Formoterol Exerts Anti-Cancer Effects Modulating Oxidative Stress and Epithelial-Mesenchymal Transition Processes in Cigarette Smoke Extract Exposed Lung Adenocarcinoma Cells

Abstract

Lung cancer frequently affects patients with Chronic Obstructive Pulmonary Disease (COPD). Cigarette smoke (CS) fosters cancer progression by increasing oxidative stress and by modulating epithelial-mesenchymal transition (EMT) processes in cancer cells. Formoterol (FO), a long-acting β2-agonist widely used for the treatment of COPD, exerts antioxidant activities. This study explored in a lung adenocarcinoma cell line (A549) whether FO counteracted the effects of cigarette smoke extract (CSE) relative to oxidative stress, inflammation, EMT processes, and cell migration and proliferation. A549 was stimulated with CSE and FO, ROS were evaluated by flow-cytometry and by nanostructured electrochemical sensor, EMT markers were evaluated by flow-cytometry and Real-Time PCR, IL-8 was evaluated by ELISA, cell migration was assessed by scratch and phalloidin test, and cell proliferation was assessed by clonogenic assay. CSE significantly increased the production of ROS, IL-8 release, cell migration and proliferation, and SNAIL1 expression but significantly decreased E-cadherin expression. FO reverted all these phenomena in CSE-stimulated A549 cells. The present study provides intriguing evidence that FO may exert anti-cancer effects by reverting oxidative stress, inflammation, and EMT markers induced by CS. These findings must be validated in future clinical studies to support FO as a valuable add-on treatment for lung cancer management.

Keywords: EMT; cigarette smoke; formoterol; inflammation; lung cancer; oxidative stress.

Figure 1

ROS, mitochondrial superoxide, and intracellular ATP production in A549 cells. A549 cells were exposed to CSE 2.5% with or without FO 10⁻⁸ M for 24 h. FO was added 30 min before CSE cell stimulation. ROS, mitochondrial superoxide, and intracellular ATP production were assessed by flow cytometry and the luminescence method. See the Section 4 for further details. ROS (A) and mitochondrial superoxide (C) levels in A549 cells are shown as % positive cells, and intracellular ATP production (E) is shown as Relative Luminescence Units (RLU) and expressed as mean ± SD (n = 5). Representative histograms of relative ROS (B) and mitochondrial superoxide (D) analysis are shown. * p < 0.05 ANOVA with Bonferroni correction.

Figure 2

Extracellular oxidative stress in A549 cells. A549 cells were exposed to CSE 2.5% with or without FO 10⁻⁸ M for 24 h. FO was added 30 min before CSE cell stimulation. Extracellular oxidative stress was evaluated by detecting hydrogen peroxide in an electrochemical sensor. See the Section 4 for further details. Levels of H₂O₂ are reported in μM and expressed as mean ± SD (n = 7). * p < 0.05 ANOVA with Bonferroni correction.

Figure 3

IL-8 release in A549 cells. A549 cells were exposed to CSE 2.5% with or without FO 10⁻⁸ M or FORSK 20 μM for 24 h. FO and FORSK were added 30 min before CSE cell stimulation. See the Section 4 for further details. IL-8 release was evaluated by ELISA. Levels of IL-8 are reported in pg/mL and expressed as mean ± SD (n = 5). * p < 0.05 ANOVA with Bonferroni correction.

Figure 4

E-cadherin protein expression in A549 cells. A549 cells were exposed to CSE 2.5% with or without FO 10⁻⁸ M (A) or FORSK 20 μM (C) for 48 h. FO or FORSK were added 30 min before CSE cell stimulation. E-cadherin protein expression was evaluated by flow cytometry. See the Section 4 for further details. E-cadherin protein expression in A549 cells is shown as % positive cells and expressed as mean ± SD (n = 5). Representative histograms relative to E-cadherin protein expression (B–D) are shown. * p < 0.05 ANOVA with Bonferroni correction.

Figure 5

CDH1 and SNAIL1 gene expression in A549 cells. A549 cells were exposed to CSE 2.5% with or without FO 10⁻⁸ M for 24 h. FO was added 30 min before CSE cell stimulation. See the Section 4 for further details. CDH1 (A) and SNAIL1 (B) gene expression were evaluated by RT-PCR. Results are shown as relative units, expressed as mean ± SD (n = 5), and normalized to the untreated sample (NT). * p < 0.05 ANOVA with Bonferroni correction.

Figure 6

Cell migration and proliferation in A549 cells. A549 cells were exposed to CSE 2.5% with or without FO 10⁻⁸ M for 24 h. FO was added 30 min before CSE cell stimulation. For the scratch assay (A,B), the results are expressed as a percentage of area reduction at 24 h (A) and 48 h (B) compared to time point 0 h (n = 6). For actin reorganization, the expression of F-actin was evaluated using a FITC-Phalloidin probe via flow cytometry (C) (n = 6). For the clonogenic assay, the results are expressed as colony number (D) (n = 4). See the Section 4 for further details. * p < 0.05 ANOVA with Bonferroni correction.