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. 2023 Feb 16;9(3):e13756. doi: 10.1016/j.heliyon.2023.e13756

Compounds originating from the edible mushroom Auricularia auricula-judae inhibit tropomyosin receptor kinase B activity

Orr Shahar a,b, Idan Pereman a,b, Hazem Khamisie a, Nirit Ezov a, Ofer Danay a,b, Ali Khattib a, Ron schweitzer a,c, Soliman Khatib a,c, Jamal Mahajna a,b,∗
PMCID: PMC9988514  PMID: 36895384

Abstract

Tropomyosin receptor kinase B (TrkB) serves as a pivotal factor in various cancers. To identify novel natural compounds with TrkB-inhibiting properties, a screening approach was applied using extracts from a collection of wild and cultivated mushroom fruiting bodies, and Ba/F3 cells that ectopically express TrkB (TPR-TrkB). We selected mushroom extracts that selectively inhibited proliferation of the TPR-TrkB cells. We then evaluated the ability of exogenous interleukin 3 to rescue growth inhibition by the selected TrkB-positive extracts. An ethyl acetate extract of Auricularia auricula-judae actively inhibited auto-phosphorylation of TrkB. LC–MS/MS analysis of this extract revealed substances that might be responsible for the observed activity. This screening approach demonstrates, for the first time, that extracts originating from the mushroom A. auricula-judae exhibit TrkB-inhibition properties that might hold therapeutic potential for TrkB-positive cancers.

Keywords: Tropomyosin receptor kinase B (TrkB), Auricularia auricula-Judae mushroom, Cancer, Kinase inhibition

1. Introduction

There are an estimated 1.5 million species of fungi worldwide, approximately 82,000 of which are described as mushrooms [1]. Fungi from the division Basidiomycota are of great interest due to the large number of biologically active compounds they contain [[1], [2], [3]]. Fungal fruiting bodies, mycelium, or the culture broth in which the mycelium has been cultivated have all been explored for biological activity. Consequently, approximately 650 species of higher Basidiomycetes have been found to possess antitumor activity [3].

Medicinal mushrooms have an established history of use in traditional oriental therapies, and medicinal effects have been demonstrated for many traditionally used mushrooms [1,4]. The effects of selected mushrooms of higher Basidiomycetes origin against cancer have been known for years. The antitumor activity of the higher Basidiomycetes was first demonstrated at 1957 [5] using extracts of fruiting bodies of Boletus edulis Bull.:Fr. and other Homobasidiomycetes. In the 1960s, calvacin was the most commonly cited natural product isolated from the medicinal mushroom and was widely used in many laboratories as an antitumor agent [6]. Moreover, many extracted mushroom compounds are commonly used as immunomodulators or as biological response modifiers. The basic strategy underlying immunomodulation is enhancement of the host response by increasing or complementing a desired immune response. Immunomodulators isolated from more than 30 mushroom species have shown anticancer activity in animals [2]. However, only a few have been taken to the next step: objective clinical assessment for anticancer potential in humans. Most of the immunomodulatory compounds from mushrooms are polysaccharide-containing β-D-glucans or β-D-glucans linked to proteins. There is evidence of β-D-glucans inducing biological responses by binding to a membrane receptor [7].

Another type of therapeutic substance of interest in mushrooms is secondary metabolite pools produced by various species that regulate the activity of signaling molecules implicated in carcinogenesis [1]. In this study, we focused on secondary metabolites produced by a variety of mushrooms to evaluate inhibitory activity against tropomyosin receptor tyrosine kinase B (TrkB).

The Trk family includes TrkA, TrkB, and TrkC proteins, which are encoded by NTRK1, NTRK2 and NTRK3, respectively [8]. Binding of neurotrophins to TRK proteins induces receptor dimerization, phosphorylation, and activation of downstream signaling cascades via PI3K, RAS/MAPK/ERK, and PLC-gamma signaling pathways [9]. The Trk family receptors play a wide variety of roles in physiological and disease processes in both neuronal and non-neuronal tissues. Trk-pathway aberrations, including gene fusions, protein overexpression, and single-nucleotide alterations, have been implicated in the pathogenesis of many types of cancer, with NTRK gene fusions being the best-validated oncogenic events to date [10]. Selective inhibition of Trk signaling may therefore be beneficial for some cancer patients. A number of Trk inhibitors have shown early promise in shrinking human tumors [11] and several Trk-targeting compounds are in clinical development [12]. Recently, the US Food and Drug Administration (FDA) approved larotrectinib as a therapeutic for neurotrophic Trk fusion-driven cancers [13]. In addition, entrectinib was also approved by the FDA to treat ROS1-positive non-small cell lung and NTRK fusion

Positive solid tumors (Rolfo, Ruiz et al., 2015).

Recently, we successfully identified mushroom substances that modulate molecular targets implicated in cancer [[14], [15], [16], [17], [18], [19]], and some that inhibit tyrosine kinases [15,20]. In this study, we focused on mushroom extracts that target TrkB. An efficient inhibitor of TrkB might have the potential to serve as a therapeutic for TrkB-positive tumors, as well as for various neuropsychiatric and degenerative conditions [21].

2. Results

2.1. Establishment of wild and cultivated mushroom screening collection

To screen for a wide array of active metabolites originating from mushroom fruiting bodies, we generated a collection of cultivated mushrooms. These were species and strains that are commonly used in mushroom agriculture, some of them highly regarded as medicinal fungi in traditional eastern medicine (such as Pholiota nameko, Ganoderma lucidum, and Grifola frondosa). The wild mushrooms used in the current study were fruiting bodies of locally foraged wild edible mushroom species, including the well-documented medicinal mushrooms Auricularia auricula-judae and Morchella galilaea (Table 1).

Table 1.

Species of cultivated and wild mushrooms used in this study.

Cultivated mushroom species Wild foraged mushroom species
Agaricus bisporus Auricularia auricula-judae
Coriolus versicolor Clavulina cinerea
Flammulina velutipes (white) Daldinia concentrica
Flammulina velutipes (yellow) Helvella sp.
Ganoderma lucidum Laccaria laccata
Grifola frondosa Lactarius deliciosus
Hericium erinaceus Lepista nuda
Hypsizygus tessellatus (brown) Morchella galilaea
Hypsizygus tessellatus (white) Schizophyllum commune
Lentinula edodes Tuber oligospermum
Pholiota aegerita
Pholiota nameko
Pleurotus eryngii
Pleurotus ostreatus
Trametes versicolor
Tuber aestivum
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2.2. Construction of Ba/F3 cells overexpressing TPR-TrkB

We utilized Ba/F3 cells (murine pro-B) which are dependent on IL-3 for proliferation and survival. Ectopic expression of constitutively active kinases has been shown to abolish the requirement for IL-3 in Ba/F3 cells engineered to carry active kinases. This system has been used to assay anti-kinase activity in substances of interest [22,23] and also by our group to identify mushroom-derived substances that inhibit kinase activity of native and mutated BCR/ABL [15]. Thus, we constructed plasmids carrying TPR-Met and TPR-TrkB to generate Ba/F3 TPR-Met and Ba/F3 TPR-TrkB cells, respectively. The TrkB construct contains amino acids 497–822 of the kinase domain. We utilized the TPR domain as a dimerization interface to generate ligand-independent activation of the fused kinases [24]. Ba/F3 TPR-TrkB cells were validated using phospho-specific antibodies (Fig. 1A), as well as growth inhibition by the relevant pharmaceutical inhibitors targeting the kinases of interest (Fig. 1B).

Fig. 1.

Fig. 1

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Validation of Ba/F3 TPR-TrkB cells. (A) Ba/F3 TPR-TrkB cells were exposed to 1 μM of the kinase inhibitors imatinib, crizotinib and GNF-5837 for 3 h. Cells were collected, lysed and separated by SDS-PAGE. Immunoblotting was performed as described in Materials and Methods using phospho-selective anti-TrkB antibody. GAPDH was used as a loading control. (B) Viability of Ba/F3 TPR-TrkB and Ba/F3 TPR-Met cells was monitored following exposure for 72 h to different concentrations of tyrosine kinase inhibitors at 0.2, 1 and 5 μM. Cell viability was monitored by XTT assay as described in Materials and Methods. Experiments were repeated twice with comparable outcomes. **p < 0.01.

Fig. 1B shows that the Abl kinase inhibitor imatinib [25] did not inhibit proliferation of TPR-Met and at the highest concentration (5 μM), only marginally inhibited proliferation of Ba/F3 TPR-TrkB cells. The multi-kinase inhibitor crizotinib [26,27] effectively inhibited both Ba/F3 TPR-Met and Ba/F3 TPR-TrkB cells. GNF-5837 [28] showed good inhibition of Ba/F3 TPR-TrkB cells, as well as of Ba/F3 TPR-Met cells, albeit with less potency, at the highest concentration (5 μM).

2.3. Mushroom extracts as selective inhibitors of Ba/F3 TPR-TrkB cell proliferation

Following the establishment of a mushroom screening library, we explored the TrkB activity-inhibiting potential of a wide array of mushroom-derived extracts. We used Ba/F3 cells ectopically expressing TPR-TrkB. Our primary screening was conducted using 59 extracts. Fig. 2 shows the IC50 values of the 10 extracts exhibiting the most potent inhibition of Ba/F3 TPR-TrkB cell proliferation (at a concentration of 388 μg/ml or less).

Fig. 2.

Fig. 2

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Inhibition of Ba/F3 TPR-TrkB proliferation by mushrooms extracts. IC50 values were determined for all mushroom extracts (the 10 most active extracts are shown in the graph).

2.4. M − 071 specifically inhibits Ba/F3 TPR-TrkB proliferation

Our data demonstrated a number of potential extracts inhibiting Ba/F3 TPR-TrkB proliferation with variable potency. We monitor ability of substances in extract M − 071 and M − 077 and others in specifically targeting TrkB activity, or also other signaling pathways that are required for Ba/F3 cell survival. We therefore attempted to rescue Ba/F3 TPR-TrkB cells exposed to M − 071 or M − 077 by adding exogenous murine IL-3 (mIL-3) and monitoring proliferation of the treated cells. As shown in Fig. 3 and Fig. S1 (supplemental data), the anti-proliferation activity of M − 077 against Ba/F3 TPR-Met cells was comparable in the presence or absence of mIL-3, suggesting that the inhibitory function against these cells is mediated by inhibition of a cellular signaling pathway required for Ba/F3 survival (Fig. 3A). In contrast, exposure of Ba/F3 TPR-TrkB to mIL-3 resulted in a reduced anti-proliferative effect of the M − 071 extract (Fig. 3B), arguing that part of the inhibitory function is mediated by TrkB inhibition. In contrast, exposure of Ba/F3 TPR-TrkB to mIL-3 in the presence of M − 077 extract did not result in a significant reduction of the anti-proliferative effect of the M − 077 extract (Fig. S2B). Thus, the results showed that M − 071 contains substances that selectively inhibit TrkB signaling as oppose to M − 077 extract. For this study, we selected the M − 071 extract derived from Auricularia auricula-judae. In addition, we are interested in this particular mushroom given its widespread medicinal use and reported beneficial effects in various medical pathologies, including cancer.

Fig. 3.

Fig. 3

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Drug specificity analysis for extract M-071. (A) Ba/F3 TPR-Met cells were cultured with and without exogenous IL-3. (B) Ba/F3 TPR-TrkB cells cultured with and without exogenous mIL-3 (10 ng/ml). Treatments were based on previously calculated IC50 multiplier.

. The extract M − 071 was derived from a mushroom initially identified as Auricularia auricula-judae. A tissue sample of the mushroom was subjected to DNA sequencing of the internal transcribed spacer (ITS) region, further confirming the proposed species identity (Supplemental data).

M − 071 extract inhibits TrkB receptor auto-phosphorylation.

Following the identification of M − 071 as a specific inhibitor of Ba/F3 TPR-TrkB cell proliferation, we examined whether this activity is mediated directly through inhibition of TrkB activity by measuring the potential of M − 071 to inhibit TrkB auto-phosphorylation. The phosphorylation status of TrkB following M − 071 application to Ba/F3 TPR-TrkB cells was determined using phospho-specific antibodies that recognize the phosphorylated form of TrkB.

The phospho-TrkB antibodies recognized a 52-kD protein, which is the expected size of the TrkB fusion protein (Fig. 4). Furthermore, exposure of Ba/F3 TPR-TrkB cells to 0.1 μM GNF-5837, a pan-Trk inhibitor [28], significantly reduced the level of phosphorylated TrkB. Treatment of Ba/F3 TPR-TrkB cells with increasing concentrations of M − 071 extract caused a dose-dependent reduction of phosphorylated TrkB. Thus, M − 071 contains phyto-substances that actively inhibit auto-phosphorylation activity of TrkB.

Fig. 4.

Fig. 4

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TrkB auto-phosphorylation assay. Ba/F3 TPR-TrkB cells were treated with increasing concentrations of M − 071 crude extract. GNF-5837 served as a positive control. Blot shows solvent-treated (DMSO 1.2%) and M-071-treated samples probed with phospho-specific anti-TrkB antibody. GAPDH was used as a control.

2.5. Identification of active fractions in whole-mushroom extract M-071

The M − 071 extract was subjected to bioassay-guided fractionation to isolate active fractions using direct preparative chromatography with a CombiFlash fractionating machine. Hexane and ethyl acetate (EtOAc) served as the mobile phase. Five consecutive fractions were collected, starting with fraction 1 (F1, obtained with 100% hexane) and ending with fraction 5 (F5, obtained with 70% hexane–30% EtOAc). Ba/F3 TPR-TrkB cells were exposed to 100 and 300 μg/ml of the different fractions and auto-phosphorylation levels were measured (Fig. 5). The levels of phospho-TrkB were significantly reduced in cells exposed to GNF-5837 (0.1 μM). No reduction in phospho-TrkB levels was observed in samples treated with 100 and 300 μg/ml of fractions F1–F3 (Fig. 5A). In contrast, treating Ba/F3 TPR-TrkB cells with fractions F4 and F5 caused a significant and dose-dependent reduction in phospho-TrkB levels (Fig. 5A). The inhibitory potential of these two fractions was further tested at lower concentrations (Figs. 5B) and 100 μg/ml of F4 was found to inhibit autophosphorylation of TrkB (Fig. 5B). Thus, we selected F4 as our active fraction and F5 with mild activity.

Fig. 5.

Fig. 5

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Inhibitory activity of M − 071 fractions. TrkB auto-phosphorylation levels following incubation with fractions 1–5 (F1–F5) (100 μg/ml and 300 μg/ml) (A), and 50 μg/ml to 100 μg/ml of F4 and F5 (B). GNF-5837 (0.1 μM) served as a positive control, and GAPDH served as a loading control on the immunoblot.

2.6. LC–MS/MS analysis of the A. auricula-judae active fraction

In an attempt to identify potential active moieties in the active fractions (F4 and F5) of A. auricula-judae extract, we compared features that became enriched in the active fractions, but not in the negative fractions (F1–F3). Thus, all fractions of A. auricula-judae were subjected to LC–MS/MS analysis, and 551 features were detected; 314 features were annotated based on MS1 analysis and isotope pattern using the ChemSpider database, and 41 features were annotated based on MS2 analysis using the mzCloud database (31 of these were annotated with fragment similarity higher than 80%). The 504 compounds identified by LC–MS/MS in the A. auricula-judae fractions were subjected to principal component analysis (PCA) (Fig. 6A) and a graphical representation of the data was plotted as a color-coded heat map to represent different values (Fig. 6B). Substances enriched in the active fractions (F4 or F5) were plotted separately and compared to their levels in the non-active fractions (Fig. 6C–E).

Fig. 6.

Fig. 6

Fig. 6

Fig. 6

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HPLC–MS/MS analysis of A. auricula-judae fractions. (A) PCA of 504 compounds identified by LC–MS/MS in A. auricula-judae fractions. (B) Heatmap and hierarchical cluster analysis graph for all metabolites that were significantly (P < 0.05) enriched in fraction 4 (F4). X-axis represents three repetitions of each fraction. (C) Relative amounts (peak area) of metabolites that were enriched in F4. (D, E) Relative amounts (peak area) of metabolites that were enriched in F5. Data represent median ±SE of three biological replicates. Significant differences were calculated by Tukey–Kramer HSD test, P < 0.05. Plots in A and B were generated using MetaboAnalyst 5.0 software. Plots in C–E were generated using GraphPad Prism 9.

A comparison of the substances in the active fraction F4 and the non-active fractions (F1, F2 and F3) demonstrated a number of compounds that were at higher levels in the former. Interestingly, among the compounds that were found in F4 but not in the non-active fractions was tiaprost (a prostaglandin F2α analogue), icosanedioic acid, γ-tocotrienol (one of the four types of tocotrienol, a type of vitamin E), methyl dehydroabietate (diterpene with preferential cytotoxic activity), C22H47NO3 (2-(bis{2-[(6-methylheptyl)oxy]ethyl}amino)ethanol), and C28H42O5 (15-hydroxy-1-(5-hydroxy-2-methoxy-3-methylphenyl)-3,7,11,15-tetramethyl-2,6-hexadecadiene-5,12-dione). Interestingly, only icosanedioic acid and tiaprost were enriched in F4 and the other were even higher in F5.

Interestingly, levels of methyl abieta-8,11,13-trien-18-oate were low in the non-active fractions F1–F3 and strikingly higher in thein the combined active fractions (F4 and F5). It is reasonable to speculate that TrkB-inhibiting moieties might be related to one or a combination of compounds found exclusively in active fractions (F4 and F5). This conjecture remains to be experimentally explored.

A significant number of compounds were at significantly higher levels in F5 compared to the other fractions (Fig. 6D and E). Among them were γ-tocotrienol, 15-hydroxy-1-(5-hydroxy-2-methoxy-3-methylphenyl)-3,7,11,15-tetramethyl-2,6-hexadecadiene-5,12-dione, methyl abieta-8,11,13-trien-18-oate, 1-(21-hydroxy-17-methoxy-21-phenylaspidospermidin-1-yl) ethenone, N-gondoylethanolamine, 3-((2-hydroxydodecyl)methylamino)propane-1,2-diol, 1-[5,8-dimethoxy-2-methyl-2-(4-methylpentyl)-3,4-dihydro-2H-chromen-6-yl]-3-(2,5-dimethoxyphenyl)-1-propanone, and 1-(1,11-dihydroxy-2,5,10a,12a-tetramethyl-7-phenyl 1,2,3,3a,3b,7,10,10a,10b,11,12,12a-dodecahydrocyclopenta [5,6]naphtho [1,2-f] indazol-1-yl)-2-hydroxyethanone (Fig. 6D and E; Table S2).

3. Discussion

We demonstrate a research approach in which extracts of whole macrofungi can be screened for beneficial activity in various health conditions and specifically with various cancers. In this study, we screened organic extracts prepared from a large number of medicinal mushrooms for inhibitory activity toward TrkB. We utilized Ba/F3 cells—murine B-lymphocytes that require the presence of mIL-3 for their survival [22]. Ba/F3 cells carrying activated kinases are becoming a popular tool for identifying kinase modulators, as they are independent of IL-3 for their survival [22,29]. Using the above screening system, we identified 10 mushroom organic extracts that showed significant inhibitory activity against Ba/F3 TPR-TrkB cell growth. The EtOAc extract M − 071 (Fig. 2) was selected for further analysis. This extract actively inhibited proliferation of Ba/F3 cells carrying activated TPR-TrkB with an IC50 of 200 μl/ml. Since Ba/F3 cells are dependent on a constitutively active tyrosine kinase for proliferation, inhibition of such kinases results in growth arrest and apoptosis, a condition that can be rescued by reintroducing mIL-3 into the growth media. We made use of this phenomenon to assess whether the bioactive substances present in M − 071 that inhibit proliferation of Ba/F3 TPR-TrkB cells actually interfere with TrkB function, or induce apoptosis, which is independent of TrkB activity [22]. The presence of IL-3 did not rescue Ba/F3 TPR-Met cells in the presence of different concentrations of M − 071 (Fig. 3), arguing that bioactive substances in M − 071 do not interfere with Met activity. In contrast, the presence of IL-3 actively rescued Ba/F3 TPR-TrkB cells and partially antagonized the anti-proliferative activity of M − 071, arguing that substances in the M − 071 extract inhibit TrkB function. To provide conclusive evidence for extract M-071's ability to inhibit the function of TrkB, we monitored the latter's auto-phosphorylation and the ability of M − 071 extract to inhibit it compared to known pan-Trk inhibitor such as GNF-5837 [28]. As expected, exposure to 0.1 μM GNF-5837 significantly reduced the level of phosphorylated TrkB (Fig. 4). Similarly, exposure to extract M − 071 inhibited auto-phosphorylation of TrkB in a dose-dependent fashion.

The active extract M − 071 was chemically fractionated into five fractions, and their ability to inhibit TrkB auto-phosphorylation was tested. F4, and to a lesser extent F5, were found to actively inhibit TrkB auto-phosphorylation. In an attempt to identify potential active substances, we performed LC–MS/MS analysis of active and non-active fractions and identified substances that correlated with activity against TrkB. A number of such compounds were present in either F4 or F5 (active fractions), but not in F2 (non-active fraction. Of special interest was methyl abieta-8,11,13-trien-18-oate, which was enriched in F4 and F5 (Fig. 6D), and has been reported to exhibit potent preferential cytotoxicity against pancreatic cancer cells (PANC-1) [30]. A recent study revealed elevated TrkB expression in 66% of pancreatic adenocarcinoma specimens compared to adjacent normal tissues [31]. Another study showed that TrkB expression in resected pancreatic samples correlates with increased rates of perineural invasion, positive margins, and development of metastatic disease [32]. Furthermore, established pancreatic cancer cell lines increased growth and invasion when incubated with increasing amounts of the Trk receptor ligands [33,34]. Thus, it is reasonable to propose the potential use of A. auricula-judae substances in pancreatic cancer with elevated levels of TrkB. Although one might expect that the identified substances are the bioactive substances that inhibit TrkB kinase activity, there may be others that were not detected by our analytical methods. Experiments are underway to identify and elucidate the chemical structure of the bioactive moiety. Nevertheless, our data show the therapeutic potential of substances in the mushroom A. auricula-judae for modulation of TrkB activity.

(Photo: Courtesy of Emanuel Sarid).

A. auricula-judae (Fig. 7) is well-known in Asian food markets for both its culinary quality and health benefits, making it the third most cultivated mushroom in the world [35]. In addition, A. auricula-judea has been widely used in traditional medicine for hundreds of years, making it a target for numerous studies which have exhibited its beneficial bioactivity in various medical pathologies, including cancer [[36], [37], [38], [39], [40], [41]]. The potential of A. auricula-judae for use as an herbal antimicrobial in the treatment of human bacterial and fungal pathogens has also been recently reported [36].

Fig. 7.

Fig. 7

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Auricularia auricula-judae growing on a tree branch in northern Israel.

Previously, A. auricula-judae was reported to exhibit potent antioxidant activity in vitro, to promote the biosynthesis of the collagen precursor procollagen, and to enhance the expression of hyaluronic acid synthase in HaCat cells [37]. Moreover, A. auricula-judae has been reported to possess immunomodulatory effects. The hot aqueous extract and the β-D-glucan-rich polysaccharide fraction stimulate increases in total lymphocyte count and neutrophil count, and show increased protection from cyclophosphamide-induced myelosuppression compared to the untreated negative control group [38]. Another study showed that a dichloromethane extract of A. auricula-judae significantly inhibits lipopolysaccharide-induced production of nitric oxide and other inflammatory cytokines (IL-6, TNF-α and IL-1β) in murine RAW 264.7 macrophages [39]. Moreover, others have reported the antitumor potential of A. auricula-judae extracts [40,41].

In conclusion, A. auricula-judae extract was identified as an inhibitor of TrkB auto-phosphorylation. Additionally, we identified potential compounds that might exert TrkB inhibition. Our objective was to identify an active moiety in the mushroom extract that mediates TrkB inhibition. However, it is reasonable that a single compound is not the sole inhibitor, but that a combination of compounds cooperates to inhibit TrkB function. Moreover, our current analysis relied on LC-MS/MS to identify active moieties; however, it is possible that the true active compound was not detected by our analytical methods. Furthermore, identifying an active moiety using in vitro experiments is only the first step; additional evidence for activity against TrkB-positive cancers in pre-clinical and clinical setting must be provided. Nevertheless, our results indicate the mushroom's potential to serve as a therapeutic for TrkB-positive cancers. Significantly, our results are the first to demonstrate anti-kinase activity in general, and against TrkB in particular, of the medicinal mushroom A. auricula-judae.

4. Materials and methods

4.1. Mushroom cultivation

The mushroom species and strains used in this study are summarized in Table 1. Mushrooms were grown using a standardized system under controlled conditions typical of commercial operations. The substrate was a sterilized mixture of 30% (d/w) fine eucalyptus wood chips, 35% (d/w) beer cereals and 35% (d/w) olive mill solid waste—the solid fraction from a three-phase olive mill. Hydration level was set to 64% for optimal growth. The substrate mixture was packed into 2.5-l polypropylene bags with a microporous filter, containing 0.8 kg wet substrate. The bags were autoclaved at 121 °C for 1 h, closed and cooled to 25 °C for inoculation with spawn, under sterile conditions. The culture was incubated at 25 °C for 14–21 days. For fruiting, the bags were opened, and the temperature was reduced to 17 °C with relative humidity of 90%, 12 h light with a Champi TC 58 bulb (HATO) and CO2 concentration of 600–800 ppm.

4.2. Whole mushroom extraction

Ethanol, EtOAc, and hexane were used as extraction solvents. Extraction was carried out using 3 g of dry material in 200 ml organic solvent with shaking at 170 rpm for 48 h. The extraction liquid was dried in a R-100 Rotavapor/Recirculating Cooler F-105 (Buchi, Flawil, Switzerland) to obtain 40.1 mg extract (1.34% yield). Dried material was dissolved in DMSO to a final concentration of 100 mg/ml and stored at 4 °C.

4.3. Ba/F3 TPR-Met and Ba/F3 TPR-TrkB cell lines

Native Ba/F3 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Prior to infection, native Ba/F3 cells were grown in a Greiner 40-ml liquid cell culture flask with 10% (w/v) RPMI 1640 medium, 5% (w/v) fetal bovine serum and (1%) penicillin–streptomycin. Every 2–3 days, the flask was inspected for cell viability using a standard light microscope, and 1 ml of culture was transferred to 9 ml of fresh media with the addition of 10 ng/ml mIL-3. After 2 weeks, Ba/F3 cells were separated into two cultures. For IL-3-dependency validation, one cell culture was isolated and given fresh mIL-3-deficient media. The second culture was subjected to Puromycin IC50 toxicity assay in standard 12-well plates using 6 wells, and the other 6 wells as duplicates. From a flask with 2.5 ml native Ba/F3 cells in 22.5 ml fresh media +10 ng/ml mIL-3, a 2-ml aliquot was transferred to each well. The plate was left to incubate for 24 h. Then wells 1–6, and 7–12 were subjected to the following concentrations of puromycin, respectively; 0, 0.5 μg/ml, 1 μg/ml, 1.5 μg/ml, 2 μg/ml, 2.5 μg/ml. The cultures were incubated for 3 days and cell number was counted in a hemocytometer using trypan blue (0.5% solution).

4.4. Trypan blue exclusion assay

Ba/F3 TPR-Met or TPR-TrkB cells were plated (4 × 105 cells/well) in 6-well plates, with each well containing 3 ml medium. After 24 h, cells were treated with the appropriate agents. Solvent-treated samples were incubated with 1% DMSO. The cells were collected 72 h later, stained with 0.4% trypan blue solution (1:1), and counted using a hemocytometer to determine IC50 values.

4.5. Rescue of growth inhibition by exogenous IL-3

Twelve-well plates were used with a total volume of 2 ml in each well, comprised of cell culture at a concentration of 1.5 × 105 cells/ml from Ba/F3 TPR-Met cell culture in one plate and Ba/F3 TPR-TrkB cell culture in another. Extract concentrations were applied twice in each plate, in wells 1–6 and wells 7–12, as follows: untreated, 14 IC50, 12 IC50, IC50, 2 x IC50, and 3 x IC50. The first 6 wells in each plate were treated with 10 ng/ml mIL-3 per well, and the last 6 wells were not. After 72 h, a sample was taken from each well for counting in a hemocytometer with 0.4% Trypan blue solution. This experiment was repeated twice for all treatments and cells.

4.6. Cell-proliferation assay

2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay was used as previously described [42] to evaluate the cytotoxicity of the different mushroom extracts. Briefly, 1.5 × 104 cells were plated in RPMI 1640 medium using 96-well plates for 24 h, and then treated for an additional 72 h with the different mushroom extracts. A total of 50 μl of XTT solution was then added to each well and incubated for 3 h at 37 °C. The optical density was measured by multiwell-plate spectrophotometer at 450 nm with a reference wavelength of 630 nm. The concentrations of all tested extracts inhibiting cell proliferation by 50% (IC50) were calculated. The experiment was performed in triplicate, and standard deviations were calculated.

4.7. Western blot

Six-well plates were seeded with 7 ml of Ba/F3 TPR-TrkB cell culture at a concentration of 5 × 105 cells/ml and incubated overnight. Cells were treated with extract M − 071 as follows: untreated, 50 μg/ml, 100 μg/ml, 200 μg/ml, 300 μg/ml, and a positive control of 1 μM GNF-5837. Similar 6-well plates were plated with Ba/F3 TPR-Met cell culture and a positive control of 5 μM crizotinib. The plates were incubated for 3 h.

The contents of each well was placed into a 15-ml tube and centrifuged at 1000 g for 5 min. The supernatant was discarded and the pellet was resuspended in 1 ml cold PBS and transferred to a 1.5-ml Eppendorf tube. The Eppendorf tubes were centrifuged at 1000 g for 5 min at 4 °C and the supernatant was again discarded and the pellet resuspended in 1 ml cold PBS, centrifuged, and the supernatant discarded. The washed pellets were kept frozen at −20 °C for 30 min. Protein analysis was performed by Western blot protocol on a 10% acrylamide gel. Cell lysate samples were prepared for loading by adding lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing protease inhibitors (Sigma, Neustadt, Germany) and phosphatase inhibitor (AG Scientific, San Diego, CA, USA) to the cell pellets. After 30 min, samples were centrifuged and protein concentration in the supernatants was determined using DC™ Protein Assay (Bio-Rad, Hercules, CA, USA) and absorbance at 630 nm. Samples with 50–60 μg protein were subjected to SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane (Schleicher and Schuell BioScience GmbH, Dassel, Germany) which was blocked with Tris Buffered Saline with Tween (TBS/T) with 5% skim milk and incubated with the following antibodies: phospho-TrkA (Tyr674/675)/TrkB (Tyr706/707) (C50F3) rabbit mAb (#4621 Cell Signaling Technology) for Ba/F3 TPR-TrkB treatments, and phospho-Met (Tyr1234/1235) (D26) XP® rabbit mAb (#3077 Cell Signaling Technology) for Ba/F3 TPR-Met treatments. Secondary antibody, HRP-linked anti-rabbit (#7074, Cell Signaling Technology), was used according to the manufacturer's instructions. Chemiluminescence was performed with SuperSignal™ West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and imaged in an HP imager LAS4000, and ChemiDoc MP Imaging System (Bio-Rad). The membranes were washed with TBS/T and incubated with antibody against the housekeeping gene GAPDH as a loading control (ab9485, Cambridge Biomedical Campus, Trumpington, UK). Secondary antibody was HRP-linked anti-rabbit (#7074, Cell Signaling Technology).

4.8. Mushroom ITS sequencing

PCR amplification of the ITS, used as a barcode for the fungal kingdom [43], was performed. Extract-N-Amp™ FFPE Tissue PCR Kit (Merck KGaA, Darmstadt, Germany) was used for DNA extraction and PCR amplification according to the manufacturer's protocol. Primers used were: NLB4 primer – 5′GGA TTC TCA CCC TCT ATG AC 3′, and NSI1 – 5′GAT TGA ATG GCT TAG TGA GG 3’ [44].

PCR conditions were: 95 °C, 4 min, 38 cycles (95 °C, 60 s; 52 °C, 45 s; 72 °C, 120 s), 72 °C for 5min. DNA was sent for sequencing to Hylabs (Park Tamar, Rehovot Israel).

4.9. Bioassay-guided fractionation of M − 071 extract

Bioassay-guided fractionation of M − 071 extract was performed using a Pure C-815 Flash (BUCHI, Flawil, Switzerland) CombiFlash fractionating machine; 133.0 mg of extract in 70 ml was mixed with about 400 mg silica gel beads (0.032–0.063 mm), then the solvent was evaporated to dryness. The dry mixture was dry-loaded into the CombiFlash machine using a direct-phase silica gel column (FlashPure EcoFlex Silica, 12 g). The extract was eluted using a gradient of hexane (A) and EtOAc (B) starting with 0% B for 3.8 min, then 5% B for 3 min, 10% B for 8.3 min, then the gradient was increased to 15%, 20%, 25% and 30% B (for 3 min at each solvent ratio). Finally, the column was eluted with 100% EtOAc for 7 min.

4.10. Monitoring phospho-TrkB auto-phosphorylation

Ba/F3 TPR-TrkB cells were treated with fractions F1–F5 for 3 h. The wells were treated as follows: untreated, 1 μM GNF-5837, and F1–F5 at 100 μg/ml and 300 μg/ml. Cell pellets were collected and subjected to immunoblotting as described above.

4.11. HPLC analysis

The samples were analyzed by injecting 5 μl of the diluted fractions (50 ppm in methanol) into a UHPLC connected to a photodiode array detector (Dionex Ultimate 3000), with a reverse-phase column (ACE Excel ®1.7 C18, 100 × 3.0 mm diameter). The mobile phase consisted of (A) DDW with 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. The gradient was started with 5% B and kept isocratic for 2 min, then increased to 98% B for 18 min and kept isocratic at 98% B for 3 min. Phase B was returned to 5% in 2 min and the column was allowed to equilibrate at 5% B for 5 min before the next injection. The flow rate was 0.4 ml/min.

4.12. MS/MS analysis

MS/MS analysis was performed with a heated electrospray ionization (HESI-II) source connected to a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Scientific). ESI capillary voltage was set to 3500 V, capillary temperature to 350 °C, gas temperature to 350 °C and gas flow to 35 ml/min. Mass spectra (m/z 100–1000) were acquired in negative- and positive-ion mode with high resolution (FWHM = 70,000). For MS2 analysis, collision energy was set to 15, 50 and 100 EV.

4.13. Data preprocessing

Peak determination and peak area integration were performed with Compound Discoverer 3.1 (Thermo Xcalibur, Version 3.1.0.305). Auto-integration was manually inspected and corrected if necessary. For some of the compounds, identification was performed based on the mzCloud database using MS2 data and ChemSpider database using HRMS.

Author contribution statement

Jamal Mahajna: Conceived and designed the experiments; Wrote the paper.Ofer Danay, Idan Pereman, Soliman Khatib, Ali Khattib, Ron Schweitzer: Contributed reagents, materials, analysis tools or data.Orr Shaharand, Nirit Ezov, Hazem Khamisie: Performed the experiments.

Funding statement

Prof. Jamal Mahajna was supported by Migal [001].

Data availability statement

Data included in article/supplementary material/referenced in article.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

None.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2023.e13756.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Multimedia component 1
mmc1.xlsx (18.3KB, xlsx)
Multimedia component 2
mmc2.xlsx (16.2KB, xlsx)
Multimedia component 3
mmc3.pptx (1.3MB, pptx)
Multimedia component 4
mmc4.docx (353.4KB, docx)

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Associated Data

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Supplementary Materials

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Data Availability Statement

Data included in article/supplementary material/referenced in article.

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