Lobaric Acid Exhibits Anticancer Potential by Modulating the Wnt/β-Catenin Signaling Pathway in MCF-7 Cells

Abstract

Lichen secondary metabolites with many remarkable biological activities are used in cancer treatments due to their low side effects and high anticancer potential. In particular, these metabolites constitute an interesting research area in cancer treatments due to their potential to induce apoptosis and suppress metastasis by inhibiting cancer-related signaling pathways. The Wnt/β-catenin signaling pathway plays a role in important biological processes such as oncogenesis, cell cycle regulation, cell proliferation, metastasis, differentiation, apoptosis, and drug resistance. Therefore, inhibition of this pathway is a potential target in cancer therapies. There is no detailed study explaining the potential anticancer molecular mechanism of the lichen secondary metabolite lobaric acid (LA) on breast cancer. Here, it is aimed to investigate the effect of LA on viability, apoptosis, and migration in MCF-7 cells and to elucidate the relationship between the potential anticancer effect and the Wnt/β-catenin signaling pathway. The dose- and time-dependent viability of LA-treated MCF-7 cells was evaluated by XTT assay, and the IC₅₀ value was determined as 44.21 μg/mL at 48 h. LA increased the apoptotic cell population, as shown by flow cytometry analysis, qPCR, and Western blot results. LA inhibited β-catenin by inducing GSK3-β protein expression, thereby suppressing Wnt/β-catenin target genes. LA might be a natural active compound candidate for breast cancer treatment.

Keywords: Wnt/β‐catenin pathway; apoptosis; breast cancer; cytotoxicity; expression.

FIGURE 1

Inhibition of MCF‐7 cell viability by LA. (A) Dose (10, 25, 37.5, 50, 75, and 100 μg/mL) and time (24 and 48 h) dependent inverted microscope images of MCF‐7 cells treated with LA. (B, C) MCF‐7 cells were treated with different LA concentrations for 24 and 48 h and cell viability was determined by XTT assay. (D, E) IC₅₀ values were calculated using GraphPad Prism. The experiment was performed in three biological and technical replicates. **p < 0.01, and ***p < 0.001 in relation to the control. Scale bar, 500 μm.

FIGURE 2

Induction of apoptosis of MCF‐7 cells by LA. Flow cytometry was performed to determine the pro‐apoptotic effect of LA (IC₅₀ concentration at 48 h) on MCF‐7 cells. (A) Cell population distributions were determined after treatment with LA at 48 h. Top left identifies necrotic cells (PI single positive); top right late apoptotic cells (annexin V and PI double positive); bottom left early apoptotic cells (annexin V single positive) and bottom right viable cells (non‐apoptotic cells). (B–E) BAX, BCL2, and P53 mRNA levels and the BAX/BCL2 ratio were analyzed by qPCR in MCF‐7 cells. (F–H) P53 and BCL2 protein expressions were determined by Western blot analysis. The experiment was performed in three biological and technical replicates. *p < 0.05, **p < 0.01, and ***p < 0.001 in relation to the control.

FIGURE 3

Effect of LA on Wnt/β‐catenin pathway in MCF‐7 cells. (A) The migration of cells treated with LA into the wound was visualized at 0, 6, 12, and 24 h under an inverted microscope, and the percentage of wound closure was evaluated statistically. (B–I) Expressions of WNT2, DVL1, AXIN1, β‐Catenin, TCF‐4, CCND1, c‐MYC, and CDK1 genes analyzed by qPCR in LA‐treated MCF‐7 cells at 48 h. (J–M) Protein expressions of WNT2, GSK3‐ β, and β‐catenin determined by Western blot analysis. The experiment was performed in three biological and technical replicates. *p < 0.05, **p < 0.01, and ***p < 0.001 in relation to the control. Scale bar, 500 μm.