Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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USE OF SIRAMESINE IN THE TREATMENT OF CANCER
Field of invention
The present invention relates to the use of Siramesine for the preparation of
medicaments useful for the treatment of cancer.
Background of the invention
to Tumor cells have often acquired resistance towards classical treatment
modalities,
such as classical caspase-mediated apoptosis. However, there is still a large
unmet
need for novel efficient drugs for the treatment of cancers.
It is known that sigma2 receptors are upregulated in cells representing many
different
types of cancers (Bem et al. 1991, Cancer Res. 51, 6558; Vilner et al. 1995,
Cancer
Res. 55, 408), and fwthermore that sigma2 receptor ligands may inhibit cell
proliferation and induce apoptosis in tumor cells (Brent & Pang, 1995, Eur. J.
Pharmacol. 278, 151; Crawford & Bowen, 2002, Cancer Res. 62, 313).
Additionally,
it has been shown that sigma 2 ligands may potentiate the activity of
antineoplastic
drugs (Crawford & Bowen, 2002, Cancer Res. 62, 313).
2o International Patent Publication No. WO 92/22554 describes a series of
sigma
receptor ligands considered useful for the treatment of a range of psychic and
neurological disorders. The structure activity relationship of these compounds
has
been further investigated by Perregaard, J. et al., J. Med. Chem., 1995, 38,
11, p.
1998-2008.
Among numerous other compounds WO 92/22554 discloses the compound 1'-[4-[1-
(4-fluorophenyl)-1H indole-3-yl]-1-butyl]-spiro[isobenzofuran-1(34'-
piperidine]
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2
(Siramesine), the novel use of which is the subject of the present invention.
We have now, surprisingly found that Siramesine is significantly more potent
in its
anti-carcinogenic activities, than reference sigma2 ligands.
Description of the invention
According to the present invention a medicament for the treatment of cancer is
provided.
Sigma2 receptor ligands have been known to induce apoptosis in cancer cells of
different origin. We have now surprisingly found that Siramesine of the
invention,
when used alone is more potent in inducing apoptosis in cancer cells than
sigma2
active reference compounds such as haloperidol. Furthermore, we have observed
a
significant synergistic effect of Siramesine when used in combination with
known
chemotherapeutic compounds such as etoposide, doxorubicin, staurosporin,
vincristine and tamoxifen. The present invention therefore demonstrate that
Siramesine may be used for the manufacture of pharmaceutical compositions for
the
treatment of cancer, and that such compositions may be used in combination
with
2o other chemotherapeutic cancer drugs, and/or in combination with
radiotherapy.
Tn the combined use of Siramesine and anticancer chemotherapeutic drugs, the
drugs
may be administered simultaneously or sequentially.
In one embodiment, the present invention relates to the use of Siramesine or a
pharmaceutically acceptable salt thereof, together with a chemotherapeutic
drug in a
synergistic effective dose for the preparation of a pharmaceutical composition
as
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3
above, which is adapted for simultaneous administration of the active
ingredients. In
particular, such pharmaceutical compositions may contain the active
ingredients
within the same unit dosage form, e.g. in the same tablet or capsule. Such
unit dosage
forms may contain the active ingredients as a homogenous mixture or in
separate
compartments of the unit dosage form.
In another embodiment, the present invention relates to the use of Siramesine
or a
pharmaceutically acceptable salt thereof together with a chemotherapeutic drug
in a
synergistic effective dose for the preparation of a pharmaceutical composition
or kit
1o as above, which is adapted for sequential administration of the active
ingredients. In
particular, such pharmaceutical compositions may contain the active
ingredients in
discrete unit dosage forms, e.g. discrete tablets or capsules containing
either of the
active ingredients. These discrete unit dosage forms may be contained in the
same
container or package, e.g. a blister pack.
As used herein the term kit means a pharmaceutical composition containing each
of
the active ingredients, but in discrete unit dosage forms.
As used herein, the term "synergistic effective dosage" means the dosages of
Siramesine and the chemotherapeutic agent at which their combined use provides
a
2o synergistic effect, preferably the maximal obtainable synergistic effect.
The pharmaceutical composition or kit of the invention may be adapted for
simultaneous administration of the active ingredients or for sequential
administration
of the active ingredients, as described above.
More specifically, the present invention relates to the novel use of
Siramesine having
the general formula
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4
0
\ N
a
i
F
or pharmaceutically acceptable salts thereof for the preparation of a
medicament for
the treatment of cancer.
Moreover, the present invention relates to a method for the treatment of
cancer
comprising administering to an individual in need thereof a pharmaceutically
acceptable amount of Siramesine or a pharmaceutically acceptable salt thereof.
In a further aspect the invention relates to a method for treatment of cancer
1o comprising administering Siramesine or a pharmaceutically acceptable salt
thereof to
an individual to be treated with or undergoing treatment with a
chemotherapeutic
agent.
According to the invention the compound 1'-[4-[1-(4-fluorophenyl)-1H indole-3-
yl]-
15 1-butyl]-spiro[isobenzo-furan-1(3I~,4'-piperidine] (Siramesine) may be used
as the
base of the compound or as a pharmaceutically acceptable acid addition salt
thereof or
as an anhydrate or hydrate of such salt. The salts of the compound used in the
invention are salts formed with non-toxic organic or inorganic acids.
Exemplary of
such organic salts are those with malefic, fumaric, benzoic, ascorbic,
succinic, oxalic,
?o bis-methylenesalicylic, methanesulfonic, ethane-disulfonic, acetic,
propionic, tartaric,
salicylic, citric, gluconic, lactic, malic, mandelic, cinnamic, citraconic,
aspartic,
stearic, palinitic, itaconic, glycolic, p-amino-benzoic, glutamic, benzene
sulfonic and
theophylline acetic acids, as well as the 8-halotheophyllines, for example 8-
bromo-
theophylline. Exemplary of such inorganic salts are those with hydrochloric,
'S hydrobromic, sulfuric, sulfamic, phosphoric and nitric acids. Preferably
the compound
is used as the fumarate or the hydrochloric salt.
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The fumarate of 1'-[4-[1-(4-fluorophenyl)-1H indole-3-yl]-1-butyl]-
spiro[isobenzo-
furan-1 (3I~,4'-piperidine] can be prepared as described in Perregaard, J. et
al., J.
Med. Claem., 1995, 38, 11, 1998-2008 (compound 14f) and the base and other
pharmaceutically acceptable salts may be obtained there from by standard
procedures.
Thus the acid addition salts according to the invention may be obtained by
treatment
of 1'-[4-[1-(4-fluorophenyl)-1H indole-3-yl]-1-butyl]-spiro[isobenzo-furan-
1(3I~,4'-
piperidine] with the acid in an inert solvent followed by precipitation,
isolation and
optionally re-crystallisation by known methods and if desired micronisation of
the
crystalline product by wet or dry milling or another convenient process, or
preparation
of particles from a solvent-emulsification process.
Precipitation of the salt is preferably carried out in an inert solvent, e.g.
an inert polar
solvent such as an alcohol (e.g. ethanol, 2-propanol and n-propanol).
According to the invention, 1'-[4-[1-(4-fluorophenyl)-1H indole-3-yl]-1-butyl]-
spiro[isobenzofuran-1(3I~,4'-piperidine] or a pharmaceutically acceptable salt
thereof
may be administered in any suitable way e.g. orally or parenterally, and it
may be
presented in any suitable form for such administration, e.g. in the form of
tablets,
2o capsules, powders, syrups or solutions or dispersions for injection.
Preferably, and in
accordance with the purpose of the present invention, the compound of the
invention
is administered in the form of a solid pharmaceutical entity, suitably as a
tablet or a
capsule or in the form of a suspension, solution or dispersion for injection.
Methods for the preparation of solid pharmaceutical preparations are well
known in
the art. Tablets may thus be prepared by mixing the active ingredients with
ordinary
adjuvants and/or diluents and subsequently compressing the mixture in a
convenient
tabletting machine. Examples of adjuvants or diluents comprise: corn starch,
lactose,
talcum, magnesium stearate, gelatine, lactose, gums, and the like. Any other
adjuvant
or additive such as colourings, aroma, preservatives, etc. may also be used
provided
that they are compatible with the active ingredients.
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As used herein, the term "composition" is intended to encompass a
product comprising the specified ingredients in the specific amounts, as well
as any
product which results, directly or indirectly, from combination of the
specific
ingredients in the specified amounts.
The pharmaceutical compositions containing the active ingredient may
be in a form suitable for oral use, for example, as tablets, troches,
lozenges, aqueous
or oily suspensions, dispersible powders or granules, emulsions, hard or soft
capsules,
or syrups or elixirs. Compositions intended for oral use may be prepared
according to
any method known to the art for the manufacture of pharmaceutical compositions
and
such compositions may contain one or more agents selected from the group
consisting
of sweetening agents, flavoring agents, coloring agents and preserving agents
in order
to provide pharmaceutically elegant and palatable preparations. Tablets
contain the
active ingredient in admixture with non-toxic pharmaceutically acceptable
excipients
which are suitable for the manufacture of tablets. These excipients may be for
example, inert diluents, such as calcium carbonate, sodium carbonate, lactose,
calcium phosphate or sodium phosphate; granulating and disintegrating agents,
for
example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or
alginic
acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or
acacia, and
lubricating agents, for example, magnesium stearate, stearic acid or talc.
The compositions of the invention may be used for treatment of cancer in
mammals,
preferably in humans.
Siramesine or a salt thereof for use of the invention is most conveniently
administered
orally in unit dosage forms such as tablets or capsules, containing the active
ingredient (calculated as the free form) in an amount from about 0.01
~g/kg/day to
100 mg/kg/day, preferably 0.01 ~,g/kg/day to 30mg/kg/day body weight, most
preferably 0.5 mg/day/kg to 7.0 mg/day/kg body weight.
When siramesine is combined with other compounds in order to obtain an
increased
effect, or in order to allow for the use of a subnormal dose of the other
compound, to
minimize side effects, then subnormal doses of siramesine and/or the other
compound
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7
may be used for the treatment. Calculation of patient specific doses is
routine practice
for those skilled in the art.
The pharmaceutical compositions and methods provided in the present invention
are
particularly deemed useful for the treatment of cancer including solid tumors
such as
skin, breast, brain, cervical carcinomas, testicular carcinomas, etc.. More
particularly,
cancers that may be treated by the compounds, compositions and methods of the
invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma,
fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma,
lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell,
undifferentiated
l0 small cell, undifferentiated large cell, adenocarcinoma), alveolar
(bronchiolar)
carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma,
mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma,
adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,
leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma,
gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma,
carcinoid tumors, I~arposi's sarcoma, leiomyoma, hemangioma, lipoma,
neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous
adenoma, hamartoma, leiomyoma); Genitourinary tract: kidney (adenocarcinoma,
Wilin's tumor [nephroblastoma] , lymphoma, leukemia), bladder and urethra
(squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma),
prostate
(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma,
teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma,
fibroma,
fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular
carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular
adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma,
malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant
lymphoma (reticulum cell sarcoma), multiple mycloma, malignant giant cell
tumor
chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma,
chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors;
Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis
deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain
(astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma],
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glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma,
congenital
tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma);
Gynecological:
uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical
dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous
cystadenocarcinoma, unclassified carcinoma] , granulosa-thecal cell tumors,
Sertoli-
Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell
carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoina,
melanoma),
vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma
(embryonal
rhabdomyosarcoma), fallopian tubes (carcinoma); Hematologic: blood (myeloid
to leukemia [acute and chronic] , acute lymphoblastic leukemia, chronic
lymphocytic
leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic
syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma];
Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma,
Karposi's
sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids,
psoriasis;
and Adrenal lands: neuroblastoma.
Thus, the term "cancerous cell" as provided herein, includes a cell afflicted
by any
one of the conditions identified above, and the term "cancer" includes but is
not
limited to any of the conditions identified above. Any of the above mentioned
conditions are to be considered as single embodiments, and the compositions
directed
to the treatment of each condition may accordingly be claimed individually or
be
included in the claimed group when the term cancer is used.
Whenever any of the above cancer indications are mentioned in relation to use
of
siramesine, a pharmaceutical composition, a kit, a method of treatment it is
intended
to be an individual embodiment. Accordingly, each of the indications specified
above
z5 may individually be claimed together with said use of siramesine,
pharmaceutical
composition, kit, method of treatment and method for the identification of
compounds
useful for treatment.
Experimental procedure
;o
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Cell culture
Marine fibrosarcoma cells WEHI-S, Wn902, Wn912, and WEHI-R4 are normal,
vector control, Hsp70 overexpressing, and hTNF resistant cells, respectively.
Other
cell types tested include: Human breast cancer cell types MCF7-S 1 and MDA-MB-
468, and non-tumorigenic immortalized breast epithelial cells HBL100. MCF7
casp3.1 and -3.3 and neo2 are single cell clones expressing caspase3 and
vector.
MCF7 pCEP, -Bcl2 WT, -Bcl2 NT, -Bcl2 Acta, and -Bcl2 Cb5 are single cell
clones
expressing vector, wildtype Bcl2, cytoplasma localized Bcl2, mitochondria
localized
Bcl2, and ER localized Bcl2 (Maria H~yer Hansen, Apoptosis Laboratory, Danish
Cancer Society). Human neuroblastoma cell line SK-N-MC cells (ATCC, USA). In
addition human cervix carcinoma cell lines HeLa (kindly provided by Dr. J.
Lukas,
Danish Cancer Society) and ME180 were tested. HEK293-A, prostata cancer cell
line
PC3, and non-tumorigenic immortalized prostata epithelial cell line PNT1A were
tested as well. Non-transformed NIH3T3 marine fibroblasts were kindly provided
by C.
Holmberg (University of Copenhagen, Denmark). Fibroblasts were transduced with
pBabe-
puro mock, -SV40LT, -v-Ha-ras, -c-src as described (Fehrenbacher et al, 2004).
Cells were
propagated in DMEM (Invitrogen, Paisly, UK) supplemented with 10 % heat-
inactivated calf serum (Biological Industries, Beit Haemek, Israel), 0,1 mM
non-
essential amino acids (Invitrogen), and antibiotics or RPMI-1640 (Invitrogen)
supplemented with 6 % heat-inactivated calf serum and antibiotics at 37
°C in a
humidified air atmosphere with 5 % C02. Cells were repeatedly tested and found
negative for mycoplasma by hoecsht staining (H-33342, Molecular Probes,
Eugene,
OR).
?5
f3HlSiramesine binding to cell membranes or tissue membranes
The presence of Siramesine-sensitive binding sites on tissue prepared from
cell lines
so as indicated above or tissue from rodent or human were demonstrated using
[3H]Lu
28-179 (Siramesine) binding assay described previously S~by, K. et al.,
Neuropharmacol., 2002, 43, 95-100. In brief, cells were cultured as described,
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harvested in phosphate buffered saline using a cell scraber and centrifuged
(1000 x g,
10 min). The resulting pellets were used for [3H]Lu 28-179 binding assays as
described in S~by et al., 2002.
5 Apoptotic stimuli
The following apoptotic stimuli were tested: Siramesine (Lu-28-179),
Siramesine
analogs: 28131M, 28134M, 292880, 32160F, 32124C, and 32168F, and haloperidol
(H. Lundbeck A/S, Copenhagen, Denmark), human TNF-a (Strathmann Biotech
10 Gmbh, Germany), thapsigargin, etoposide, doxorubicin, staurosporine,
vincristine,
tamoxifen (SIGMA-Aldrich, St Louis, MO), concanamycinA (Alexis Biochemicals,
San Diego, CA).
Pharmacological inhibitors and drugs
The following protease inhibitors were used: zVAD-fmk, DEVD-fink
(Bachem Bubendorf, Switzerland), DEVD-CHO (Neosystems, Strasbourg, France),
LEND-CHO, zFA-fmk (Enzyme System Products, Livermore, CA), CA-074-Me
(Peptides International, Louisville, K~, APC11138 (Celera Applied Biosystems,
Foster City, CA, USA), Pepstatin A, PD150606, and calpain inhibitor I (CI)
(Calbiochem, La Jolla, CA), TPCI~ (Boehringer Mannheim), pefabloc (AEBSF)
(Ruche Diagnostics, DID).
The following antioxidants were used: Butylated hydroxyanisole (BHA), a-
tocopherol, y-tocopherol, glutathione ethyl ester (GSH), N-acetyl-cysteine
(NAC)
(SIGMA-Aldrich, St Louis, MO). Cells were pre-incubated with inhibitors and
antioxidants 1h prior to drug.
In addition, the effect on siramesine cytotoxicity of the following drugs were
tested
sigma2-receptor antagonists BD1047 (Tocris) and AC927 (gift of W. Bowen, Brown
University, USA), 3-methyl-adenine, ActinomycinD, cyclohexamide and
cholesterol.
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Detection of cell death
Cell viability
Cell viability was measured using the MTT reduction assay. Conversion of the
tetrazolium salt 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrasodiumbromide
(MTT,
SIGMA-Aldrich) into the blue-colored formazan product was assessed
spectrophotometrically by absorption at 570 nm using a Versaznax microplate
reader
(Molecular Devices). The survival rate was determined as the percentage of
untreated
cells or inhibitor-treated cells: Cells were seeded into 96-well plate,
containing 200
~.L media, and treated the following day with drugs. The effect on cell
viability was
assessed 24-60h after drug addition by removal of 100 ,uL media and addition
of 25
~.L MTT solution (1 mg/mL in PBS, sterile filtered). After 3h incubation 37
°C in
darkness, the cells were permeabilized using 100 ~tL solubilization buffer (20
% SDS
in 50 % dimethylformamide solution) and analyzed on spectrophotometer the
following day.
Cell death ahd cytotoxicity
Cell death and cytotoxicity was estimated using the lactate dehydrogenase
(LDH)
release assay (Roche, Mannheim, Germany). Rupture of the plasma membrane
2o releasing cytoplasmic LDH into the culture media is a measure of
cytotoxicity or cell
lysis. The enzymatic acticity of LDH resulting in oxidation of lactate to
pyruvate and
reduction of NAD+ to NADH + H+ was measured spectrophotometrically by the
conversion of tetrazolium salt (yellow) to formazan salt (red). Cells were
seeded and
treated as described for MTT assay. Upon analysis, 50 ,uL media was removed
and
and mixed with 50 p,L reaction buffer. The remaining media was removed and
cells
lysed in 1 % Triton X-100 for 20 min at 37 °C. The LDH content was
measured in the
cell lysate as well, and an estimate of % LDH release was calculated:
CytOtOXlclty (% LDH release= LDHin medial ~DHin media+ LDHin lysate) x 100
Nuclea~~ cohderasatiofz
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The cell death mode was assessed by the type of nuclear condensation and
performed
by hoechst staining (cell permeable Hoechst 33342, SIGMA-Aldrich) of drug-
treated
cells. Non cell permeable Sytox Green (Molecular Probes) was used to assay
loss of
membrane integrity and compared with nuclear morphological changes observed
with
Hoechst. Cells were incubated with 10.000 x dilution of 25 mg/mL hoechst and
/or
10.000x dilution of 5 mM sytox green for 5 min at 37°C where after type
of nuclear
condensation and possible co-stain was analyzed using an inverted Olympus
microscope IX-70.
to
Gel elect~ophor~esis fog detection ofDNA ladde~~ihg. Cells were harvested and
incubated at 50 °C overnight in 120 ~L lysis buffer (100 mM NaCl, 100
mM Tris-
HCl (pH ~.0), 25 mM EDTA, 0,5% SDS and 100 ~.g/mL proteinase K). The samples
were precipitated with 6M NaCl and centrifuged at 13.000 rpm for 5 min at
4°C.
Genomic DNA was subsequently precipitated from the supernatant by 2,5 volumes
of
96 % ethanol. After centrifixgation at 20.000 rpm for 10 min at 4° C
and rinsing with
70 % ethanol, the pellets were dissolved in 10 mM Tris-HCl (pH ~.0) and 1 mM
EDTA containing 1 ~,g/mL Rnase A. The samples were incubated at 37° C
for 1,5 h,
and the DNA concentration was estimated from absorbance (A) at 260 nm. DNA (5
zo ~.g/lane) was electrophorezed on 1,5% agarose gel and visualized by
ethidium
bromide staining.
Caspase and Cathepsin activity
?5
To measure cytosolic cystein cathepsin enzyme activity, subconfluent cells
seeded in
24-well plates were treated with an extraction buffer (250 mM sucrose, 20 mM
HEPES, 10 mM KCI, 1,5 mM MgCl2, 1mM EDTA, 1 mM EGTA, 1mM pefablock;
pH 7,5) containing 20 ,ug/mL digitonin for 12-15 min on ice. To measure total
cellular
.o cystein cathepsin enzyme activity, cells were treated with the above
extraction buffer
containing 200 ~glmL digitonin for 12-15 min on ice. For analysis of caspase
3!7-like
activity subconfluent cells were treated with caspase extraction buffer (0,5 %
Triton
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13
X-100, 25 mM HEPES, 5 mM Mg2Cl, 1 mM EGTA, 1 mM pefablock, pH 7,5) for 20
min on ice. The caspase 3/7-like activity and cystein cathepsin activities
were
estimated by adding one volume of 20 ~,M Ac-DEVD-AFC (Biomol) in caspase
reaction buffer (100 mM HEPES, 20 % glycerol, 0,5 mM EDTA, 0,1 % CHAPS, 5
mM DTT, 1 mM pefablock, pH 7,5) or 20 ~,M zFR-AFC (Enzyme System Products)
in cathepsin reaction buffer (50 mM sodium acetate, 4 mM EDTA, 8 mM DTT, 1 mM
pefablock, pH 6,0), respectively. The VmaX of the liberation of AFC
(excitation 400
nm, emission 489 nm) was analyzed over 20 min at 30°C using a
Spectramax Gemini
fluorometer (Molecular Devices, Sunnyvale, CA).
Lysosomal stability assay, cell culture
Cells were exposed to the lysomotropic weak base and metachromatic
fluorochrome
acridine orange (AO, Molecular Probes) that accumulates in acidic
compartments.
When highly concentrated in acidic lysosomes AO shows red fluorescence, but
upon
relocalozation green fluorescence in cytoplasma. In order to monitor lysosomal
integrity, subconfluent drug-treated cells were exposed to 0,1-0,5 q,g/mL AO
for 3h at
37 °C. Cells were either evaluated using an inverted Olympus microscope
IX-70 or
detached from the substratum and analyzed by flow cytometry using a FACSort
(Becton Dickinson, San Jose, CA) with an argon ion laser with an output
wavelength
of 488 nm and analyzed using CELLQuest software.
Lysosomal stability iiz vitro assay
MCF-7 cells seeded in 14 cm plates were loaded with Fe-dextran for 9h,
followed by
16 h clearance in culture medium. Subsequently, the cells were washed in PBS
and
detached from the substratum. The cell pellet was resuspended in SCA buffer
(20 mM
Hepes KOH, 10 mM KCI, 1,5 mM MgaCl, 1 mM EDTA, 1 mM EGTA, 250 mM
sucrose, pH 7,5) and equilibrated on ice for 20 min. The cell pellet was
homogenized
by 150-200 strokes using a Teflon pestle. After centrifugation for 5 min at
750g, the
supernatant was isolated and the centrifugation step was repeated. The
remaining
supernatant was transferred to a column contained in a magnetic field. The
lysosomal
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14
fraction was washed twice and eluted from the column. The release of lysosomal
cathepsins was subsequently measured in 25 ,uL lysosome solution in black
costar 96
well plates as described for cathepsin activity measurements. After 1,5 hour
reaction
at 37 C, the VmaX of the liberation of AFC was analyzed over 20 min at
30°C using a
Spectramax Gemini fluorometer (Molecular Devices, Sunnyvale, CA).
Immunoblot analysis and Immunofluorescence
to
The primary antibodies used included mouse monoclonal antibodies against
cathepsin
B (Oncogene Research Products, Boston, MA), cathepsin L (Transduction
Laboratories, Lexington, KY), cytochrome c (BD Pharmingen), and glyceraldehyde-
3-phosphate dehydrogenase (GAPDH, Biogenesis, Poole, UI~.), Hsp70 (2H9; kindly
15 provided by Boris Margulis, St. Petersbrug, Russia) and rabbit polyclonal
cathepsin D
(DAKO corporation, CA), and AIF (apoptosis lab, Danish Cancer Research). For
immunoblot analysis, proteins were separated by 6-12% SDS-PAGE and transferred
to a nitrocellulose membrane. After primary antibody incubation 1h at RT or
4°C
overnigh the blot was incubated with secondary horseradish-peroxidase-
conjugated
2o goat anti-mouse or -rabbit IgG antibody 1h at RT. Subsequent protein
detection was
performed using ECL or ECL plus (Amersham Biosciences, Buckinghamshire,
England).
To visualize cathepsins, AIF, and cyt C, cells were fixed and permeabilized in
-20°C
25 methanol for 10 min at 25°C. After preblocking with 5% goat serum
(in 1% BSA,
0,3% Triton X-100 in PBS) for 20 min, cells were incubated with primary
antibodies
(in 0,25% BSA, 0,1% Triton X-100 in PBS) as indicated for 1,5h. After 1h
incubation
with secondary antibodies Alexa Fluor-488 or-546-conjugated anti-mouse IgG or
anti-rabbit IgG (Molecular Probes) cells were washed three times in 0,05%
Tween 20
3o in PBS and mounted using Prolong Antifade Kit (Molecular Probes).Confocal
analysis was conducted using a Zeiss Axiovert 100M microscope with LSM 510
software.
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In vivo experiments
WEHI-R4 cells (5 x 106) were implanted subcutaneously in the back of
5 immunocompetent female BALB/c mice. Siramesine treatment commenced two days
prior to tumor implant. Mice were divided into groups (5-9/group) and
administered
peroral 200 ~L 1) vehicle (0,5 % methylcellulose 15 in 0,9 % NaCl solution),
2) 100
mg/kg/day Siramesine in suspension (in 0,5 % methylcellulose 15 in 0,9 % NaCI
solution) 7 days per week. For combinational in vivo studies, 50 mg/kg
siramesine
to was administered 7 days/week in combination with a single dose of etoposide
(i.p.) up
to 30 mg/kg (administered on day one). Tumor volumes were estimated by the use
of
a caliper. The effect of the drugs on the tumor growth was monitored over 14-
21 days
before the mice were sacrificed.
Results
is
In a dose dependent manner, Siramesine effectively induces programmed cell
death in
cultured tumor cell lines of various origins, including cell lines originating
from
tumors of the prostate, breast and cervix. The sensitivity of cells towards
Siramesine
is increased upon the oncogenic transformation by ras or src, indicating that
2o siramesine has the desired ability of a cancer drug to induce its effects
selectively on
the cancer cells with only limited effects on normal cells.
Furthermore, when tested against reference sigma ligands such as Haloperidol
and
pentazocine in WEHI-S and MCF7 cells, siramesine was a significantly more
potent
inducer of apoptosis than Haloperidol and pentazocine was.
The mode of death induced by siramesine is caspase-independent based on the
nuclear
morphological changes during the death process, the absence of protection by
pharmacological caspase inhibitors, and the absence of effector caspase
activation
Instead, release of lysosomal cathepsins into the cytosol seems to be involved
in the
execution of the death process. This finding is based on imrnmunohistochemical
3o stainings of lysosomal cathepsin B and L as well as the in vitro release of
cathepsins
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16
from purified lysosomes. Also, the use of pharmacological inhibitors of
cathepsins
can attenuate the death induced by siramesine.
Tumor cells that were protected against most other anti-cancer drugs by
ectopic
expression of Bcl-2 were effectively killed by siramesine.
Whereas some chemotherapeutics activate a p53-dependent death pathway,
siramesine does not activate p53. This indicates that the death pathway
differs
significantly from that induced by DNA damaging agents (such as etoposide), a
result
that supports the data showing a broad cancer indication range for siramesine,
since
the p53-dependent death pathway is often compromised in cancer. This is
further
1o supported by the above mentioned observation that Siramesine induced tumor
cell
death is not inhibited by Bcl-2.
Importantly, siramesine was well tolerated in vivo and showed an anti-
tumorigenic
effect in a syngenic tumor xenograft model in BALB/c mice. In addition, a
15 combinational effect of siramesine and etoposide was observed in vivo as
compared
to groups single-treated with siramesine and etoposide respectively.
Furthermore,
siramesine worked in a synergistic manner together with etoposide,
doxorubicin,
staurosporin, vincristine and tamoxifen in induction of cell death in WEHI-S
cells.
These results show that siramesine is a novel anti-cancer drug which is
especially
2o effective as compared to other reference sigma ligands, and which may be
used alone
or in combination with conventional chemotherapeutics for the treatment of
cancer.
