U.S. patent application number 11/841379 was filed with the patent office on 2008-01-24 for mushroom extracts having anticancer activity.
This patent application is currently assigned to Gavish-Galilee Bio Applications Ltd.. Invention is credited to Jamal A. MAHAJNA, Solomon P. WASSER, Majed YASSIN.
Application Number | 20080019996 11/841379 |
Document ID | / |
Family ID | 35943482 |
Filed Date | 2008-01-24 |
United States Patent
Application |
20080019996 |
Kind Code |
A1 |
MAHAJNA; Jamal A. ; et
al. |
January 24, 2008 |
MUSHROOM EXTRACTS HAVING ANTICANCER ACTIVITY
Abstract
The invention relates to a method for treatment of chronic
myelogenous leukemia (CML), Ph+ acute lymphoblastic leukemia (ALL),
prostate cancer and .beta.-globin disorders such as sickle cell
anemia and .beta.-thalassemia, comprising administration of a
mycelium extract from at least one higher Basidiomycetes medicinal
mushroom selected from the group consisting of Ganoderma adspersum,
Hypsizygus ulmarium, Kuehneromyces mutabilis, Omphalotus olearius,
Panus conchatus, Piptoporus betulinus, Pleurotus eryngii, and
Trametes zonata.
Inventors: |
MAHAJNA; Jamal A.;
(Nazareth-lllit, IL) ; YASSIN; Majed; (Tamara,
IL) ; WASSER; Solomon P.; (Nesher, IL) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
Gavish-Galilee Bio Applications
Ltd.
Kiryat Shmona
IL
Carmel-Haifa University Economic Corp. Ltd.
Mount Carmel
IL
|
Family ID: |
35943482 |
Appl. No.: |
11/841379 |
Filed: |
August 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11257128 |
Oct 25, 2005 |
7258862 |
|
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11841379 |
Aug 20, 2007 |
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10925224 |
Aug 25, 2004 |
|
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11257128 |
Oct 25, 2005 |
|
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Current U.S.
Class: |
424/195.15 |
Current CPC
Class: |
A61K 36/07 20130101;
Y10S 514/908 20130101; A61P 37/00 20180101; A61K 36/074
20130101 |
Class at
Publication: |
424/195.15 |
International
Class: |
A61K 36/07 20060101
A61K036/07; A61P 37/00 20060101 A61P037/00 |
Claims
1. A method for treatment of prostate cancer, comprising
administering to a patient in need thereof a therapeutically
effective amount of a mycelium extract from the medicinal mushroom
Trametes zonata, wherein said mycelium extract is obtained by
extraction of a dry mycelium of said mushroom with an extraction
solvent comprising one or more organic solvents selected from the
group consisting of methanol, ethanol, acetonitrile, ethyl acetate,
chloroform, hexane, cyclohexane, isooctane and dichloromethane.
2. A method for treatment of a disease selected from the group
consisting of a Philadelphia Chromosome-positive Leukemia, prostate
cancer, and a .beta.-globin disorder, comprising administering to a
patient in need thereof a therapeutically effective amount of a
mycelium extract from at least one higher Basidiomycetes medicinal
mushroom selected from the group consisting of Ganoderma adspersum,
Hypsizygus ulmarium, Kuehneromyces mutabilis, Omphalotus olearius,
Panus conchatus, Piptoporus betulinus, Pleurotus eryngii, and
Trametes zonata, wherein said mycelium extract is obtained by
extraction of a dry mycelium of said mushroom with an extraction
solvent comprising one or more organic solvents selected from the
group consisting of methanol, ethanol, acetonitrile, ethyl acetate,
chloroform, hexane, cyclohexane, isooctane and dichloromethane.
3. The method according to claim 2, wherein said extraction solvent
is a non-aqueous organic solvent.
4. The method according to claim 3, wherein said organic solvent is
selected from the group consisting of methanol, ethanol,
dichloromethane and ethyl acetate.
5. The method according to claim 2, wherein said extraction solvent
is an aqueous organic solvent.
6. The method according to claim 5, wherein said extraction solvent
is 70% ethanol or a mixture of ethyl acetate and methanol.
7. The method according to claim 2, wherein said Philadelphia
Chromosome-positive Leukemia is chronic myelogenous leukemia (CML)
or Ph+ acute lymphoblastic leukemia (ALL).
8. The method according to claim 2, wherein said mycelium extract
is further concentrated and purified to obtain a concentrated and
purified mycelium extract.
9. The method according to claim 8, wherein the concentrated
mycelium extract is purified by column chromatography, fractional
distillation, preparative thin layer chromatography (TLC),
preparative high performance liquid chromatography (HPLC), or
centrifugal partition chromatography (CPC).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional application of U.S.
application Ser. No. 11/257,128, filed Oct. 25, 2005, now U.S. Pat.
No. 7,258,862, which is a divisional application of U.S.
application Ser. No. 10/925,244, filed August 25, 2004, now
abandoned, the entire contents of each and both these applications
being hereby incorporated by reference herein in their entirety as
if fully disclosed herein.
FIELD OF THE INVENTION
[0002] The present invention relates to extracts of medicinal
mushrooms, to compositions comprising them and to methods for
treatment of chronic myelogenous leukemia, Ph+ acute lymphoblastic
leukemia, prostate cancer and .beta.-globin disorders by
administration of said extract or composition.
ABBREVIATIONS
[0003] ABL: Abelson leukemia virus; ALL: acute lymphoblastic
leukemia; AR: androgen receptor; BCR: breakpoint cluster region;
CML: chronic myelogenous leukemia; ALL: acute lymphoblastic
leukemia; CV: coefficient of variance; DMSO: dimethyl sulfoxide;
ELISA: enzyme-linked immunoabsorbent assay; ERK: extracellular
signal-regulated kinase; HBM: higher Basidiomycetes mushrooms; HDP:
host defense potentiators; HMW: high molecular weight; HSP: heat
shock protein; IC50: inhibitory concentration; JNK: c-Jun
N-terminal kinase; LMW: low molecular weight; MAPK:
mitogen-activated protein kinase; PARP: poly(ADP-ribose)
polymerase; PBS: phosphate-buffered saline; Ph: Philadelphia
cromosome; Ph+: Ph positive; PSA: prostate-specific antigen.
BACKGROUND OF THE INVENTION
[0004] Higher Basidiomycetes mushrooms (HBM) represent a major and
still largely untapped source of potent new pharmaceutical
products. Of approximately 15,000 known species, 2,000 are safe for
human consumption, and about 650 of them possess medicinal
properties (Wasser et al., 2000; Hawksworth, 2001; Kirk et al.,
2001; Wasser, 2002). Of about 650 mushroom species with known
medicinal properties, only about 20 species are in use at the
present (Chang, 2001). Most traditional knowledge about medicinal
properties of HBM comes from the Far East (China, Japan, Korea,
Russian Siberia). Many pharmaceutical substances with potent and
unique properties have recently been extracted from mushrooms and
have made their way all around the world. Unique anticancer
medicines were prepared from these extracts such as polysaccharides
lentinan, krestin, and schizophyllan (Mizuno, 1999).
[0005] Present studies suggest that HBM are probiotic, i.e., they
help the body to strengthen itself and fight off illness by
maintaining physiological homeostasis, restoring the body's balance
and natural resistance to disease. The compounds they contain have
been classified as Host Defense Potentiators (HDP), which can have
immune system enhancement properties. That is one of the reasons
why they are currently used as adjuncts to cancer treatments in
many countries (Tomatis et al., 2001). In Japan, Russia, China, and
the USA, several polysaccharide anticancer and immunomodulating
agents have been developed from the fruiting body, mycelia, and
culture medium of various medicinal mushrooms (Lentinus edodes,
Ganoderma lucidum, Schizophyllum commune, Trametes versicolor,
Inonotus obliquus, Hypsizygus marmoreus, and Flammulina velutipes)
(Ikekawa, 2001).
[0006] Mushroom HDP include hemicellulose (AHCC), polysaccharides,
polysaccharide-peptides, nucleosides, triterpenoids, complex
starches, and other metabolites. It is believed that combinations
of these products target the human immune system, and also aid
neuron transmission, metabolism, hormonal balance, and the
transport of nutrients and oxygen. Through a host-mediated (T cell)
immune mechanism, they help the body regulate the development of
lymphoid stem cells and other important defense responses (Mizuno,
1999).
[0007] Chronic myelogenous leukemia (CML) is a member of a group of
diseases classified as myeloproliferative disorders, which account
for 20% of all leukemias. CML is a clonal disorder that is usually
easily recognized because the leukemia cells of more than 95% of
patients suffering from CML have a distinctive cytogenetic
abnormality, the Philadelphia chromosome (Ph), that results from a
reciprocal translocation between the long arms of chromosomes 9 and
22. This translocation results in the transfer of the Abelson (Abl)
oncogene on chromosome 9 to an area of chromosome 22 that includes
the breakpoint cluster region (Bcr) gene. This results in the
presentation of a leukemia-specific fusion gene (Bcr-Abl) which
gives rise to an abnormal tyrosine kinase protein, p210 (Bcr-Abl),
with increased activity (Clarkson et al., 1997; Cortez et al.,
1997). In addition, the Ph chromosome is also found in a sizeable
portion of acute lymphoblastic leukemia (ALL) patients (25-30%) and
in a small number of acute myeloid leukemia (AML) cases (Drexler et
al., 1999). Bcr-Abl expressing leukemic blasts are highly resistant
to different classes of chemotherapeutic drugs. K562 cells, derived
from patients with CML in blast crisis (Lozzio and Lozzio, 1975),
which express p210.sup.Bcr-Abl, have been shown to be highly
resistant to apoptosis induced by many chemotherapeutic agents
(McGahon et al., 1994). Overexpression of Bcr-Abl has been
implicated in inhibiting apoptosis induced by cytokine deprivation,
DNA damage, and a variety of chemotherapeutic drugs (Cortez et al.,
1997). Thus, the Bcr-Abl fusion protein has been suggested to
function as an antiapoptotic factor, and overexpression of the
Bcr-Abl protein in K562 cells may, in part, account for the
resistance of these cells to apoptosis, thereby leading to the
accumulation of leukemic blasts in patients with chronic myeloid
leukemia (Urbano et al., 1998).
[0008] Gleevec (imatinib mesylate, also known as STI-571), is being
used as oral treatment for patients with CML. It is a
protein-tyrosine kinase inhibitor that inhibits the Bcr-Abl
tyrosine kinase.
[0009] Apoptosis, programmed cell death, is a genetically
controlled ablation of cells during development. It is
characterized by chromatin condensation, nuclear fragmentation,
cell membrane blebbing, apoptotic body formation, and mitochondrial
changes, including enhanced membrane permeability, fall of
mitochondrial membrane potential (.DELTA..psi.m), and release of
cytochrome c into the cytosol. Induction of apoptosis is the
principal mechanism by which the majority of chemotherapeutic
agents exert their function. Consequently, failure to undergo
apoptosis is the likely mechanism mediating drug resistance in
tumors.
[0010] Antitumor and anticancer properties have been studied in
mushrooms. The three mushrooms which have the broadest range of
action are Shiitake (Lentinus edodes), Reishi (Ganoderma lucidum)
and Maitake (Grifola frondosa). Another popular mushroom is
Coriolus versicolor, also known as Trametes versicolor.
[0011] Among the main active substances found in medicinal
mushrooms are:
[0012] (i) lentinan, a highly purified polysaccharide fraction
extracted from Shiitake mushrooms, is an approved drug in Japan. It
is generally administered by injection and has been used as an
agent to prolong survival of patients in conventional cancer
therapy, for example, in bowel cancer, liver cancer, stomach
cancer, ovarian cancer and lung cancer; it also stimulates the
production of T-lymphocytes and natural killer cells and can
potentiate the effect of AZT in the antiviral treatment of
AIDS;
[0013] (ii) a substance known as activated hexose-containing
compound (AHCC) or 1,3-beta glucan is an active fraction found in
shiitake mushrooms which has shown anti-cancer properties in some
human, animal and lab studies in Japan;
[0014] (iii) polysaccharide-peptide or PSP, a proteoglycan from
Trametes versicolor, also known as Coriolus versicolor, has been
widely used in China as anticancer and immunomodulatory agent in
the treatment of patients with cancer of the stomach, esophagus,
lung, ovary and cervix;
[0015] (iv) the immunostimulating polysaccharide krestin
polysaccharide-K or PSK is a popular Japanese extract made from
Trametes versicolor. PSK has been shown in several studies to help
cancer patients undergoing chemotherapy, and significantly extended
survival at five years or beyond in cancers of the breast, liver,
prostate, stomach, colon-rectum, esophagus, nasopharynx, and lung
(non-small cell types). PSK acts directly against tumor cells as
well as indirectly in the host to boost cellular immunity by
increasing white cell activity and increasing natural killer cell
function. The list of cancers for which it is known to be useful in
animals includes adenosarcoma, fibrosarcoma, mastocytoma,
plasmacytoma, melanoma, sarcoma, carcinoma, mammary cancer, colon
cancer, and lung cancer; and
[0016] (v) a dietary supplement prepared from extracts of Trametes
versicolor is in use for general health purposes. Ethanol extracts
(70%) of Trametes versicolor dietary supplement reduced LNCaP cell
growth and down-regulated the levels of secreted prostate specific
antigen (PSA), raising the possibility of chemopreventive potential
for hormone-refractory prostate cancer (Hsieh and Wu, 2001).
[0017] We have not found in the literature any publication
disclosing the activity of mushroom extracts on CML cells.
[0018] Although most bioactive substances isolated from mushrooms
are high-molecular-weight (HMW) polysaccharides, our interest is in
low-molecular-weight (LMW) compounds capable of exhibiting
antitumor activity. We have thus focused on the search for novel
compounds that induce apoptosis in CML cells and might be useful in
the therapy of patients with CML.
SUMMARY OF THE INVENTION
[0019] It has now been found, in accordance with the present
invention, that mycelium extracts of some higher Basidiomycetes
mushrooms selectively inhibit the growth, promote apoptosis, and
induce erythroid differentiation of K562 cells, a human CML cell
line, and also inhibit the growth of LNCaP cells, a human
hormone-responsive prostate cancer cell line.
[0020] The present invention thus relates, in one aspect, to a
composition comprising a mycelium extract from at least one higher
Basidiomycetes medicinal mushroom selected from the group
consisting of Ganoderma adspersum, Hypsizygus ulmarium,
Kuehneromyces mutabilis, Omphalotus olearius, Panus conchatus,
Piptoporus betulinus, Pleurotus eryngii, and Trametes zonata, said
composition having selective antiproliferative activity, or
selective apoptosis-inducing activity on the human chronic
myelogenous leukemia K562 cells and on the human prostate cancer
LNCaP cells.
[0021] The compositions may be in the form of pharmaceutical
compositions or they may be comprised within a food or
beverage.
[0022] Further provided are methods for the treatment of a patient
suffering from CML, ALL, prostate cancer or a .beta.-globin
disorder consisting of sickle cell anemia and .beta.-thalassemia,
which comprises administration of a composition of the invention to
said patient in need.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1 shows the effect of mycelium crude extracts on the
viability of K562 cells. K562 cells were plated in 6-well plates at
1.times.10.sup.5 cells/ml (Day -1). Twenty-four hours later (Day
0), 500 .mu.g/ml of mycelium crude extracts Meth114, MH428, MC293,
MH161, Meth178, MH210, MH17, and Meth134 (see Tables 1 and 3 for
mushroom species) were added and cell numbers were monitored for 4
days by trypan blue exclusion assay as described in Materials and
Methods. Experiments were carried out in duplicate. Changes in
duplicate samples were minimal with % CV below 10% in all
experiments. This experiment was repeated twice with similar
outcomes.
[0024] FIGS. 2A-2B depict Western blots showing cleavage of PARP by
mushroom extracts. K562 cells were plated in T25 flasks at
1.times.10.sup.5 cells /ml. Twenty-four hours later mycelium crude
extracts MC293, MH428, tyrphostin AG957 (2A), MH210, Meth178, MH17,
and Meth134 (2B) were added at 500 .mu.g/ml and 250 .mu.g/ml for 48
hours. Cell lysates were resolved into 8% SDS-polyacrylamide gel
electrophoresis SDS-PAGE) followed by transfer to nitrocellulose
filters. Western Blotting was performed as described in Materials
and Methods in which monoclonal anti-PARP antibody was used (Santa
Cruz, USA).
[0025] FIGS. 3A-3B depict Western blots showing the effect of
mushroom extracts on the expression of p210.sup.Bcr-Abl in K562
cells. K562 cells were plated in T25 flasks at 1.times.10.sup.5
cells/ml. Twenty-four hours later, tyrphostin AG957, mushroom
extracts MH210, Meth178, MH17 (3A), tyrphostin AG957, Meth134,
MC293, and MH428 (3B) were added at 500 .mu.g/ml and 250 .mu.g/ml
for 48 hr. Western Blotting was performed as described in Materials
and Methods, in which monoclonal c-Abl antibody was used (Santa
Cruz, USA). Filters were stripped and re-probed with loading
control, monoclonal .beta.-actin antibody (Santa Cruz, USA).
[0026] FIG. 4 depicts Western blots showing the effect of Gleevec
on the phosphorylation of wild-type Bcr-Abl and mutated Bcr-Abl
T315I and E255K.
[0027] FIG. 5 depicts Western blots showing inhibition of Bcr-Abl
phosphorylation in BaF3 cell lines by DCM540.
[0028] FIG. 6 depicts Western blots showing inhibition of Bcr-Abl
phosphorylation in K562 cell line by DCM540.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention provides novel extracts of higher
Basidiomycetes medicinal mushrooms selected from the group
consisting of Ganoderma adspersum, Hypsizygus ulmarium,
Kuehneromyces mutabilis, Omphalotus olearius, Panus conchatus,
Piptoporus betulinus, Pleurotus eryngii, and Trametes zonata, said
extracts being obtained from dry mycelium of the mushrooms by
solvent extraction. Preferred mushrooms are Omphalotus olearius,
more preferably the strain Omphalotus olearius (DC.:Fr.) Fr.,
Piptoporus betulinus, more preferably the strain Piptoporus
betulinus (Bull.:Fr.) P. Karst., and Trametes zonata, more
preferably the strain Trametes zonata (Nees.:Fr.) Pilat.
[0030] The extraction is performed with an extraction solvent
comprising one or more organic solvents, and optionally comprising
water. Examples of organic solvents that can be used according to
the invention are, without being limited to, methanol, ethanol,
acetonitrile, ethyl acetate, chloroform, hexane, cyclohexane,
isooctane and dichloromethane.
[0031] In one embodiment, the extraction solvent is a sole organic
solvent used alone, e.g. dichloromethane (DCM), methanol or
ethanol, or together with water, preferably ethanol, more
preferably 70% ethanol. In another embodiment, the solvent is a
mixture of two organic solvents, optionally with water, such as
methanol and ethyl acetate, preferably 10-60% methanol and 20-40%
ethyl acetate, more preferably, 11% methanol and 22% ethyl acetate
or 50% methanol and 33% ethyl acetate, or methanol and chloroform,
preferably 50% methanol and 33% chloroform.
[0032] The mushrooms are grown initially in agar plates at
27.degree. C. and are then transferred to liquid media and grown in
a suitable medium in submerged conditions for about 2-3 weeks. Dry
mycelium is extracted with the solvents of choice and tested for
biological activity.
[0033] As used herein in the specification, the terms and phrases
set out below have the meanings which follow:
[0034] For the extraction solvents, "Eth" means 70% ethanol, "E"
means a mixture of 33% ethyl acetate and 50% methanol, "H" means a
mixture of 22% ethyl acetate and 11% methanol, "C" means a mixture
of 33% chloroform and 50% methanol (the remainder in these cases is
water), and "DCM" means 100% dichloromethane. The letters C, E, H
Eth, and DCM also appear in the designation of the extracts as
defined below and in the examples.
[0035] "MH161" means the crude mycelium extract of Kuehneromyces
mutabilis, strain 18 in Table 1 hereinafter, in solvent H.
[0036] "MH210" means the crude mycelium extract of Pleurotus
eryngii, strain 13 in Table 1 hereinafter, in solvent H.
[0037] "Meth178" means the crude mycelium extract of Omphalotus
olearius, strain 12 in Table 1 hereinafter, in solvent Eth.
[0038] "MH17" means the crude mycelium extract of Piptoporus
betulinus, strain 11 in Table 1 hereinafter, in solvent H.
[0039] "Meth134" means the crude mycelium extract of Ganoderma
adspersum, strain 8 in Table 1 hereinafter, in solvent Eth.
[0040] "Meth114" means the crude mycelium extract of Panus
conchatus, strain 7 in Table 1 hereinafter, in solvent Eth.,
[0041] "MC293" means the crude mycelium extract of Hypsizygus
ulmarium, strain 4 in Table 1 hereinafter, in solvent C.
[0042] "MH428" means the crude mycelium extract of Trametes zonata,
strain 1 in Table 1 hereinafter, in solvent H.
[0043] "Meth162" means the crude mycelium extract of Kuehneromyces
mutabilis, strain 18 in Table 1 hereinafter, in solvent Eth.
[0044] "Meth215" means the crude mycelium extract of Pleurotus
eryngii, strain 13 in Table 1 hereinafter, in solvent Eth.
[0045] "Meth194" means the crude mycelium extract of Pholiota
Aurivella, strain 31 in Table 1 hereinafter, in solvent Eth.
[0046] "Meth327" means the crude mycelium extract of Trametes
zonata, strain 1 in Table 1 hereinafter, in solvent Eth.
[0047] "DCM540" means the crude mycelium extract of Trametes
zonata, strain 1 in Table 1 hereinafter, in solvent DCM.
[0048] "K562 cells" means the human chronic myelogenous leukemia
cell line.
[0049] Once obtained, the extracts are tested for antiproliferative
activity on the K562 cells. Extracts that inhibit the growth of
K562 cells, but do not inhibit the growth of other tumor or normal
cells, are specific to the K562 cells and are suitable candidates
for treatment of CML. The extracts may be further tested to see
whether they promote apoptosis in K562 cells, in which case they
will be more preferred candidates for treatment of CML
patients.
[0050] The extracts selective for K562 cells are also tested to see
whether they induce erythroid differentiation in K562 cells.
Extracts that induce erythroid differentiation are suitable
candidates for treatment of a .beta.-globin disorder such as sickle
cell anemia and .beta.-thalassemia,
[0051] The extracts are also tested for antiproliferative activity
on human prostate cancer LNCaP cells. Extracts that inhibit the
growth of LNCaP cells, but do not inhibit the growth of other tumor
or normal cells, are specific to the LNCaP cells and are suitable
candidates for treatment of prostate cancer. The extracts may be
further tested to see whether they promote apoptosis in LNCaP
cells, in which case they will be more preferred candidates for
treatment of prostate cancer patients.
[0052] Thus, according to the present invention, 42 species of HBM
were cultivated as pure cultures in submerged conditions, and dry
mycelium were used to prepare 168 different crude extracts. The
crude extracts were used to evaluate antiproliferative activity
against a number of cancer cell lines, including K562, Jurkat
(human T lymphoblast cells), HT29 (human colon adenocarcinoma
cells), MH3924A (rat Morris hepatoma), and ABAE (adult bovine
aortic endothelial cells) using XTT proliferation assay.
[0053] Forty-four different crude extracts were selected with
antiproliferative effect against K562 cells and eight mycelium
extracts were K562-selective compared with MH3924A, HT29, ABAE, and
Jurkat cells. Growth inhibition against K562 ranged from 51% to 78%
compared with solvent-treated samples. Most crude extracts
exhibited a complete or partial cytostatic effect against K562
cells.
[0054] The antiproliferative effect observed by the selected crude
extracts was attributed to the induction of apoptosis pathway as
determined by Apostain ELISA assay and by monitoring PARP cleavage.
Interestingly, crude extract MH428 was the most active extract in
inducing apoptosis of K562 cells.
[0055] In addition, expression levels of p210.sup.Bcr-Abl were
affected by the presence of the selected crude extracts. Our data
revealed a significant inhibition of p210.sup.Bcr-Abl expression by
MH428 extract, a moderate effect by MH17, and minor changes by the
other extracts.
[0056] Furthermore, mycelium crude extracts were active in inducing
erythroid differentiation in K562 cells. Crude extracts Meth178,
MH17, and MH428 show significant ability to induce hemoglobin
production in K562 cells as indicative of erythroid
differentiation.
[0057] Data presented in accordance with the present invention
illustrate the potential of the mycelium extracts, particularly the
MH428 extract, in CML therapy. This extract was prepared from
Trametes zonata and was also active against LNCaP cells, indicating
that Trametes zonata extract is active in inhibiting
hormone-refractory prostate cancer cells.
[0058] The mycelium extracts obtained as described above are
concentrated and purified for human use. Concentration can be
carried out by conventional techniques such as thermal,
decompressing thermal, activated carbon or ion exchange resin
methods. The concentrated extract is then purified to yield a
purified extract of one or more purified compositions using
standard techniques such as column chromatography, fractional
distillation, preparative TLC (thin layer chromatography),
preparative HPLC (high performance liquid chromatography), CPC
(centrifugal partition chromatography) or other techniques known to
those skilled in the art. After concentration and purification, the
product is dried by any conventional technique such as air-dry,
hot-blast drying, spray dry, and freeze-dry methods.
[0059] The invention also provides a composition comprising a
mycelium extract of the invention. The composition may be a
pharmaceutical composition, in which case the extract is in
admixture with one or more pharmaceutically acceptable carriers.
The composition may also be in the form of food or beverage.
[0060] For formulation of the compositions of the invention,
powders of the extract may be used in that form directly as a loose
powder or encapsulated powder, or may be formulated into capsules,
caplets, tablets and similar dosage forms. Further, powders may be
formulated within liquid previous membranes such as filters, meshes
and the like, such as a tea bag-type infuser, for generating
liquids containing the dissolved extract. The powder form of the
extract may also be incorporated into liquids, formulated as
solutions, dispersions or suspensions by dissolving the extract,
for example as a drink, tincture, or drop. The extract may be
administered alone, or with a carrier.
[0061] The extract can be prepared alone or as an active ingredient
in pharmaceutical compositions including non-toxic,
pharmaceutically acceptable carriers, diluents and excipients, as
are well known in the art (see, for example Merck Index, Merck
& Co., Rahway, N.J.; and Gilman et al., (eds) (1996) Goodman
and Gilman's: The Pharmacological Bases of Therapeutics, 8.sup.th
Ed., Pergamon Press). For standard dosages of conventional
pharmacological agents, see, e.g., Physicians Desk Reference (1997
Edition); and U.S. Pharmacopeia National Formulary (1995) United
States Pharmacopeial Convention Inc., Rockville, Md. Compositions
may also include flavors, colorings, coatings, etc. All agents must
be non-toxic and physiologically acceptable for the intended
purpose, and must not substantially interfere with the activity of
the extract so as to deleteriously affect the desired biological
activity thereof. Ingredients are thus only included in
therapeutically acceptable amounts
[0062] The dosage of the extract to be administered depends upon
many factors that are well known to those skilled in the art, for
example, the particular form of the extract; the stage of the
disease; the age, weight and clinical condition of the patient; any
concurrent therapeutic treatments; and the experience and judgment
of the clinician or practitioner administering the therapy. The
extract may be administered orally, intraperitoneally, or
intravenously at a dosage range and frequency (e.g., at least once
daily) such that the level of active extract is maintained in the
body. The dosage range varies with the route of administration, and
the form and potency of the extract; for example, one dose of the
extract in a capsule taken orally may contain for example 100-2000
mg of the extract, preferably 200-1500 mg, more preferably 250-1000
mg, still more preferably 500-800 mg. The extract is preferably
administered in spaced dosages throughout the day to maintain the
level of active extract in the body.
[0063] The present invention further provides food or beverage
containing a composition of the invention. Thus, for example, the
extract may be added to fruit juice, vegetable juice, all kinds of
tea and nutrient drinks possibly containing nutraceuticals of
choice such as vitamins, minerals, antioxidants and the like.
[0064] The invention further provides a method of treating a
Philadelphia chromosome-positive (Ph.sup.+) leukemia patient such
as CML and ALL Ph.sup.+ leukemia patients, comprising administering
to a patient in need a therapeutically effective amount of a
composition of the invention, preferably a composition comprising a
mycelium extract of Piptoporus betulinus or Trametes zonata.
[0065] Further provided by the invention is a method of treating
sickle cell anemia comprising administering to a patient in need a
therapeutically effective amount of a composition of the invention,
preferably a composition comprising a mycelium extract of of
Omphalotus olearius, Piptoporus betulinus, or Trametes zonata.
[0066] The invention still further relates to a method of treating
.beta.-thalassemia comprising administering to a patient in need a
therapeutically effective amount of a composition of of the
invention, preferably a composition comprising a mycelium extract
of of Omphalotus olearius, Piptoporus betulinus, or Trametes
zonata.
[0067] The invention yet further provides a method of treating
prostate cancer comprising administering to a patient in need a
therapeutically effective amount of a composition of the invention,
preferably a composition comprising a mycelium extract of Trametes
zonata.
[0068] The invention will now be illustrated by the following
non-limiting Examples.
EXAMPLES
Materials and Methods
(i) Mushroom Species, Cultivation and Extraction
[0069] The strains used in the present invention are from a culture
collection of HBM (acronym HAI) of the Institute of Evolution,
University of Haifa, Israel (Wasser et al., 2002), that contains
presently over 1000 strains of edible and medicinal mushrooms.
About 200 species were collected in different ecological regions of
Israel, and new strains have been permanently introduced into the
collection. The collection also contains many diverse strains of
well-known medicinal mushrooms from North America, Europe, and
other parts of the world. Table 1 hereinafter shows a list of
mushroom species used to prepare the mycelium crude extracts tested
herein. Mushrooms were grown initially in agar plates at 27.degree.
C., and then transferred to liquid media to generate a starter
culture. Large-scale growth was carried out in 500 ml liquid medium
in 2-liter Erlenmeyer for 2-3 weeks at 27.degree. C. with shaking
at 180 rpm.
[0070] Medicinal mushrooms were grown in submerged condition, and
mycelium was dried and used to prepare crude extracts using various
mixtures of aqueous organic solvents, including Eth (70% ethanol),
E (33% ethyl acetate, 50% methanol), H (22% ethyl acetate, 11%
methanol), and C (33% chloroform, 50% methanol). Obtained yield of
crude extract with the various mixtures of organic solvents is
shown in Table 1 in mg/gram of dried mycelium used. ND indicates
`not determined`.
(ii) Mushroom Growth Medium
[0071] Mushrooms were grown in liquid or solid media containing 2%
glucose, 0.1% bacteriological peptone, 0.1% yeast extract, 0.1%
KH.sub.2PO.sub.4, 0.1% MgSO.sub.4, and 1.7% of a Bacto-Agar in agar
plates. The media were also supplemented with 10 ml per 1 liter of
trace solution (18 mM FeSO.sub.4, 3.7 mM MnSO.sub.4, 1.5 mM
ZnSO.sub.4, 0.8 mM CuSO.sub.4).
(iii) Preparation of Mycelium Crude Extracts
[0072] Dry mycelium was extracted with the four solvents Eth, C, E
and H (see (i) above) (1 gram of material used for each
condition).
(iv) Cell Lines and Cell Cultures
[0073] Human K562, Jurkat, and HT-29 cell lines were grown in RPMI
1640 medium with L-glutamine supplemented with 10% fetal bovine
serum. Bovine aortic arch-derived endothelial cell (ABAE) and rat
Morris hepatoma MH3924A cells were grown in DMEM and supplemented
with 10% fetal bovine serum. Human LNCaP prostate cells were
maintained in RPMI 1640 medium supplemented with 10% fetal calf
serum. Penicillin at 100 U/ml and streptomycin at 100 .mu.g/ml were
added to the culture media. All cell lines were grown at 37.degree.
C. in a humidified atmosphere with 5% CO.sub.2. Adherent cell lines
were transferred with 0.025% trypsin and 0.02% EDTA.
(v) Cell Viability
[0074] To determine cell growth and viability, K562 cells
(2.times.10.sup.5 cells/ml) in triplicate were incubated in 2 ml of
RPMI 1640-10% fetal calf serum (FCS) containing mycelium crude
extracts or DMSO. The volume of the DMSO was kept at 0.6% of the
medium volume. At the indicated times, cell viability was
determined by the trypan blue exclusion assay (Freshney, 1987). At
least 200 cells were examined in each sample. Data were expressed
as % of inhibition relative to solvent-treated samples.
(vi) Erythroid Differentiation
[0075] Erythroid differentiation was determined by monitoring
levels of hemoglobin production in treated cells. K562 cells were
plated in 6-well plates at 2.times.10.sup.5 cells/ml, followed by
treatment with various concentrations of mycelium extracts for 5
days. Cells were washed with phosphate-buffered saline (PBS) and
cell pellet was re-suspended in 100 .mu.L lysis buffer (100 mM
potassium phosphate pH 7.8, 0.2% Triton X-100) and incubated for 10
min at room temperature. Intracellular hemoglobin levels were
determined by means of the plasma hemoglobin kit from Sigma (USA)
according to the manufacturer's instructions. Levels of hemoglobin
were normalized to protein concentration found in each sample.
Protein concentration was determined by DC Protein Assay Kit
(Bio-Rad, USA) according to manufacturer's instructions. Relative
hemoglobin levels were calculated in relation to solvent-treated
sample, which was designated 1.0.
(vii) XTT Cell Proliferation Assay
[0076] The XTT assay for fungal viability is based on the MTT assay
(Mosmann, 1983) used to monitor cell proliferation growing in
suspension. In brief, K562 cells were seeded in 96-well plates at
1.5.times.10.sup.4 cells/well; 24 hours later cells were treated
with mycelium crude extracts at 1 mg/ml and 250 .mu.g/ml for an
additional 24 hours. 50 .mu.L of XTT solution at 1.5 .mu.g/ml were
added to each well and were incubated for three hours at 37.degree.
C. The optical density was measured by multiwell plate
spectrophotometer at 405 nanometers.
(viii) PARP Cleavage
[0077] To analyze poly(ADP-ribose) polymerase (PARP) cleavage (Dou
et al., 1999), cells (2.times.10.sup.5 cells/ml) were treated with
mycelium crude extracts or DMSO for the indicated time. Cells were
collected, washed once with cold PBS, and lysed in buffer [10 mM
Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20
mM Na.sub.4P.sub.2O.sub.7, 2 mM Na.sub.3VO.sub.4, 1% Triton X-100,
10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM
phenylmethylsulfonyl fluoride] for 30 min at 4.degree. C. Cell
lysate supernatants (40 .mu.g protein/each) were resolved on 8%
SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by
immune-blotting with an anti-PARP antibody (Santa Cruz Co.,
USA).
(ix) Apostain ELISA Assay
[0078] K562 cells (1.times.10.sup.5 cells/ml) were treated with
mycelium crude extracts or DMSO for 48 hours. Samples were
collected, washed with PBS, resuspended in 100 .mu.l of 80%
methanol, and kept at -20.degree. C. for 2-3 days. Between 2500 and
5000 cells were used for Apostain ELISA assay according to
manufacturer's instructions (Alexis Biochemical, USA).
(x) Bcr-Abl Phosphorylation Assay
[0079] K562, BaF3/Bcr-Abl wild-type (WT), BaF3/Bcr-Abl T315I and
BaF3/Bcr-Abl E255K cell lines were seeded 4 mls/well in 6-well
plates at 5-6.times.10.sup.5 cells/ml. Control cells were treated
with 2% DMSO. Gleevec-treated cells were treated with 1 .mu.M
Gleevec and dichloromethane (DCM) extract of T. zonata (#540) at
two concentrations: (A) at 500 .mu.g/ml and (B) at 1000 .mu.g/ml,
that were added after 24 hours. Cells were treated for 60 min, then
collected and centrifuged at 5000 rpm for 5 min. Cell pellets were
lysed in lysis buffer (10 mM Tris, pH 7.4; 100 mM NaCl; 1 mM EDTA;
1 mM EGTA; 1 mM NaF; 20 mM Na.sub.4P.sub.2O.sub.7; 2 mM
Na.sub.3VO.sub.4; 1% Triton-X100; 10% glycerol; 0.1% SDS; 0.5%
deoxycholate; 1 mM PMSF; 10 .mu.l protease inhibitor cocktail and
phosphatase inhibitor cocktail were added to every 1 ml Iysate}.
After that, 40 .mu.g protein from each sample were separated on 8%
SDS-PAGE, then Western blot was performed according to the
manufacturer's instructions using phospho-c-Abl (Tyr245) Antibody
(Cell Signaling Technology Co.) and c-Abl monoclonal antibody
(Santa Cruz Biotechnology).
Example 1
Identification of Mycelium Crude Extracts that Inhibit
Proliferation of K562 Cells
[0080] A large number of medicinal mushrooms (42) shown in Table 1
were cultivated in submerged conditions, and extracted with various
extraction solvents (4), as described in Materials and Methods,
resulting in the preparation of 168 crude mycelium extracts. The
mycelium was dried and used to prepare crude extracts using various
mixtures of organic solvents including Eth (70% ethanol), E (33%
ethyl acetate, 50% methanol), H (22% ethyl acetate, 11% methanol),
and C (33% chloroform, 50% methanol). The obtained yield of crude
extract with the various organic solvents is shown in Table 1 in mg
of crude extract/gram of dried mycelium. ND indicates not
determined.
[0081] Crude extracts were screened for their ability to inhibit
the growth of K562, a human chronic myelogenous leukemia blast
cell. Growth inhibition was evaluated by XTT assay as described in
Materials and Methods. Cleavage of the tetrazolium salt XTT, sodium
3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)ben-
zene-sulfonic acid hydrate, by dehydrogenase enzymes of
metabolically active cells yields a highly colored formazan
product, which is water soluble. This feature obviates the need for
formazan crystal solubilization prior to absorbance measurements,
as required with the use of other tetrazolium salts such as MTT,
and offers a simple method for evaluating proliferation of cells
growing in suspension such as K562 cells (Roehm et al., 1991).
TABLE-US-00001 TABLE 1 List of mushroom species tested and yield of
various crude extracts prepared from mycelium of submerged cultures
mg crude extract/g dry mycelium HAI strain No. Species, authors
number C E H Eth 1 Trametes (T.) zonata (Nees.:Fr.) Pilat 540 183
64 16 110 2 T. multicolor (Schaeff.) Julich 428 100 90 70 50 3 T.
hirsutus (Wulf.:Fr.) Pilat 598 376 340 462 418 4 Hypsizygus (H.)
ulmarium (Bull.:Fr.) 812 55 88 107 96 Redhead 5 H. decastes (Fr.)
Sing 510 117 107 93 107 6 H. marmoreus (Peck) Bigel. 609 144 91 82
96 7 Panus conchatus (Bull.:Fr.) Fr. 396 100 100 100 80 8 Ganoderma
adspersum (S. Schulz.) Donk 349 245 100 230 180 9 G. applanatus
(Pers.: Wallr.) Pat. 604 210 160 200 215 10 G. resinaceum Boud. 142
220 170 210 220 11 Piptoporus betulinus (Bull.:Fr.) P. Karst. 241
240 ND 200 ND 12 Omphalotus olearius (DC.:Fr.) Fr. 173 110 100 115
140 13 Pleurotus (P.) eryngii (DC.:Fr.) Quel. 202 156 131 169 100
14 P. ostreatus (Jacq.:Fr.) Kumm. 592 200 80 70 100 15 P. salignus
(Fr.) Kumm. 571 75 44 75 69 16 P. cystidiosus O. K. Miller 140 169
146 133 108 17 P. pulmonarius (Fr.) Quel. 573 169 108 75 116 18
Kuehneromyces mutabilis (Schaeff.:Fr.) 114 100 70 100 120 Sing et
A. H. Sm. 19 Rigidoporus ulmarius (Sow.: Fr.) Imazeki 439 60 60 70
60 20 Spongipellis litshaueri Lohw 444 100 100 60 100 21 Panellus
(P.) stipticus (Bull.:Fr.) P. Karst. 520 100 90 90 70 22 P.
serotinus (Pers.:Fr.) Kuhn 498 169 119 125 156 23 Gloeophyllum
odoratum (Wulf.) Imazeki 337 100 70 90 90 24 Schizophyllum commune
Fr.: Fr. 632 90 60 80 50 25 Oudemansiella radicata (Relh.:Fr.) Sing
773 170 130 180 150 26 O. mucida (Schrad.:Fr.) Hoehn. 181 170 100
140 130 27 Leucoagaricus (L.) carneifolius (Gill.) S. Wasser 344
110 65 100 98 28 L. leucothitus (Vitt.) S. Wasser 282 185 160 155
160 29 Marasmius scorodonius (Fr.) Fr. 784 290 160 70 150 30
Polyporus squamous Huds.:Fr. 242 180 75 140 80 31 Pholiota
aurivella (Batsch.:Fr.) Kumm. 236 125 80 65 88 32 Grifola frondosa
(Dicks.:Fr.) S. F. Gray 270 173 152 193 133 33 Fomes fomentarius
(L.:Fr.) Fr. 383 163 138 63 125 34 Lentinus edodes (Berk.) Sing 313
187 193 200 207 35 Phellinus ignarius (L.:Fr.) Quel. 785 131 113
138 103 36 Merulius tremellosus Fr. 267 286 120 320 ND 37 Irpex
lacteus (Fr.:Fr.) Fr. 532 178 168 176 152 38 Inonotus levis P.
Karst. 796 174 144 115 118 39 Flammulina velutipes (Curt.:Fr.) Sing
105 153 64 125 58 40 Oxyporus obducens (Fr.) Donk 824 97 95 109 108
41 Agaricus langei (Moell.) Moell. 295 88 102 46 86 42 Funalia
trogii (Berk. upud. Trog.) Bond. 352 200 130 150 60 et Sing
[0082] Antiproliferative activity of crude mycelium extracts was
evaluated against K562 cells. K562 cells were plated in 96-well
plates at 1.5.times.10.sup.4 cells/well and 24 hours later were
treated with mycelium crude extracts at 250 .mu.g/ml and 1 mg/ml
(with addition of 10 .mu.l of stock solution to each well
containing 100 .mu.l of K562 cells). Twenty-four hours later, XTT
assay was preformed as described in Materials and Methods.
[0083] The obtained results are summarized in Table 2. A total of
44 crude extracts from the 168 screened extracts were active in
inhibiting the growth of K562 cells by more than 50%. The 44 crude
mycelium extracts represent about 26% of the screened extracts. The
number of active extracts among the various extraction methods
varied significantly. With solvent mixtures H (22% ethyl acetate,
11% methanol), E (33% ethyl acetate, 50% methanol), C (33%
chloroform, 50% methanol), and Eth (70% ethanol), the number of
K562 active extracts were 5, 13, 12, and 14, respectively (Table
2). TABLE-US-00002 TABLE 2 Distribution of K562 active mycelium
crude extracts among the different organic mixtures Number %
Extracts Total H E C Eth Total H E C Eth Screened 168 41 43 42 42
100 24.4 25.6 25 25 Active 44 5 13 12 14 26 11.4 29.5 27.3 31.8
(>50%) Selective 8 4 0 1 3 18.2 50 0 12.5 37.5
Example 2
Identification of Mycelium Crude Extracts that Selectively Inhibit
Proliferation of K562 Cells
[0084] The K562 active crude extracts of Example 1 were subjected
to selectivity evaluation by monitoring growth inhibition of the
selected extracts against other cell lines, including Jurkat (human
T lymphoblasts), HT29 (human colon adenocarcinoma cells), MH3924A
(rat Morris hepatoma), and ABAE (adult bovine aortic endothelial
cells).
[0085] Cells were plated in 96-well plates at 1.5.times.10.sup.4
cells/well. Twenty-four hours later mycelium crude extracts were
added for an additional 24 hours followed by XTT determination
according to manufacturer's instructions (Biological Industries,
Israel). Percentage of growth inhibition was calculated in relation
to solvent-treated samples. Experiments were carried out in
duplicate. The results are shown in Table 3. Changes in %
coefficient of variance (CV) among duplicate samples were minimal.
For example, % CV in growth inhibition of K562 using duplicate
samples treated with mycelium crude extracts MH161, MH210, Meth178,
MH17, Meth134, Meth114, MC293, MH428 were 0.4, 28, 6.4, 1.1, 4.3,
1.9, 17.9, 4.1, respectively. This experiment was repeated twice
with similar outcomes.
[0086] Mycelium crude extracts that inhibited K562 by more than 50%
and showed minimal growth inhibition (less than 30% inhibition)
against other cell lines were designated as selective K562
inhibitors. Table 3 shows a list of selective mycelium crude
extracts (with their designations and identification of the
mushroom strain in the first two columns) with percentage of growth
inhibition applied to a variety of cell lines. Growth inhibition
was calculated compared with solvent-treated samples.
TABLE-US-00003 TABLE 3 Effect of K562-selective mycelium crude
extracts on the growth of K562, MH3924A, ABAE, HT-29 and Jurkat
cell lines % of Growth Inhibition HT- Ext Mushroom Strain Solvent
K562 MH3924A ABAE 29 Jurkat MH161 Kuehneromyces 114 H 61.3 16.6
-30.1 -19.5 16.2 mutabilis MH210 Pleurotus eryngii 202 H 53.8 3.4
-34.0 -27.2 8.8 Meth178 Omphalotus 173 Eth 56.9 -14.5 -56.2 -11.3
-36.4 olearius MH17 Piptoporus 241 H 68.8 15.2 28.2 -7.6 -2.9
betulinus Meth134 Ganoderma 349 Eth 59.2 -15.5 -44.7 23.1 -18.4
adspersum Meth114 Panus conchatus 396 Eth 56.0 11.5 27.8 4.3 2.4
MC293 Hypsizygus 812 C 56.7 14.4 -28.9 -7.6 7.3 ulmarium MH428
Trametes zonata 540 H 69.0 -11.4 -5.1 1.9 30.1
[0087] Table 3 shows that a total of 8 mycelium crude extracts
exhibited selective activity against K562 cells. The 8 positive
crude extracts were distributed among the various extraction
methods as follows: 4, 0, 1, and 3 using organic mixture H, E, C,
and Eth, respectively. It is interesting that most of the selective
extracts were extracted by means of the H and Eth organic
solvents.
Example 3
Effect of the Mycelium Crude Extracts on the Viability of K562
Cells
[0088] It is important to note that XTT assay does not distinguish
between cytostatic and cytotoxic effects in continuously
proliferating cultures. Therefore, the effect of the selected
mycelium crude extracts on the viability of K562 cells was
evaluated. Although cell viability can be reflected by a variety of
different parameters, integrity of the outer cell membrane is often
used. The vital dye trypan blue, which is usually excluded from
viable cells, was used to assess whether mycelium crude extracts
function as a cytotoxic or a cytostatic compound against K562
cells. Cells were treated with 500 .mu.g/ml of the appropriate
crude extracts shown in Table 3 and viable cells were monitored for
several days.
[0089] K562 cells were plated in 6-well plates at 1.times.10.sup.5
cells/ml (day -1). Twenty-four hours later (day 0), 500 .mu.g/ml of
mycelium crude extracts Meth114, MH428, MC293, MH161, Meth178,
MH210, MH17, and Meth134 (see Table 3 for mushroom species) were
added and cell numbers were monitored for 4 days by trypan blue
exclusion assay as described in Materials and Methods.
[0090] The results shown in FIG. 1 illustrate that most mycelium
crude extracts exhibit a complete (MH210, Meth134, Meth114, MH326)
or partial (Meth178, MH17, MC293) cytostatic effect. In contrast,
crude extract MH161 exhibited cytotoxic effect at the concentration
used.
Example 4
Involvement of the Apoptosis Pathway in Mediating the Growth
Inhibition of K562 Cells by Mycelium Crude Extracts
[0091] Apoptosis, programmed cell death, is a genetically
controlled ablation of cells during development. Furthermore,
induction of apoptosis is the principal mechanism by which the
majority of chemotherapeutic agents exercise their function.
Accordingly, we evaluated whether our mycelium crude extracts
affected the apoptosis pathway in K562 cells. Our data show that 8
mycelium crude extracts exhibited selective antiproliferative
effect against K562 cells as determined by XTT assay (Table 3) and
trypan blue exclusion assay (FIG. 1).
[0092] To evaluate whether the antiproliferative effect of mycelium
crude extracts is mediated by induction of the apoptosis process,
we monitored changes in chromatin condensation as a marker of
apoptosis (Allera et al., 1997). A recent report illustrated that
formamide, a gentle denaturing agent, denatured DNA in apoptotic
cells, but not in necrotic cells (Frankfurt and Krishan, 2001a, b).
Apostain ELISA kit (Alexis Biochemicals, USA) uses the increased
sensitivity of DNA in condensed chromatin of apoptotic cells to
denaturation by formamide (Frankfurt and Krishan, 2001a), which is
attributed, in part, to changes in the DNA-histone interactions.
The increased sensitivity of cells with denatured DNA is detected
by a monoclonal antibody specific for single-strand DNA (Mab F7-26)
in an ELISA format (Frankfurt and Krishan, 2001b).
[0093] K562 cells were treated with the appropriate mycelium crude
extracts and samples were collected 48 hours post-treatments.
Apostain ELISA assay was performed according to manufacturer's
instructions and as detailed in Materials and Methods.
[0094] K562 cells were plated in 6 well-plates at 1.times.10.sup.5
cells/ml. Twenty-four hours later cells were treated with the
mycelium crude extracts MH210, Meth178, MH17, Meth134, MC293, and
MH428 at 250 .mu.g/ml and 500 .mu.g/ml (see Table 3). The
tyrphostin AG957 at 10 .mu.M and 20 .mu.M was included as a
positive control. In addition, a solvent-treated sample was
included, in which DMSO was added to 0.6%. Forty-eight hours post
treatment cells were counted and washed with cold PBS and subjected
to Apostain ELISA Assay according to manufacturer's instruction. To
calculate relative Apostain values, empty wells were subtracted
from absorbance values obtained from cells treated with the
different extracts. The solvent-treated sample was assigned the
value 1, and relative values were calculated.
[0095] The data shown in Table 4 illustrate the ability of the
various mycelium crude extracts to induce an increase in DNA
condensation. Almost all crude extracts were able to induce a more
than twofold increase in DNA condensation above the solvent-treated
sample. Mycelium crude extracts Meth178, MH17, Meth134, MC293, and
MH428 induced increases in DNA condensation to values of 4.3, 3.5
3.6, 5.3, and 2.8, respectively, after 48-hour treatments.
[0096] Furthermore, AG957, a known p210.sup.Bcr-Abl inhibitor (Kaur
et al., 1994), also significantly induced an increase in DNA
condensation at 10 .mu.M. It is interesting that a higher dose of
mycelium crude extracts caused a decrease in the measured signal,
suggesting that at higher concentrations of mycelium extracts, cell
death is mediated, in part, by necrosis. TABLE-US-00004 TABLE 4
Apostain values from K562 cells treated with mycelium crude
extracts Relative Apostain Value Mycelium Extract 250 .mu.g/ml 500
.mu.g/ml MH210 1.9 1.9 Meth178 4.3 1.4 MH17 3.5 1.6 Meth134 3.6 3.2
MC293 5.3 6.5 MH428 2.8 10.9 AG957 6.2 (10 .mu.M) 3.9 (20
.mu.M)
Example 5
Cleavage of PARP by the Mycelium Extracts
[0097] The execution of apoptosis requires specific molecular
machinery, the central component of which is a family of proteases
called caspases, which are cysteine proteases that cleave proteins
after specific aspartate residues, in response to proapoptotic
signals (Nicholson and Thomberry, 1997). During apoptosis, caspases
activated in an amplifying proteolytic cascade, cleave one another
in sequence (Raff, 1998). One of the most widely studied caspases,
caspase 3, is classified as an effector caspase and cleaves death
substrates such as the structural protein lamin and the nuclear
protein PARP (McGowan et al., 1996).
[0098] K562 cells treated with the appropriate concentration of the
mycelium crude extracts MH210, Meth178, MH17, Meth134, MC293, and
MH428 for 48 hours and used to monitor cleavage of PARP as an
indication of the activation of the apoptosis pathway. Presence of
cleaved PARP was monitored by means of anti-PARP. Treatment with
AG957, a known Bcr-Abl inhibitor (Kaur et al., 1994), significantly
activates PARP cleavage, attesting that AG957 promotes apoptosis in
K562 cells.
[0099] Thus, K562 cells were plated in T25 flasks at
1.times.10.sup.5 cells/ml. Twenty-four hours later, mycelium crude
extracts MC293, MH428, tyrphostin AG957, MH210, Meth178, MH17, and
Meth134 were added at 500 .mu.g/ml and 250 .mu.g/ml for 48 hours.
Cell lysates were resolved into 8% SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) followed by transfer to nitrocellulose
filters. Western blotting was performed (as described in Materials
and Methods) in which monoclonal anti-PARP antibody was used (Santa
Cruz Co., USA).
[0100] The results are shown in FIGS. 2A (MC293, MH428 and AG957)
and 2B (MH210, Meth178, MH17, Meth134). Although most mycelium
extracts shown failed to activate cleavage of PARP, extract MH428
significantly activated PARP cleavage.
Example 6
Mycelium Crude Extracts Induce Terminal Differentiation of K562
Cells
[0101] Leukemic blasts expressing p210.sup.Bcr-Abl display arrested
differentiation as well as resistance to apoptosis, even when
exposed to high doses of anti-leukemic drugs (Bedi et al., 1995;
Ray et al., 1996). K562 is a human erythroleukemia cell line
derived from a patient with chronic myelogenous leukemia (Lozzio
and Lozzio, 1975). These cells are pluripotent in that they are
able to differentiate along a megakaryocytic, erythroid, or, to a
lesser extent, monocytic lineage (Leary et al., 1987). Erythroid
differentiation can be induced by a number of compounds, including
hemin and butyric acid (Rowley et al., 1981). However, TPA (or PMA)
treatment induces macrophage-like morphology and promotes the
expression of proteins associated with megakaryocytes (Leary et
al., 1987; Burger et al., 1992). Erythroid differentiation is
frequently monitored by induced expression of hemoglobin, while
megakaryocyte differentiation can be assessed by monitoring of the
ability of treated K562 cells to reduce nitroblue tetrazolium (NBT)
(Sutherland et al., 1986).
[0102] To assess erythroid differentiation, we monitored levels of
hemoglobin production in treated cells using a commercially
available kit (Sigma, USA). K562 cells were plated at
2.times.10.sup.5 cell/ml in 6-well plates. Twenty-four hours later
mycelium crude extracts at appropriate concentration were added.
Growth of K562 cells in the presence of crude extracts was
monitored by trypan blue exclusion assays (as described in
Materials and Methods). Numbers of viable cells were monitored
after 2 and 4 days post-treatment. On Day 5, cells were washed with
PBS, cell pellet was lysed, and levels of hemoglobin were
determined (as described in Materials and Methods). Hemoglobin
levels were normalized to protein concentration in each sample.
Relative hemoglobin levels were calculated in relation to
solvent-treated sample, which was designated 1.0. The experiment
was carried out in duplicate with minimal variations. This
experiment was repeated twice with similar outcomes.
[0103] Levels of expressed hemoglobin assayed at day 5
post-treatment were normalized to the amount of total proteins
present in each sample. Relative units of hemoglobin were
calculated in relation to levels of hemoglobin found in
solvent-treated samples (DMSO at 0.6%). The results summarized in
Table 5 show that butyric acid was active in inducing hemoglobin
expression in K562, which is in agreement with published data
(Villeval et al., 1983). Furthermore, three mycelium crude extracts
(Meth178, MH17 and MH428) were active in inducing hemoglobin
expression above the levels of DMSO-treated cells. Extracts MH17
and MH428 induced hemoglobin expression 4.2 and 2.1 times higher,
respectively, than the solvent-treated cells. It was interesting
that extract Meth178 was the most potent extract (about 12 times
higher than DMSO-treated cells) and was more active than butyric
acid. TABLE-US-00005 TABLE 5 Induction of hemoglobin expression in
K562 cells by mycelium crude extracts Mushroom Concentration
Relative Species Strain Extract (.mu.g/ml) Hemoglobin Kuehneromyces
114 MH161 300 0.9 mutabilis Pleurotus 202 MH210 500 0.8 eryngii
Omphalotus 173 Meth178 500 11.9 olearius Piptoporus 241 MH17 500
4.2 betulinus Ganoderma 349 Meth134 500 0.7 adspersum Panus 396
Meth114 300 0.2 conchatus Hypsizygus 812 MC293 300 0.8 ulmarium
Trametes 540 MH428 250 2.1 zonata Butyric Acid 1 mM 9.4
Example 7
Involvement of MAP Kinase p38 in Mediating Growth Inhibition of
Mycelium Crude Extracts
[0104] Previous reports indicated that proliferation and
differentiation of K562 cells are mediated by mitogen-activated
protein kinase (MAPK) pathway (Cobb, 1999; Cross et al., 2000). We
examined the involvement of p38 MAPK in mediating the
antiproliferative effect of the mycelium crude extracts. This was
achieved by pretreatment of K562 cells with SB203580, a specific
inhibitor of p38 kinase pathway, in the presence of the various
mycelium crude extracts.
[0105] K562 cells were plated on 96 well-plates at
1.5.times.10.sup.4 cells/well. Cells were pretreated with 10 .mu.M
of SB203580 (Calbiochem, USA) 24 hours later. Mycelium crude
extracts (see Table 3) were added at 1 mg/ml hours 3 later and
incubated for additional 24 hours. XTT assay was carried out as
previously described. Growth inhibition was calculated as before
and relative to solvent-treated samples. The experiment was
performed in duplicate with minimal variations.
[0106] The data shown in Table 6 indicate that pretreatment of
SB203580 resulted in a moderate relief of the growth inhibition
caused by MH161, Meth178, and MH210 and no influence in MC293 and
MH428 extracts. These results indicate that the antiproliferative
function of Meth114, MH161, Meth178, MH17, and MH210 are partially
dependent on p38. Furthermore, the p38 kinase pathway is not
involved in mediating the antiproliferative effect of mycelium
extracts MC293 and MH428. TABLE-US-00006 TABLE 6 Involvement of MAP
kinase p38 in the growth inhibition of K562 cells by mycelium
extracts Inhibition of K562 Extract Strain -- SB203580 (10 .mu.M)
Meth114 396 56 27.9 MH161 114 61.3 34.3 Meth178 173 56.9 40.2 MH210
202 53.8 43.3 MH17 241 68.8 46.3 Meth134 349 59.2 47.6 MC293 812
56.7 70.7 MH428 540 69 80.3
Example 8
Effect of Mycelium Crude Extracts on the Expression of
p210.sup.Bcr-Abl in K562 Cells
[0107] Expression of p210.sup.Bcr-Abl hybrid protein is correlated
with carcinogenesis in CML. A number of CML inhibitors have been
found to exert their effect by down-regulating the expression of
p210.sup.Bcr-Abl. We examined the ability of our mycelium crude
extracts to affect p210.sup.Bcr-Abl expression levels.
[0108] K562 cells were plated in T25 flasks at 1.times.10.sup.5
cells/ml. Twenty-four hours later, tyrphostin AG957 (a small
molecule inhibitor of the protein tyrosine kinase p145(abl) and its
oncogenic derivative p210(bcr-abl)) and mushroom extracts MH210,
Meth178, MH17 (3A), tyrphostin AG957, Meth134, MC293, and MH428
(3B) were added at 500 .mu.g/ml and 250 .mu.g/ml for 48 hours.
Western blotting was performed (as described in Materials and
Methods), in which monoclonal c-Abl antibody was used (Santa Cruz
Co., USA). Filters were stripped and reprobed with loading control,
monoclonal .beta.-actin antibody (Santa Cruz Co., USA).
[0109] FIGS. 3A and 3B demonstrate that exposure of K562 cells to
AG957 (10 .mu.M and 20 .mu.M), Meth134 (500 .mu.g/ml and 250
.mu.g/ml), MH428 (500 .mu.g/ml and 250 .mu.g/ml), and MH17 (500
.mu.g/ml and 250 .mu.g/ml) caused a reduction in Bcr-Abl protein
levels in K562 cells with varying potency (Meth 134 shows a
moderate reduction). In contrast, no significant effect on
p210.sup.Bcr-Abl levels was observed with MH210, MC293, and Meth178
mycelium extracts. Interestingly, mycelium crude extract MH428 was
the most potent of our selected crude extracts and caused a
dramatic reduction in both p210.sup.Bcr-Abl and normal p145 c-Abl
levels. Moreover, the reduction of p210.sup.Bcr-Abl and p145 c-Abl
was specific, because no effect on levels of .beta.-actin was
detected.
Example 9
Identification of Mycelium Crude Extracts that Selectively Inhibit
Proliferation of LNCaP Cells
[0110] Mushroom crude extracts were tested for their ability to
inhibit the growth of prostate cancer cells. We used the LNCaP cell
line established from a metastatic lesion of human prostatic
adenocarcinoma (Horoszewicz et al., 1983) as a representative of
prostate cancer cell line--it is androgen-dependent for
proliferation and expresses several markers of prostate cancer
including the prostate-specific antigen (PSA).
[0111] LNCaP cells were plated in 96-well plates at
1.5.times.10.sup.4 cells/well. Twenty-four hours later, mycelium
crude extracts were added for an additional 24 hours followed by
XTT determination according to manufacturer's instructions
(Biological Industries, Israel). Percentage of growth inhibition
was calculated in relation to solvent-treated samples. Experiments
were carried out in duplicate. The results are shown in Table 7.
Changes in % coefficient of variance (CV) among duplicate samples
were minimal. TABLE-US-00007 TABLE 7 Effect of LNCaP-selective
mycelium crude extracts on the growth of MH3924A, ABAE, HT-29 and
cell lines % of Growth Inhibition Ext Mushroom Strain Solvent LNCaP
MH3924A ABAE HT-29 Meth162 Kuehneromyces 114 Eth 60.4 26.0 25.1
18.9 mutabilis Meth215 Pleurotus eryngii 202 Eth 64.2 19.5 41.7
-1.5 Meth194 Pholiota aurivella 236 Eth 70.0 -7.0 -75.6 22.0
Meth134 Ganoderma 349 Eth 50.0 -15.5 -44.7 23.1 adspersum Meth114
Panus conchatus 396 Eth 52.4 11.5 27.8 4.3 Meth327 Trametes zonata
540 Eth 49.9 39.8 33.6 7.4
[0112] Mycelium crude extracts that inhibited LNCaP cell lines by
50% or more and also exhibited minimal growth inhibition against
other cell lines were designated as selective LNCaP inhibitors.
Table 7 shows a list of selective mycelium crude extracts (with
their designations and identification of the mushroom strain in the
first two columns) with percentage of growth inhibition applied to
a variety of cell lines. Growth inhibition was calculated compared
with solvent-treated samples.
[0113] Table 7 shows that a total of 6 mycelium crude extracts
exhibited selective activity against LNCaP cell line with varying
potency and selectivity. It is of interesting that all mycelium
extracts active against LNCaP were prepared using 70% ethanol
extraction.
Example 10
Resistance to Imatinib Mesylate (Gleevec) in CML Treatment
[0114] The tyrosine kinase p210 Bcr-Abl is the principal driving
force in CML development, therefore modulators of its activity or
function are expected to serve as CML therapeutics. Recently,
imatinib mesylate (STI571, Gleevec, Novartis, Basel, Switzerland)
was introduced as a powerful inhibitor of the tyrosine kinase
activity of p210 Bcr-Abl and, thereby, as an effective therapy for
CML. Although imatinib mesylate produces high rates of complete
clinical response in the chronic phase, resistance is universal and
clinical relapse develops rapidly in the advanced phase of CML
(Gorre et al., 2001). The majority of patients resistance to
imatinib therapy coincides with reactivation of the tyrosine kinase
activity of the Bcr-Abl fusion oncoprotein. This can result from
gene amplification and, more importantly, from point mutations that
disrupt the binding of imatinib to Bcr-Abl itself. More than 50%,
and perhaps as many as 90%, of patients with CML relapse have
Bcr-Abl point mutations in different amino acids scattered
throughout the Abl kinase domain such as mutations of Thr-315 in
the Abl kinase domain to Ile (T315I), the Tyr-253 to Phe (Y253F) or
the E255K mutation.
[0115] The murine wild-type (WT) BaF3 pro-B-lymphocyte cell line
depends on IL-3 for growth and viability, whereas the BaF3/p185,
expressing the oncogenic p185Bcr-Abl tyrosine kinase, became factor
independent. The p185 Bcr-Abl (WT) as well as p185 Bcr-Abl carrying
point mutations E255K or T315I were cloned into PSLXBcr-Abl vectors
and used to transform BaF3 cells. Stable BaF3/p185bcr-abl clones
were selected using appropriate antibiotics that were growing
adherent on ECM proteins (van der Kulp et al., 2001). BaF3 cell
lines were kindly provided by Dr. J. Duyster (Munich, Germany).
[0116] BaF3 cell lines carrying WT Bcr-Abl were sensitive to
treatment with Gleevec for both growth and phosphorylation of
Bcr-Abl. However, BaF3 cells carrying mutated Bcr-Abl were
resistant to Gleevec (Table 8 and FIG. 4). Table 8 shows that the
IC.sub.50 of Gleevec for BaF3 cell lines carrying WT Bcr-Abl was
about 0.7 .mu.M. However, the IC.sub.50 using the mutant Bcr-Abl
T315I was higher by about twenty fold (14 .mu.M). In contrast,
using extract of Trametes zonata mushroom in 100% dichloromethane
(DCM540), the IC.sub.50 with the two cell lines were comparable,
indicating that the mushroom extract is effective in inhibiting the
growth of both the wild-type and the mutation-carrying cell lines.
FIG. 4 shows that treatment with 1 .mu.M of Gleevec for 60 min
causes significant reduction in phosphorylated WT Bcr-Abl, while no
reduction in the level of phosphorylation was seen in mutated
Bcr-Abl. In contrast, DCM540 was equally effective in inhibiting
the phosphorylation of WT as well as mutated Bcr-Abl., indicating
that the extract of Trametes zonata may be useful for treatment of
CML and of Gleevec-refractory CML. TABLE-US-00008 TABLE 8 Growth
inhibition of BaF3/Bcr-Abl cell lines with organic extract of
Trametes zonata IC.sub.50 BaF3/p185 Bcr-Abl Compound BaF3/p185
Bcr-Abl (WT) T315I mutant Trametes zonata DCM organic 211 .mu.g/ml
180 .mu.g/ml extract (#540) Gleevec (STI-571) 0.7 .mu.M 14
.mu.M
[0117] K562, BaF3/Bcr-Abl WT, BaF3/Bcr-Abl T315I and BaF3/Bcr-Abl
E255K cell lines were seeded 4 mls/well in 6-well plates
5-6.times.10.sup.5 cells/ml. 24 hours post-plating, different
concentrations of the appropriate extract or drug were added.
Control cells were treated with 2% DMSO. Thus, cells were treated
with 1 .mu.M Gleevec and dichloromethane (DCM) extract of T. zonata
#540 at two concentrations: (A) at 500 .mu.g/ml and (B) at 1000
.mu.g/ml were added after 24 hours. Cells were treated for 60 min,
then collected and centrifuged at 5000 rpm for 5 min. Cell pellets
were lysed in lysis buffer {10 mM Tris, pH 7.4; 100 mM NaCl; 1 mM
EDTA; 1 mM EGTA; 1 mM NaF; 20 mM Na.sub.4P.sub.2O.sub.7; 2 mM
Na.sub.3VO.sub.4; 1% Triton-X100; 10% glycerol; 0.1% SDS; 0.5%
deoxycholate; 1 mM PMSF; 10 .mu.l protease inhibitor cocktail and
phosphatase inhibitor cocktail were added to every 1 ml lysate}.
After that, 40 .mu.g protein from each sample were separated on 8%
SDS-PAGE, and Western blot was performed according to the
manufacturer's instructions using phospho-c-Abl (Tyr245) Antibody
(Cell Signaling Technology Co.) and c-Abl monoclonal antibody
(Santa Cruz Biotechnology). FIG. 5 shows the inhibition of Bcr-Abl
tyrosine kinase phosphorylation in BaF3 cells expressing the
Bcr-Abl WT or the mutants Bcr-Abl T315I and Bcr-Abl E255K treated
with the tyrosine kinase inhibitor Gleevec or the #540 extract at
two different concentrations A and B. FIG. 6 shows the inhibition
of Bcr-Abl tyrosine kinase phosphorylation in K562 cells treated
with Gleevec (STI571) or the #540 extract at the same two different
concentrations as in FIG. 5.
[0118] Cell viability was determined using trypan blue exclusion
assay. Number of cells obtained relative to untreated samples at 48
hours post-treatment were used to calculate percentage of
inhibition and IC50 was calculated.
Discussion
[0119] CML is a malignancy of pluripotent hematopoietic cells
characterized by the presence of the Philadelphia (Ph) chromosome,
which results from reciprocal translocation between the long arms
of chromosomes 9 and 22 {(t(9;22) q34;q11)} resulting in the
creation of the fusion gene p210.sup.Bcr-Abl. The bcr-abl fusion
gene encodes a phosphoprotein (p210) that functions as a
disregulated (abnormal) protein tyrosine kinase and predisposes the
cell to become neoplastic. The presence of active p210.sup.Bcr-Abl
renders CML cells resistant to apoptosis and delays
differentiation.
[0120] Medicinal mushrooms have been an important source of
therapeutic substances for the treatment of various human diseases.
Antitumor activities from mushrooms were described by many reports
(Mizuno, 1999; Wasser, 2002). In most cases, activity was due to
high-molecular-weight polysaccharides with a molecular weight of
200-400,000 D. The antitumor activity of HMW polysaccharides was
attributed to the immune-modulation function or enhancement
properties of the immune system, and not to direct influence on the
tumor cells. In the present invention, we attempted to identify
mycelium crude extracts that directly show antitumor effects
against cancer cells. Furthermore, we used various organic mixtures
to prepare our mycelium crude extracts in an attempt to enrich them
with low molecular weight moieties that can easily penetrate the
cell wall.
[0121] Mycelium crude extracts prepared from our culture collection
of Higher Basidiomycetes (these mushrooms are also available
elsewhere) were evaluated for their ability to selectively inhibit
the growth of the human CML 562 cell line. Using submerged
conditions, we cultivated 42 species of Higher Basidiomycetes
mushrooms and prepared 168 mycelium crude extracts by a variety of
extraction methods (Table 1). Initially, we evaluated the ability
of our mycelium crude extracts to inhibit the growth of K562 cells
by more than 50% compared with the solvent-treated cells. Data
shown in Table 2 illustrate that 44 extracts were active in
inhibiting K562 cells. However, only 8 were found to exhibit a
selective effect against K562 cells (Table 3). IC.sub.50 values for
the K562-selective extracts ranged from 250 to 500 .mu.g/ml. Most
of the selective extracts showed partial or complete cytostatic
activity and only MH161 showed cytotoxic activity against K562
cells (FIG. 1).
[0122] The observed antiproliferative activity of the selective
K562 mycelium crude extracts was attributed to induction of
apoptosis by most extracts, as determined by Apostain ELISA assay.
Our results indicate that growth inhibition was caused by the
induction of the apoptosis pathway and not as a result of necrosis.
However, higher concentration or longer exposure time can also
cause death from necrosis, as indicated by a reduction in DNA
condensation determined by the Apostain ELISA assay (Table 4). Data
obtained by monitoring PARP cleavage as an indication of apoptosis
illustrated that only our control AG957 and higher concentration of
MH428, and not the other mycelium crude extracts could cause a
significant cleavage of PARP. This might be explained by the fact
that Apostain ELISA assay is a more sensitive measurement than the
PARP cleavage assay. In addition, PARP cleavage assay measures a
very late event in the apoptosis pathway, while Apostain measures a
much earlier event. Thus, longer exposure times might be required
to observe signs of apoptosis by the PARP cleavage assay.
[0123] Table 9 summarizes various characteristics of the extracts
herein designated MH428, Meth178. MH17, MH210, MC293, Meth134,
Meth114 and MH161. TABLE-US-00009 TABLE 9 Characteristics of
K562-selective mycelium crude extracts MH428 Meth178 MH17 MH210
MC293 Meth134 Meth114 MH161 Selective Growth + + + + + + + +
Inhibition of K562 cells Down-regulation ++ - +/- - - - NT NT of
Bcr-Abl Induction + +++ ++ - - - - - of erythroid differentiation
PARP cleavage + - - - - - NT NT Apostain ++++ + + + +++ ++ NT NT
Viability Cytostatic Cytostatic Cytostatic Cytostatic Cytostatic
Cytostatic Cytostatic Cytostatic "+": positive activity; "-":
negative activity by. NT: not tested. "++" and "+++" "indicate
greater potency of positive response.
[0124] K562 cells are pluripotent cells that are able to
differentiate along a megakaryocytic, erythroid, or, to a lesser
extent, monocytic lineage. Erythroid differentiation can be induced
by a number of compounds including hemin and butyric acid (Villeval
et al., 1983). K562 selective mycelium crude extracts were
evaluated for their ability to induce erythroid differentiation in
K562 cells. Three extracts, Meth178, MH17, and MH428, showed
significant ability to induce hemoglobin production by a factor of
11.9, 4.2, and 2.1, respectively.
[0125] It is interesting that there was no correlation between the
ability to induce apoptosis and to induce differentiation (Table
8). Mycelium crude extract Meth178 was the most potent in inducing
erythroid differentiation. However, it was only a moderate inducer
of apoptosis as determined by Apostain, and it failed to activate
PARP cleavage. Conversely, MH428 extract showed weak activity in
inducing erythroid differentiation, while it displayed the most
significant apoptosis-inducing activity, as determined by both
Apostain ELISA and PARP cleavage assays. Furthermore, mycelium
extracts MC293 and Meth134 showed significant apoptosis activity as
determined by Apostain ELISA assay, but failed to induce erythroid
differentiation. Thus, the two activities are separable. At this
stage, the molecular mechanism by which these mycelium extracts
cause the induction of differentiation is not clear. It is worth
noting that the hemoglobin produced in K562 is a fetal hemoglobin,
mainly .gamma.-globin. Increased expression of the endogenous
.gamma.-globin gene is a realistic approach to therapy of
.beta.-globin disorders such as sickle cell anemia and
.beta.-thalassemia (Nagel et al., 1985; Labie et al., 1985). Thus,
our mycelium extracts, especially Meth178, has the potential to
serve as a therapy for .beta.-globin disorders.
[0126] The fusion protein p210.sup.Bcr-Abl plays a principal role
in CML carcinogenesis. Consequently, down-regulation of
p210.sup.Bcr-Abl is an appealing strategy for developing
chemotherapeutics for the treatment of CML. We evaluated the
ability of our mycelium crude extracts to lower expression levels
of p210.sup.Bcr-Abl. Our data revealed that MH428 significantly
inhibited the expression of p210.sup.Bcr-Abl, which was moderately
inhibited by MH17. In contrast, the other extracts exerted a minor
effect on the expression levels of p210.sup.Bcr-Abl. The mechanism
leading to down-regulation of p210.sup.Bcr-Abl by MH428 and MH17 is
unclear. However, it is worth investigating whether this
downregulation is caused by inhibition of the transcription of
p210.sup.Bcr-Abl or by affecting protein degradation. A number of
compounds such as geldanamycin and radicicol exhibited
antiproliferative effect against K562 cells, mediated in part by
downregulation of p210.sup.Bcr-Abl (Nimmanapalli et al., 2001;
Shiotsu et al., 2000). Immune precipitation analysis showed that
p210.sup.Bcr-Abl formed multiple complexes with heat shock protein
90 (Hsp90), some containing p23 and others Hsp70. The presence of
geldanamycin (GA) decreased the association of p210.sup.Bcr-Abl
with Hsp90 and p23 and increased its association with the
chaperones Hsp70 and p60Hop. Loss of Hsp90/p23 association and
acquisition of Hsp70/p60Hop preceded GA-induced degradation of
p210.sub.Bcr-Abl (An et al., 2000).
[0127] The MAPK pathway includes the extra cellular
signal-regulated kinase (ERK), the c-Jun N-terminal kinase (JNK),
and the p38 kinase modules (Cross et al., 2000). Such signaling
pathways regulate multiple biological activities, including cell
proliferation, differentiation, and survivals (Cobb, 1999). The
bulk of the evidence suggests that activation of the ERK pathway
increases the cell death threshold (Ishikawa and Kitamura, 1999).
Conversely, activation of the JNK and p38 kinase cascades is
generally associated with enhanced activation of the apoptosis
program (Ichijo et al., 1997). In an attempt to elucidate the
mechanism of action of our mycelium crude extracts, we used potent
and selective pharmacological inhibitors to investigate the role of
p38 in mediating the antitumor effect of our mycelium crude
extracts. Data shown in Table 6 argue that inhibition of MAPK p38
partially relieves growth inhibition caused by some mycelium crude
extracts such as Meth178, MH17, Meth114, MH161, and MH210 but not
by MC293 and MH428. This argues that MAPK p38 is not the principle
mediator of the antiproliferative function of our K562-selective
mycelium crude extracts. Similar analysis using additional
pharmaceutical inhibitors targeting other second messenger pathways
are required for the elucidation of the molecular pathways involved
in mediating the anti-CML effect of our mycelium crude
extracts.
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