Cancer Res Treat.  2015 Jan;47(1):90-100. 10.4143/crt.2013.194.

Synergistic Effect of Sulindac and Simvastatin on Apoptosis in Lung Cancer A549 Cells through AKT-Dependent Downregulation of Survivin

• Kim YS
• Seol CH
• Jung JW
• Oh SJ
• Hwang KE
• Kim HJ
• Jeong ET
• Kim HR

• KMID: 2380394
• DOI: http://doi.org/10.4143/crt.2013.194

Abstract

PURPOSE
Non-steroidal anti-inflammatory drugs (NSAIDs) and statins are potential chemopreventive or chemotherapeutic agents. The mechanism underlying the deregulation of survivin by NSAIDs and statins in human non-small cell lung cancer cells has not been elucidated. In this study, we investigated the synergistic interaction of sulindac and simvastatin in lung cancer A549 cells.
MATERIALS AND METHODS
Cell viability was measured by an MTT assay, while the expression of apoptotic markers, AKT, and survivin in response to sulindac and simvastatin was examined by Western blotting. DNA fragmentation by apoptosis was analyzed by flow cytometry in A549 cells. Reactive oxygen species (ROS) generation was measured by flow cytometry using H2DCFDA and MitoSOX Red, and the effects of pretreatment with N-acetylcysteine were tested. The effects of AKT on survivin expression in sulindac- and simvastatin-treated cells were assessed. Survivin was knocked down or overexpressed to determine its role in apoptosis induced by sulindac and simvastatin.
RESULTS
Sulindac and simvastatin synergistically augmented apoptotic activity and intracellular ROS production in A549 cells. Inhibition of AKT by siRNA or LY294002 inhibited survivin, while AKT overexpression markedly increased survivin expression, even in the presence of sulindac and simvastatin. Moreover, survivin siRNA enhanced sulindac- and simvastatininduced apoptosis. In contrast, survivin upregulation protected against sulindac- and simvastatin-induced apoptosis.
CONCLUSION
Combined treatment with sulindac and simvastatin augmented their apoptotic potential in lung cancer cells through AKT signaling-dependent downregulation of survivin. These results indicate that sulindac and simvastatin may be clinically promising therapies for the prevention of lung cancer.

Keyword

Sulindac; Simvastatin; Apoptosis; Lung neoplasms; Oncogene protein AKT; Survivin

MeSH Terms

Acetylcysteine
Anti-Inflammatory Agents, Non-Steroidal
Apoptosis*
Blotting, Western
Carcinoma, Non-Small-Cell Lung
Cell Survival
DNA Fragmentation
Down-Regulation*
Flow Cytometry
Humans
Hydroxymethylglutaryl-CoA Reductase Inhibitors
Lung Neoplasms*
Oncogene Protein v-akt
Reactive Oxygen Species
RNA, Small Interfering
Simvastatin*
Sulindac*
Up-Regulation
Acetylcysteine
Anti-Inflammatory Agents, Non-Steroidal
Oncogene Protein v-akt
RNA, Small Interfering
Reactive Oxygen Species
Simvastatin
Sulindac

Figure

• 

  Fig. 1. Effect of combined treatment with sulindac and simvastatin on the growth of A549 cells. (A) Cells were treated with different concentrations of sulindac and simvastatin for 48 hours, after which viability was determined by an MTT assay. Survival relative to that of control cells is presented. (B) Cells were incubated with 300 μM sulindac and/or 5 μM simvastatin for 48 hours, after which apoptosis was quantified by DNA content analysis. The percentage of hypodiploid DNA content is indicated for each test condition. Bars represent the mean±standard deviation of three independent experiments. Sim, simvastatin; Sul, sulindac. *p < 0.05 compared to control.

• 

  Fig. 2. Contribution of reactive oxygen species (ROS) generation to the anticancer activity of combination therapy in A549 cells. Cells were treated with 300 μMsulindac and/or 5 μM simvastatin for 48 hours, then loaded with H2DCFDA (blue bars) and MitoSOX Red (green bars). Fluorescence measurements were carried out using a FACSCalibur flow cytometer. The data represent the mean±standard deviation of three independent experiments. Sim, simvastatin; Sul, sulindac. *p < 0.05 compared to the control.

• 

  Fig. 3. Effect of N-acetylcysteine (NAC) on sulindac- and simvastatin-induced apoptosis and survivin expression. (A) Cells were treated with 300 μM sulindac and/or 5 μM simvastatin for 48 hours. (B) Cells were pretreated with or without the free radical scavenger NAC. The expression of phosphorylated AKT, AKT, survivin, X-linked inhibitor of apoptosis protein (XIAP), cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, -8, -9, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured in the cell lysates. Data are representative of two individual experiments. Sim, simvastatin; Sul, sulindac.

• 

  Fig. 4. Effect of AKT on survivin expression in sulindac- and simvastatin-treated cells. (A, B) Effect of AKT inhibition on survivin expression. Cells were pretreated with the phosphoinositide-3 kinase inhibitor LY294002 or transfected with AKT siRNA and then further incubated with sulindac and/or simvastatin for 48 hours. Scrambled RNA containing the same number of each nucleotide found in AKT siRNA was used as the transfection control. (C) Effect of AKT activation on survivin expression. Cells were transiently transfected with constructs expressing constitutively active AKT and then incubated with sulindac and simvastatin for 48 hours. The expression of phosphorylated AKT, AKT, cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, -8, -9, and survivin was measured in the cell lysates. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Data are representative of two individual experiments.

• 

  Fig. 5. Downregulation of survivin with siRNA augments apoptosis induced by sulindac and simvastatin. (A) Cells were transfected with siRNA specific for survivin, and survivin protein expression was assessed by immunoblot analysis 24 hours after transfection. Scrambled RNA containing the same number of each nucleotide found in survivin siRNA was used as the transfection control. The expression of survivin, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase-9 was measured in the cell lysates. (B) Apoptosis was evaluated by green fluorescent protein–annexin V+propidium iodide. Bars represent the mean±standard deviation of three independent experiments. *p < 0.05 for survivin-siRNA and combined treatment with sulindac and simvastatin versus either scrambled siRNA and combined treatment or combined treatment alone. Sul, sulindac; Sim, simvastatin; siSCR, scrambled siRNA; siSUR, survivin-siRNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 6. Effect of survivin overexpression on apoptosis induced by sulindac and simvastatin in A549 cells. (A) Cells were transfected with a survivin gene expression vector, pcDNA3-myc-survivin, and survivin protein expression was assessed by immunoblot analysis 48 hours after transfection. The expression of survivin, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase-3, -8, and -9 was measured in the cell lysates. (B) Apoptosis was evaluated by green fluorescent protein–annexin V+propidium iodide. The data represent the mean±standard deviation of three independent experiments. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Sul, sulindac; Sim, simvastatin; SUR, survivin. *p < 0.05 for the indicated comparisons.

Reference

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