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(11) EP 2 576 761 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 1/14 (2006.01) A61K 36/07 (2006.01) 08.02.2017 Bulletin 2017/06 (86) International application number: (21) Application number: 11738493.3 PCT/IL2011/000434

(22) Date of filing: 02.06.2011 (87) International publication number: WO 2011/151831 (08.12.2011 Gazette 2011/49)

(54) EXTRACTS OF STRIATUS MUSHROOMS, PHARMACEUTICAL COMPOSITIONS COMPRISING THEM AND A NEW STRAIN EXTRAKTE AUS CYATHUS-STRIATUS-PILZEN, PHARMAZEUTISCHE ZUSAMMENSETZUNGEN DAMIT UND NEUER CYATHUS STRIATUS-STAMM EXTRAITS DE CHAMPIGNONS CYATHUS STRIATUS, COMPOSITIONS PHARMACEUTIQUES LES COMPRENANT ET NOUVELLE SOUCHE DE CYATHUS STRIATUS

(84) Designated Contracting States: (74) Representative: Dennemeyer & Associates S.A. AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Postfach 70 04 25 GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO 81304 München (DE) PL PT RO RS SE SI SK SM TR (56) References cited: (30) Priority: 03.06.2010 US 351078 P • ROUMYANA D PETROVA ET AL: "Fungal substances as modulators of NF-[kappa]B (43) Date of publication of application: activation pathway", MOLECULAR BIOLOGY 10.04.2013 Bulletin 2013/15 REPORTS ; AN INTERNATIONAL JOURNAL ON MOLECULAR AND CELLULAR BIOLOGY, (73) Proprietor: Carmel-Haifa University Economic KLUWER ACADEMIC PUBLISHERS, DO, vol. 34, Corporation Ltd no. 3, 9 November 2006 (2006-11-09), pages 31905 Haifa (IL) 145-154, XP019531902, ISSN: 1573-4978 cited in the application (72) Inventors: • KANG H S ET AL: "Cyathuscavins A, B, and C, • FARES, Fuad new free radical scavengers with DNA protection 25155 Hourfish (IL) activity from the Basidiomycete Cyathus • SHARVIT, Lital stercoreus", BIOORGANIC & MEDICINAL 26317 Qiryat Motzkin (IL) CHEMISTRY LETTERS, PERGAMON, ELSEVIER • WASSER, Solomon P. SCIENCE, GB, vol. 18, no. 14, 15 July 2008 36812 Nesher (IL) (2008-07-15) , pages 4047-4050, XP022852894, ISSN: 0960-894X, DOI: 10.1016/J.BMCL.2008.05.110 [retrieved on 2008-06-03]

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 576 761 B1

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Description etes vesicolor, Inonotus obliquus, Hypsizigus mar- moreus, and Flammulina velutipes) (Ikekawa, 2001). TECHNICAL FIELD [0005] Many edible mushrooms were used in tradition- al folk medicine includingLentinus edodes (shiitake [0001] The present invention relates to a new and dis- 5 mushroom). Grifola frondosa (maitake), Hericium eri- tinct variety of higher Basidiomycetes mushroom Cyat- naceus, Flammulina velutipes, Pleurotus ostreatus, and hus striatus HAI-1302, deposited under The Budapest Tremellamesenterica which are also sources of relatively Treaty with the Centralbureau voor Schimmelcultures pure bioactive compounds for medical usage, while other (CBS)under Accession No. CBS 126585 (hereinafter Cy- non-edible , such as Ganoderma lucidum, Schiz- athus striatus CBS 126585), to extracts thereof, to phar- 10 ophyllum commune, and Trametes versicolor, are used maceutical compositions comprising the extracts and to only for their medicinal properties. the extracts for use in treatment of pancreatic and other [0006] Fungal metabolites have been gaining scientific types of cancer. The present invention further relates to interest as they were found to possess medicinal prop- a pure submerged mycelial culture ofCyathus striatus erties. Many studies on medicinal mushrooms proved CBS 126585. 15 their potential not only as dietary supplements and im- munoenhancers (Wasser & Weis, 1999a, b) but also as BACKGROUND ART modulators of various cellular responses (Zaidman et al., 2005). [0002] Natural products have been the most significant [0007] Thediversification ofmedicinal higher fungirep- source of drugs and drug leads in history. Many conven- 20 resents great potential for new drugs. As known, cancer tional therapies have been developed from nature-de- chemotherapy has relied mostly on cytotoxic drugs, rived materials. Their dominant role in cancer chemo- which inhibit tumor cell proliferation and cause cell death. therapeutics is clear, with about 74% of anticancer com- Cytotoxic activities against various tumor cell lines of pounds being either natural products or - higher fungi secondary metabolites were widely investi- derived. It is estimated that approximately 25% of the 25 gated in the past decades. drugsprescribed worldwide, at present, come from plants [0008] Low-molecular-weight (LMW) substances with and 60% of anti-tumor/anti-infectious drugs, already on fungal origin can penetrate the cell membrane and inter- the market or under clinical investigations, are of natural fere, in particular, with cellular pathways linked to proc- origin (Wang et al., 2007). esses such as , carcinogenesis, cell differ- [0003] Higher Basidiomycetes mushrooms (HBM) rep- 30 entiation and survival, metastasis, etc. On the basis of resent a major and still largely unexploited source of new these facts, medicinal mushrooms were established as pharmaceutical products. Of approximately 15,000 a novel and promising source for natural therapeutics known species, 2,000 are safe for human consumption, that can be successfully applied in the treatment of dif- and about 650 of them possess medicinal properties. Of ferent diseases, including cancer (Smith et al., 2002). about 650 mushroom species with known medicinal35 Extracts from many mushrooms, as well as specific ac- properties, only about 20 species are in use at the tive substances isolated from such extracts (e.g. Yassin present. Most traditional knowledge about medicinal et al., 2003), have been shown to exhibit anti-cancer ac- properties of HBM arrives from the Far East (China, Ja- tivity. pan, Korea, and Russian Siberia). Many pharmaceutical [0009] For example, Muller et al. (2006) found that substances with potent and unique properties have re- 40 methanol:water extracts from fruiting bodies of G. luci- cently been extracted from mushrooms. Unique antican- dum induce apoptosis for each of the 26 panel of human cer remedies were prepared from these extracts such as cancer cell lines examined, in a dose- dependent man- lentinan, krestin, and schizophyllan (Mi- ner. zuno, 1999). [0010] Wu et al. (2006) used ethyl acetate extracts [0004] Presentstudies suggestthat extracts and active 45 from the of Cordyceps sinensis to show the agents obtained from HBM act as immunomodulators or inhibitory effect on various cancer cell lines. They pro- as biological response modifiers (BRM). They help the jected the inhibitory effect to ergosterol and related com- body to strengthen itself and fight off illness by maintain- pounds present in the extract. ing physiological homeostasis and restoring the body’s [0011] Lu et al. (2004) used ethanol:water extracts of Host Defense Potentiators (HDP), which can have im- 50 the Coriolus vesicolor fruiting bodies to inhibit the prolif- mune system enhancement properties. This benefit is eration of Burkitt’s lymphoma B- cell line (HL- 60) and one of the reasons why they are currently used as ad- humanacute promyelocytic leukemia celllines (NB- 4) ,in juncts to cancer treatment in many countries. In Japan, a dose-dependent manner, with more than 90% suppres- Russia, China, and the USA several an- sion, and no significant suppression on the proliferation ticancer and immunomodulating agents have been de- 55 of normal liver cell lines (WRL). veloped from the fruiting body, mycelia, and culture me- [0012] Pancreatic ductal adenocarcinoma (PDAC) is dium of various medicinal mushrooms ( Lentinus edodes, one of the deadliest of all solid malignancies. It is the Ganoderma lucidum, Schizophyllum commune, Tram- thirteenth most common cancer worldwide and one of

2 3 EP 2 576 761 B1 4 the leading causes of cancer death with 232,000 new Consequently, the biomass and extracts of this novel cases each year and 213,000 deaths. In the United mushroom is efficacious in treating cancer, in particular States and Israel, pancreatic cancer is the fourth leading pancreatic cancer, and can be used in pharmaceutical cause of cancer related mortalities. In the year 2010 an products. estimated 43,140 patients were diagnosed with pancre- 5 [0017] The present invention relates therefore, in one atic cancer; 36,800 patients died of their disease in the aspect, to the new and distinct variety of higher Basidi- US alone. omycetes mushroom Cyathus striatus HAI-1302, depos- [0013] PDAC is a disease with very poor prognosis ited under The Budapest Treaty with the Centralbureau mainly because of insensitivity to most standard thera- voor Schimmelcultures (CBS) under Accession No. CBS pies including chemotherapy, radiotherapy, and immu- 10 126585 (hereinafter Cyathus striatus CBS 126585), an notherapy. Therefore, surgical resection offers, at the extract obtained from Cyathus striatus CBS 126585, and moment, the only potential prospect for a cure. a pharmaceutical composition comprising a pharmaceu- [0014] For all stages combined, the 1- and 5-year rel- tically acceptable carrier and said extract. In certain em- ative survival rates of PDAC are 24% and 5%, respec- bodiments, the extract is for use in treating cancer, in tively. Even for those people diagnosed with local dis- 15 particular pancreatic cancer. In a further aspect, the ease, the 5-year survival is merely 20%. These unfortu- present invention relates to a pure submerged mycelial nate statistics reflect the advanced stage at which most culture of Cyathus striatus CBS 126585. patients with pancreatic cancer are diagnosed and the [0018] The disclosure describes a method for treating paucity of effective chemotherapeutic regimens for ad- pancreatic cancer, comprising administering to a patient vanced disease. 20 in need a therapeutically effective amount of an extract [0015] To our knowledge, no previous publication dis- obtained from higher Basidiomycetes mushroom Cyat- closing the activity of Cyathus striatus mushroom ex- hus striatus. tracts on pancreatic cancer cells exists. We have found [0019] The disclosure describes a method for treating some data on other members of the Cyathus, e.g., cancer such as pancreatic cancer, breast cancer, chronic Cyathus bulleri (Kang et al., 2007), , 25 myelogenous leukemia (CML) and prostate cancer, in , Cyathus africanus, Cyathus colensoi, Cy- particular pancreatic cancer, comprising administering athus gansuensis, Cyathus sp.39, Cyathus pallidus, Cy- to a patient in need a therapeutically effective amount of athus intermedius,Cyathus sp.34, Cyathus sp. 37, Cyat- an extract obtained from Cyathus striatus CBS 126585. hus nigroalbus, Cyathus sp. 73, and Cyathus luxiensis (Liu & Zhang, 2004). Most of the data pertains to antimi- 30 BRIEF DESCRIPTION OF DRAWINGS crobial or antifungal properties. The mushroom Cyathus striatus was investigated in our lab for the first time for [0020] its anticancer properties. In an extensive screening of 242 crude mushroom extracts, Petrova et al. (2007) from Figs. 1A-B show the effect of C. striatus C.L ethyl our lab demonstrated a potent growth-inhibitory effect of 35 acetate (EAC) extract on the viability of ( A) HPAF-II some of the extracts, e.g. from C.striatus, on MCF7- and (B) PL45 cells by XTT assay. Cells were seeded human breast cancer cell line, PC3 and DU145- human in a 96-well plate (10 4 cells/ml), after 24 h cells were androgen-independent prostate cancer, 9L- rat glioblas- treated with different extract concentrations and in- toma, and Baf3/p185 Bcr-Abl -B-lymphocytes, a labora- cubated for 24, 48, and 72 hours. At the end of treat- tory model of CML. Petrova also found C. striatus to mod- 40 ment, cell viability was measured using XTT (Biolog- ulate IκBα levels even in the lowest concentration used ical Industries, Israel). All results presented are an (5 and 1 mg/ml) and demonstrated the ability to inhibit average of three independent experiments: four re- both IκBα phosphorylation and degradation (Petrova et peats each (mean 6 SEM) and expressed as per- al., 2007). centages of control (non-treated cells). Statistical 45 significance was determined by one way ANOVA SUMMARY OF INVENTION P<0.05. Each letter above bars represents relations to fellow concentrations in the same treatment peri- [0016] A novel and distinct variety of the higher Basid- od. iomycetes mushroom Cyathus striatus, designated Cy- Figs. 2A-B depict the effect of C. striatus C.L EAC athus striatus CBS 126585, has been isolated by the in- 50 on the viability of ( A) HPAF-II and ( B) PL45 cells by ventorsand it is shownherein that an extract ofthis mush- Crystal violet assay. Cells were seeded in a 24-well room is effective in inhibiting growth of, and inducing ap- plate (5x104 cells/ml). After 24 h, cells were treated optosis in, pancreatic cancer cells. Other varieties of C. with different extract concentrations and incubated Striatus are known to be rich in nutraceutical agents and for 24, 48, and 72 hours. At the end of treatment, cell biologically active compounds and extracts thereof have 55 viability was measured using the Crystal violet assay been shown to be effective in killing breast cancer cells, (see Example 2, Materials and Methods). All results human androgen-independent prostate cancer cells, rat presented are an average of three independent ex- glioblastoma cells, and a laboratory model of CML cells. periments: four repeats each (mean6 SEM) and

3 5 EP 2 576 761 B1 6 expressed as percentages of control (non-treated extract on apoptosis induction using TUNEL and cells). Statistical significance was determined by one DAPI staining of A ( ) HPAF-II and B( ) PL45 cells. way ANOVA P<0.05. Each letter above bars repre- Cells were counted and seeded on chamber slides sents relations to fellow concentrations in the same (Nunc, ) (25x10 3cells/ml). On the next day, treatment period. 5 cells were treated with C. striatus C.L extract for 24 Fig. 3 shows the effect of C. striatus C.L EAC on the h at a concentration of 10 mg/ml. At the end of treat- cytotoxicity of HPAF-II and PL45 cells by LDH leak- ment, cells were stained with TUNEL and DAPI and age. Cells were seeded in a 24-well plate (5x104 analyzed under a fluorescent microscopy (x 400 cells/ml). After 24 h, cells were treated with different magnification). 1. DAPI control; 2. TUNEL control; extract concentrations and incubated for 24 h. At the 10 3. DAPI with 10 mg/ml C. striatus C.L EAC extract; end of treatment, LDH leakage was measured using 4. TUNEL with 10 mg/ml C. striatus C.L EAC extract. a colorimetric assay (see Example 2, Materials and Figs. 8A-B show the effect of C. striatus C.L EAC Methods). All results were expressed as percentag- extract on apoptosis induction according to Annexin es of control (non-treated cells). V-PI double staining by flow cytometry of HPAF-II Figs. 4A-B depict the effect of C. striatus C.L EAC 15 cells (A) and PL45 cells ( B). Cells were treated with on DNA synthesis of ( A) HPAF-II and (B) PL45 cells different extract concentrations (Control (0 mg/ml), by the BrdU assay (see Example 2, Materials and 2.5, 5 or 10 mg/ml) for 4 h, and 5x105 cells/ml were Method). Cells were seeded in a 96-well plate (104 counted and collected for the analysis. Apoptotic cells/ml). After 24 h, cells were treated with different cells as percent of total cells observed is presented. extract concentrations and incubated for 24, 48, and 20 All results represent means 6 SEM of three inde- 72 hours. At the end of treatment, cell DNA synthesis pendent experiments. was measured using the BrdU assay. All results pre- Figs. 9A-B show the effect of C. striatus C.L EAC sented are averages of three independent experi- extract on caspase 9 activity of HPAF ( A) and PL45 ments; four repeats each (mean6 SEM) and ex- (B) cells by flow cytometry. 1x106 cells/ml were in- pressed as percentages of control (non-treated25 cubated with different concentrations (Control (0 cells). Statistical significance was determined by one mg/ml), 2.5 or 5 mg/ml) of C. striatus extract for 2 or way ANOVA P<0.05. Each letter above bars repre- 4 h. Caspase-9 positive cells as percent of total cells sents relations to fellow concentrations in the same observed is presented. All results represent mean treatment period. 6 SEM of three independent experiments. Figs. 5A-D show the effect of C. striatus C.L EAC 30 Figs. 10A-B show the effect of C. striatus C.L EAC extract on cell cycle using FACS Flow cytometry extract on caspase 8 activity of HPAF ( A) and PL45 analysis. C. striatus C.L EAC extract induces cell (B) cells by flow cytometry. 1x106 cells/ml were in- accumulation in SubG1-phase of the cell cycle in cubated with different concentrations (Control (0 both HPAF-II and PL45 cell lines. Exponentially mg/ml), 2.5 or 5 mg/ml) of C. striatus extract for 2 or growing cells were exposed to either medium (con- 35 4 hrs. Caspase-8 positive cells as percent of total trol) or C. striatus C.L EAC extract (10 mg/ml) for 24 cells observed is presented. All results represent h. Cells were then harvested, washed in PBS, and mean 6 SEM of three independent experiments. fixed in 70% ethanol. DNA content was evaluated Fig. 11 shows the effect of C. striatus C.L EAC on with propidium iodide staining and fluorescence was apoptosis induction according to expression of pro- measured and analyzed as described in Materials 40 caspase-9 and cleaved caspase-9. Different con- and Methods (Example 2). Data are representative centrations (C (0 mg/ml), 2.5, 5 and 10 mg/ml) of C. of three independent experiments. ( A) HPAF-II con- striatus C.L extract were added to 2x10 6 HPAF and trol; (B) 24 h 10 mg/ml C. striatus C.L EAC extract PL45 cells for 12 h. At the end of treatment, treated treatment; (C) PL45 control; (D) 24 h 10 mg/ml C. and control untreated cells were collected, proteins striatus C.L EAC extract treatment. The data shown 45 were extracted, and identical samples of 60mg pro- in the upper right corner of each panel show the rel- tein were subjected to Western blot analysis. Data ative number (%) of cells found in each phase of the are representative of three independent experi- cell cycle (SubG1, G1, S, and G2/M). ments. Fig. 6 shows the effect of C. striatus C.L EAC extract Fig. 12 shows the effect of C. striatus C.L EAC on on apoptosis induction by agarose gel electrophore- 50 apoptosis induction according to expression of pro- sis of DNA fragmentation. HPAF-II and PL45 cells caspase-3 and cleaved caspase-3. Different con- were treated with 10 mg/ml C. striatus C.L extract centrations (C (0 mg/ml), 2.5, 5, and 10 mg/ml) of C. (c.s) for 24 h before being harvested. Left to right: striatus C.L extract was added to 2x10 6 cells for 12 Lane 1: DNA marker: 100 bp ladder; Lane 2: HPAF- h. At the end of treatment, treated and control un- II control untreated cells, Lane 3: HPAF-II treatment, 55 treated cells were collected, proteins were extracted, Lane 4: PL45 control untreated cells, Lane 5: PL45 and identical samples of 60 mg protein were subject- treatment. ed to Western blot analysis. Data are representative Figs. 7A-B depict the effect of C. striatus C.L EAC of three independent experiments.

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DETAILED DESCRIPTION OF THE INVENTION roform. [0028] The extracts obtained as described above are [0021] The disclosure describes inter alia to extracts concentrated and may be purified. Concentration can be and active agents obtained from Cyathus striatus mush- carried out by conventional techniques such as thermal, rooms. 5 decompressing thermal, activated carbon or ion ex- [0022] Although most bioactive substances isolated change resin methods. The concentrated extract is then from mushrooms are high-molecular-weight (HMW) purified to yield a purified extract of one or more purified polysaccharides, our interest is in low-molecular-weight compositions using standard techniques such as column (LMW) compounds capable of exhibiting antitumor ac- chromatography, fractional distillation, preparative TLC tivity. The search for such compounds was focused on 10 (thin layer chromatography), preparative HPLC (high the higher Basidiomycetes mushroom Cyathus striatus, performance liquid chromatography), CPC (centrifugal in view of the inventors’ previous experience with this partition chromatography) or other techniques known to mushroom. For example, Petrova et al. (2007) demon- those skilled in the art. After concentration and purifica- strated a potent growth-inhibitory effect of extracts from tion, the product is dried by any conventional technique C. striatus, on breast cancer cells, human androgen-in- 15 such as air-drying, hot-blast drying, spray drying, and dependent prostate cancer cells, rat glioblastoma cells, freeze-drying methods. The concentrated extract is dis- and a laboratory model of CML cells. Petrova et al. also solved in dimethyl sulfoxide (DMSO). found C. striatus to modulate IκBα levels and demon- [0029] The extracts obtained from the C. striatus CBS strated the ability to inhibit both κIBα phosphorylation 126585 mushroom are rich in low-molecular weight com- and degradation. 20 pounds such as , terpenoids, glycosides, flavo- [0023] Presently, a new and distinct variety of the noids, terpenes and phenols, obtained from culture liquid mushroom was isolated and characterized (see Example or mycelium of the mushrooms. Low-molecular-weight 1). Extracts obtained from this mushroom were shown mushroom organic substances have their origins as de- herein to be extremely toxic to pancreatic cancer cells rivatives from many intermediates in primary metabo- (see Example 2). 25 lism. The upper molecular weight limit for a small mole- [0024] Thus, in some aspects, the present invention cule is approximately 800 Daltons. relates to a new and distinct variety of higher Basidio- [0030] It has been found in accordance with the mycetes mushroom Cyathus striatus HAI-1302, depos- present invention that the extract obtained from the cul- ited under The Budapest Treaty with the Centralbureau ture liquid of a submerged mycelium culture of the novel voor Schimmelcultures (CBS) under Accession No. CBS 30 mushroom Cyathus striatus CBS 126585 contains most 126585 (hereinafter Cyathus striatus CBS 126585), ex- of the growth-inhibiting or cytotoxic activity of the mush- tracts obtained from Cyathus striatus CBS 126585, and room and that the extraction of the culture liquid with ethyl to a pharmaceutical composition comprising the extract acetate results in the most active extract. Consequently, and a pharmaceutically acceptable carrier. in one embodiment, the organic extraction solvent used [0025] In other aspects, the present invention relates 35 to extract the culture medium of the mushroom mycelium to an extract obtained from higher Basidiomycetes mush- is ethyl acetate. room Cyathus striatus CBS 126585 for use in the treat- [0031] It has also been found in accordance with the ment of cancer, in particular pancreatic cancer, and to a present invention that the active agents comprised in the pharmaceutical composition comprising a pharmaceuti- extract exert their activity by arrest of the cell cycle, re- cally acceptable carrier and the extract, wherein the phar- 40 duction in DNA synthesis in cancer cells, and induction maceutical composition is for treatment of cancer, in par- of apoptosis in cancer cells by activation of both the cas- ticular pancreatic cancer. pase 8 and the caspase 9 pathways followed by the ac- [0026] The term "treating cancer" as used herein refers tivation of the executing caspase 3 pathway. to the inhibition of the growth of cancer cells. Preferably [0032] Thus, in certain embodiments, the extract inhib- such treatment also leads to the regression of tumor45 its growth of cancer cells, arrests cancer cell cycle, re- growth,i.e. to thedecrease insize or complete regression duces DNA synthesis in cancer cells, and/or induces ap- of the tumor. The term refers to treatment and alleviation optosis in cancer cells. This is of great importance be- or complete cure of disseminated tumors, namely, of me- cause it is well known that characteristic changes such tastases. as deregulation of the cell cycle machinery and acquire- [0027] In certain embodiments, the extracts of the50 ment of self-sufficiency in growth signals, insensitivity to present invention are obtained from the culture liquid of growth inhibitory signals, ability to evade apoptosis, in- a submerged mycelium culture of the mushroom. The vade tissue form metastases, and to sustain angiogen- extraction is performed with an extraction solvent com- esis are hallmarks of the majority of cancers and can also prising one or more organic solvents such as, without be found in Pancreatic ductal adenocarcinoma. being limited to, ethyl acetate, ethanol, chloroform, meth- 55 [0033] One of these routes of changes, the evasion of anol, acetonitrile, hexane, cyclohexane, isooctane and apoptosis, is a fundamental mechanism in cancer treat- dichloromethane. In certain embodiments, the organic ment. Apoptosis, or , is a central solvent is selected from ethyl acetate, ethanol and chlo- regulator of normal tissue homeostasis. The physiologi-

5 9 EP 2 576 761 B1 10 cal "cell suicide" program is essential for the elimination cer such as ductal adenocarcinomas, acinar cell carci- of redundant, damaged, and infected cells. Disturbed ap- nomas, adenosquamous carcinomas, colloid carcino- optosis is involved in the pathogenesis of multiple dis- mas, giant cell tumors, hepatoid carcinomas, intraductal eases, especially cancer. The acquisition of mechanisms papillary-mucinous neoplasms, mucinous cystic neo- to evade apoptosis is a hallmark of cancer, with both the 5 plasms, pancreatoblastomas, serous cystadenomas, loss-of function of pro-apoptotic signals and gain-of func- signet ring cell carcinomas, solid and pseudopapillary tion of anti-apoptotic mechanisms contributing to tumor- tumors, undifferentiated carcinomas, undifferentiated igenesis and the cancer phenotype. carcinomas with osteoclast-like giant cells and pancre- [0034] Most chemotherapies act by the induction of ap- atic endocrine tumors. optosis. Therefore, the evasion of apoptosis is mainly 10 [0040] In other certain embodiments, the extract ob- responsible for the insufficiency of current therapies. Tu- tained from the novel and distinct variety Cyathus striatus mor cells use multiple pathways to escape apoptosis. CBS 126585, is for treating cancer, such as pancreatic Defective apoptotic mechanisms allow genetically unsta- cancer, breast cancer, chronic myelogenous leukemia ble cancer cells to avoid elimination and confer resist- (CML) and prostate cancer. In particular, the pharmaceu- ance to chemotherapy. 15 ticalcomposition comprisingan extract obtainedfrom Cy- [0035] As such, modulating the apoptotic pathways athus striatus CBS 126585 is for use in treating pancre- likely represents a propitious strategy for inducing tumor- atic cancer. cell death and increasing responses to chemotherapy, [0041] The disclosure describes methods for treating radiotherapy, and even targeted therapies. Therefore, cancer, such as pancreatic cancer, breast cancer, chron- agents that can restore apoptosis in cancer cells hold 20 ic myelogenous leukemia (CML) and prostate cancer, promise for therapy and have been the focus of many comprising administering to a cancer patient a therapeu- preclinical drug discovery studies. tically effective amount of an extract disclosed herein or [0036] Execution of apoptosis relies on a group of a pharmaceutical composition comprising this extract. cysteine proteases, the caspases (Degterev et al., 2003). [0042] The pharmaceutical compositions, depending Caspases are synthesized as proforms and become ac- 25 on their intended use, may be adapted for oral, intrave- tivated by cleavage next to aspartate residues. Since nous, subcutaneous, intraarticular, intramuscular, inha- caspases cleave and activate each other, an amplifica- lation, intranasal, intrathecal, intraperitoneal, intrader- tion mechanism through a protease cascade exists, as- mal, transdermal or enteral administration. suring proper execution of apoptotic cell death (Degterev [0043] The present invention is further directed to a et al., 2003). 30 pure submerged mycelial culture of Cyathus striatus CBS [0037] There are two alternative pathways to initiate 126585. The disclosure describes a biomass and an ex- apoptosis through the initiator caspases (caspase-8, -9, tract obtained from the pure submerged mycelial culture, and -10), and both ultimately activate the executioner which are rich in nutraceutical agents and biologically caspases-3, - 6, and -7. The first pathway is the intrinsic active compounds including , , or mitochondrial pathway, which involves an imbalance 35 proteins rich in essential amino acids, vitamins, lipids rich of pro- and anti-apoptotic members of the BCL-2 protein in essential fatty acids, agents and minerals. family. The second pathway is the extrinsic pathway and [0044] The disclosure describes a composition com- is mediated by different death receptors on the cell sur- prising the biomass or an extract obtained from the pure face (Debatin et al., 2004; Fas et al., 2006; Wajant et al., submerged mycelial culture, or a mixture of said bio- 2006). 40 masses or extracts, and food supplement, pharmaceuti- [0038] A most beneficial feature of the extract, as cal, prebiotic, nutraceutical, beverage and cosmetic shown in Example, 2, is that it is capable of inducing products comprising this composition. Furthermore, a apoptosis in pancreatic cancer cells. Since a multitude pharmaceutical composition comprising a pharmaceuti- of active agents are comprised in the extract of the cally acceptable carrier and the composition comprising present invention that may induce apoptosis by different 45 the biomass or an extract obtained from the pure sub- pathways, it is likely that the cells in a cancer tumor ex- merged mycelial culture is disclosed herein. posed to the extract would not have enough time to de- [0045] The disclosure describes the use of the extracts velop mutated proteins that would render all these path- and biomasses in mixtures with biomasses and/or ex- ways resistant to the active agents in the extract. In view tracts of other medicinal mushrooms such as, but not of this, and the fact that extracts from otherC . striatus 50 limited to, Shitake, Coprinus and Tremella. strains have been shown to be efficacious against a [0046] The invention will now be illustrated by the fol- number of cancer types, the extracts and pharmaceutical lowing examples: compositions disclosed herein are useful for treating can- cer, such as pancreatic cancer, breast cancer, chronic myelogenous leukemia (CML) and prostate cancer. 55 [0039] In certain embodiments, the extract obtained from the higher Basidiomycetes mushroomCyathus striatus CBS 126585 is for use in treating pancreatic can-

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EXAMPLES ation percentage of the respective control. [0051] Crystal violet assay. Crystal violet assay was Materials and Methods performed for the evaluation of the effect of fungal ex- tracts on cell viability as well. HPAF-II and PL45 cells (6 Preparations of mushroom extracts 5 x104) were seeded in 500 ml of medium, using 24-well plates. After 24 h, fungal extracts were added in several [0047] Mycelial culture of the investigated mushroom concentrations: 1, 2.5, 5, 7.5, 10, 25, and 50m g/ml for species was obtained from the culture collection (HAI) of 24, 48 and 72 hours. Subsequently growth medium and the Institute of , University of Haifa, Israel. The fungal extracts were washed with 0.9% NaCl, and then mushroom strain was grown first on a solid medium and 10 cells were fixed with 96% ethanol for 10 minutes. Crystal then transferred onto submerged conditions, following violet dye (0.05%) in 20% ethanol was added for 30 min- Yassin et al. ((2003) Int J Med Mushr, 5:261-276). The utes. After cells were dyed, the residue dye was removed, strain was grown in submerged conditions for biomass and plates were washed 5-7 times with tap water and left production for 10 days, in order to reach the growth stage for evaporation of residue water. Then, 400 ml 0.1% ace- of secondary metabolite production. Biomass extraction 15 tic acid in 50% ethanol was added to each well for the was performed according to Yassin et al. (2003; see elicitation of the dye from the cell’s nucleus. The number above), with the exception of the solvents used. In this of living cells is proportional to the amount of dye. study, the obtained fungal biomass was extracted with Strength of dye was measured using Elisa reader (Bi- the following organic solvents (Frutarom, Israel): ethyl oTek) at 595 nm. Experiments were repeated 3 times alcohol (EAL), ethyl acetate (EAC), and chloroform20 independently and conducted in at least 3 replicates. Da- (CHL). Culture liquid (CL) was extracted with EAC in the ta are presented as average percentage of the respective ratio of 1 (CL):500 ml (EAC) (Khan et al., (2001) Pakistan control. J Biol Sci, 4(11):1374-1376). [0052] Lactate dehydrogenase (LDH) leakage assay. [0048] The dried extract was diluted at a concentration LDH is a cytoplasmic that catalyzes the oxidation of 50 mg/ml with 99.9% dimethyl sulfoxide (DMSO) (Sig- 25 of L lactate to pyruvate with NAD + as a hydrogen accep- ma-Aldrich, St Louis, MO, USA) and kept at -20°C. tor, the final step in the metabolic chain of anaerobic gly- colysis. The extracellular appearance of LDH serve as a Cell cultures marker for tissue lysis since cell damage, such as necro- sis, causes a rise of LDH in the cells’ medium (Moran [0049] The human pancreatic cancer cell lines HPAF- 30 and Schnellmann, 1996). In order to exclude the possi- II and PL45 (ATCC, Rockville, MD, USA) were main- bility for necrotic effects of the extract on the cell lines, tained in MEM-EAGLE and DMEM medium, respective- lactate LDH leakage into the medium was measured in ly, with 1% L-glutamine supplemented, 10% fetal calf se- aliquots of the extracellular fluid of each sample, as de- rum (FCS), and 1% PenStrep (penicillin + streptomycin) scribed previously (Goeptar et al., 1994). (Biological industries, Kibbutz Beit Haemek, Israel). The 35 [0053] DNA synthesis. 5-Bromo-2-uridine Labeling Kit HPAF-II cell line was supplemented with an additional (BrdU) (Roche Applied Science, USA) is a method based 1% sodium pyruvat, and PL45 cell line was supplement- on the incorporation of BrdU to proliferating cells. BrdU ed with an additional 1% L-glutamine. Cells were grown was added to the cell’s culture and was incorporated into in a humidified incubator at 37°C with 5% CO 2 in air and newly synthesized DNA of replicating cells, substituting fed twice a week with fresh medium. 40 for thymidine during DNA replication. Monoclonal anti- bodies labeled with peroxidase specific for BrdU are used Cytotoxicity assays to detect the incorporated BrdU. The enzyme peroxidase catalyzes the cleavage of the peroxidase substrate that [0050] XTT assay. Evaluation of fungal extracts’ effect was added to the cells’ culture and produces a color re- on cell line viability was performed by XTT ((sodium45 action, thus indicating cells that were actively replicating 3’-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4- their DNA. The color reaction can be measured by Elisa methoxy-6-nitro) benzene sulfonic acid hydrate)) assay reader, 492 nm and a background absorption of 690 nm. (Biological Industries). HPAF-II and PL45 cells (104) HPAF-II and PL45 cells (104) were seeded in 100 ml of were seeded in 100 ml of medium, using 96-well plates. medium, using 96-well plates. After 24 h, fungal extracts After 24 hours, fungal extracts were added in several 50 were added in several concentrations: 1, 2.5, 5, 7.5, 10, concentrations: 1, 2.5, 5, 7.5, 10, 25, and 50m g/ml for 25, and 50 mg/ml for 24, 48, and 72 hours. On the day 24, 48, and 72 hours. Control wells were medium treated of examination 10 ml of BrdU reagent was added to each wells. After 24 hours, viability levels were determined ac- well, and the plates were incubated in a CO2 incubator cording to the manufacturer’s instructions using an Elisa for 4 hours. Next, cells were fixed with 0.5M ethanol/HCL reader (BioTek) at 450 nm wave and subtracted from the 55 for 30 minutes in-20°C.Afterwards, plates were washed reference absorbance at 620 nm. Experiments were re- 3 times to remove residue solution. Then, for the dena- peated 2-5 times independently and conducted in at least turation of DNA partly, plates with cells were incubated 3 replicates. Data are presented as the average prolifer- with nuclease solution for 30 minutes in 37°C wrapped

7 13 EP 2 576 761 B1 14 with parafilm to avoid CO2 leakage from the incubator. Cell morphology characterization DNA synthesis was determined according to the manu- facturer’s instructions using an Elisa reader (BioTek) at [0057] Pancreatic cancer cells (HPAF-II and PL45) 492 nm wave and subtracted from the reference absorb- were counted (25x103cells/ml) and seeded on chamber ance at 690 nm. Experiments were repeated 2-5 times 5 slides (Nunc, Denmark). The next day, growth medium independently and conducted in at least 3 replicates. Da- was replaced with medium containing 10 mg/ml C. stria- ta are presented as average DNA synthesis percentage tus C.L. EAC extract. After 24 h, cell morphology was of the respective control. examined using DAPI and TUNEL (In Situ Cell Death Detection Kit, Roch) staining. Cell growth medium and Cell cycle and apoptosis assays 10 mushroom extracts were removed, cells were washed twice with PBS, fixed for 60 minutes, and permeabilized. [0054] Cell cycle analysis. 106 HPAF-II and PL45 cell Subsequently, cells were incubated with the TUNEL re- was treated with 10 mg/ml c. striatus extract for 24 h at action mixture that contains TdT and fluorescein-dUTP. the end of that time cells was trypsinized and collected During this incubation period, TdT catalyzes the addition with the growth medium, centrifuged, and washed with 15 of fluorescein-dUTP at free 3’-OH groups in single- and PBS and fixed with 70% ethanol for one hour. This was double-stranded DNA. After washing, the label incorpo- then followed by the incubation with 0.1% NP-40 for 5 rated at the damaged sites of the DNA is visualized by minutes in 4°C and then incubated on ice with 100 mg/ml fluorescence microscopy. DAPI stain was added on top RNase for 30 minutes. Finally, 50 mg/ml PI was added of TUNEL treated cells. for 20 minutes. Cell cycle phase distributions were de- 20 termined by Fluorescence Activated Cell Sorter (FACS) Annexin V-FITC staining flow cytometry (Becton Dickinson); 10,000 cells were counted for each control and treatment group. [0058] Pancreatic cancer cells (HPAF-II and PL45) [0055] Apoptosis induction. Apoptosis induction was were treated with different extract concentrations for 4 h, examined biochemically and morphologically. 25 and 5x105 cells/ml were counted and collected for the analysis. Cells were resuspended in 500 ml of 1X binding DNA fragmentation buffer, 5 ml of Annexin V-FITC and 5m l of propidium iodide were added. Cells were incubated for 5 minutes [0056] Genomic DNA was extracted from treated and at room temperature in the dark. Cell apoptosis was untreated control cells of both cell lines. Briefly, at the 30 measured using Annexin V-FITC Apoptosis Detection Kit end of treatment, the cells’ medium was collected into 50 (MBL, USA) and determined using a BD Facscalibor flow ml tubes and cells were trypsinized and added to 50-ml cytometer. tubes following centrifugation at 2000 rpm. Subsequent- ly, cells were washed with PBS twice and suspended in Caspase activation 1 ml lysis buffer that contains: 0.5% SDS, 0.1 M NaCl, 35 50 Mm Tris-buffer, 1 mM EDTA, pH=7.5, and 200 mg/ml [0059] The mammalian caspases play distinct roles in proteinase K. Next, an eppendorf tube with lysis buffer apoptosis and inflammation. In apoptosis, caspases are and cells were incubated at 55°C overnight. On the next responsible for a proteolytic cleavages that lead to cell day, samples were mixed with equal volumes of phe- disassembly (effector caspases), and are involved in up- nol:chloroform:isoamyl alcohol, vortexed for a minute, 40 stream regulatory events (initiator caspases). An active and centrifuged for 15 minutes at 13,000 rpm. The upper caspase consists of two large ∼(20 kD) and two small phase was carefully collected into a new eppendorf tube (∼10 kD) subunits that form two heterodimers which as- and mixed again with phenol:chloroform:isoamyl. This sociate in a tetramer (Nicholson & Thornberry, 1997). step was repeated twice. RNAse (5 ml) was added and [0060] FLICA Apoptosis Detection Kits use a Fluoro- cells were incubated for 60 minutes at 37°C. Next, phe- 45 chrome Inhibitor of Caspases (FLICA). Once inside the nol:chloroform:isoamyl was repeated following centrifu- cell, the FLICA inhibitor binds covalently to the active gation at 13,000 rpm for 15 minutes. DNA precipitation caspase and retained in cells undergoing the was carried out using 0.3 M sodium acetate and ice cold process of apoptosis. HPAF-II and PL45 cells (1x106 absolutealcohol was addedto an equal volume of sample cells/ml) were incubated with different concentrations andincubated over nightat -20°C. The nextday, samples 50 (2.5 or 5 mg/ml) of C. striatus extract for 2 or 4 h. At the were centrifuged for 15 minutes at 4°C and 13,000 rpm; end of treatment, cells were labeled with FLICA FAM- all supernatants were removed and samples were left to LETD-FMK for caspase 8 detaction or FLICA FAM-LE- dry. DNA was re-suspended in dionized H 20 and kept in HD-FMK for caspase 9 detaction using carboxyfluores- 37°C overnight. DNA concentration was determined us- cein FLICA Apoptosis Detection Kit Caspase Assay (Im- ing Nanodrop. Fragments were observed by gel electro- 55 munochemistry Technologies, LLC, USA). The green flu- phoresis (1.5% agarose TBEx0.5) using 60V for 3 hours. orescent signal is a direct measure of the number of ac- Gel was analyzed using ChemiDoc XRS (Bio-Rad). tive caspase enzymes that were present in the cell at the time the reagent was added. Caspase activity was de-

8 15 EP 2 576 761 B1 16 tected using a BD Facscalibor flow cytometer. knots appeared upon the mycelium. Clamp connections, crystals, and anastamoses are present on hyphae. Western blotting [0066] A novel strain was isolated and deposited at Haifa University Culture Collection (HAI) under the name [0061] HPAF-II and PL45 cell lines were induced for 5 Cyathus striatus (HAI-1302). It was also deposited under 12h with medium containing the indicatedconcentrations The Budapest Treaty with the Centralbureau voor Schim- of C. striatus extract. After induction, 2x106 cells were melcultures (CBS) under Accession No. CBS 126585 lysed in 0.2 ml RIPA buffer and 20 ml of protease inhibitors (hereinafter Cyathus striatus CBS 126585). The novel stock (325) per 0.5 ml RIPA buffer was also added, sub- strain was isolated as follows: jected to SDS-PAGE, and blotted with the following an- 10 tibodies: Caspase-9 and caspase-3 (Cell Signaling Tech- Fruiting bodies of Cyathus striatus were collected nology, Beverly, MA) The detection of immobilized pro- from their natural habitat in Israel. The mushroom teins was performed using the EZ-ECL Chemilumines- submerged culture mycelium (SCM) production in- cence Detection Kit for HRP (Biological Industries, Beit- cluded 5 steps of culture growth: Museum culture (I) Haemek, Israel) according to manufacturer’s instruc-15 → Intermediate culture (II) → Pre-inoculums culture tions. The proteins were visualized using the Chemi- (III) →Inoculums culture(IV) →Fermentation culture DocTMXRS Gel Documentation System (BioRad). (V).

Statistics [0067] Morphological characters of Cyathus striatus 20 (HAI-1302): Colony dense, first cream, later becoming [0062] All results displayed as means6SE were ex- pale cream-brownish with small exudate drops on aerial pressed as percentages of control (XTT, Crystal violet, mycelium. Around central zone with well developed, BrdU). ONE WAY ANOVA was used for the evaluation dense, downy aerial mycelium. From central zone to of the differences between treatment groups and control edge of margin with radial branched brown hyphae. Mar- groups. P<0.05 was considered significant, and SPSS 25 gin even, reverse achromatic. Plate covered within one software was used for the calculation of differences. All month. experiments were performed at least three times and with [0068] Hyphal characteristics: Marginal mycelium four replicates. hyphae hyaline to pale brown, brownish pigmented, sim- ple septate, thick to thin -walled, 3.96-5.94 m m wide, Example 1. A novel Cyathus striatus strain 30 branched. Many clamp connections, anastamoses, and crystals are present. [0063] FRUIT BODY 7-15 mm tall, 6-8 mm wide, strongly infundibuliform with narrow, tapering base. Example 2. Effect of selected mushroom extract on PERIDlUM at first entirely covering the gasterocarp then cell viability. apically fragmenting to reveal the epifragm, outer surface 35 rusty brown to dark fuscous brown, shaggy-tomentose Cytotoxicity assays to hairy. Inner surface grayish, vertically ridged or fluted. PERIDIOLES 12-16 in number, 1-2 mm in diam., lenticu- [0069] XTT assay. EAC extracts of the mycelial bio- lar, pale grayish each attached by a fine, threadlike fu- mass of C. striatus and its culture liquid (C.L.) were tested niculus to the inner peridial surface. 40 for their ability to affect cell viability (not shown). The C. 12-21 3 7.0-12 mm, oblong ellipsoid to ellipsoid, hyaline, striatus C.L. extract showed a most potent growth-inhib- smooth, thick-walled. PERIDIOPELLIS of brown, con- itory effect, which inhibited the growth of HPAF-II and stricted hyphae, with clamp connections, and fusoid ter- PL45 with IC50 of 6 and 4 mg/ml, respectively, for 24 h minal elements, 35-75 3 8-15 mm. treatment, 2 mg/ml for both cell lines for 48 h treatment, [0064] HABITAT & GENERAL DISTRIBUTION: Fruit 45 and 8 and 4 mg/ml for 72 h treatment (not shown). bodies are gregarious on fallen branches, twigs and other [0070] Pancreatic cancer cell lines treated with 10 and debris, often in large numbers, nearly always in wood- 5 mg/ml C. striatus C.L. EAC extract for 24 h induced land, rarely in gardens. AFRICA: Cameroon. ASIA: and more than 70% reduction in cell viability of HPAF-II and IsraelJapan, Korea. AUSTRALASIA: . EU- 65% reduction in cell viability of PL45 cells 50 ROPE: Austria, Belgium, Czech Republic, Denmark, Es- (f(9,81)=38.783, p<0.001 and f (9,67)=33.626, p<0.001), re- tonia, France, Germany, Ireland, Italy, Norway, Poland, spectively (Figs. 1A-B). The decline in cell viability was Portugal, Romania, Slovenia, Spain, Sweden, UK. found to be dose-dependent and the decrease in living NORTH AMERICA: Canada, USA. SOUTH AMERICA: cells of the cell line HPAF-II was found significant in all Costa Rica, Ecuador, Venezuela. concentrations leave out treatment concentrations of 1 [0065] VEGETATIVE MYCELIUM IN PURE CUL-55 and 2.5 mg/ml. Interestingly, the reduction in cell viability TURE: The mycelium was at first white, but soon became was maximal at 10 mg/ml and it was not further reduced a dirty brownish color, felty, dense in the center of colony, (at 15, 20, 25, and 50 mg/ml). Regarding cell line PL45, with many strong mycelial strands. In a few cases small the 24 h treatment also induced a decrease in living cells,

9 17 EP 2 576 761 B1 18 and the cell viability was not further reduced above con- the proliferation of surviving cells. To test this possibility, centrations of 5 mg/ml. Treatment for 48 and 72 h pro- we examined whether C. striatus C.L. extract hinders duced similar results: a significant decrease in living cells DNA synthesis. The effect of the extract on both cell lines (f(9,94)=139.055, p<0.001; f(6,63)=182.779, p<0.001) for was tested by the incorporation of BrdU. DNA synthesis 48 and 72 h treatment, respectively, for both the cell line 5 was measured for HPAF-II and PL45 cell lines following

HPAF-II and (f(9,109)=40.985, p<0.001; (6,73) f =27.311, 24, 48, and 72 h treatment with different concentrations p<0.001) for the cell line PL45. The treatment with con- of the C. striatus C.L. extract (1, 2.5, 5, 7.5, 10, 15, and centrations above 5 and 7.5 mg/ml for cell lines HPAF-II 20 mg/ml). and PL45, respectively, induced more than 80% reduc- [0076] DNA synthesis measurement was conducted tion in cell viability that did not change significantly as the 10 to further support the evidence of inhibition of growth in- concentration increased above those recorded at 48 h duced by the C. striatus C.L. extract of HPAF-II and PL45 treatment. However, treatment for 72 h induced smaller pancreatic cell lines. A decrease in DNA synthesis as reduction in cell viability; only 50% decline in cell viability measured by BrdU Labeling Kit for HPAF-II cell line was was found for cell line HPAF-II. For the cell line PL45 the found to be significant for all treatment hours (24, 48 and 15 effect was the same (69- 86% for concentrations of 5, 72 hours; (8,111) f =7.776, p<0.001; (7,111 f )=4.130, 10, 25, and 50 mg/ml). p<0.001; f (7,84)=2.92, p=0.00, respectively) ( Figs. 4A-B). [0071] In all treatment periods (24, 48, and 72 hours) DNA synthesis as measured for HPAF-II cell line was wedid notfind a significant differencebetween theeffects decreased by 35%, 22%, and 16% following 24, 48, and of treatment with 10 mg/ml C. striatus C.L. EAC extract 72hours treatment, respectively, with 10 mg/mlof C. stria- that induced a 70-80% decrease in HPAF-II and PL45 20 tus C.L. extract. There was no significant difference cell lines viability (f(2,35)=1.978, p=0.154; f(2,35)=0.970, among the effects of different treatment hours (p=0.098) p=0.389), respectively. For this reason, the 10 mg/ml C. at the 10 mg/ml concentration of the extract. For the PL45 striatus C.L. extract and 24 h treatment were chosen for cell line, a significant decrease was shown for the same further evaluation (FACS, DNA fragmentation, and ap- treatment concentration (10m g/ml) at 24 h 25 optotic morphology) in spite of the different IC 50 values. (f(8,114)=5.857, p<0.001). The synthesis declined by 20% The effect of the extract in the specific concentration (10 for all treatment concentrations. The treatment at 48 h mg/ml) was also not found to be significantly different did not induce a significant reduction in DNA synthesis from the effect of higher concentrations (15, 20, 25, and of treated cells compared to control cells (10.5%). In con-

50 mg/ml) in both cell lines (HPAF-II;(2,35) f=1.978, trast, 72-h treatment revealed a 40% decrease of DNA 30 p=0.154 and PL45; f(2,35)=0.97, p=0.389). synthesis (f(7,84)=3.874, p=0.001). [0072] Crystal violet assay. The same trend seen in XTT assays was shown with Crystal violet assay. The Apoptosis and cell cycle assays Crystalviolet resultsemphasize the finding that there was no significant difference between the effect of 24, 48, and [0077] Cell cycle analysis. In order to further evaluate 72 treatment hours with 10 mg/ml of C. striatus C.L. EAC 35 the effect of C. striatus C.L. extract on the pancreatic extract for cell line HPAF-II (f(2,33)=3.119, p=0.057). For cancer cell lines, we examined whether C. striatus C.L. the PL45 cell line, we found a slight difference (p=0.047) extract affects cell cycle progression. HPAF-II and PL45 between the effect of 24 h treatment with the extract and cells were treated with 10 mg/ml of C. striatus C.L. extract 72 h treatment (37% and 54% inhibition, respectively). (approximate IC80). As shown in Figure 5, C. striatus Generally, the reduction in viability ranged between40 C.L. extract caused both cell lines to accumulate in 40-60% for all treatment concentrations and all treatment SubG1-phase (47.24% and 26.66% in C. striatus C.L. hours for both cell lines (Figs. 2A-B). extract treated vs. 0.86% and 2.42% in control HPAF-II [0073] LDH leakage. LDH is a cytoplasmic enzyme and PL45 cells, respectively) within 24 h at the expense constantly expressed in most mammalian cells. It is well of cells mainly in G1 phase for HPAF-II cells and G2/M accepted to use the amount of LDH in extracellular space 45 for PL45 cells. This was associated with the induction of to assess plasma membrane integrity (Korzeniewski & apoptosis as represented by the large sub-G1 peak. On Callewaert, 1983). The capability of LDH assay to detect the other hand, induction of apoptosis by C. striatus C.L. cytotoxic effects is limited to the agents causing direct extract was less dramatic in PL45 cells where cellular damage on cellular membranes. accumulation in sub-G1 was slightly lower (26.66%) than [0074] LDH assay was not able to detect any toxic ef- 50 HPAF-II cells (47.24%). fects caused by all concentrations exhibited in Figure 3, [0078] DNA fragmentation. To determine whether the and, therefore, C. striatus C.L. extract was found not to decrease in viable cells occurs after exposure to C. stria- damage the cell membranes of HPAF-II and PL45 cell tus C.L. extract we investigated whether the effect of this lines. extract was mediated through the induction of apoptosis. [0075] DNA synthesis. Notwithstanding, it was appar- 55 Following treatment with 10 mg/ml of C. striatus C.L. ex- ent that the C. striatus C.L. extract impaired the viability tract, DNA was extracted from HPAF-II and PL45 cells of the pancreatic cancer cell lines; the sustained effects and placed onto a 1.5% agarose gel, as described in on culture growth raised suspicion that it might also affect Materials and Methods. The results presented in Figure

10 19 EP 2 576 761 B1 20

6 show a DNA ladder in HPAF-II cells after 24 h treatment optosis, we examined caspase activation by western (a dose causing 75% decrease in cell growth). The typical blotting also. As shown in Fig. 11, caspase-9 activation DNA ladder was observed only for the HPAF-II cell line. occurred in both cell lines. A decrease in pro caspase-9 For the PL45 cell line there was no obvious fragmentation and an increase in caspase-9 cleaved forms was ob- shown by DNA ladder. 5 served after treatment with different concentrations (2.5, [0079] Cell morphology characterization. Apoptotic 5, and 10 mg/ml) for 12 h. Western blotting was also per- cells were also characterized by morphological changes formed for caspase-3 activity (Fig. 12); a decrease in pro such as cell shrinkage and the generation of apoptotic caspase-3 and an increase in caspase-3 cleaved forms bodies. Cells were counted and seeded on chamber was observed after treatment with different concentra- slides (Nunc, Denmark) (25*103cells/ml). On the next 10 tions(2.5, 5, and10 mg/ml) for 12 h. These results indicate day cells were treated with C. striatus C.L. extract for 24 that the caspases play an essential role in C. striatus-in- h at a concentration of 10 mg/ml and stained with TUNEL duced apoptosis in HPAF-II and PL45 pancreatic cancer or DAPI. The results in Figure 7 show treated cells with cells. typical apoptotic morphology, condensation, and frag- mentation of the nucleus in comparison to untreated15 Example 3. In vivo studies cells. [0080] Annexin V-FITC staining. Shortly after initiating [0083] The anti-cancer activity of the C. Striatus C.L apoptosis, most cell types translocate the membrane EAC extract is investigated in vivo using six-week-old phospholipid phosphatidylserine (ps) from the inner face athymic nude male mice (Harlan Laboratories, Indiana- of the plasma membrane to the cell surface. Once on the 20 polis, USA). Animals are housed in air-conditioned quar- cell surface, ps can be easily detected by staining with a ters with a 12 h light/dark cycle. Standard chow and water fluorescent conjugate of annexin V, a protein that has a are available ad labium. strong natural affinity for ps (koopman et al., 1994; martin [0084] The effect of extract on Pancreatic cancer cell et al., 1995). Control untreated cells and cells treated growth in animals. PL45 human pancreatic adenocarci- with 2.5, 5, and 10 mg/ml of C. striatus extract for 4 h was 25 noma cells are suspended at a concentration of 1x107 stained with Annexin V-FITC and propidium iodide and cells/ml medium. 0.1ml aliquots (1x10 6 cells) are injected analyzed for Annexin V-FITC binding by flow cytometry. subcutaneously into the flank of the mice, using a 27- [0081] Flow cytometry-based annexin V apoptosis as- gauge needle. One week after tumor cell transplantation, say revealed a significant increase in apoptotic cells due the mice are treated 3 times a week by i.p. (intraperito- to treatment with C. stiatus C.L extract. Apoptosis induc- 30 neal) injection, for three weeks (9 doses), with either sa- tion in treated HPAF-II and PL45 cells was found to be line or C. Striatus C.L EAC extract. Tumor size is meas- dose dependent and a dose of 10m g/ml induced a re- ured biweekly with a digital caliber and the volumes are markable effect, 85% of both cell lines displayed a con- calculated using the formula length x width 2 x 0.52 (Sau- siderable increase compared with the apoptotic ratio of ter et al., 2000). At the end of the treatment period, the control untreated cells (Figs. 8A-B). This finding further 35 mice are sacrificed; tumors are collected, weighed, verifies the results of cell cycle analysis, TUNEL and measured, and tested for histological studies. Blood sam- DAPI staining which also showed a prominent apoptosis ples are collected from the mice for analysis of liver and induction by the C. striatus extract. kidney function also. [0082] Caspase activation. Execution of apoptosis re- [0085] Histological studies. Tumors from control and lies on a group of cysteine proteases, the caspases40 treated groups are kept in formalin, and paraffin blocks (Degterev et al., 2003). To determine whether C. striatus are prepared. Four-micron sections are cut and fixed onto extract-induced apoptosis involves the activation of cas- slides for histological staining. pase cascade, we examined the activations of caspase- [0086] Ki-67 staining. Examination of cell proliferation 8 (flow cytometry), caspase-9 (flow cytometry and west- in the tumor is conducted using an immunohistochemical ern blotting), and caspase -3 (western blotting). It was 45 reaction with anti-mouse Ki-67 antigen antibody, accord- found that the activation of caspase-8, and -9 was ob- ing to the manufacturer’s instructions. Ki-67 is a large served at 2 h after C. stiatus extract treatment at a con- nuclear protein, preferentially expressed during all active centration of 2.5 mg/ml. The cell line PL45 exhibited 40% phases of the cell cycle (G1, S, G2, and M), but absent caspase-8 activation in contrast to 11% seen in control from resting cells (Go). The proliferation index of the cells untreated cells (Fig. 9B) and 42% caspase-9 activation 50 is determined in the central and peripheral areas of the in contrast to 24% seen in control untreated cells (Fig. tumors. The index is calculated as the ratio of Ki-67-pos- 10B). Activation of caspases in HPAF-II treated cells was itive tumor cells to all counted tumor cells x 100. The also observed; 46% caspase-8 activation in contrast to results are presented as the mean 6 SE, calculated ac- 26% seen in control untreated cells (Fig. 9A), and 57% cording to the cell number in three different fields at x caspase-9 activation in contrast to 22% seen in control 55 400 magnification. untreated cells were evident after 4 h treatment with 5 [0087] DAPI and TUNEL staining. DAPI is a fluores- mg/ml C. striatus extract (Fig. 10A). To further confirm cent compound that specifically binds to the DNA and the involvement of caspases in C. striatus-induced ap- creates a stable complex providing a 20-fold higher flu-

11 21 EP 2 576 761 B1 22 orescence than DAPI alone, and allows visualization of and breast cancer cells in vitro. Oncology reports, DNA morphology. DAPI staining of the slides is conduct- 12:659-662 ed as described previously (Almog et al., 2002). Tunel Maitra, A. & Hruban, R.H. (2008) Pancreatic Cancer. (in situ cell death detection kit, Roch) is used to measure Annu Rev Pathol, 3:157-188. and quantitate cell death (apoptosis) by labeling and de- 5 Martin SJ, Reutelingsperger CP, McGahon AJ, Rad- tection of DNA strand breaks in individual cells. During er JA, van Schie RC, LaFace DM, Green DR. (1995) apoptosis, DNAse activity introduces strand breaks Early redistribution of plasma membrane phosphati- ("nicks") into the DNA. The assays use an optimized ter- dylserine isa generalfeature of apoptosis regardless minal transferase (TDT) to label free 3’OH ends in ge- of the initiating stimulus: inhibition by overexpression nomic DNA with TMR-dUTP. Slides are photographed 10 of Bcl-2 and Abl. J Exp Med. 1995 Nov usinga fluorescencemicroscope at x 200 magnifications. 1;182(5):1545-56. Mizuno, T. (1999) The extraction and development REFERENCES of antitumoractive polysaccharides from medicinal mushrooms in Japan. Int J Med Mushr, 1:9-29 [0088] 15 Moran, J.H. & Schnellmann, R.G. 1996. A rapid beta- NADH-linked fluorescence assay for lactate dehy- Debatin, K.M. & Krammer, P.H. (2004) Death recep- drogenase in cellular death. J Pharmacol Toxicol tors in chemotherapy and cancer. Oncogene, Methods. 36:41-44. 23:2950-2966 Muller, C.I., Kumagai, T., O’kelly, J., Seeram, N.P., Degterev, A., Boyce, M. & Yuan, J. (2003) A decade 20 Heber, D., & Koeffler, H.P. (2006) Ganoderma luci- of caspases. Oncogene, 22:8543-8567. dum causes apoptosis in leukemia, lymphoma and Fas,S.C., Fritzsching, B., Suri-Payer, E.& Krammer, multiple myeloma cells. Leuk Res, 30:841-848 P.H.(2006). Death receptor signalingand itsfunction Nicholson, D.W. & Thornberry, N.A. 1997. Caspas- in the immune system. Curr Dir Autoimmun, 9:1-17 es: killer proteases. Trends Biochem. Sci. Goeptar, A.R., Groot E.J., Scheerens H., Comman- 25 22:299-306 deur J.N., & Vermeulen N.P. (1994) Cytotoxicity of Okada, H. & Mak, T.W. (2004) Pathways of apoptotic mitomycin C and adriamycin in freshly isolated rat and non-apoptotic death in tumour cells. Nat Rev hepatocytes: the role of cytochrome P450. Cancer Cancer, 4:592-603 Res. 54:2411-2418 Petrova R.D., Mahajna, J., Reznick, A Z., Wasser, Hager, J.H.,& Hanahan,D. (1999) Tumorcells utilize 30 S.P., Denchev, C.M. & Nevo E. (2007) Fungal sub- multiple pathways to down-modulate apoptosis. Les- stances as modulators of NF-jB activation pathway. sons from a mouse model of islet cell carcinogene- Mol Biol Rep, 34:145-154 sis. Ann N Y Acad Sci, 887:150-163 Schneider, G., Saur, D., & Schmid, R.M. (2007) Pan- Hezel, A.F., Kimmelman, A.C., Stanger, B.Z., Bar- creatic cancer - Molecular alterations. The Chinese- deesy, N. & Depinho, R.A. (2006) Genetics and bi- 35 German Journal of Clinical Oncology, 6:102-106 ology of pancreatic ductal adenocarcinoma. Genes Schulze-Bergkamen, H. & Krammer, P.H. (2004) Dev, 20:1218-1249. Apoptosis in cancer-implications for therapy. Semin Ikekawa, T. (2001) Beneficial effects of edible and Oncol, 31:90-119 medicinal mushrooms in health care. Int J Med Smith, J.E., Rowan, N.J., & Sullivan, R. (2002) Me- Mushr, 3:291-298 40 dicinal mushrooms: their therapeutic properties and Kang, H.S., Jun, E.M., Park, S.H., Heo, S.J., Lee, current medical usage with special emphasis on can- T.S., Yoo, I.D. & Kim, J.P. (2007) Cyathusals A, B, cer treatments. Cancer Research UK. University of and C, from the fermented mushroom Strathclyde, Glasgow Cyathus stercoreus. J Nat Prod 70:1043-1045 Wajant, H.(2006). CD95L/FasL and TRAILin tumour Koopman, G., Reutelingsperger, C.P.M., Kuijten, 45 surveillance and cancer therapy. Cancer Treat Res, G.A.M., Keehnen, R.M.J., Pals, S.T. &. van Oers 130:141-165 M.H.J. (1994) Annexin V for Flow Cytometric Detec- Wang, M.W., Hao, X. & Chen, K. (2007) Biological tion of Phosphatidylserine Expression on B Cells Un- screening of natural products and drug innovation in dergoing Apoptosis. Blood 84:1415-1420. China. Phil. Trans. R. Soc. B, 362:1093-1105 Korzeniewski, C. & Callewaert, D.M. (1983) An en- 50 Wasser, S.P., and Weis, A.L. (1999a) Medicinal zyme-release assay for natural cytotoxicity. J. Im- properties of substances occurring in higher Basid- munol. Methods 64: 313 iomycetes mushrooms: current perspectives (Re- Liu, Y.J. & Zhang, K.Q (2003) Antimicrobial activities view). Int J Med Mushr, 1:31-62 of selected Cyathus species, Mycopath, 157: Wasser, S.P., and Weis, A.L. (1999b) Therapeutic 185-189. 55 effects of substances occurring in higher Basidio- Lu, Q.Y., Sartippour, M.R., Brooks, M.N., Zhang, Q., mycetes mushrooms: a modem perspective. Crit Hardy, Go, V.L., Li, F.P. & Heber, D. (2004) Gano- Rev Immunol, 19:65-96 derma Lucidum extract inhibits endothelial Wu, J.Y., Zhang, Q.X., and Leung, P.H. (2006) In-

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hibitory effects of ethyl acetate extract of Cordyceps tus CBS 126585. sinensis mycelium on various cancer cells in culture and B16 melanoma in C57BL/6 mice. Phytomedi- 11. The extract for use in treatment of pancreatic cancer cine, 14:43-49 according to claim 8, obtained from the culture liquid Yassin M, Mahajna JA and Wasser SP (2003) Sub- 5 of a submerged mycelium culture of the mushroom. merged cultured mycelium extracts of higher Basid- iomycetes mushrooms selectively inhibit prolifera- 12. The extract for use in treatment of pancreatic cancer tion and induce differentiation of K562 human chron- according to claim 8, obtained from an organic ex- ic myelogenous leukemia cells. Int J Med Mushr 5: traction solvent, preferably ethyl acetate. 261-276. 10 Zaidman, B., Yassin, M., Mahajna, J., and Wasser, 13. The extract for use in treatment of pancreatic cancer S.P. (2005) Medicinal mushroom modulators of mo- according to claim 8, wherein said pancreatic cancer lecular targets as cancer therapeutics. Appl Micro- is selected from ductal adenocarcinomas, acinar cell biol Biotechnol, 67(4):453-468 carcinomas, adenosquamous carcinomas, colloid 15 carcinomas, giant cell tumors, hepatoid carcinomas, intraductal papillary-mucinous neoplasms, muci- Claims nous cystic neoplasms, pancreatoblastomas, se- rous cystadenomas, signet ring cell carcinomas, sol- 1. A new and distinct variety of higher Basidiomycetes id and pseudopapillary tumors, undifferentiated car- mushroom Cyathus striatus HAI-1302, deposited 20 cinomas, undifferentiated carcinomas with osteo- under The Budapest Treaty with the Centralbureau clast-like giant cells and pancreatic endocrine tu- voor Schimmelcultures (CBS) under Accession No. mors. CBS 126585 (hereinafterCyathus striatus CBS 126585). 25 Patentansprüche 2. An extract obtained fromCyathus striatus CBS 126585 according to claim 1. 1. Neue und eigenständige Varietät des höheren Ba- sidiomycetes-Pilzes Cyathus striatus HAI-1302, un- 3. The extract according to claim 2, obtained from the ter dem Budapester Vertrag mit dem Centralbureau culture liquid of a submerged mycelium culture of 30 voor Schimmelcultures (CBS) unter der Hinterle- the mushroom. gungs-Nr. CBS 126585 hinterlegt (im Folgenden Cy- athus striatus CBS 126585 genannt). 4. The extract according to claim 3, obtained from an organic extraction solvent, preferably ethyl acetate. 2. Extrakt, das aus Cyathus striatus CBS 126585 nach 35 Anspruch 1 erhalten ist. 5. The extract according to claim 4, rich in low-molec- ular weight compounds such as alkaloids, terpe- 3. Extrakt nach Anspruch 2, das aus der Kulturflüssig- noids, glycosides, flavonoids, terpenes and phenols. keit einer Myzelsubmerskultur des Pilzes erhalten ist. 6. The extract according to any one of claims 2 to 5, 40 which (a) inhibits growth of cancer cells; (b) arrests 4. Extrakt nach Anspruch 3, das aus einem organi- cancer cell cycle; (c) reduces DNA synthesis in can- schen Extraktionslösungsmittel, vorzugsweise cer cells; and/or (d) induces apoptosis in cancer Ethylacetat, erhalten ist. cells. 45 5. Extrakt nach Anspruch 4, das reich an Verbindungen 7. The extract according to claim 6, for use in treating mit niedrigem Molekulargewicht wie z. B. Alkaloiden, cancer, preferably a cancer selected from pancreatic Terpenoiden, Glykosiden, Flavonoiden, Terpenen cancer, breast cancer, chronic myelogenous leuke- und Phenolen ist. mia (CML) or prostate cancer. 50 6. Extrakt nach einem der Ansprüche 2 bis 5, das (a) 8. The extract for use according to claim 7, wherein das Wachstum von Krebszellen hemmt; (b) einen said cancer is pancreatic cancer. Krebszellzyklus stoppt; (c) eine DNA-Synthese in Krebszellen verringert; und/oder (d) eine Apoptose 9. A pharmaceutical composition comprising a phar- in Krebszellen induziert. maceutically acceptable carrier and the extract ac- 55 cording to any one of claims 2 to 8. 7. Extrakt nach Anspruch 6 zur Verwendung bei der Behandlung von Krebs, vorzugsweise eines Kreb- 10. A pure submerged mycelial culture of Cyathus stria- ses, der aus Bauchspeicheldrüsenkrebs, Brust-

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krebs, chronischmyeloischer Leukämie (CML) oder 5. Extrait selon la revendication 4, riche en composés Prostatakrebs ausgewählt ist. de faible masse moléculaire tels que des alcaloïdes, des terpénoïdes, des glycosides, des flavonoïdes, 8. Extrakt zur Verwendung nach Anspruch 7, wobei der des terpènes et des phénols. Krebs Bauchspeicheldrüsenkrebs ist. 5 6. Extrait selon l’une quelconque des revendications 2 9. Pharmazeutische Zusammensetzung, die einen à 5, qui (a) inhibe la croissance de cellules pharmazeutisch annehmbaren Träger und das Ex- cancéreuses ; (b) arrête le cycle de cellules trakt nach einem der Ansprüche 2 bis 8 umfasst. cancéreuses ; (c) réduit la synthèse d’ADN dans des 10 cellules cancéreuses ; et/ou (d) induit une apoptose 10. Reine Myzellsubmerskultur von Cyathus striatus dans des cellules cancéreuses. CBS 126585. 7. Extrait selon la revendication 6, pour utilisation dans 11. Extrakt zur Verwendung bei der Behandlung von le traitement d’un cancer, de préférence d’un cancer Bauchspeicheldrüsenkrebs nach Anspruch 8, das 15 choisi parmi un cancer pancréatique, un cancer aus der Kulturflüssigkeit einer Myzelsubmerskultur mammaire, une leucémie myélogène chronique des Pilzes erhalten ist. (CML) ou un cancer de la prostate.

12. Extrakt zur Verwendung bei der Behandlung von 8. Extrait pour utilisation selon la revendication 7, dans Bauchspeicheldrüsenkrebs nach Anspruch 8, das 20 lequel ledit cancer est un cancer pancréatique. aus einem organischen Extraktionslösungsmittel, vorzugsweise Ethylacetat, erhalten ist. 9. Composition pharmaceutique comprenant un véhi- cule pharmaceutiquement acceptable et l’extrait se- 13. Extrakt zur Verwendung bei der Behandlung von lon l’une quelconque des revendications 2 à 8. Bauchspeicheldrüsenkrebs nach Anspruch 8, wobei 25 der Bauchspeicheldrüsenkrebs aus duktalen Ade- 10. Culture mycélienne submergée pure deCyathus nokarzinomen, Azinuszellkarzinomen, adenosqua- striatus CBS 126585. mösen Karzinomen, kolloiden Karzinomen, Riesen- zelltumoren, hepatoiden Karzinomen, intraduktalen 11. Extrait pour utilisation dans le traitement d’un cancer papillärmuzinösen Neoplasmen, muzinösen zysti- 30 pancréatique selon la revendication 8, obtenu à par- schen Neoplasmen, Pankreatoblastomen, serösen tir du liquide de culture d’une culture de mycélium Zystadenomen, Siegelringzellkarzinomen, soliden submergée du champignon. und pseudopapillären Tumoren, undifferenzierten Karzinomen, undifferenzierten Karzinomen mit os- 12. Extrait pour utilisation dans le traitement d’un cancer teoklastenähnlichen Riesenzellen und endokrinen 35 pancréatique selon la revendication 8, obtenu à par- Bauchspeicheldrüsentumoren ausgewählt ist. tird’un solvantd’extraction organique,de préférence l’acétate d’éthyle.

Revendications 13. Extrait pour utilisation dans le traitement d’un cancer 40 pancréatique selon la revendication 8, dans lequel 1. Variété nouvelle et distincte du champignon supé- ledit cancer pancréatique est choisi parmi les adé- rieurBasidiomycetes Cyathusstriatus HAI-1302, dé- nocarcinomes canalaires, les carcinomes à cellules posé selon les termes du Traité de Budapest au Cen- acinaires, les carcinomes adéno-squameux, les car- tralbureau voor Schimmelcultures (CBS) sous le nu- cinomes colloïdes, les tumeurs à cellules géantes, méro d’accès CBS 126585 (ci-après Cyathus stria- 45 les carcinomes hépatoïdes, les néoplasmes papil- tus CBS 126585). laires-mucoïdes intra-canalaires, les néoplasmes cystiques mucoïdes, les pancréato-blastomes, les 2. Extrait obtenu à partir deCyathus striatus CBS cystadénomes séreux, les carcinomes à cellules en 126585 selon la revendication 1. bague à chaton, les tumeurs pseudo-papillaires et 50 solides, les carcinomes indifférenciés, les carcino- 3. Extrait selon la revendication 2, obtenu à partir du mes indifférenciés avec cellules géantes analogues liquide de culture d’une culture de mycélium submer- à des ostéoclastes, et les tumeurs endocriniennes gée du champignon. pancréatiques.

4. Extrait selon la revendication 3, obtenu à partir d’un 55 solvant d’extraction organique, de préférence l’acé- tate d’éthyle.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description

• YASSIN et al. Int J Med Mushr, 2003, vol. 5, 261-276 •LU,Q.Y.; SARTIPPOUR, M.R. ; BROOKS, M.N. ; [0047] ZHANG, Q. ; HARDY, GO, V.L. ; LI, F.P. ; HEBER, • KHAN et al. Pakistan J Biol Sci, 2001, vol. 4 (11), D. Ganoderma Lucidum spore extract inhibits en- 1374-1376 [0047] dothelial and breast cancer cells in vitro. Oncology • DEBATIN, K.M. ; KRAMMER, P.H. Death receptors reports, 2004, vol. 12, 659-662 [0088] in chemotherapy and cancer. Oncogene, 2004, vol. • MAITRA, A. ; HRUBAN, R.H. Pancreatic Cancer. 23, 2950-2966 [0088] Annu Rev Pathol, 2008, vol. 3, 157-188 [0088] • DEGTEREV, A. ; BOYCE, M. ; YUAN, J. A decade • MARTIN SJ ; REUTELINGSPERGER CP ; MCGA- of caspases. Oncogene, 2003, vol. 22, 8543-8567 HON AJ ; RADER JA ; VAN SCHIE RC ; LAFACE [0088] DM ; GREEN DR. Early redistribution of plasma • FAS, S.C. ; FRITZSCHING, B. ; SURI-PAYER, E. ; membrane phosphatidylserine is a general feature of KRAMMER, P.H. Death receptor signaling and its apoptosis regardless of the initiating stimulus: inhibi- function in the immune system. Curr Dir Autoimmun, tion by overexpression of Bcl-2 and Abl. J Exp Med., 2006, vol. 9, 1-17 [0088] 01 November 1995, vol. 182 (5), 1545-56 [0088] • GOEPTAR, A.R. ; GROOT E.J. ; SCHEERENS H. ; • MIZUNO, T. The extraction and development of an- COMMANDEUR J.N. ; VERMEULEN N.P. Cytotox- titumoractive polysaccharides from medicinal mush- icity of mitomycin C and adriamycin in freshly isolated rooms in Japan. Int J Med Mushr, 1999, vol. 1, 9-29 rat hepatocytes: the role of cytochrome P450. Cancer [0088] Res., 1994, vol. 54, 2411-2418 [0088] • MORAN, J.H. ; SCHNELLMANN, R.G. A rapid be- • HAGER, J.H. ; HANAHAN, D. Tumor cells utilize ta-NADH-linked fluorescence assay for lactate dehy- multiple pathways to down-modulate apoptosis. Les- drogenase in cellular death.J Pharmacol Toxicol sons from a mouse model of islet cell carcinogenesis. Methods, 1996, vol. 36, 41-44 [0088] Ann N Y Acad Sci, 1999, vol. 887, 150-163 [0088] • MULLER, C.I. ; KUMAGAI, T. ; O’KELLY, J. ; SE- • HEZEL, A.F. ; KIMMELMAN, A.C. ; STANGER, ERAM, N.P. ; HEBER, D. ; KOEFFLER, H.P. Gan- B.Z. ; BARDEESY, N. ; DEPINHO, R.A. Genetics oderma lucidum causes apoptosis in leukemia, lym- and biology of pancreatic ductal adenocarcinoma. phoma and multiple myeloma cells. Leuk Res, 2006, Genes Dev, 2006, vol. 20, 1218-1249 [0088] vol. 30, 841-848 [0088] • IKEKAWA, T. Beneficial effects of edible and medic- • NICHOLSON, D.W. ; THORNBERRY, N.A. Caspas- inal mushrooms in health care.Int J Med Mushr, es: killer proteases. Trends Biochem. Sci., 1997, vol. 2001, vol. 3, 291-298 [0088] 22, 299-306 [0088] • KANG, H.S. ; JUN, E.M. ; PARK, S.H. ; HEO, S.J. ; • OKADA, H. ; MAK, T.W. Pathways of apoptotic and LEE, T.S. ; YOO, I.D. ; KIM, J.P. Cyathusals A, B, non-apoptotic death in tumour cells. Nat Rev Cancer, and C, antioxidants from the fermented mushroom 2004, vol. 4, 592-603 [0088] Cyathus stercoreus. J Nat Prod, 2007, vol. 70, • PETROVA R.D. ; MAHAJNA, J. ; REZNICK, A Z. ; 1043-1045 [0088] WASSER, S.P. ; DENCHEV, C.M. ; NEVO E. Fungal • KOOPMAN, G. ; REUTELINGSPERGER, C.P.M. ; substances as modulators of NF-jB activation path- KUIJTEN, G.A.M. ; KEEHNEN, R.M.J. ; PALS, way. Mol Biol Rep, 2007, vol. 34, 145-154 [0088] S.T. ; VAN OERS M.H.J. Annexin V for Flow Cyto- • SCHNEIDER, G. ; SAUR, D. ; SCHMID, R.M. Pan- metric Detection of Phosphatidylserine Expression creatic cancer - Molecular alterations.The Chi- on B Cells Undergoing Apoptosis. Blood, 1994, vol. nese-GermanJournal of Clinical Oncology, 2007, vol. 84, 1415-1420 [0088] 6, 102-106 [0088] • KORZENIEWSKI, C. ; CALLEWAERT, D.M. An en- • SCHULZE-BERGKAMEN, H. ; KRAMMER, P.H. zyme-release assay for natural cytotoxicity. J. Immu- Apoptosis in cancer-implications for therapy. Semin nol. Methods, 1983, vol. 64, 313 [0088] Oncol, 2004, vol. 31, 90-119 [0088] •LIU,Y.J.; ZHANG, K.Q. Antimicrobial activities of •SMITH,J.E.; ROWAN, N.J. ; SULLIVAN, R. Medic- selected Cyathus species. Mycopath, 2003, vol. 157, inal mushrooms: their therapeutic properties and cur- 185-189 [0088] rent medical usage with special emphasis on cancer treatments. Cancer Research UK. University of Strathclyde, Glasgow, 2002 [0088]

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• WAJANT, H. CD95L/FasL and TRAIL in tumour sur- •WU,J.Y.; ZHANG, Q.X. ; LEUNG, P.H. Inhibitory veillance and cancer therapy.Cancer Treat Res, effects of ethyl acetate extract of Cordyceps sinensis 2006, vol. 130, 141-165 [0088] mycelium on various cancer cells in culture and B16 • WANG, M.W. ; HAO, X. ; CHEN, K. Biological melanoma in C57BL/6 mice. Phytomedicine, 2006, screening of natural products and drug innovation in vol. 14, 43-49 [0088] China. Phil. Trans. R. Soc. B, 2007, vol. 362, • YASSIN M ; MAHAJNA JA ; WASSER SP. Sub- 1093-1105 [0088] merged cultured mycelium extracts of higher Basid- • WASSER, S.P. ; WEIS, A.L. Medicinal properties of iomycetes mushrooms selectively inhibit proliferation substances occurring in higher Basidiomycetes and induce differentiation of K562 human chronic my- mushrooms:current perspectives (Review). IntJ Med elogenous leukemia cells. Int J Med Mushr, 2003, Mushr, 1999, vol. 1, 31-62 [0088] vol. 5, 261-276 [0088] • WASSER, S.P. ; WEIS, A.L. Therapeutic effects of • ZAIDMAN, B. ; YASSIN, M. ; MAHAJNA, J. ; substances occurring in higher Basidiomycetes WASSER, S.P. Medicinal mushroom modulators of mushrooms: a modem perspective. Crit Rev Immu- molecular targets as cancer therapeutics. Appl Micro- nol, 1999, vol. 19, 65-96 [0088] biol Biotechnol, 2005, vol. 67 (4), 453-468 [0088]

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