential was detected by flow cytometry. Significant differences from the untreated group show <i>P</i><0.05 (*).</p

Evaluation of the intracellular shikonin concentration in shikonin-treated glioma cells.

<p>U87MG cells were treated with 2, 8, 50 and 100 μM of shikonin for 2 h. After treatment, cells were shattered by sonication and then centrifuged at 12,000 g for 10 min at 4°C. The cytosolic substances (supernatant) were evaluated the shikonin concentration detected by HPLC.</p