Alpha-Lipoic Acid-Mediated Inhibition of LTB4 Synthesis Suppresses Epithelial-Mesenchymal Transition, Modulating Functional and Tumorigenic Capacities in Non-Small Cell Lung Cancer A549 Cells

Abstract

Background: Leukotriene B₄ (LTB₄) plays a crucial role in carcinogenesis by inducing epithelial-mesenchymal transition (EMT), a process associated with tumor progression. The synthesis of LTB₄ is mediated by leukotriene A₄ hydrolase (LTA₄H), and it binds to the receptors BLT₁ and BLT₂. Dysregulation in LTB₄ production is linked to the development of various pathologies. Therefore, the identification or design of inhibitors of LTB₄ synthesis or receptor antagonists represents an ongoing challenge. In this context, our laboratory previously demonstrated that alpha-lipoic acid (ALA) inhibits LTA₄H. The objective of this study was to evaluate the effect of ALA on the expression of canonical EMT markers and the functional and tumorigenic capacities induced by LTB₄ in A549 cells.

Methods: The expression of cPLA₂, 5LOX, FLAP, LTA₄H, BLT1, and LTB₄ production in human adenocarcinomic alveolar basal epithelial A549 cells was assessed using Western blot, RT-qPCR, and ELISA, respectively. Subsequently, the expression of canonical EMT markers was evaluated by Western blot. Functional assays were performed to assess cell viability, proliferation, invasion, migration, and clonogenicity using MTT, Western blot, Transwell assays, and colony formation assays, respectively. Results were expressed as median with interquartile range (n≥3) and analyzed using the Kruskal-Wallis or Tukey multiple comparisons tests.

Results: A549 cells express key proteins involved in LTB₄ synthesis and receptor binding, including LTA₄H and BLT₁, and ALA inhibits the production of LTB₄. Additionally, LTA₄H and BLT1 were detected in lung adenocarcinoma tissue samples. LTB₄ was found to induce EMT, whereas ALA treatment enhanced the expression of epithelial markers and reduced the expression of mesenchymal markers. Furthermore, ALA treatment resulted in a decrease in LTB₄ levels and attenuated the functional and tumorigenic capacities of A549 cells, including their viability, migration, invasion, and clonogenic potential.

Conclusions: These findings suggest that ALA may offer therapeutic potential in the context of lung cancer, as it could be integrated into conventional pharmacological therapies to enhance treatment efficacy and mitigate the adverse effects associated with chemotherapy. Further studies are warranted to confirm the clinical applicability of ALA as an adjunctive treatment in lung cancer.

Keywords: Alpha lipoic acid (ALA); Epithelial Mesenchymal Transition (EMT) and Functional and tumor capacities; Leukotriene A4 Hydrolase (LTA4H); Leukotriene B4 (LTB4); Small cell lung carcinoma and non-small cell lung cancer (NSCLC).

Figure 1

Protein expression of the 5-lipoxygenase pathway and the BLT1 receptor in A549 non-small lung cancer cells. (A) mRNA levels of cPLA₂, 5-LOX, FLAP, LTA₄H and BLT1 normalized with housekeeping (pumilio) in the cell line. (B) The protein lysate from A549 cells were analyzed by immunoblot and the membranes were incubated with primary antibodies against cPLA₂, 5-LOX, FLAP, LTA₄H, BLT1 and β-actin. (C) Semi-quantification of the relative protein expression of cPLA₂, 5-LOX, FLAP, LTA₄H and BLT1, normalized with β-actin. The values 110, 78, 15, 70, 37, and 43 correspond to mass units (kDa). cPLA₂, 5-LOX, LTA₄H, BLT1; n = 3; FLAP; n = 4

Figure 2

Localization of LTA₄H and BLT1 in samples from patients with lung adenocarcinoma. Images indicate the localization of LTA₄H and BLT1 in samples from patients with lung adenocarcinoma. (A) Immunohistochemistry analysis of LTA₄H. Scale bar: 50 µm. (B) Immunohistochemistry analysis of BLT1. Scale bar: 50 µm.

Figure 3

Effect of ALA on LTB₄ secretion in A549 non-small lung cancer cells. Cells were pre-incubated with SC (0.01 µM), ALA (0.01 µM), or ALA (10 µM) for 30 minutes, and then stimulated with PMA (5 nM) for 15 minutes. LTB₄ secretion was determined by ELISA Kit. Data are presented as the median with interquartile range (n = 3 independent experiments; *P ≤ 0.05; Kruskal-Wallis Test).

Figure 4

Effect of LTB₄ on the expression of canonical markers of epithelial mesenchymal transition in non-small cell lung cancer A549. (A) The cells were incubated with IL-6 or different concentrations of LTB₄ for 48 hours, subsequently the protein lysate was analyzed by immunoblot and the membranes were incubated with primary antibodies against E-cadherin, Vimentin, ZEB-1, and b-actin. (B) Semi-quantification of the relative protein expression of E-cadherin normalized with β-actin and the control. (C) Semi-quantification of the relative protein expression of Vimentin normalized with β-actin and the control. (D) Semi-quantification of the relative protein expression of ZEB-1 normalized with β-actin and the control. The values 100, 55, 180, and 43 correspond to mass units (kDa). Data are presented as the median with interquartile range (ZEB-1 (n = 3); Vimentin and E-cadherin (n = 4) independent experiments; **P ≤ 0.01; Kruskal-Wallis Test).

Figure 5

Effect of ALA-mediated LTB₄ decrease on the expression of canonical markers of epithelial mesenchymal transition in non-small cell lung cancer A549. (A) Cells were pre-incubated with SC (0.01 µM) or ALA (0.01 or 10 µM) for 30 minutes, and then stimulated with PMA (5 nM), for 15 minutes, LTB₄ (100 nM), 50 ng/mL IL-6 or IL-6/LTB₄ for 48 hours, subsequently the protein lysate was analyzed by immunoblot and the membranes were incubated with primary antibodies against E-cadherin, Vimentin, ZEB-1 and β-actin. (B) Semi-quantification of the relative protein expression of E-cadherin normalized with β-actin and the control. (C) Semi-quantification of the relative protein expression of Vimentin normalized with β-actin and the control. (D) Semi-quantification of the relative protein expression of ZEB-1 normalized with β-actin and the control. The values 70, 55, 180, and 43 correspond to mass units (kDa). Data are presented as the median with interquartile range (n = 3 independent experiments; *P ≤ 0.05; Kruskal-Wallis Test).

Figure 6

Effect of LTB₄ and ALA-mediated LTB₄ decrease on the viability and proliferation in non-small cell lung cancer A549. (A) The cells were stimulated with different concentrations of LTB₄ for 12 hours, subsequently the percentage of viable cells was evaluated through MTT. Cells were pre-incubated with SC (0.01 µM) or ALA (0.01 or 10 µM) for 30 minutes, and then stimulated with PMA (5 nM) for 15 minutes. (B) Percentage of viable cells by MTT. (C) Protein lysate was analyzed by immunoblot and the membranes were incubated with primary antibodies against PCNA and β-actin. (D) Semi-quantification of the relative protein expression of PCNA normalized with β-actin and the control. The values 36 and 43 correspond to mass units (kDa). Data are presented as the median with interquartile range (viability (n = 3); proliferation (n = 4) independent experiments; *P ≤ 0.05 and **P ≤ 0.01; Kruskal-Wallis Test).

Figure 7

Effect of ALA-mediated LTB₄ decrease on invasive capacities, motility, and colony formation in non-small cell lung cancer A549. Cells were pre-incubated with SC (0.01 µM) or ALA (0.01 or 10 µM) for 30 minutes, and then stimulated with PMA (5 nM) for 15 minutes. (A) Illustration of the migration transwell test per 6 hours, Scale bar; 1 mm. (B) Number of cells migrated per 6 hours. (C) Illustration of the invasion transwell test per 20 hours, Scale bar; 1 mm. (D) Number of cells invaded per 20 hours. (E) Illustration of the colony formation assay after 6 days, Scale bar; 1 cm and 1 mm (zoom). (F) Quantification of the number of colonies after 6 days. (G) Illustration of the colony formation test at 9 days, Scale bar; 1 cm and 1 mm (zoom). (H) Quantification of the number of colonies after 9 days. Data are presented as the median with interquartile range (migration and invasion n = 3 independent experiments; *P ≤ 0.05; Kruskal-Wallis Test). colony formation (6 days; n = 3 independent experiments; *P ≤ 0.05; Mann-Whitney and 9 days; n = 4 independent experiments; *P ≤ 0.05; Mann-Whitney).

Figure 7

Effect of ALA-mediated LTB₄ decrease on invasive capacities, motility, and colony formation in non-small cell lung cancer A549. Cells were pre-incubated with SC (0.01 µM) or ALA (0.01 or 10 µM) for 30 minutes, and then stimulated with PMA (5 nM) for 15 minutes. (A) Illustration of the migration transwell test per 6 hours, Scale bar; 1 mm. (B) Number of cells migrated per 6 hours. (C) Illustration of the invasion transwell test per 20 hours, Scale bar; 1 mm. (D) Number of cells invaded per 20 hours. (E) Illustration of the colony formation assay after 6 days, Scale bar; 1 cm and 1 mm (zoom). (F) Quantification of the number of colonies after 6 days. (G) Illustration of the colony formation test at 9 days, Scale bar; 1 cm and 1 mm (zoom). (H) Quantification of the number of colonies after 9 days. Data are presented as the median with interquartile range (migration and invasion n = 3 independent experiments; *P ≤ 0.05; Kruskal-Wallis Test). colony formation (6 days; n = 3 independent experiments; *P ≤ 0.05; Mann-Whitney and 9 days; n = 4 independent experiments; *P ≤ 0.05; Mann-Whitney).