Ganoderiol A-Enriched Extract Suppresses Migration and Adhesion of MDA-MB-231 Cells by Inhibiting FAK-SRC-Paxillin Cascade Pathway

<div><p>Cell adhesion, migration and invasion are critical steps for carcinogenesis and cancer metastasis. <i>Ganoderma lucidum</i>, also called Lingzhi in China, is a traditional Chinese medicine, which exhibits anti-proliferation, anti-inflammation and anti-metastasis properties. Herein, GAEE, <i>G. lucidum</i> extract mainly contains ganoderiol A (GA), dihydrogenated GA and GA isomer, was shown to inhibit the abilities of adhesion and migration, while have a slight influence on that of invasion in highly metastatic breast cancer MDA-MB-231 cells at non-toxic doses. Further investigation revealed that GAEE decreased the active forms of focal adhesion kinase (FAK) and disrupted the interaction between FAK and SRC, which lead to deactivating of paxillin. Moreover, GAEE treatment downregulated the expressions of RhoA, Rac1, and Cdc42, and decreased the interaction between neural Wiskott-Aldrich Syndrome protein (N-WASP) and Cdc42, which impair cell migration and actin assembly. To our knowledge, this is the first report to show that <i>G.lucidum</i> triterpenoids could suppress cell migration and adhesion through FAK-SRC-paxillin signaling pathway. Our study also suggests that GAEE may be a potential agent for treatment of breast cancer.</p></div

GAEE suppressed FAK signaling in MDA-MB-231 cells.

<p>(A) Immunoblots of FAK signaling proteins and other related proteins after 24 h of treatment with GAEE in MDA-MB-231 cells. Decreased expression of FAK, p-FAK (Y397), and p-FAK (Y925) were shown, while there is no alter on the expression of SRC, p-SRC, integrin β1, and integrin β4. (B) The relative densities of FAK, p-FAK (Y397), integrin β1 and integrin β4 were normalized against GAPDH by densitometric analysis. The values represented as the mean ± SEM of four independent experiments compared with control group. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (one-way ANOVA with Tukey's multiple comparison test) (C) IP-Western confirmation for the disruption of interaction between FAK and SRC after treatment with GAEE.</p

Effects of GAEE on cell migration, adhesion and invasion.

<p>(A, B) GAEE inhibited cell motility as tested by wound-healing assay in MDA-MB-231 cells. Cell monolayers were scraped using a sterile 100 microliter tip, and the cells were treated indicated concentrations of GAEE for 24 h. The migratory effects of cells were quantified by measuring the distance between the edges (White lines indicate the wound edge). (C, D) GAEE inhibited cell motility as determined by Transwell assay in MDA-MB-231 cells. The migratory cells were detected by crystal violet staining and photographed. (E, F) GAEE inhibited cell adhesion in MDA-MB-231 cells. The adhesive cells were fixed and photographed by 4% PFA and crystal violet staining. Quantitative assessment of the number of adhesive cells was performed by MTT assay. (G, H) Effect of GAEE on the invasion of MDA-MB-231 cells. In the Transwell chamber invasion assay, cells were treated with GAEE for 24 h and the invasion ability of cells was quantified by counting the number of cells that invaded the underside of the porous polycarbonate membrane using crystal violet staining. The quantification result from three independent experiments is shown on the right. All data were shown as mean ± SEM of more than triplicates compared with the untreated control. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (one-way ANOVA with Tukey's multiple comparison test).</p

Effects of GAEE on cell cycle progression and apoptosis.

<p>(A, B) The cell cycle distribution in MDA-MB-231 cells after treatment with GAEE. Data were shown as mean ± SEM of four independent experiments. (C) Cells were exposed to various concentration of GAEE for 24 h, nuclear morphology was observed by the Hoechst-33342 staining. (D) MDA-MB-231 cells were treated with GAEE for 24 h and the protein levels of Bcl-2, Bax, PARP, and caspase-9 were examined using Western blot analysis. GAPDH was used as a loading control.</p

Effects of GAEE on Rho GTPases expression and actin assembly.

<p>(A) Cells were treated with indicated concentrations of GAEE for 24 h, the downregulation of RhoA, Rac1, and Cdc42 was observed, and the expression of N-WASP was not affected. (B) MDA-MB-231 cells were treated with GAEE (10 or 20 µg/ml) for 24 h, and then cell lysates were prepared and subjected to immunoprecipitation with N-WASP, and followed by immunoblotting with Cdc42 and actin. The samples were blotted with anti-N-WASP as a control.</p