CC BY
0
EXTRACTS OF DAEDALEOPSIS CONFRAGOSA F-1368 INHIBIT IN VITRO AND IN VIVO THE PROLIFERATION OF HUMAN CANCER CELLS
O.I. Solovieva1'2*, I.A. Razumov1'2, T.V. Teplyakovd, E.L. Zavjalov1'2
1 Federal Research Center Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, 10 Lavrentyev Prospekt, Novosibirsk, 630090, Russian Federation;
2 Novosibirsk State University, 2 Pirogov St., Novosibirsk, 630090, Russian Federation;
3 State Scientific Center of Virology and Biotechnology «Vector», Koltsovo, Novosibirsk Oblast, 630559, Russian Federation.
* Corresponding author: solovieva@bionet.nsc.ru
Abstract. The aim of this study was to evaluate the effects of two variants of the Daedaleopsis confragosa fungus extract, F-1368 strain, differing in polysaccharide concentration, on five human cancer cell lines (U-87 MG, C-33 A, SK-Mel-28, MDA-MB-231, SW620) in vitro. Standard cytotoxicity assessment methods, including MTT-assay and clonogenic assay, were employed. Additionally, an experiment was conducted on laboratory SCID mice with heterotopic xenografts of human glioblastoma U-87 MG, where the test preparations were administered subcutaneously into the tumor area. Tumor sizes were measured using a caliper, and upon euthanasia, xenografts were histologically examined with hematoxylin-eosin staining under a light microscope. Results from MTT and clonogenic assays demonstrated that F-1368 extracts reduced the viability, mitochondrial function, and proliferative activity of tumor cells in vitro. However, a threefold increase in polysaccharide concentration in one of the extracts did not significantly enhance its cytotoxicity against tumor cells in vitro. Furthermore, one extract was tested on U-87 MG cell xenografts, revealing a reduction in tumor growth in SCID mice. The maximum tumor growth inhibition index for U-87 MG cells reached 50.7% at 21 days post-commencement of extract injections. These findings suggest that the D. confragosa F-1368 strain holds promise for investigating both in vitro and in vivo models of antitumor activity and identifying potential bioactive molecules or compounds.
Keywords: antitumor activity, anticancer activity, Daedaleopsis confragosa, F-1368, mycotherapy, U-87 MG, glioblastoma.
List of Abbreviations
Daedaleopsis confragosa F-1368 - D. confragosa F-1368 or F-1368
Introduction
Mortality from cancer diseases is known to increase steadily. The World Health Organization predicts a more than double increase in cancer cases by 2030 (WHO report on cancer, 2020). Therefore, the development of new antitumor remedies is one of the priority areas of modern science.
In general, natural resources serve as valuable raw materials to prepare pharmaceuticals in current medicine and public health service industries. Along with medicinal plants, the representatives of the kingdom of fungi more and more often act as these raw materials, which is explained by their pharmaceutical potential (Zeb & Lee, 2021; Gargano et al., 2017; Blagodatski et al.., 2018; Thomson et al., 2018).
Higher fungi from two divisions, namely Asco-mycota and Basidiomycota, represent a source of polysaccharides (especially, P-glucans) (Vetvicka et al., 2021) and polysaccharide-pro-tein complexes with antitumor and im-munostimulating properties (Venturella et al., 2021; Culliao, 2020; Jeitler et al., 2020). It is shown that fungal polysaccharides appreciably increase the cytokine production by T-cells and macrophages, induce the tumor necrosis factor expression in vitro and in vivo, enhance the phagocytic activity of human neutrophils (Wasser, 2017; Chang et al., 2017). Moreover, most Basidiomycota contain various types of other biologically active high- and low-molecular compounds (triterpenes, lectins, steroids, phenols, polyphenols, lactones, statins, alkaloids, and antibiotics) in fruit bodies and cultivated mycelium (Wasser, 2017).
The object of the study - Daedaleopsis con-fragosa - belongs to a vast group of polyporous
fungi, genus Daedaleopsis Schroet (Vladykina et al., 2020). There are data that the extracts of these fungi exhibit the antibacterial, antioxidant, and gene-protective properties, as well as the antiviral effect (Knezevic et al., 2017; Fakoyal et al., 2013; Vidovic & Zekovic, 2011; Teplyakova et al., 2021). Some D. confragosa strains have long been considered as a source of dietary protein. Thus, even more than 30 years ago, it was shown that 1 kg of the protein product from the mycelium of the D. confragosa G-115 strain contained the same amount of protein as 1 kg of meat (Reshetnikova, 1989). Until now, this type of fungi has been not applied in pharmaceutics, prevention or therapy of any diseases, although there are references indicating the high content of biologically active compounds in these fungi (Culliao, 2020; Manjusha, 2020).
The new D. confragosa strain registered under number F-1368, further - F-1368 (Teplyakova, 2018), was isolated from the natural habitat. Preliminary in vitro experiments demonstrated the cytostatic activity of its proteins against IM-9 human bone marrow myeloma and human Namalva lymphoma cells (Teplyakova, 2018). Furthermore, the in vivo antitumor activity of proteins from this strain was noted in the tumor model of xenografts of A-431 human skin epidermoid carcinoma when a solution of lyophilically dried proteins was introduced in a concentration of 1.4 mg/mL (or 1400 p,g/mL) (Kochneva et al., 2019). At the same time, a similar activity of polysaccharides of the F-1368 strain has been practically unexplored and not shown in in vivo models.
The purpose of this study was to analyze in vitro the antitumor activity of extracts of the D. confragosa F-1368 strain against human U-87 MG, C-33 A, SK-Mel-28, MDA-MB-231, SW620 tumor cells and the growth in xenografts of U-87 MG cells in SCID mice, and also to reveal the role of increasing concentration of polysaccharides in this activity.
Materials and Methods
Extracts
Purified extracts obtained by the procedures described previously in (Lebedev et al., 2019)
from the D. confragosa F-1368 strain from the «Vector» collection were used as objects of the study. Two variants of the extract of the F-1368 strain (Table 1) designated as K-3 and K-4 were investigated. These variants insignificantly differ in the protein concentration, however, the difference in the polysaccharide concentration was almost threefold. In our opinion, this fact implied the possibility of determining the role of increasing concentration of polysaccharides in the anticancer activity of extracts of the F-1368 strain.
Cell cultures
Tumor cell cultures stored in the cryobank of the Multi-Access Center "SPF-Vivarium", Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences were unfrozen and cultured in 1:1 DMEM/F12 (Biolot, Russia) supplemented with 10% fetal bovine serum (Invitrogen, USA). In the work, we used the following human cell lines: U-87 MG (ATCC HTB 14™) glioblastoma, C-33 A (ATCC HTB-31™) cervical carcinoma, SK-Mel-28 (ATCC HTB-72™) melanoma, MDA-MB-231 (ATCC CRM-HTB-26™) breast carcinoma, SW620 (ATCC CCL-227™) intestinal carcinoma. The diploid culture of HEF-15 cells (untransformed or normal human embryonic fibroblast cells) was selected as the control one (Radaeva, 2009).
Cell biological reactions for studying cyto-toxicity after the action of the extracts were assessed by the effect on the mitochondrial function (with the use of the MTT-assay) as well as cell growth and proliferation (clonogenic assay) (Simone et al., 2013; Coccini et al., 2014; Coc-cini et al., 2015; Franken & Rodermond, 2006).
MTT-assay
Cells were seeded in 96-well plates at a density of 20*103 cells/well. The next day, the extracts under study were added and titrated (8 reps for each concentration) together with the commercial drug cisplatin (TeVA PHARMA-CHEMIE B.V., Netherlands) as a positive control of cytotoxicity. Wells with cells in the growth medium without any additions were used as the negative control. They were incu-
Table 1
Concentrations (C) of proteins and polysaccharides in aqueous extracts used in the study
Name C of proteins, ^g/mL C of polysaccharides, ^g/mL Polysaccharide/ protein ratio
K-3 500 2900 5.8
K-4 730 1000 1.4
bated for three days at 5% CO2, then the medium with drugs was replaced by 90 pl of the medium without serum, and 10 pl of the MTT (Dia-M, Russia) solution was added in a concentration of 5 mg/mL. The cells with MTT were incubated for 4 h. Formazan crystals formed were dissolved with dimethylsulfoxide (Khimmed, Russia) and the absorbance was measured at 595 nm on a Thermo Fisher Scientific Inc. Multiskan SkyHigh (USA) spectro-photometer.
The obtained data (absorbance in wells) were recalculated and presented as a survival rate relative to the control (average absorb-ance in the wells with the negative control was taken to be 100%). The concentration causing the death of 50% of cells relative to the control was taken to be LD50; correspondingly, the concentration causing the death of 100% cells relative to the control was taken to be LD100 (Franken & Rodermond, 2006; Baumann et al., 2008).
Clonogenic assay
The cells were seeded in 24-well plates at a density of 100 cells/well (four reps for each concentration), incubated for 14 days at 5% CO2, with the state of colonies being daily monitored. When the colonies consisted of at least 50 cells, they were fixed with 10% formalin (Biovitrum, Russia) and colored with Gimsa (Biovitrum, Russia). The colonies were counted in a Zeiss Primo Vert (Germany) inverted microscope. The cell cloning efficiency (number of clones) in the control group was assumed to be 100%. In the experimental group, the cloning efficiency was determined as the percentage of cloning in the control group (Simone et al., 2013).
Animals
The study was performed with SPF mice in the Multi-Access Center "SPF-Vivarium", Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences. In the experiment, we used SCID (SHO-Prkdcs~ cidHrhr) mice (17 males). All manipulations were carried out in accordance with European Communities Counsil Directive 86/609 EEC.
Xenografts
The model of human glioblastoma as one of the most lethal and intractable forms of cancer was selected for the in vivo study. Subcutaneous xenografts of SCID mice were obtained by injecting 100 pl of the cell suspension containing 5 millions of U-87 MG cells in the region of the right scapula of each mouse. The cells were prepared for the injection by the previously described procedure (Razumov et al., 2020).
Injection of the extract to mice and in vivo data
The K-3 extract was injected subcutaneously to mice near the tumor in the amount of 200 pl to each mouse, which corresponds to 100 pg of proteins and 580 pg of polysaccharides. The same volume of sterile water was injected to the control group. The following treatment scheme was used: the extract was injected for 30 days in series for five consecutive days with breaks of two days.
To assess the antitumor action the linear sizes of U-87 MG xenografts of SCID mice were regularly measured and statistically processed; the volumes were calculated by the standard formula (Tomyako & Reynolds, 1989).
Tumor growth inhibition indices (TGII) TGII = (Vk-Vo)/Vk*100%, where Vk is the mean tumor volume in the control group, Vo is the mean tumor volume in the treatment group, were also calculated (Khabriev, 2005). The tumor growth index (TGI) TGI = Vi/Vo, where Vi is the tumor volume on a certain day of the study, Vo is the tumor volume on the first day of the study (before the injection of the extract) was determined.
Histological analysis of xenografts
After euthanasia the xenograft was excised in each mice and fixed in a 10% buffer formalin (Biovitrum, Russia). Then 4 p,m thick paraffin sections were prepared. To this end, we used a Thermo scientific CITADEL 2000 (USA) tissue processor, Thermo scientific HistoStar (USA) embedding workstation, and a Thermo scientific Microm HM340E (USA) rotary microtome. The obtained sections were colored with hematoxylin and eosine. Histological preparations were analyzed in a Leica DM 2500 (Germany) microscope (magnification x100) with Nikon (Japan) immersion oil. To estimate TGII we calculated the number of cells with mitoses in the field of view (20 fields of view per animal) (Meuten et al., 2016).
Statistical processing of results
Statistical differences were assessed using the STATISTICA.10 program package. To compare the survival of cells treated with the drugs under study and control cells the t-crite-rion was applied. To compare the tumor growth in the xenografts of mice we utilized the non-parametric Mann-Whitney U-test. Differences were considered to be statistically significant at p < 0.05. The data are represented as the mean (M) ± standard error of the mean (SE). To compare the number of mitoses we used the x2 criterion.
Results
To evaluate the safety of extracts in the MTT-assay we investigated the effect of K-3 and K-4 on HEF-15 human diploid cells (Fig. 1). The results of the MTT-assay with maximum concentrations of K-3 (polysaccha-
rides 290 mg/mL, proteins 50 mg/mL) and K-4 (polysaccharides 100 mg/mL, proteins 73 mg/mL) revealed that the viability of HEF-15 cells was no less than 79.9 ± 0.3% and 89.6 ± 1.6%. When minimum concentrations of K-3 (polysaccharides 2.3 mg/mL, proteins 0.4 mg/mL) and K-4 (polysaccharides 0.8 mg/mL, proteins 0.6 mg/mL) were added to the incubation medium, the viability of HEF-15 cells was no less than 94.1 ± 3.4% and 96.6 ± 4.2% relative to the control (Fig. 1). These values reliably differed from the values obtained for cisplatin (t = -16.2, p < 0.01 for K-3; t = -15.7, p < 0.01 for K-4) in high concentrations, which was used as the positive control for toxicity.
Finally, the dose-dependent effect of the drugs on the viability of HEF-15 cells was not noted. Both variants of the extract in the maximum doses seem to be low toxic (<LD25) for HEF-15 cells and are practically non-toxic in minimal doses, The toxic effects observed can be due to the residual content of ammonium sulfate in the extracts (Lebedev et al., 2019).
The K-3 and K-4 extracts under study were tested for cytotoxicity by MTT and clonogenic assays for five human tumor cell lines (U-87 MG, C-33 A, SK-Mel-28, MDA-MB-231, SW620) in vitro. Results of the determination of the cytotoxic activity of the extracts being studied, which are expressed through sublethal (LD50) and lethal (LD100) doses, are summarized in Tables 2 and 3.
MTT-assay
The assessment of the cytotoxicity of the extracts (K-3 and K-4) against human tumor cells in vitro by MTT indicated that three cell lines (U-87 MG, C-33 A, SK-Mel-28) proved to be most subject to the toxic action of F-1368 extracts.
The best result was obtained for U-87 MG cells, for which LD50 was 1.3 p,g/mL of proteins and 7.3 p,g/mL of polysaccharides when K-3 was used and 2.4 p,g/mL of proteins and 3.3 ^g/mL of polysaccharides when K-4 was used. The SW620 and MDA-MB-231 cells appeared to be the most resistant to polysaccha-rides and proteins of two variants of the extract (Table 2). The LD100 effect in the MTT assay with the used tumor cell lines was not revealed.
150
125
0
1/1280 1/640 1/320 1/160 1/80 1/40 1/20 1/10
Dilution
-•—Control -«-Cisplatine K-3 -*-K-4
Fig. 1. Viability of normal human HEF-15 cells after 3 days incubation with K-3 and K-4 drugs, M ± SE
Table 2
Determination of toxicity for K-3 and K-4 in MTT-assay on cell lines
Cell line LD50 (^g/mL), polysaccharides / proteins
K-3 K-4
U-87 MG 7.3 / 1.3 3.3 / 2.4
C-33 A 47.3 / 8.2 5.0 / 3.7
SK-Mel-28 58.0 / 10.0 5.0 / 3.7
SW620 > 58.0 / > 10.0 20.0 / 14.6
MDA-MB-231 290.0 / 50.0 66.7 / 48.7
The data obtained show that K-4 had the most pronounced cytotoxic effect against all the cell lines used (Table 2). A relatively high concentration of polysaccharides in the K-3 extract compared to that of K-4 did not enhance the cytotoxic effect against all cell lines in a short period.
Clonogenic assay
Apart from MTT-assays, we made longer-term clonogenic assays in which the incubation of cells with the drugs lasted for 14 days.
During a long-term action of F-1368, U-87 MG and C-33 A cells, as well as SW620 and MDA-MB-231, were the most susceptible to the concentration of polysaccharides and proteins in the compositions of K-3 and K-4 (Table 3). The SK-Mel-28 human melanoma cells
proved to be the most resistant. The maximum effect (LD100) was revealed for the K-3 and K-4 extracts against U-87 MG and MDA-MB-231 cells and against SW620 cells for K-3. For U-87 MG cells the LD100 effect was noted at the minimum concentrations of proteins (800 ng/mL (0.8 p,g/mL) and
1.1 ^g/mL) and polysaccharides (4.5 and 1.6 ^g/mL).
For the in vivo study we used SCID mice with heterotopic xenografts of U-87 MG cells. We began to inject K-3 when the average volume of xenografts was 104 ± 18 mm3 (0 days, Figure 2). The growth of xenografts in the K-3 group gradually slowed down, and although the differences maintained until the end of the experiment, they were statistically significant only on the 16th (M-W test, U = 13, p = 0.03),
Fig. 2. Variation of the growth index of subcutaneous xenografts of U-87 MG cells in SCID mice after the injections of K-3, M ± SE. * are the statistically significant differences (M-W U-test). Arrows mark the days of the first and last injections
Table 3
Clonogenic assay for the cytotoxicity of K-3 and K-4 against cell lines
Cell line K-3, polysaccharides / proteins K-4, polysaccharides / proteins
LD50 fag/mL) LD100 fag/mL) LD50 fag/mL) LD100 fog/mL)
U-87 MG 2.3 / 0.4 4.5 / 0.8 0.8 / 0.6 1.6 / 1.1
C-33 A 18.1 / 3.1 > 290.0 / > 50.0 2.1 / 1.5 > 100.0 / > 73.0
SW620 < 18.1 / < 3.1 290.0 / 50.0 6.3 / 4.6 > 100.0 / > 73.0
MDA-MB-231 36.3 / 6.3 290.0 / 50.0 25.0 / 18.3 50.0 / 36.5
SK-Mel-28 145.0 / 25.0 > 290.0 / > 50.0 33.3 / 24.3 > 100.0 / > 73.0
21st (U = 14, p = 0.04), 30th (U = 8, p = 0.02), and 32nd (U = 10, p = 0.04) days. In 21 days after the beginning of injections, the maximum TGII value of 50.7% was determined, with the mean tumor volume being 2340 ± 390 mm3 in the control group and almost two times less (1153 ± 165 mm3) in the experimental group when K-3 was introduced.
On the 30th day, the growth of xenografts became more intense, and TGII decreased from 50.7% to 48% in 30 days, then to 23.3% in 43 days, and to 10.8% in 54 days. On the 54th day euthanasia was performed for the animals from the control group.
Histological assessment of the proliferation of tumor cells in xenografts
The assessment of histological preparations showed that in the control group, the average
number of mitotic cells (Fig. 3) was 0.58 in the field of view, while in the F-1368 group, it was 0.42 (x2 = 49.2, df = 99, p = 0.9). However, there is a downward trend in the proliferation rate of xenografts of the mice injected with F-1368. Since the xenografts were extracted from the animals and fixed immediately after euthanasia (for the K-3 group, starting from the 39th day and later), the obtained data were consistent with the results illustrated in Figure 2 because in 21 days, TGII decreased. This seems to be the reason why the histological analysis did not reveal statistically significant differences.
Discussion
From the comparison of data on the cytotoxicity of drugs against tumor cells in the in vitro system (Tables 2 and 3), the U-87 MG and
Fig. 3. Histological preparations of U-87 MG xenografts colored with hematoxylin-eosin, eyepiece x20, lens x100. A - control; B - F-1368. The arrow marks the mitotic cell
C-33 A cell lines can be considered to be the most subject to the toxic effect of the components of K-3 and K-4 extracts of the F-1368 strain, whereas the MDA-MB-231 line, on the contrary, is the most resistant. The in vitro studied extracts of the F-1368 strain have the pronounced antitumor effect against all five human tumor cell lines tested. Both variants of the extract demonstrated the violation of the mito-chondrial function, and hence, the metabolic activity of cancer cells (Simone et al., 2013) as well as the inhibition of their ability to divide.
At the same time, the extracts were low toxic and non-toxic relative to normal HEF-15 cells, human embryonic fibroblasts. The cells known to be resistant to the in vitro action of the drug are most likely to be resistant to it in the organism, too (Baumann et al., 2008). Consequently, these results imply the possible application of F-1368 extracts in in vivo experiments.
The results of clonogenic assays enable the prediction of the cytotoxicity of the drugs under study against various tumor cells because we used cell lines of different tissue origin. Both tested variants of the F-1368 extract completely limited the proliferative potential (exhibited LD100) of MDA-MB-231 human breast carcinoma cells and U-87 MG human glioblastoma cells. Despite their high proliferation rate, the glioblastoma cells turned out to be the most susceptible: both extracts completely inhibited the cell growth in extremely low nanogram doses.
The increased concentration of polysaccha-rides does not seem to play the major role in the antitumor activity in vitro. Thus, it may be supposed that during the short-term incubation with extracts of the F-1368 strain in the MTT-assay, high concentrations of polysaccharides might not have the expected effect, however, the long-term incubation in the clonogenic assay, also did not reveal the dependence of the antitumor effect on the total amount of polysac-charides. This result enables the conclusion that further it is needed to study the qualitative composition of both the total F-1368 extract and separate fractions of polysaccharides and proteins.
In our study, we also for the first time discovered the antitumor effect of the K-3 extract of D. confragosa F-1368 in vivo against xenografts of U-87 MG human glioblastoma cells. The growth rate of heterotopic U-87 MG xenografts of SCID mice twice decreased after three weeks of K-3 injections (Figure 2). When the injections were regular, the tumor inhibition effect was retained, however, without injections this effect gradually slowed down. It is worth noting that even in the absence of therapy for two weeks (from the 30th to the 43rd day), U-87 MG xenografts grew slower by 23.3% in the K-3 group than the control xenografts. The results obtained indicate that the long and continuous action of F-1368 on tumor xenografts is required to reach the maximum TGII.
Conclusion
In our opinion, the obtained results demonstrate that F-1368 has pronounced antitumor effects in vitro against different types of carcinoma, and slows down the growth of human glioblastoma xenografts. All these facts make the D. confragosa F-1368 strain promising for the development of therapy of cancers of various etiologies, including glioblastoma.
Funding
The studies are supported by the budget project (No. FWNR-2022-0023) and implemented using the equipment of the Center for Genetic
Resources of Laboratory Animals «SPF-vivar-ium» at the FRC Institute of Cytology and Genetics SB RAS, supported by the Ministry of Education and Science of Russia (RFMEFI62119X0023).
Acknowledgements
We would like to thank the Center for Genetic Resources of Laboratory Animals «SPF-vivarium» at the Federal Research Center Institute of Cytology and Genetics and the Laboratory of mycology at the State Scientific Center of Virology and Biotechnology «Vector» for equipment and materials.
References
BAUMANN M., KRAUSE M. & HILL R. (2008): Clonogens and cancer stem cell. Nature Reviews Cancer 8, 990.
BLAGODATSKI A., YATSUNSKAYA M., MIKHAILOVA V., TIASTO V., KAGANSKY A. & KAT-ANAEV V.L. (2018): Medicinal mushrooms as an attractive new source of natural compounds for future cancer therapy. Oncotarget 9, 29259-29274.
CHANG S.T. & WASSER S.P. (2017): The Cultivation and Environmental Impact of Mushrooms, Environmental Science.
COCCINI T., GRANDI S., LONATI D., LOCATELLI C. & DE SIMONE U. (2015): Comparative cellular toxicity of titanium dioxide nanoparticles on human astrocyte and neuronal cells after acute and prolonged exposure. Neurotoxicology 48, 77-89.
COCCINI T., MANZO L., BELLOTTI V. & DE SIMONE U. (2014): Assessment of cellular responses after short- and long-term exposure to silver nanoparticles in human neuroblastoma (SH-SY5Y) and astrocytoma (D384) cells. The Scientific World Journal 13, 259765.
CULLIAO A.G.L. (2020): Preliminary Bioactivity Screening of Crude Extracts of Six Wild Macrofungi from Pine Forests in Benguet and Mt. Province, Philippines. Manila Journal of Science 13, 74-88.
DE SIMONE U., MANZO L., FERRARI C., BAKEINE J., LOCATELLI C. & COCCINI T. (2013): Short and long-term exposure of CNS cell lines to BPA-f a radiosensitizer for boron neutron capture therapy: safety dose evaluation by a battery of cytotoxicity tests. Neurotoxicology 35, 84-90.
FAKOYAL S., ADEGBEHINGBE K T. & OGUNDIIMU A.A. (2013): Biopharmaceutical assessment of active components of Deadaleopsis confragosa and Ganoderma lucidum. Open Journal of Medical Microbiology 3(2), 135-138.
FRANKEN N.A.P. & RODERMOND H.M. (2006): Clonogenic assay of cells in vitro. Nature Protocols 5, 2315-2319.
GARGANO ML., VAN GRIENSVEN L.J.L.D., ISIKHUEMHEN O S., LINDEQUIST U., VEN-TURELLA G., WASSER S.P. ET AL. (2017): Medicinal mushrooms: Valuable biological resources of high exploitation potential. Plant Biosystems 151(3), 548-565.
JEITLER M., MICHALSEN A., FRINGS D., HÜBNER M., CHER M., KOPPOLD-LIEBSCHER DA. ET
AL. (2020): Significance of Medicinal Mushrooms in Integrative Oncology: A Narrative Review. Frontiers in Pharmacology 11, 580656.
KHABRIEV R.U. (2005): Guidelines for the experimental (preclinical) study of new pharmacological substances. Moscow: Medicine, 832 pp.
KNEZEVIC A., STAJIC M. & ZIVCOVIC L. (2017): Antifungal, Antioxidative, and Genoprotective Properties of Extracts from the Blushing Bracket Mushroom, Daedaleopsis confragosa (Agaricomycetes). International Journal of Medicinal Mushrooms 19(6).
KOCHNEVA G., GRAZHDANTSEVA A., LEBEDEV L. & TEPLYAKOVA TV. (2029): Antitumor activity of the proteins from biomass of fungus Daedaleopsis confragosa F-1368 in relation to human epidermoid carcinoma. Abstract of the 10th International Medicinal Mushroom Conference, 266-267.
LEBEDEV L.R., TEPLYAKOVA TV., VYAZOVAYA E.A. & DANILENKO E D. (2019): Method for obtaining extracts from Daedaleopsis confragosa F-1368 with antitumor activity. Biotechnology 35(1), 1-5.
MANJUSHA P.G.S. (2020): Molecular Identification and Isolation of Antimicrobial, Phytochemical, Antioxidant Properties of Different Solvent Extracts from Wild Mushrooms. Saudi Journal of Pathology and Microbiology 5(2), 98-107.
MEUTEN D.J., MOORE F.M. & GEORGE J.W. (2016): Mitotic Count and the Field of View Area: Time to Standardize. Veterinary Pathology 53(1), 7-9.
RADAEVA I F., NECHAEVA E.A. & DROZDOV I.G. (2009): The Cell Culture Collection of the FSRISRC VB «Vector» Rospotrebnadzor. Novosibirsk: Tseris, 251 pp.
RAZUMOV I.A., TROITSKII S.Y., SOLOV'EVA O.I., BOLDYREV N.D. & ZAVJALOV E.L. (2020): Manganese Oxide Nanoparticles Inhibit the Growth of Subcutaneous U-87 MG Glioblastoma Xenografts in Immunodeficient Mouse. Bulletin of Experimental Biology and Medicine 3, 148-153.
RESHETNIKOVA I.A. (1989): Mushroom mycelium as a source offeed and food protein. Moscow: Publishing House of Moscow State University, 55 pp.
TEPLYAKOVA T V., ILYICHEVA T.N., DANILENKO E D. & LEBEDEV L.R. (2021): Antiviral activity of extracts of the basidiomycete Daedaleopsis confragosa F-1368. Biotechnology 37(1), 21-25.
TEPLYAKOVA TV., LEBEDEV L.R., NECHAEVA E.A., DUMCHENKO N.B., ILICHEVA T.N. & GILEVA I.P. (2018): A strain of the basidial fungus Daedaleopsis confragosa containing proteins exhibiting antitumor and antiviral activity. Patent RU 2663111, Bulletin 22.
THOMSON P.J., WARREN C., ARSHAD A.P., SAMANA B., SHAO L.Q., MINTAO Z. ET AL. (2018): A Preclinical Evaluation of the Antitumor Activities of Edible and Medicinal Mushrooms: A Molecular Insight. Integrative Cancer Therapies 17(2), 200-209.
TOMAYKO M.M. & REYNOLDS C P. (1989): Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemotherapy and Pharmacology 24(3), 148-154.
VENTURELLA G., FERRARO V., CIRLINCIONE F. & GARGANO ML. (2021): Medicinal Mushrooms: Bioactive Compounds, Use, and Clinical Trials. International Journal of Molecular Sciences 22(2), 634.
VETVICKA V., TEPLYAKOVA T V., SHINTYAPINA A.B. & KOROLENKO T.A. (2021): Effects of Medicinal Fungi-Derived ß-Glucan on Tumor Progression. Journal of Fungi 7, 250.
VIDOVIC S. & ZEKOVIC Z. (2011): The antioxidant properties of polypore mushroom Daedaleopsis confragosa. Central European Journal of Biology 6, 575-582.
VLADYKINA V.D., MUKHIN V.A. & BADALYAN S.M. (2020): Daedaleopsis Genus in Siberia and the Far East of Russia. Proceedings BDI, 17-26.
WASSER S.P. (2017): Medicinal Mushrooms in Human Clinical Studies, Part I. Anticancer, Oncoimmuno-logical, and Immunomodulatory Activities: A Review. International Journal of Medicinal Mushrooms 19(4), 279-317.
WHO (2020): WHO report on cancer: setting priorities, investing wisely and providing care for all. World Health Organization, 1-60.
ZEB M. & LEE C.H. (2021): Medicinal Properties and Bioactive Compounds from Wild Mushrooms Native to North America. Molecules 26(2), 251.
