Ganoderma lucidum Extract Reduces the Motility of Breast Cancer Cells Mediated by the RAC⁻Lamellipodin Axis

Abstract

Breast cancer (BC) is the second leading cause of cancer death among women worldwide. The main cause of BC morbidity and mortality is the invasiveness capacity of cancer cells that may lead to metastasis. Here, we aimed to investigate the therapeutic efficacy of Ganoderma lucidum extract (GLE)-a medicinal mushroom with anticancer properties-on BC motility via the Rac/Lamellipodin pathway. GLE treatment effects were tested on MDA-MB-231 breast cancer cells. The effects were tested on cell viability, migration and invasion. Pulldowns, immunoblotting, and immunofluorescence were used to measure Rac activity and the expression of proteins involved in cell migration and in lamellipodia formation, respectively. As a result, GLE suppressed BC cell viability, migration, and invasion capacity. GLE impaired Rac activity, as well as downregulated Lamellipodin, ENA/VASP, p-FAK (Tyr925), Cdc42, and c-Myc expression. Lamellipodia formation was significantly reduced by GLE. In conclusion, we demonstrate that GLE reduces Rac activity and downregulates signaling molecules involved in lamellipodia formation. These novel findings serve as basis for further studies to elucidate the potential of GLE as a therapeutic agent regulating the Rac/Lamellipodin pathway in BC metastasis.

Keywords: Ganoderma lucidum; Rac; breast cancer; cancer cell migration; lamellipodin.

Figure 1

Ganoderma lucidum extract (GLE) decreases the viability of MDA-MB-231 breast cancer cells. (A,B) MDA-MB-231 (1 × 10⁵) and MCF-10A (1 × 10⁵) cells were treated with 0.062, 0.125, 0.250, 0.500, and 1.000 mg/mL of GLE for 48 h. Cell viability was calculated as described in materials and methods. IC₅₀ was calculated from dose response curve fittings using nonlinear regression parameters. (C) MDA-MB-231 cells were treated with same concentrations of GLE as in (A) for 48 h. Treatment was removed after 48 h and the cells were incubated for an additional 72 h to assess recovery by a washout assay. Data are expressed as mean ± SEM. Experiments represent data obtained from three independent biological replicates. Statistically significant differences are shown at ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to vehicle.

Figure 2

GLE decreases the migration and invasion of MDA-MB-231 breast cancer cells. (A,B) MDA-MB-231 (2 × 10⁴) cells were seeded onto two-well silicone insert with a defined cell-free gap wound healing assay plates and grown in 5% FBS media before treatment with vehicle (0.1% DMSO) or GLE (0.96 mg/mL) for 24 h. Cells were fixed and stained with rhodamine phalloidin and DAPI as described in materials and methods. We quantified 36 micrographs that were taken at a magnification of 20X. The migratory effect of the cells was quantified by measuring the distance between the edges of the wound with ImageJ V.1.50i. (C,D) Quiescent MDA-MB-231 (7.5 × 10⁴) cells were seeded in Transwell^® chambers and treated with vehicle, 0.25 mg/mL, and 0.96 mg/mL GLE for 24 h. Cells were fixed and PI-stained. The invasion ability of the cells was quantified by counting the number of cells that invaded through the extracellular matrix. Approximately, 36 micrographs were taken at a magnification of 20X and quantified using ImageJ. Data are expressed as mean ± SEM. Experiments represent data obtained from three independent biological replicates. Statistically significant differences are shown at * p < 0.05, ** p < 0.01 compared to vehicle. Scale bars = 20 μm.

Figure 3

GLE decreases Rac activity in MDA-MB-231 breast cancer cells. (A) MDA-MB-231 cells were treated with vehicle or GLE (0.96 mg/mL) for 24 h. Total lysates were obtained and incubated with PAK-PDB beads to precipitate active Rac by a pulldown activation assay. The total protein lysates and pulldowns were analyzed by Western blot. GTP was used as a positive control to test activity of the beads. (B) Densitometry analysis of western blot bands. Quantification was done using integrated density units and Rac activity was calculated relative to total protein expression. Data are expressed as mean ± SEM. Experiments represent data obtained from three independent biological replicates. Statistically significant difference is shown at * p < 0.05 compared to vehicle.

Figure 4

GLE modulates the expression of proteins involved in cell migration in MDA-MB-231 breast cancer cells. (A) MDA-MB-231 cells were grown in 5% FBS media for 24 h prior to treatment with vehicle (0.1% DMSO) or GLE (0.96 mg/mL, 0.50 mg/mL) for 24 and 48 h, respectively, before lysis. Equal amount of protein (30 µg) from each sample was used for western blot analysis. β-actin was used as a loading control. (B–F) Densitometry analysis of Western blot bands. Data are expressed as mean ± SEM. Experiments represent data obtained from three independent biological replicates. Statistically significant difference is shown at * p < 0.05 compared to vehicle.

Figure 5

GLE inhibits Lpd expression in MDA-MB-231 breast cancer cells. MDA-MB-231 (1.4 × 10⁴) cells were grown in 5% FBS media for 24 h prior to treatment with vehicle or GLE (0.25 mg/mL, 0.96 mg/mL) for 24 h. Cells were fixed and stained with DAPI (blue) and anti-Lamellipodin (green) per manufacturer’s instructions. White arrows show lamellipodia. Experiments represent data obtained from three independent biological replicates. Zoom in each image is shown in white squares, and each share equal dimensions. Scale bars = 30 μm.