Studying Ganoderma lucidum as a source of molecular inducers of autophagy

Autophagy is one of the three main mechanisms of programmed cell death[1]. One of the basis of cancer therapy is to induce death of tumour cells. There are several clinically approved molecules that induce cell death[2,3], but unfortunately not all tumours highly resistant to cell death respond to the existing therapies. Therefore, the search for new molecules capable of inducing tumour cell death is an area of growing interest. In this context, modulators of autophagy have become very attractive in recent years. Previous work performed by some of us has shown that methanolic extracts of the medicinal mushroom Ganoderma lucidum were modulators of cellular autophagy[4]. The aim of the present work was to further understand the cellular mechanisms involved in the autophagy modulation and to identify bioactive compounds responsible for this modulation. A methanolic extract obtained by cold extraction (-20ºC) from G. lucidum was studied with respect to its ability to induce autophagy in a gastric adenocarcinoma cell line (AGS). The presence of autophagic vacuoles was observed following transfection of cells with a mCherry-LC3 expression vector and the levels of some autophagic proteins were analysed by Western Blot. Cells were also treated with the lysossomal protease inhibitors E-64d/pepstatin together with the extract, in order to confirm if the extract induced autophagy or a decrease in the autophagic flux. The levels of the autophagic proteins after this treatment were also evaluated by Western Blot. An increase in the autophagic vacuoles was observed in cells treated with the extract (concentrations corresponding to the GI50 and 2×GI50) 24h before transfection with the mCherry-LC3 vector. In addition, treatment of cells with the same concentrations of the extract for 48h induced an increase in the expression of LC3-II together with a slight reduction in the levels of p62, particularly with the highest concentration. Additionally, cells treated with the extract together with E-64d/pepstatin expressed higher levels of LC3-II and p62, when compared with cells treated only with the extract, indicating that G. lucidum caused an induction of autophagy rather than a decrease in the autophagic flux. With these results we can conclude that the methanolic extract from the mushroom G. lucidum may be a source of compounds capable of inducing autophagy. Further studies to identify the bioactive compounds present in the tested extract and responsible for such activity are still ongoing.