2.2. Chemical Characteristic of M-0969 Strain Determine Their Biological Properties
The chemical analysis of the crude extract was performed via engagement of different chemical techniques including spectral–FTIR (Fourier-transform infrared spectroscopy) and spectroscopic–AAS (atomic absorption spectroscopy) as well as chromatographic analysis including GC-MS and HPLC. It is very important due to the fact that there is evidence that the bioactive potential of the tested mushroom is determined by the chemical composition and has direct effects on their anticancer activity. The FTIR spectroscopy indicated the presence of intense bands at 2940–2840 cm−1
, which corresponds to the stretching mode of C-H bonds. The medium size bands at 1460–1445 cm−1
and 1380–1370 cm−1
can be ascribed to the C–H deformations modes. Intense signals at 1740–1700 cm−1
were characterized as C=O vibrations. The signals at 1245–1010 cm−1
can be attributed to the C-O and C-N different types of vibrations (Figure 1
). These signals are associated with the presence of different compounds, including carbohydrates, sterols, carboxylic acid, amino acids, etc., which were deeply characterized by other techniques and described in the next section. Interesting is the presence of the peak at 1246 cm−1
, which is also characteristic for succinate and so-called “Baltic amber shoulder”. However, aforementioned signal might be also prescribed for N-Ar band in tryptophan molecules. This can indicate the presence of plant resin compounds that might have been absorbed by the fungus from its host.
Preliminary chemical analysis of the extract using GC-MS techniques showed the presence of compounds belonging to the groups carbohydrates (6.82% of TIC), sterols (1.82% of TIC), and carboxylic acids (1.74% of TIC) (Table 1
and Figure S1
). All these chemical groups are common when compared to chemical profile of best known medicinal mushrooms and are also responsible for their biological activity [20
In the next step, identification of individual compounds from specific groups, including indoles, phenolic acid, and sterols, was performed by High-Performance Liquid Chromatography. Indole compounds present in mushrooms can be divided into two groups: tryptamine derivatives with hallucinogenic properties and bioactive indole derivatives without hallucinogenic properties [26
]. Among the non-hallucinogenic indole derivatives indicated in HA, L-tryptophan and 5-hydroxy-L-tryptophan have soporific properties and are supportive in depression treatment. They are also precursors of serotonin and melatonin—endogenous substances responsible for regulating the circadian cycle of our body. Serotonin has also proven antioxidant and anticancer properties. Moreover, it can be used to treat depression and migraine [27
]. In the fruiting bodies, three indole compounds have been quantified: 5-hydroxy-L-tryptophan, L-tryptophan, and 6-methyl-D,L-tryptophan (Table 1
and Figure S2
). Besides, small amounts of melatonin were also qualitatively determined in the extract. From the indole compounds, 5-hydroxy-L-tryptophan (39.11 mg/100 g d.w.) was determined in the highest amounts in fruiting bodies. Significant amounts of L-tryptophan have also been obtained (34.95 mg/100 g d.w).
Analyses of several dozens of species of cultivated and wild growing mushrooms of the Ascomycota and Basidiomycota divisions, i.e., Agaricus bisporus, Aleuria aurantia, Boletus edulis, Cantharellus cibarius, Cortinarius varius, Cortinarius collinitus Fr., Gymnopilus penetrans, Gymnopilus spectabilis, Lactarius deliciosus, Leccinum scabrum, Lentinula edodes, Mycena pura, Mycena rosea, Paneolus spinctrinus, Pleurotus ostreatus, Russula ochroleuca
, have demonstrated the presence of numerous non-hallucinogenic indole derivatives [29
]. Analysis of the indole compounds content in Phellinus
, a genus of mushrooms growing on wood, demonstrated the presence of three indole metabolites including L-tryptophan. The content of L-tryptophan was 8.32 mg/100 g d.w., which is less compared to extract from wild growing HA [31
]. In ethanolic extracts from mycelial cultures of the edible Tricholoma equestre
and Imleria badia
, the content of 8 indole derivatives has also been determined. Three common metabolites in fruiting bodies of both species were found, L-tryptophan, tryptamine, and serotonin, while in the biomass from in vitro cultures 2 compounds, L-tryptophan and tryptamine. The relatively high content of L-tryptophan, serotonin, and tryptamine was noticeable [32
]. These results are similar to extract from HA because high amounts of indole compounds have been quantified in fruiting bodies. Analysis of indole compound content in other fruiting bodies and mycelial cultures of edible mushroom C. cibarius
demonstrated the considerable content of 5-hydroxy-L-tryptophan (similar amounts to 12.52 mg/100 g d.w.) [29
]. Furthermore, in this case, the HA species proved to be a better source of 5-hydroxy-L-tryptophan.
It is established that the most commonly occurring phenolic acids in sporocarps of Basidiomycota are protocatechuic, gallic, p
-hydroxybenzoic, gentisic, caffeic, syringic, and vanillic acids [33
]. Phenolic acids determined in the fruiting bodies of HA were protocatechuic acid in the amount of 2.24 mg/100 g d.w. and gentisic acid in the amount of 76.47 mg/100 g d.w. (Table 1
). Both of them are important for the survival of fungal organisms, as evidenced by the various biogenetic pathways leading to their formation. These compounds play an important role against fungal, parasites, and microorganisms invasion. Thus, their insecticidal, anti-bacterial, and anti-fungal properties are known. It should be emphasized that the aforementioned properties are directly associated with their antioxidant activity and affect the ability to help the endogenous defense system and regulation of host microbiota. These immunomodulatory properties are crucial during the prevention and treatment of different pathologies, including cancer [34
]. Moreover, these compounds play an important role in the case of arboreal species. During decomposition processes of wood where arboreal mushrooms exist, catalyzed by several enzymes, i.e., ligninase, hemicellulose, cellulose, oxidase, and peroxidase (Lignin peroxidase–LIP, Mn-peroxidase–MnP), there is an increased production of free oxygen radicals that induce the process of fatty acids autooxidation. Probably to avoid oxidative damage, polyporoid fungi, during the evolution process, have developed the ability to synthesize antioxidants and their main representatives are phenolic acids [35
]. Gentisic acid is a well-known and used anti-inflammatory and analgesic drug [36
]. It is obtained as a result of Kolbe reaction from hydroquinone, but it also can be obtained biotechnologically by the strains Penicillium patulum
and Polyporus tumulosus
. The protocatechuic acid (3,4-dihydroxybenzoic acid) is a natural polyphenol found in many edible and medicinal plants and fungi. Studies conducted in recent years indicate that it can be used in the prevention of cardiovascular disease and cancer. The mechanism of action of protocatechuic acid is based on its antioxidant properties, i.e., inhibiting the generation of free radicals, the ability to scavenge them, and increasing the catalytic activity of endogenous enzymes involved in the neutralization of free radicals. The significant effect of protocatechuic acid on the enzymes involved in the first and second stages of biotransformation of some carcinogens should also be highlighted. Their mode of action probably based on possibly blocking specific binding sites of metabolized carcinogens with the DNA molecule, which consequently prevents the formation of adducts that can cause mutations and cancer transformations. However, the other aspects of the chemopreventive activity of protocatechuic acid, i.e., influence on the activity of both inducible isoenzyme of cyclooxygenase and nitric oxide synthase or cell cycle regulating proteins, are not fully proven. Nevertheless, they can be helpful during the explanation of observed anticancer efficacy.
Among the determined sterols, ergosterol and ergosterol peroxide were identified (in quantities 9.48 and 23.85 mg/100 g d.w. of extract, respectively) (Table 1
and Figure S3
). Sterols are common ingredients of fruiting bodies of most Basidiomycota representatives. The most common is ergosterol. It has been proven that ergosterol and its peroxide are essential for the proper development of hyphae of higher fungi. Ergosterol (provitamin D2
) is one of the main components of fungal cell membranes. It is also a precursor of cortisol, an adrenal hormone with anti-inflammatory activity, exerting anticancer and immunostimulatory effects [37
]. Moreover, numerous studies have shown that ergosterol and its peroxidation products (ergosterol peroxide) display a therapeutic effect in the reduction of pain associated with inflammation, reduce the incidence of cardiovascular disease, and inhibit the action of the enzyme cyclooxygenase (COX) [38
]. Additionally, ergosterol peroxide has a broad spectrum of biological properties, such as antioxidant, anticancer, anti-inflammatory, or antimicrobial activity [39
]. Besides, it is believed that ergosterol peroxide is present as an intermediate in the H2
-dependent enzymatic oxidation reaction in the steroid biosynthetic pathway or as a detoxification product of reactive oxygen species. Its content in cells depends on many factors, including from the level of reactive oxygen species and the individual relationship between the formation of ergosterol peroxide and its re-conversion to ergosterol. It was also indicated that the presence of substances with antioxidant properties in biomass may affect the ratio of ergosterol to ergosterol peroxide [39
According to results from F-AAS analysis in the mushroom material, four bioelements important for human health (Cu, Fe, Mg, and Zn) were identified. Magnesium was determined in the highest amount (186.66 mg/100 g d.w.) (Table 1
). In addition, it is proved that the fruiting bodies of HA are a good source of Fe (14.21 mg/100 g d.w.) and slightly worse source of Zn (4.20 mg/100 g d.w.). Cu was found in the lowest amounts. The amount of Zn detected in another mushroom I. badia
and its mycelial cultures was found to be around 12.13 mg/100 g d.w., which is much higher quantity compared to HA content. It shows the specific properties of mycelial cultures and mushrooms and their ability to absorb specific bioelements from the medium on which they grow [40
]. In T. equestre
fruiting bodies, content of bioelements such as Mg (28.6 mg/100 g d.w.), Cu (4.9 mg/100 g d.w.), Fe (98 mg/100 g d.w.), and Zn (17 mg/100 g d.w.) were a bit different also. Only in the case of Mg, higher amounts were determined in fruiting bodies of HA compared to the T. equestre
Taking together, detailed physicochemical analysis of the crude methanolic extract of HA performed in this study might explain the observed anticancer activity. Since there is evidence of the bioactive potential of medical fungi, the chemical and biological importance of these is thus enhanced. According to their pleiotropic properties including immunomodulatory, anticancer, and antimicrobial properties, mushrooms might be used directly in diet and promote health or as adjuvant therapy, taking advantage of the additive and synergistic effects of all the present bioactive compounds.
2.3. Hemocompatibility and Cytotoxic Activity of HA Extract at In Vitro and In Vivo Level
Tumor diseases are one of the main causes of death worldwide. Subsequently, cancer has become the most intense field in life science, thus it is one of the main conditions where medicinal mushrooms have been used. Few extracts got a license as adjuvant nutrition in cancer therapy [42
]. Wide range of immunomodulatory effects that make medicinal mushrooms ideal for support in cancer cases comprises the capability to facilitate more effective immune response to cancer cells, ability to increase apoptosis of tumor cell and to impede tumor growth and metastasis, and ultimately the possibility to increase the efficacy with side-effects reduction of conventional therapy [43
]. Nowadays, it is expected that medicinal mushrooms and their synthetic derivatives would play a more significant role in the development of innovative products in cancer prevention. Current anticancer drugs have been shown to exert numerous side effects. This underlines the necessity of the implementation of novel and less toxic agents, such as from natural products.
With regard to all of this, our research was performed to establish a novel approach to HA. This basidiomycete fungus is a very important, necrotrophic pathogen on Pinaceae; however, its potential antitumor effect has not been described yet.
2.3.1. High Compatibility and Restriction of CRC Cell Viability after Exposure to HA Extract
The study was performed with the use of HA extract, in which concentrations were selected based on multiple assays using many concentrations due to lack of such data in literature. In the first step, hemocompatibility of tested extract was evaluated. Data in Figure 2
A indicate that HA extract does not affect membrane permeability of red blood cells (RBCs) at a concentration range of 1–50 μg/mL. Determination of hemolytic activity showed that tested extract applied at different concentration range exerts high hemocompatibility against the representative of host cells, RBCs. In addition, in our study, we also assessed the pH-dependent hemolysis assay that is generally engaged as a model for screening pharmaceutical agents designed for intracellular delivery of biologic drugs. Results indicate that there was no hemolytic activity of HA extract at any concentration and different pH. However, results indicated the increasing hemolytic potential in tested concentration as the pH decreases. This could suggest that, in the tested extract, some compounds with endosomolitic potential might exist. The above results are important as they signalize high level of safety in case of potential intravenous application of the product. DLD-1 is an epithelial, adherent cell line derived from a colorectal adenocarcinoma (Dukes type C). This cell line possesses unique features, including high tumorigenicity, and is appropriate to in vivo evaluation in a xenograft model in nude mice. Moreover, it is characterized by positive expression of different genes, such as myc
, which determine their resistance for treatment by classical chemotherapy [46
]. Viability of DLD-1 cell line, which is representative of CRC, after exposure to three different concentrations of HA extract (0.1, 1 and 5 μg/mL), presents Figure 2
B as a percent of control. For this purpose, neutral red assay was employed. Significant decrease in viability of DLD-1 cells in concentration-dependent manner was noted. To support the observed anticancer potential, significant suppression of cell proliferation was indicated in DLD-1 cell line at concentrations 1 and 5 µg/mL of HA extract (Figure 2
C). For this purpose, [3H]-thymidine incorporation test was engaged. These results clearly show that even micro concentrations of tested extract exhibit high cytotoxic activity. Unfortunately, there is very little data in the literature to discuss; however, due to the rising evidence about the development of cancer cells resistance, increasing interest in searching for alternative therapeutic options has been noted. Tomasi et al. screened extracts of 58 mushroom species for their cytotoxic activities against two murine cancer cell lines, L1210—lymphocytic leukemia (ATCC CCL 219) and 3LL—Lewis lung carcinoma (CRL-1642). Among tested methanol extracts, HA was also included. Nonetheless, the authors claimed that extract from HA was inactive and exhibited IC50 > 100 µg/mL against both cell lines [47
]. According to the standards of the National Cancer Institute (NCI), extract may be recognized as significantly cytotoxic with IC50 < 20 µg/mL [48
2.3.2. Exposition to HA Extract Induces Apoptosis via the Complex Mode of Action
Currently, anticancer drugs available on the market have numerous adverse effects. In view of the above, the search for new strategies such as the application of extract from natural sources where interaction between multiple compounds with different modes of action takes place, is the current focus of research. To evaluate the HA extract’s mode of action, which is responsible for observed cytotoxic and antiproliferative efficacy, a number of techniques including study of cells metabolic activity, mitochondrial transmembrane potential, lactate dehydrogenase (LDH) release assay, distribution of thiol levels as well as expression of specific apoptotic-associated proteins in DLD-1 cell culture were carried out. In the first step, the impact of the extract on cells’ metabolic activity has been performed. Figure 2
D shows that the addition of HA extract caused depletion of cells metabolism in concentration-dependent manner. Importantly, a significant reduction of metabolic activity (over 8-fold) was observed in cells with HA extract in a concentration of 5 µg/mL, compared to control. Results from evaluation of mitochondrial transmembrane potential showed the highest percentage of cells with properly polarized mitochondria in the control group (94.2%) (Figure 2
E). An increase in the percentage of cells with depolarized mitochondrial membrane was observed after administration of 0.1 μg/mL and 1 μg/mL of HA extract, 17.9% and 42.6%, respectively. In turn, as presented in Figure 2
F, the addition of tested extract at the concentration range 0.1–5 μg/mL strongly increased the release of LDH from treated cancer cells. This is a result of the disruption of the plasma membrane and leakage of cytoplasmatic content from the treated cells. The abovementioned HA extract activity might suggest that, in the mixture of active agents, membrane-active agents might be presented. This provides the non-specific mode of action, against which the creation of the resistance phenomena is strongly restricted. This finding confirms the results obtained in the vitality assay (Figure 2
G–I). About 96.3% of healthy cells and 3.3% of apoptotic cells were observed in the control group. Addition of HA extract (0.1 and 1 μg/mL) evoked a growth in rate of apoptotic cells (4.4% and 32.9%, respectively). Taking together, performed complex studies in DLD-1 cell line revealed increasing percentage of apoptotic cells with increscent concentrations of tested extract. It is associated with the fact that the decrease in cellular glutathione (GSH) concentration is an early hallmark in the progression of cell death in response to apoptotic stimuli.
To prove the obtained results, immunofluorescence microscopy was used. Results presented in Figure 3
showed increased expression of caspase-3 and translocation of caspase-3 and p53 protein from cytoplasm to cell nucleus in cells exposed to increasing concentrations of HA extract (0.1, 1, and 5 µg/mL).
It is established that antitumor effect might be associated with different pathway including inhibition of proliferation, induction of cell cycle arrest, increased apoptosis, or regulation of signal transduction pathways [49
]. In agreement with our studies, Youn et al. showed that water extract from Inonotus obliquus
could significantly inhibit the viability and proliferation, as well as induce apoptosis in human hepatoma cells (HepG2) [50
]. Other authors treated HT-29 human colon cancer cells with 1.0 mg/mL of I. obliquus
water extract. Maximum inhibitory effect that they could observe was 56%, with concomitant Bcl-2 downregulation and Bax and caspase-3 upregulation. Ultimately, this led to inducing apoptosis of cancer cells. Others also found a decrease in Bcl-2 expression with a simultaneous increase in caspase-3 and Bax or p53 expression. However, this was observed in different extract concentrations and cancer cell lines [51
]. The above results, though applied to different mushrooms, are in agreement with our findings concerning caspase-3 and p53 protein. We showed increased expression of caspase-3 and translocation of caspase-3 and p53 protein from cytoplasm to cell nucleus in cells exposed to increasing concentrations of HA extract. Due to lack of data, this outcome could not be compared with similar results featuring HA extract.
2.3.3. Effects of HA Extract in Xenograft CRC Model
The in vitro results were confirmed by our subsequent xenografted tumor model in vivo. The studies performed on animals with inoculated DLD-1 colorectal cancer cells showed that growth of tumors in studied groups was approximate up to 18th day, and the differences, however not statistically significant, became noticeable in the second half of experiment. We noticed lower tendency of tumor growth in the group of mice receiving HA extract compared to the control group. The smallest tumor growth was noted in the group of animals receiving 5-fluorouracil (5-FU), as a positive control, intraperitoneally (Figure 4
). 5-FU belongs to the group of anti-metabolites that interferes with the synthesis and stability of DNA and RNA of nucleic acids. Its mode of action causes the conversion of fluorodeoxyuridine monophosphate (FdUMP), which forms a stable complex with thymidylate synthase (TS), and thus inhibits deoxythymidine mono-phosphate (dTMP) production, which is essential for DNA replication, its repair, and depletion, therefore, causing cytotoxicity [54
There are no data involving HA in terms of its biological activity and thus our study is innovative. To date, the influence of medicinal mushrooms on the growth of colon cancer is described in limited number of papers. Li et al. tested Hericium erinaceus
extracts against HT-29 colon cancer, HepG2 and Huh-7 liver cancer cells, and NCI-87 gastric cancer cells in vitro and tumor xenografts bearing in SCID mice in vivo [55
]. Their results showed that extracts are active against cancer cells in vitro and also more effective and less toxic compared to 5-FU in in vivo tumor models [55
]. In another study by Kang et al., beneficial effect of active compounds from Inonotus obliquus
was presented on Azoxymethane/Dextran sulfate sodium colon cancer model [56
]. Anticancer effects of various medicinal mushrooms are presented on other tumor models. Wang et al. showed apoptotic potential of Phellinus igniarius
extract (TPI) on gastric cancer cells. Published results showed that the application of TPI markedly inhibited the viability of human cancer lines A549, HepG-2, Hela, AGS, and SGC-7901 in vitro, which was consistent with in vivo results performed on xenograft mouse model. After two weeks of TPI treatment of nude mice bearing SGC-7901 cells, the tumor size and weight in the groups that received TPI at doses 400 and 800 mg/kg were significantly reduced compared to the control group [57
]. Antitumor activity of polysaccharides from Inonotus obliquus
was noted by Fan et al. [58
]. The growth of gastric carcinoma in in vivo was significantly inhibited by tested polysaccharides compared to control [58
]. Won et al. presented that polysaccharides isolated from fruiting bodies of Inonotus obliquus
reduced melanoma tumor growth in tumor-bearing mice [59
Additionally, only a few studies focus on the synergistic effects of mushrooms with 5-FU. Yang L. et al. examined the activity of 5-FU in hepatocellular carcinoma (HCC) [60
]. Due to the significant resistance of tumor cells to standard chemotherapy regimens, researchers added to 5-FU anagrafolide (ANDRO), a bicyclic diterpenoid isolated from Andrographis paniculata
. Synergistic induction of apoptosis was observed under the influence of 5-FU + ANDRO polytherapy, which was confirmed by increased activity of caspase-8, p53, and significant changes in Bax conformation in cancer cells [60
]. Opattova et al. analyzed simultaneous effect of Ganoderma lucidum
(GLC) with 5-FU in mice xenograft model of colorectal cancer (CT26.WT cells) [61
]. GLC positively influenced the cytotoxic effect of 5-FU on tumor size, and better survival was observed in the group of mice treated with GLC and 5-FU together [61
A tendency to decrease tumor size after HA administration observed in the present study suggests that the experiments should be extended. Our goal is to develop a dose of HA to achieve a reduction in the growth of colorectal tumors to the values obtained after 5-FU administration. Similar to above-cited papers, scheme of synergistic action of HA and 5-FU is under investigation.
2.3.4. HA Extract Applied in Doses up to 2000-Fold Effective Dose Caused Mild or Moderate Acute Toxicity
In the evaluation of acute oral toxicity, HA extract in doses of 175, 560, 1792, and 2000 mg/kg body weight (b.w.) was used. Some toxicity up to 24 h after administration has been noted. The majority of these changes were mild or moderate in intensity (e.g., depression, hypopnea, gait disturbance, hypersalivation) and resolved from 6 to 10 h or even sooner. Five animals were sacrificed on the first day or later, however, 2 of them were from the control group. One mouse which received an extract in concentration 560 mg/kg b.w. and two after 1792 mg/kg b.w., and also 1 from the control group, survived 14 days of the experiment without any toxic effects. Mice that were sacrificed due to toxicity or survived 14 days of the experiment have undergone macroscopic and microscopic observations. The most frequent changes included organ hyperemia and petechiae.
Due to oral administration of HA extract, macroscopic and microscopic analysis of the gastrointestinal tract have been performed. As microscopic images (Figure 5
) of intestinal and stomach mucosa of animals from acute toxicity studies show, there are no pathological changes after treatment of mice with the highest doses of HA extract (1792 and 2000 mg/kg b.w.).
Therefore, we cannot say if there was a toxic effect of HA extract or DMSO, especially when there was no correlation between extract dose and toxic effect. On the basis of the obtained results, it is not possible to establish any clear conclusions or to determine the maximum tolerated dose (MTD) of HA extract. It must be noted that concentrations used in the acute toxicity experiment exceeded up to 2000-fold effective dose used in the experiment, which suggests that even if the results do not point specific toxic doses, in therapeutic doses, none of the toxic symptoms were observed.
As a conclusion, it should be emphasized that the obtained results suggest that precise dose selection is obligate, especially concerning the fact that HA is not classified as an edible mushroom.