Leccinum vulpinum Watling induces DNA damage, decreases cell proliferation and induces apoptosis on the human MCF-7 breast cancer cell line

The current work aimed to study the antitumour activity of a phenolic extract of the edible mushroom Leccinum vulpinum Watling, rich essentially in hydroxybenzoic acids. In a first approach, the mushroom extract was tested against cancer cell growth by using four human tumour cell lines. Given the positive results obtained in these initial screening experiments and the evidence of some studies for an inverse relationship between mushroom consumption and breast cancer risk, a detailed study of the bioactivity of the extract was carried out on MCF-7 cells. Once the selected cell line to precede the work was the breast adenocarcinoma cell line, the human breast non-malignant cell line MCF-10A was used as control. Overall, the extract decreased cellular proliferation and induced apoptosis. Furthermore, the results also suggest that the extract causes cellular DNA damage. Data obtained highlight the potential of mushrooms as a source of biologically active compounds, particularly with antitumour activity.

Investigating mushroom LMW compounds as potential Bcl-2 inhibitors: docking studies using AutoDock4

The B cell CLL/lymphoma-2 (Bcl-2) family is functionally classified as either anti-apoptotic or pro-apoptotic, and the regulation of its interactions dictates survival or commitment to apoptosis. Bcl-2 family is also implicated in a wide range of diseases. In some types of cancers, including lymphomas and epithelial cancers, protein overexpression of anti-apoptotic Bcl-2 family, such as the Bcl-2 protein is indicative of cancer in an advanced stage, with a poor prognosis and resistant to chemotherapy [1]. Several reports indicate that mushrooms have the ability to promote apoptosis in tumour cell lines, but the mechanism of action is not fully understood. Inhibition of the interaction between Bcl-2 (anti-apoptotic protein) and proapoptotic proteins could be an important step in the mechanism of mushroom induced apoptosis. Therefore, the discovery of compounds with the capacity to inhibit Bcl-2 is an ongoing research topic on cancer therapy. In this work, docking studies were performed using a dataset of 40 low molecular weight (LMW) compounds present in mushrooms. The docking software AutoDock 4 was used and docking studies were performed using 5 selected Bcl-2 crystal structures as targets. Compounds with the lowest predicted binding energy (predΔG) are expected to be the more potent inhibitors. Among the tested compounds, steroids presented the lowest predΔG with several exhibiting values below -9 kcal/mol. The results are corroborated by several reports that state that steroids induce apoptosis in several tumor cells. It is thus feasible that they might act by preventing Bcl-2 from forming complexes with the respective proapoptotic protein interaction partners, namely Bak, Bax, and Bim. Moreover, previous studies on our research group demonstrated that 48 h treatment of MCF-7 cells (breast carcinoma) with Suillus collinitus methanolic extract caused a decrease in Bcl-2, highlighting the antitumor potential of this mushroom species [2]. In conclusion, the process of apoptosis promoted by mushroom extracts may be related to the inhibition of Bcl-2 by the steroid derivatives herein studied. However, further studies are needed to confirm this hypothesis.

Study of antioxidant, antiproliferative and apoptosis-inducing properties of wild mushrooms from the Northeast of Portugal

Mushrooms are known as a powerful source of bioactive compounds including antioxidants, inhibitors of human tumour cell lines growth, inducers of apoptosis and enhancers of immunity. Indeed, many pre-clinical studies have been conducted in human tumour cell lines and in some cases a number of compounds isolated from mushrooms have followed to clinical trials. The Northeast of Portugal is one of the European regions with higher wild mushrooms diversity. However, to our knowledge, no studies had been conducted so far to verify their bioactivities. The main aim of this work was the evaluation of the bioactive properties (antioxidant properties and growth inhibitory potential on human tumour cell lines) of wild edible mushrooms collected in the Northeast of Portugal. Once properly identified, methanolic, ethanolic and boiling water extracts were prepared from thirty eight wild mushroom species collected in that region. Chemical characterization was obtained by high performance liquid chromatography (HPLC) coupled to a photodiode array detector (DAD) or to a refraction index detector (RI). Antioxidant activity assays were carried out in those extracts, including evaluation of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging capacity, reducing power and inhibition of β-carotene bleaching. Extract-induced cell growth inhibition was assessed with the sulforhodamine B assay in four human tumour cell lines (NCI-H460 - lung cancer, MCF-7 -breast cancer, HCT-15 -colon cancer and AGS - gastric cancer). The effects on cell cycle profile and apoptosis were evaluated by flow cytometry and the effect on the expression levels of proteins related to cell cycle and apoptosis was further investigated by Western blotting. Three wild edible mushroom species revealed growth inhibitory activity in the studied human tumour cell lines: Clitocybe alexandri ethanolic extract, Lepista inversa methanolic extract and Suillus collinitus methanolic extract. C. alexandri ethanolic extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells, in the NCI-H460 cell line. The analysed mushroom species also provided interesting antioxidant potential, mainly the boiling water extract of L. inversa which showed the highest DPPH radical scavenging activity, reducing power and β-carotene bleaching inhibition. S. collinitus methanolic extract induced a slight increase in the number of cells in G1, with a concomitant decrease in the percentage of cells in the S phase of the cell cycle and an increase in the percentage of apoptotic cells, in the MCF-7 cell line. The combined use of the S. collinitus methanolic extract and etoposide caused a greater decrease in the percentage of cell growth, when compared to either of them used individually, indicating the potential benefit of this combination. The tested extracts were chemically characterized and protocatechuic, p-hydroxybenzoic, p-coumaric and cinnamic acids were the main compounds identified on the phenolic (methanolic and ethanolic) extracts, while mannitol, trehalose and arabinose were the main sugars found in the polysaccharidic (boiling water) extracts after hydrolysis. The individual compounds identified in the extracts were submitted to a screening of tumour cells growth inhibitory activity, but only the phenolic acids and a related compound, cinnamic acid, presented activity. This compound was found to be the most potent one regarding cell growth inhibition in the NCI-H460 cell line. The effect of the individual and combined treatment with the identified compounds was also evaluated. Cinnamic and protochatequic acids caused a statistically significantly reduction in the number of viable cells. In addition, p-hydroxybenzoic acid did not show any significantly reduction in the viable cell number. Nevertheless, it was verified that the concomitant use of the three compounds provided the strongest decrease in the viable cell number, suggesting a possible concomitant effect of those compounds. Overall, the present work has contributed to further understand the bioactive potential of wild edible mushrooms from the Northeast of Portugal. This study allowed to identify some species with antioxidant or tumour cell growth inhibitory potential.