Meripilus giganteus ethanolic extract exhibits pro-apoptotic and anti-proliferative effects in leukemic cell lines

Abstract

Background: The interest towards botanicals and plant extracts has strongly risen due to their numerous biological effects and ability to counteract chronic diseases development. Among these effects, chemoprevention which represents the possibility to counteract the cancerogenetic process is one of the most studied. The extracts of mushroom Meripilus giganteus (MG) (Phylum of Basidiomycota) showed to exert antimicrobic, antioxidant and antiproliferative effects. Therefore, since its effect in leukemic cell lines has not been previously evaluated, we studied its potential chemopreventive effect in Jurkat and HL-60 cell lines.

Methods: MG ethanolic extract was characterized for its antioxidant activity and scavenging effect against different radical species. Moreover, its phenolic profile was evaluated by HPLC-MS-MS analyses. Flow cytometry (FCM) analyses of Jurkat and HL-60 cells treated with MG extract (0-750 μg/mL) for 24-72 h- allowed to evaluate its cytotoxicity, pro-apoptotic and anti-proliferative effect. To better characterize MG pro-apoptotic mechanism ROS intracellular level and the gene expression level of FAS, BAX and BCL2 were also evaluated. Moreover, to assess MG extract selectivity towards cancer cells, its cytotoxicity was also evaluated in human peripheral blood lymphocytes (PBL).

Results: MG extract induced apoptosis in Jurkat and HL-60 cells in a dose- and time- dependent manner by increasing BAX/BCL2 ratio, reducing ROS intracellular level and inducing FAS gene expression level. In fact, reduced ROS level is known to be related to the activation of apoptosis in leukemic cells by the involvement of death receptors. MG extract also induced cell-cycle arrest in HL-60 cells. Moreover, IC₅₀ at 24 h treatment resulted 2 times higher in PBL than in leukemic cell lines.

Conclusions: Our data suggest that MG extract might be considered a promising and partially selective chemopreventive agent since it is able to modulate different mechanisms in transformed cells at concentrations lower than in non-transformed ones.

Keywords: Apoptosis; Chemoprevention; Cytotoxicity; Flow-cytometry; Leukemic cell lines; Meripilus giganteus.

Fig. 1

Effect of MG on viability of Jurkat and HL-60 cells. Cell viability was determined as described in Methods section. IC₅₀ obtained by curve fitting of viable cells after 24 h treatment with MG for Jurkat cells (a) and HL-60 cells (b). Data are presented as means ± SEM of five independent experiments

Fig. 2

Increase in apoptotic Jurkat cell fraction after MG treatment. Apoptosis was evaluated at 24 h (a), 48 h (b) and 72 h (c), as reported in Methods section. Each bar represents means ± SEM of five independent experiments. Data were analysed using repeated ANOVA followed by Bonferroni post-test.^**p < 0.01 vs control; ^***p < 0.001 vs control

Fig. 3

Increase in apoptotic Jurkat cell fraction after MG treatment. Apoptosis was evaluated at 24 h (a), 48 h (b) and 72 h (c), as reported in Methods section. Each bar represents the mean ± SEM of five independent experiments. Data were analysed using repeated ANOVA followed by Bonferroni post-test. ^**p < 0.01 vs control; ^***p < 0.001 vs control

Fig. 4

Fluorescence microscopy analysis of Jurkat and HL-60 after MG treatment. Nuclear condensation and fragmentation associated to apoptotic process on Jurkat and HL-60 cells was evaluated by fluorescence microscopy at 100x magnification after 24 h of 250 μg/mL MG treatment (b, d) respect to control culture (a, c). White arrows indicate condensed and/or fragmented nuclei as marker of apoptosis

Fig. 5

Cell-cycle analysis of Jurkat cells treated with MG extract. Fraction of Jurkat cells in the different phases of the cell cycle after MG treatment for 24 h (a) 48 h (b) and 72 h (c) was evaluated as reported in Methods section. Each bar represents the mean ± SEM of five independent experiments. Data were analysed using repeated ANOVA followed by Bonferroni post-test and revealed no statistically significant differences

Fig. 6

Cell-cycle analysis of HL-60 cells treated with MG extract. Fraction of HL-60 cells in the different phases of the cell-cycle after MG treatment for 24 h (a) 48 h (b) and 72 h (c) was evaluated as reported in Materials and Methods section. Each bar represents the mean ± SEM of five independent experiments. Data were analysed using repeated ANOVA followed by Bonferroni post-test. ^*p < 0.05 vs control; ^**p < 0.01 vs control; ^***p < 0.001 vs control

Fig. 7

Effect of MG extract treatment on FAS, BAX and BCL2 expression level in Jurkat (a, b) and HL-60 (c, d) cells. Total RNA was isolated, and the mRNA level of target genes was quantified using RT-PCR normalized to 18S rRNA and GAPDH as reference genes. Triplicate reactions were performed for each experiment. Each bar represents the mean ± SEM of three independent experiments. Data were analysed by one-way ANOVA followed by Bonferroni’s test. ^*p < 0.05 vs control; ^**p < 0.01 vs control;^***p < 0.001 vs control

Fig. 8

Intracellular ROS level in Jurkat and HL-60 cells treated with MG at 250 μg/mL for 24 h. Each bar represents the mean ± SEM of three independent experiments. Data were analysed by t-test, ^**p < 0.01 vs control

Fig. 9

Effect of MG on viability of PBL. IC₅₀ was obtained by curve fitting of viable cells after 24 h treatment. Data are presented as mean ± SEM of five independent experiments

Fig. 10

Hypothesised pro-apoptotic mechanism of MG extract in leukemic cell lines. Due to its phenolic content and antioxidant activity, MG ethanolic extract induces a decrease of ROS intracellular level. In leukemic cell, ROS decrease has been related to FAS recruitment leading to the activation of the extrinsic apoptotic pathway. A possible explanation of the observed increase of BAX/BCL-2 ratio consists in the fact that FAS ligands lead to apoptosis through caspase-8 activation which on one hand promote the extrinsic apoptotic pathway, while on the other promote mitochondrial membrane potential loss through BAX/BCL2 ratio increase