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. 2015 Sep 29;20(10):17872-82.
doi: 10.3390/molecules201017872.

Methanolic Extract of Ganoderma Lucidum Induces Autophagy of AGS Human Gastric Tumor Cells

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Free PMC article

Methanolic Extract of Ganoderma Lucidum Induces Autophagy of AGS Human Gastric Tumor Cells

Filipa S Reis et al. Molecules. .
Free PMC article

Abstract

Ganoderma lucidum is one of the most widely studied mushroom species, particularly in what concerns its medicinal properties. Previous studies (including those from some of us) have shown some evidence that the methanolic extract of G. lucidum affects cellular autophagy. However, it was not known if it induces autophagy or decreases the autophagic flux. The treatment of a gastric adenocarcinoma cell line (AGS) with the mushroom extract increased the formation of autophagosomes (vacuoles typical from autophagy). Moreover, the cellular levels of LC3-II were also increased, and the cellular levels of p62 decreased, confirming that the extract affects cellular autophagy. Treating the cells with the extract together with lysossomal protease inhibitors, the cellular levels of LC3-II and p62 increased. The results obtained proved that, in AGS cells, the methanolic extract of G. lucidum causes an induction of autophagy, rather than a reduction in the autophagic flux. To our knowledge, this is the first study proving that statement.

Keywords: Ganoderma lucidum; antitumor potential; autophagy; methanolic extract; programmed cell death (PCD).

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Phenolic acids profile of Ganoderma lucidum cold extract (-----) and room temperature extract (_____). 1—p-Hydroxybenzoic acid; 2—p-coumaric acid; 3—cinnamic acid.
Figure 2
Figure 2
Analysis of the presence of autophagic vacuoles following treatment with G. lucidum methanolic extract, by fluorescence microscopy. Cells were transfected for 24 h with LC3 mCherry vector (red) and further treated for 48 h with: medium only (Blank), with the methanolic extract (at GI50 and 2 × GI50 concentrations) or with the extract solvent (DMSO) corresponding to the higher concentration of the extract. Nuclei were stained with DAPI (blue). Images are representative of two experiments. Bar corresponds to 20 μm.
Figure 3
Figure 3
Analysis of the levels of the autophagy-related proteins Vps34, Beclin-1, LC3-I, LC3-II and p62, following treatment with G. lucidum methanolic extract, analyzed by Western blot. Cells were treated for 48 h with: medium only (Blank), with the methanolic extract (at GI50 and 2 × GI50 concentrations) or the corresponding volumes of the extract solvent (DMSO). Actin was used as loading control. Image is representative of three independent experiments.
Figure 4
Figure 4
Effect of co-treatment with G. lucidum methanolic extract together with E-64d/pepstatin in LC3-II and p62 levels, analyzed by Western blot. Cells were treated for 48 h with medium (Blank), with the solvent (DMSO) or with the extract (GI50 and 2 × GI50 concentrations), together with (or without) E-64d/pepstatin (15 µg/mL). Actin was used as loading control. Results are representative of 3 independent experiments.
Figure 5
Figure 5
Analysis of the levels of the autophagy marker LC3II in AGS cells following 48 h treatment with p-coumaric acid, p-hydroxybenzoic acid or cinnamic acid, analyzed by Western blot. Cells were treated for 48 h with medium (Blank), with the solvent (DMSO:water; 1:1 v/v; controls 1 and 2) or with the compounds (at 250 and 500 µM). Actin was used as loading control. Blot is representative of two independent experiments.

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