Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMPOSITIONS AND METHODS FOR CANCER TREATMENT
FIELD OF THE INVENTION
The present invention relates to compositions comprising arsenic for
antineoplastic treatment.
BACKGROUND OF THE INVENTION
Arsenic has been used as a therapeutic agent for more than 2,400 years.
Until the 1930s, arsenic was used as a treatment for patients with chronic
myelogenous leukemia. More recently, the use of arsenic in leukemia has
resurfaced after reports from China that arsenic induced a high remission rate
in acute promyelocytic leukemia (APL), including those who were resistant to
therapy with all-traps retinoic acid.
The activity of arsenic (As203) in APL is in part related to the disappearance
of the PML-RARa fusion protein, the gene product of the chromosomal
translocation t(15,17) characteristic of APL, and the induction of
differentiation. As203 can also induce apoptosis through a variety of
mechanisms, which appear to be independent of PML-RARa degradation. In
addition to causing mitochondrial toxicity, impairing microtubule
polymerization, and deregulating a number of proteins and enzymes through
binding to sulfhydryls groups, considerable evidence suggests that As203
induces the accumulation of reactive oxygen species (ROS) and
subsequently, induces oxidative stress. Indeed, the intracellular redox status
has been shown to be important in predicting whether a cell will respond to
arsenic.
Recently it has been shown that As203 stimulates apoptosis in additional
malignant cells including acute myeloid leukemia, chronic myeloid leukemia,
myeloma and various solid tumor cells. However, higher concentrations of
As203 are required to induce apoptosis in non-APL tumor cells, suggesting
that higher, more toxic doses might be needed for clinical efficacy. Clinical
trials are currently testing arsenic in the treatment of lymphoma and myeloma,
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but clear evidence of clinical benefit has, thus far, been largely restricted
to
patients with APL.
It would be highly desirable to increase the therapeutic index of arsenic.
SUMMARY OF THE INVENTION
In accordance with the present invention there is provided compositions
comprising a vitamin E analog and an arsenic compound for treating
hyperproliferative cells disorders such as cancer.
In another embodiment of the invention there is provided a method for
inhibiting hyperproliferative cells in a mammal comprising administering to
the
mammal a pharmaceutically effective amount of a composition of the
invention. There is also provided a method for treating a mammal in need
thereof with composition of the invention. In particular, mammals suffering
from a neoplastic disease can be treated with a composition of the invention.
In an aspect of the invention vitamin E or analog thereof is used to protect
non-cancerous cells from toxicity induced by an arsenic compound.
In yet another embodiment Trolox radicals can be used in cancer treatment of
patient in need thereof.
Compositions of the invention can also be used for inducing apoptosis in
neoplastic cells, said composition comprising vitamin E or an analog thereof
and an arsenic compound.
There is also provided a method for inducing apoptosis in neoplastic cells
said
method comprising providing neoplastic cells and contacting said cells with a
composition of the invention.
For the purpose of the present invention the following terms are defined
below.
The term therapeutic index is intended to mean the relative dose/efficacy
ratio
of a compound or composition.
The term vitamin E broadly encompasses tocopherols and tocotrienols
compounds and by vitamin E analogs it is meant derivatives of vitamin E and
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more particularly derivatives retaining the anti-oxydant properties of Vitamin
E. By vitamin E analog it is also meant compounds having a modified phytyl
chain. Preferably the modification renders the analog more hydrophilic (i.e.
more water soluble). In a preferred embodiment the vitamin E analog is
Trolox.
By arsenic compounds it is meant molecules including arsenic in their
composition such as monomethyl arsenic, dimethyl arsenic, trimethyl arsenic,
arsenic sulfides, arsenic chlorides, arsenic oxides. In a preferred embodiment
arsenic is linked to one or more electron affinic atoms, preferably oxygen.
Most preferably the molecule used for treating patients in need thereof is
arsenic trioxide.
All documents referred herein are hereby incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Trolox enhances As203-induced growth inhibition in NB4, AR2 and
IM9 cells. NB4 (A), AsR2 (B) and IM9 cells (C) were treated with trolox, As203
or the combination. Cell viability was evaluated on day 1, 3, and 6 using
trypan blue exclusion. Values are the mean of three independent experiments
each performed in triplicates. Standard deviation bars are shown. *, ** and
***
indicate a significant difference p<0.05, p<0.01 and p<0.001, respectively
from As203-treated cells.
Figure 2: Trolox enhances arsenic-mediated apoptosis in NB4, AR2 and IM9
cells. (A, B) NB4, AsR2 and IM9 cells were treated with As203 and trolox (T)
for 48 hours. Apoptosis was detected by PI-staining. Flow cytometric
histograms are shown in (A). Quantitation of PI-positive cells in a hypotonic
fluorochrome solution was pertormed. Apoptotic cells were also stained with
Annexin-V-FITC and Propidium Iodide in binding buffer and quantified (B).
Each bar represents an average of three independent samples, and standard
deviation bars are shown. Asterisks indicate significant differences from
As203-treated cells (** p<0.01; *** p<0.001 ). (C) Cells were treated as
indicated for 48 hours. Caspase 3 activation was measured using Red-DEVD-
FMK. Its binding to activated caspase 3 was analyzed by flow cytometry.
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Asterisks indicate significant differences (p<0.001) from As203-treated cells.
(D) Western blotting was performed to determine PARP protein levels after 48
hours treatments. ~i-actin was used to show equal loading of lanes. Results
are representative of three independent experiments each performed in
duplicate. (E) NB4 cells were treated with doxorubicin, AraC or etoposide with
or without trolox (T) for 48 hours. Apoptosis was detected by PI-staining as
described above. Each bar represents an average of three independent
samples, and standard deviation bars are shown.
Figure 3: Trolox potentiates As203-mediated oxidative stress. (A) NB4, IM9
and AsR2 cells were treated with As203 and trolox for 24 hours. Western blot
was used to determine HO-1 protein levels. ~i-actin was used as a loading
control. These data represent three independent experiments. (B) Protein
carbonyl content was detected by ELISA in NB4 cells treated with As20s
alone, trolox or the combination for 3 days with the concentrations indicated.
Data depicted are representative of three independent experiments each
performed in duplicate. Asterisks indicate significant differences from AS2O3
treated cells. (* p<0.05; *** p<0.001 ). (C) 8isoPGF2a was detected in whole
cells extracts from NB4 cells treated with the indicated compounds for three
days. Asterisks indicate significant differences from As203-treated cells
(p<0.001).
Figure 4: The synergistic effects of trolox on arsenic-mediated apoptosis are
not related to extracellular H20z production. Cells were treated with As203
(1 ~,M) and trolox or ascorbic acid (100iuM) for 48 hours. Catalase (500U/mL,
Cat) was added as indicated to degrade the extracellular H202 generated.
Apoptosis was detected by PI-staining, and quantitated by flow cytometric
measurement of PI-positive cells. Each bar represents an average of three
independent samples, and standard deviation bars are shown. Asterisks
indicate significant differences from As203+AA-treated cells (p<0.001).
Figure 5: Trolox enhances As203-mediated JNK activation. Immune complex
kinase assays were performed to measure JNK activity with extracts from
NB4 (A) or AsR2 cells (B) treated with As203 and trolox for 16 hours as
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described in materials and methods. Data depicted are representative of three
independent experiments.
Figure 6. Electronic Paramagnetic Resonance detection of the trolox
phenoxyl radical. EPR spectra of trolox in the reaction system containing 1mM
Trolox, 5% (v/v) DMSO (A) and As203 0.02mM (B) or 0.2p.g/ml doxorubicin
(C). (D) Computer simulation of spectrum in (B) obtained using the hyperfine
splitting constants: a" (CH3) = 4.56 G; a" (CH3) = 4.86 G; a" (CH3) = 0.23 G;
a" (CH2) = 0.37 G; a"~ (CH2) = 0.76 G.
Figure 7: The synergistic effects of trolox on arsenic-mediated apoptosis are
unique to cancer cells. (A) Normal human PBMC were isolated from three
normal donors using a Ficoll gradient. Colony forming ability of PBMC treated
with As20s and trolox was assessed by counting CFU-E, CFU-GM and BFU-E
after 15 days. Results are representative of three independent experiments
each performed in triplicate. (B) Mouse embryonic fibroblasts were treated
with As20s with or without trolox for three days. Apoptosis was detected by PI-
staining, and quantitated by flow cytometry measurement of PI-positive cells.
Each bar represents an average of three independent samples. Asterisks
indicate significant differences from As203-treated cells. (p<0.001).
Figure 8: Trolox enhances As203-induced growth inhibition in murine P388
cells. P388 cells were treated with media (~), Trolox (a), As203 2~,M (~),
4p.M
(~) and the combination of trolox with Asz03 2~.M (0) and 4~,M (o). Cell
viability was evaluated on day 1, 3, and 6 using trypan blue exclusion. Values
are the mean of three independent experiments each performed in triplicates.
Standard deviation bars are shown. Asterisks indicate a significant difference
(p<0.001) from As203-treated cells.
Figure 9: Trolox enhances arsenic-mediated apoptosis in murine P388 cells.
(A) P388 cells were treated with As203 and trolox for 48 hours. Apoptosis was
detected by AnnexinV-FITC and PI staining. The fluorescent signals of FITC
and PI were detected on a FACScan. Apoptotic cells (Annexin V positive/ PI
negative) were quantified using the CeIIQUEST software. Each bar represents
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an average of three independent samples, and standard deviation bars are
shown. Asterisks indicate significant differences from As20s-treated cells.
(p<0.001). (B) DNA fragmentation assay, a qualitative index of apoptotic cell
death, was performed using agarose gel. (C) Changes in ~4~m were
determined with the fluorochrome JC-1. Data were analyzed and the ratio of
mean florescence intensity was calculated. Standard deviation bars are
shown. * and *** indicate a significant difference p<0.05 and p<0.001,
respectively from As203-treated cells. (D) S-100 fractions were isolated and
cytochrome c release into the S-100 fraction for each condition was assessed
by Western blot analysis. (3-actin was used to show equal loading of lanes.
Results are representative of three independent experiments each performed
in duplicate.
Figure 10 is a schematic representation of the animal experiment design
Figure 11: Trolox protects mice against As20s-mediated liver toxicity. Animals
were treated as indicated in Figure 10 A. One day after the last dose of
arsenic animals were killed and the livers were weighted (A). Blood was
collected by cardiac puncture. Serum was separated. Serum activities of
alanine aminotransferase (B) and aspartate aminotransferase (C) were
assayed using commercially available kits. Standard deviation bars are
shown. Asterisk and number sign indicate a significant difference (p<0.05)
from control group and As20s- treated group respectively. (D) Western blot
was used to determine HO-1 protein levels. ~i-actin was used as a loading
control. These data represent three independent experiments. Asterisks
indicate significant differences from As203-treated group.
Figure 12: Trolox increases the life span of BDF1 mice treated with As203.
(A)Animals were treated as indicated in Figure 10 B. Percent of survival was
calculated and Kaplan-Meyer curve is depicted. Asterisks indicate significant
differences from As203-treated groups. (* p<0.05; ** p<0.01 ). (B) The
increase
in life span between the groups was also calculated.
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DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, some of the shortcomings
associated with the therapeutic use of arsenic have been overcome by the
discovery that the therapeutic index of arsenic is significantly increased
when
arsenic is provided in conjonction with an analog of vitamin E.
It has been particularly found that the compositions of the present invention
induce growth inhibition in cancer cells. It has also been found that the
compositions of the invention potentiate arsenic mediated apoptosis in cancer
cells. Furthermore, the synergistic effect of arsenic and vitamin E analog is
specific for cancer cells thereby providing an increased therapeutic index.
It has also been found that vitamin E analogs can protect against arsenic-
mediated liver toxicity. This beneficial effect provides a mean to increase
arsenic doses while keeping side effects to a minimum.
Therapeutic methods of the invention comprise identifying mammalian cells or
a mammalian subject presenting neoplastic diasease characteristics and
administering to the cells or the subject effective amounts of the composition
of the invention.
Thus, pharmaceutical compositions, comprising arsenic and vitamin E or a
vitamin E analog, are useful for the treatment of mammals, particularly
humans, suffering from neoplastic diseases. More particularly, compositions
of the invention are capable of increasing arsenic toxicity towards cells.
Compositions of the invention are particularly useful for treating cancer in
patients including but not limited to leukemias, lymphomas, myelomas and
carcinomas. For example, the composition can be used for treating acute
promyelocytic leukemia (APL), multiple myeloma and breast cancer.
The compositions of the present invention can be administered to patients in
need thereof for treating neoplasias. The need of a patient for arsenic-
vitamin
E analog therapy can be determined by those skilled in the art. For example,
neoplasias such as the ones mentioned above can be diagnosed by analysis
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of the blood formula, imaging techniques, detection of cancer specific
antigens, physical examination and the like.
The compositions of the invention can be administered with other
pharmaceutical compounds or compositions. In particular the compositions of
the invention may be administered together with chemotherapeutic drugs to
improve their therapeutic ratios.
The compositions can be administered using a pharmaceutically acceptable
carrier which can be a preservative solution, a saline solution, an isotonic
saline solution, an albumin solution, suspension, sterile water, phosphate
buffered saline, and the like. Other buffering agents, dispersing agents, and
inert non-toxic substances suitable for delivery to a patient may be included
in
the compositions of the present invention. The compositions may be
solutions, suspensions or any appropriate formulation suitable for
administration, and are typically sterile and free of undesirable particulate
matter. The compositions may be sterilized by conventional sterilization
techniques.
In accordance with the present invention, the compounds or compositions
may be administered to the patient by any biologically suitable route. For
example, they may be introduced into the patient by intravenous,
subcutaneous, intraperitoneal, intrathecal, intravesical, intradermal,
intramuscular, or intralymphatic routes. The compounds or compositions may
be in solution, tablet, aerosol, or multi-phase formulation forms. Liposomes,
long-circulating liposomes, immunoliposomes, biodegradable microspheres,
micelles, or the like may also be used as a carrier, vehicle, or delivery
system.
The incorporation can be carried out according to known liposome preparation
procedures, e.g. sonication and extrusion. Suitable conventional methods of
liposome preparation are also disclosed in e.g. A. D. Bangham et al., J. Mol.
Biol., 23:238-252 (1965); F. Olson et al., Biochim Biophys. Acta, 557:9-23
(1979); F. Szoka et al., Proc. Nat. Acad. Sci., 75:4194-4198 (1978); S. Kim et
al., Biochim. Biophys. Acta, 728:339-348 (1983); and Mayer et al., Biochim.
Biophys. Acta, 858:161-168 (1986) all incorporated herein by reference.
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The invention should not be limited to any particular method of introducing
the
compounds into the patient.
It will be appreciated that the actual preferred amounts of active compounds
in a given therapy will vary according to the specific compound or composition
being utilized, the particular compositions formulated, the mode of
application, the particular site of administration, the condition and age of
the
recipient, etc. Optimal administration rates for a given protocol of
administration can be readily ascertained by those skilled in the art using
conventional dosage determination tests.
Dosages of arsenic compounds may be generally in the range of about 0.1 to
50 mg/kg body weight. Preferred dosages of arsenic are 0.1 to 10 mg/kg,
particularly preferred dosages or arsenic are 1 to 10mg/kg. Dosages of
vitamin E analogs may be generally in the range of about 1 to about 100
mg/kg body weight. Preferred dosages of vitamin E analogs are 5 to 50
mg/kg.
Thus the present invention provides compositions comprising an analog of
vitamin E and arsenic for the treatment of neoplastic diseases.
In one embodiment of the invention the combination of vitamin E analogs and
arsenic enhances arsenic mediated apoptosis in neoplastic cells.
Advantageously, the combination can induce apoptosis in neoplastic cells
resistant to arsenic alone. Thus there is also provided a method to induce
apoptosis in neoplastic cells by contacting the cells with a composition
comprising an arsenic compound and a vitamin E analog.
In another aspect of the invention the vitamin E analogs may be utilized to
prevent or reduce the toxicity of arsenic towards non-neoplastic cells. More
specifically, vitamin E analogs can reduce the hepatotoxicity of arsenic
thereby increasing the therapeutic index of arsenic. This effect is
independent
of the increase tumor cell killing and can therefore be used for increasing
the
therapeutic index of arsenic for any arsenic based treatment.
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The present invention will be more readily understood by referring to the
following examples which are given to illustrate the invention rather than to
limit its scope.
EXAMPLE 1
MATERIALS AND METHODS
Cell lines
The arsenic trioxide-resistant APL cell line, NB4-M-AsR2 (AsR2), was
generated by culturing NB4 cells in the presence of As203 at concentrations
that were gradually increased over time'3. NB4 (provided by Dr. M Lanotte),
AsR2 and multiple myeloma IM9 (ATCC) were maintained in RPMI 1640
media. MCF-7 and MDA-231 were obtained from ATCC and maintained in
alpha MEM. T47D (ATCC) was cultured in D-MEM/F12. All media were
purchased from Life Technologies, Inc and supplemented with 10% fetal
bovine calf serum (FBS). AsR2 was routinely grown in RPMI containing 2~M
As203. In experiments examining the response of AsR2, the cells were first
washed thoroughly to remove As20s, and then cultured 24 hours in media
alone prior to initiating the experiment. All cells were grown in a humidified
chamber at 37° C with a 5% C02 environment.
G rowth assay
NB4, IM9 and AsR2 cells were seeded at 1x105 cells/ml in 24-well plates.
Cells were treated with various concentrations of As203 or doxorubicin, alone
or in combination with 100~M trolox for six days. Viable cells were counted
by trypan blue exclusion on day 1, 3 and 6. All cells were maintained at a
density lower than 1x106 cells/ml through dilution as required, and media +/-
treatment was replaced every third day. MCF-7, MDA-231 and T47D were
seeded in 24-well plates at a density of 4000 cells/well. The next day, fresh
media containing As203 +/- trolox was added. On the days indicated, cells
were fixed in 10% trichloroacetic acid and subsequently stained with
sulforhodamine B (SRB). Bound SRB was solubilized in 10mM unbuffered
Tris and optical density was measured at 570 nm in a microplate reader.
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Propidium Iodide Staining
Quantitation of apoptotic cells was performed as previously described (Hardin
et al. J. Immunol. Methods. 1992; 154:99-107). Cells were treated, washed in
buffer (PBS/ 5% FBS/ 0.01 M NaN3) at 4°C, pelleted, and resuspended in
0.5
ml of hypotonic fluorochrome solution containing 50 p.g/ml propidium iodide
(PI), 0.1 % sodium citrate, and 0.1 % Triton X-1 OOT"". Fluorescence was
measured on a Becton-DickinsonT"" FACS Calibur. Cells undergoing DNA
fragmentation and apoptosis (those in which PI fluorescence was weaker than
the typical Go-G~ cell cycle peak) were quantified using CeIIQUESTTM
software.
Annexin V staining
Cells were stained with Annexin-V-FITC and Propidium Iodide in binding
buffer according to the manufactures recommendations (BD Pharmigen, San
Diego, CA). The fluorescent signals of FITC and PI were detected by FL1 at
518 nm and FL2 at 620 nm, respectively, on a FACScanT"" (Becton Dickson,
San Jose, CA). Apoptotic cells (Annexin V positive/ PI negative) were
quantified using the CeIIQUEST software.
Western Blotting and immune kinase assays
Cell extracts were washed with cold PBS and resuspended in 0.1 ml lysis
buffer (5mM NaH2P04, 1 mM DTT, 10% glycerol, 1 mM PMSF, 1 O~,g/ml each
aprotinin and leupeptin, pH 7.4) at 4°C. Extracts were centrifuged at
14,000
rpm at 4°C, and supernatants were transferred to fresh tubes. Protein
concentration was determined with the Bio-RadT"" protein assay (Bio-Rad,
Mississauga, Ontario, Canada). To detect HO-1 or PARP, 50~g of protein
was added to an equal volume of 2x sample buffer and run on a 10% SDS-
polyacrylamide gel. Proteins were transferred to nitrocellulose membranes
(Bio-Rad), stained with Ponceau S in 5% acetic acid to ensure equal protein
loading, and blocked with 5% milk in PBS containing 0.5% Triton X-100T"" for
1 hour at room temperature. The membrane was hybridized overnight at
4°C
with antibody against PARP (1:1000; Oncogene) or 3 hours with an antibody
against HO-1 (1:1000; StressGen). Following three washes with PBS and
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0.5% Triton X-100T"", blots were incubated with a goat anti-rabbit antibody
(1:10,000; PharMingen) for one hour at room temperature. Bands were
visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech,
Baie d'Urfe, Quebec, Canada). Immunostaining for ~-actin was used to
confirm equal protein loading. Immune complex kinase assays for c-jun
kinase activity were performed as previously described (Davison et al. Blood.
2004; 103: 3496-3502).
Caspase-3 Activity Assay
Activation of caspase-3 was detected using a fluorescent caspase-3 inhibitor,
Red-DEVD-FMKT"" (Oncogene Research Products, San Diego, CA), which
irreversibly binds to activated caspase-3 in apoptotic cells. Cells were
treated
for two days and harvested into microcentrifuge tubes. The cells were
incubated with 1 ~I of Red-DEVD-FMKT"" for 1 hour at 37°C in 5% C02.
Subsequently, cells were washed twice, resuspended and analyzed by flow
cytometry, using the FL-2 channel.
Protein Carbonyls
Oxidized and reduced BSA were prepared and its carbonyl content was
quantitated by a colorimetric carbonyl assay described previously (Buss et al.
Free Radic Biol. Med. 1997; 23: 361-366). NB4 cells were treated for 3 days
with As203, trolox or the combination. Protein samples were adjusted to 4mg
protein/ml. The standards and protein samples were incubated with 3 volumes
10mM 2,4-dinitrophenylhydrazine (DNP) in 6 M guanidine-HGI, U.5 M
potassium phosphate, pH 2.5 for 45min at room temperature (mixing every
10-15min). Aliquots of cell proteins and standards were diluted in PBS and
adsorbed to a 96-well immunoplate by incubation overnight at 4°C. After
washing with PBS, non-specific sites were blocked with 0.1 % Tween 20 in
PBS for 1.5 hours at room temperature. After further washing with PBS the
wells were incubated with biotinylated anti-DNP antibody (Molecular Probes,
1:10000 dilution) for 1 hour at 37°C. Wells were washed and incubated
with
streptavidin-biotinylated horseradish peroxidase (Amersham International,
1:3000 dilution), After further washing, o-phenylenediamine/peroxide solution
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was added. The reaction was stopped after 7 min with 2.5 M sulfuric acid and
the absorbance was read with a 490 nm filter. A six point standard curve of
reduced and oxidized BSA was incubated with each plate.
Quantification of 8-iso PGF~,,
NB4 cells were treated for 3 days with As203, trolox or the combination. Cells
were washed twice with PBS containing 0.005% BHT, and 101ug/ml
indomethacin. The intracellular and membrane bound 8-iso PGF2,x, a specific
marker for lipid peroxidation, was measured using a competitive ELISA kit
from Cayman Chemical Company following the manufacturer instructions.
Detection of trolox phenoxyl radicals and measurement of intracellular
GSH
Electronic spin resonance spectroscopy reactions contained 0.02mM As203,
1mM Trolox, 5% (v/v) DMSO and 0.2~.g/ml doxorubicin. Following the final
addition of As203, reaction mixtures were transferred immediately to a quartz
ESR flat-cell positioned and pre-tuned within the cavity of a BrukerT"" ESP
300
spectrometer using a rapid delivery device and recording commenced using
the following instrument settings: modulation frequency, 100kHz; centre field,
3471.506; sweep width, 50.06; modulation amplitude, 9.51x10-~G; receiver
gain, 6.30x104; scan time, 20.97s; time constant, 10.24msec; power, 20mW.
Spectra was simulated using WinSIM program available for use at the
NIEHS/NIH website (http://epr.niehs.nih.govn33. Intracellular reduced GSH
levels were assessed enzymatically with glutathione reductase as previously
reported (Davison et al. Leukemia. 2003; 17: 931-940).
Peripheral blood mononuclear cell purification and Colony Forming Unit
Assay
Peripheral blood mononuclear cells (PBMC) were obtained from two healthy
normal donors after obtaining informed consent and were collected into tubes
containing 7.2mg K2EDTA. The blood was diluted 1:3 in PBS, layered onto an
equal volume of Ficoll-PlaqueT"" PLUS (Amershan Biosciences, Piscataway,
NJ) and centrifuged at 1500 rpm for 30 minutes. The mononuclear cell layer
was collected and washed twice in PBS. Methylcellulose media was prepared
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by combining IMDM, 30% FBS, 1 % bovine serum albumin, 10-4M 2-
mercaptoethanol, 2mM L-glutamine, 0.1 U penicillin, 0.1 ug/ml streptomycin,
granulocyte-macrophage colony-stimulating factor (GM-CSF; 10ng/ml),
interleukin-3 (IL-3; 10ng/m10) and erythropoietin (EPO; 3U/ml). PBMC were
seeded in this media at a concentration of 300,000 cells/ml and treated with
or
without As203, trolox or the combination. Cultures were performed in
triplicate
in 35mm2 dishes and incubated at 37°C in 4% C02. Colonies derived from
colony-forming units-erythrocyte (BFU-E) and CFU-GM were counted on day
7 and 13.
Statistical Analysis
The significance of data was determined using Prism version 3Ø Analysis of
variance followed by Newman-Keuls post-tests were used to determine if cell
treatments produced significant changes.
RESULTS
Trolox significantly enhances the inhibitory effects of As203 on APL,
multiple myeloma and breast cancer cells
We examined the effects of As203 and trolox, both separately and in
combination, on the growth of different cell lines. Figure 1A shows that
treatment of NB4 cells for six days with 0.5 or 1 pM As203 reduced viable cell
number by 25%~4.7 and 70%~5.6 of control, respectively. 100NM trolox alone
had no effect on cell number at any time point. However, if the cells were
treated with 0.5 or 1 pM As203 and 100pM trolox in combination, 57% ~3.5
and 97% ~4.2 reductions of cell number were observed. In all cases, trypan
blue positive cells were less than 3%. A difference was also seen between
As203 and As203+trolox after 72 hours, with 1 ~,M As20s decreasing cell
number by 30% and the combination by 50% (p<0.001 ). We next determined
whether trolox could sensitize arsenic-resistant cells. We used an NB4-
derived, arsenic-resistant subclone (AsR2), which has an ICSO value roughly
10-times higher than its parental NB4 cell line and the multiple myeloma IM9
cell line, which is also less sensitive to As203 than NB4 cells. An enhancing
effect of trolox on As203-mediated growth inhibition was observed in both cell
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lines (Fig 1 B and C), although trolox did not restore the sensitivity to
lower
concentrations of As203 in the highly resistant AsR2 cell line. Some solid
tumor cells have been shown to be more resistant to As20s than APL cells, so
we tested the combined effect of As203 and trolox in breast cancer cell lines.
As shown in Table 1, Asz03-mediated cytotoxicity was enhanced by trolox in
all tested cell lines.
Table 1
Effect of trolox on Asz03 mediated growth inhibition in breast cancer
cells
Cell lines IC50AS2~3 IC50 ~2~3 + Trolox
MCF-7 2.07-0.02 1.02 0.09
T47D 3.2210.07 1.56 0.03
MDA-MB-231 2.2710.08 0.98 X0.02
Cells were treated with 1 ~,M As203 and 100~.M trolox for 3 days. Viable cell
number was determined using the trypan blue exclusion method. IC50
indicates concentration of drug needed to inhibit 50% of cell growth. Values
are the mean of three independent experiments.
Trolox enhances Asz03-mediated apoptosis in Asz03 sensitive and
resistant malignant cells
To evaluate whether the growth inhibitory effect observed upon combined
treatment of As203 and trolox in NB4, AsR2 and IM9 cells was due to the
induction of apoptosis, cells were treated for 48 hours, subsequently stained
with PI and analyzed by flow cytometry. As shown in Figures 2A and B, trolox
enhanced Asz03-mediated apoptosis in the cell lines studied, at all
concentrations of arsenic tested, while trolox alone had no effect on the
apoptotic rate. To confirm an enhanced induction of apoptotic death, FITC-
labeled Annexin V, which detects phosphatidylserine residues appearing on
the external surface of early apoptotic cells, was used. Consistent with the
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increase in the subGo subpopulation after PI staining, trolox augmented the
percentage of cells positive for Annexin V (Figure 2B lower panels). To
further
confirm the induction of apoptosis by the combination of As203 and trolox, we
evaluated caspase 3 activation and PARP cleavage. Trolox significantly
enhanced the percentage of cells with activated caspase 3 (Figure 2C) and
cleaved PARP (Figure 2D). These results support the hypothesis that the
combined treatment with As203 and trolox induced apoptosis in NB4 cells in a
dose dependent fashion. Similar results were obtained with AsR2 and IM9
cells.
The combination of As203 and trolox results in increased cellular
oxidative stress
Oxidative damage has been postulated to be a key mechanism by which
arsenic initiates the apoptotic process. Because trolox potentiates AS2O3-
induced apoptosis, it is possible that the combination treatment increases
cellular oxidative stress. Therefore, we determined whether As203 affected
various markers for oxidative stress and whether trolox could augment this
effect. Heme oxygenase-1 (HO-1), which is the rate-limiting enzyme for heme
degradation and has been widely described as a stress responsive protein,
was not detected when trolox was used alone (Figure 3A). However, the
combined treatment markedly enhanced As203-mediated HO-1 induction in all
the cell lines tested, suggesting that this combination increased the cellular
oxidative stress. To document oxidative damage to cellular components, we
analyzed lipids and proteins isolated from NB4 cells treated with As203 or
As203+trolox for 3 days. Proteins carbonyls are generated by a variety of
mechanism and are sensitive indices of oxidative injury. Isoprostanes are
chemically stable prostaglandin-like compounds that are produced
independent of the cyclooxygenase (COX) enzyme by free radical-catalyzed
peroxidation of arachidonic acid (AA) in situ in membrane phospholipids. F2-
isoprostanes are a reliable marker of lipid peroxidation in vivo. Figures 3 B
and C show that As20s alone induces protein oxidation and, to a lesser extent,
lipid peroxidation. Oxidative damage to both proteins and lipids was found to
CA 02522411 2005-10-05
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be significantly higher when trolox and As203 where combined. Similar results
were obtained in AR2 and IM9 cells.
The cytotoxic effects observed when trolox and Asz03 are combined are
not due to generation of extracellular H2Oz
Several reports have demonstrated that ascorbic acid (AA), a known
antioxidant compound, enhances Asz03-induced cytotoxicity in multiple
myeloma cells. Clement et al. (Clement et al. Antioxid Redox Signal. 2001;
3:157-163), reported that ascorbate-mediated killing in HL60 cells depends on
the levels of H202 produced by the reaction of AA within the cell culture
medium, and direct addition of H202 to the cells reproduced these results.
Further, degradation of extracellular H202 by the addition of catalase, which
catalyzes the decomposition of H202 to H20 and Oz, blocked any additional
toxicity from AA24. They concluded that the extracellular H202 generated plays
a major role in the synergy observed in vitro by As203 and AA. We asked
whether the synergy observed between trolox and As203 was influenced by
the generation of extracellular H202. If so, we would expect that the addition
of catalase could block the generation of extracellular H202 and consequently
decrease the apoptotic rate. Therefore, we treated NB4 cells for three days
with As203, trolox, AA and catalase as indicated in Figure 4. The addition of
catalase (500U/ml) prevented the induction of apoptosis by HZOz, suggesting
that even a very large extracellular production of H202 by Asz03 and trolox
could be blocked. Catalase significantly blunted the synergy of Asz03 with AA,
confirming previous reports. In contrast, the addition of catalase did not
protect cells treated with Asz03+trolox.
Trolox enhances Asz03 -mediated c jun terminal kinase (JNK) activation
It has been demonstrated that JNK is activated in response to oxidative
stress. We have reported that JNK activation is necessary for As203-induced
apoptosis of NB4 cell (Davison et al. Blood. 2004; 103: 3496-3502).
Therefore, we asked whether the activation of JNK in NB4 cells treated with
As203 and trolox for 16 hours might play a role in the synergistic effect of
these compounds. We used an immune complex assay with GST-c-jun as an
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exogenous substrate. Figure 5A shows that a 24 hour treatment of NB4 cells
with as little as 0.5uM As203 induces significant JNK activation leading to
phosphorylation of c-jun. As expected, higher As203 concentrations increased
JNK activation. Consistent with the idea that trolox enhances As203-mediated
oxidative stress, we observed a further increase in JNK activity when cells
are
co-treated with As203 and trolox. As expected, the arsenic resistant cell line
AsR2 cells showed little activation of JNK following treatment with As203,
even at doses sufficient to elicit robust activation of NB4 cells (Figure 5B).
However, when trolox was added to the media, a considerable JNK activation
was observed which correlated with apoptotic induction.
As203 induces the formation of trolox phenoxyl radicals
Electron paramagnetic resonance (EPR) is an important tool in experimental
studies of systems containing unpaired electrons. We used EPR to directly
assay the generation of trolox radicals. As shown in Figure 6B, addition of
trolox to reaction mixtures containing As203 resulted in the observation of an
intense seven-line EPR signal. The g-value (3477.530 G), the relative
intensities, and the splittings all confirm the presence of the trolox
phenoxyl
radical. Its identity is further confirmed by the simulated spectrum (Figure
6C),
which is based on the published coupling constants for this radical. This
signal
is not generated by trolox alone (Figure 6A) nor in the presence of
doxorubicin
(Figure 6D) suggesting the requirement of As203 and its hydration products
for the formation of this radical.
Trolox does not potentiate As203 effects in non-malignant cells
We sought to determine the effects of As203 combined with trolox in normal
human hematopoietic colony forming cells and mouse embryonic fibroblasts.
Normal human PBMCs were isolated, grown in methylcellulose, and treated
with As203, trolox or the combination for 2 weeks. Figure 7A shows that 1 ~.M
of Asz03 inhibited CFU-E by approximately 62%, but had minimal effect on
CFU-GM or BFU-E colony formation. Treatment with trolox alone did not
inhibit colony formation and trolox did not enhance As203-inhibition of CFU-
GM, BFU-E or CFU-E. Mouse embryonic fibroblasts were treated with
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different concentrations of As203 for three days, stained with PI and analyzed
by flow cytometry. Interestingly, trolox significantly decreased As203-
mediated
apoptosis at all doses studied (Figure 7B).
EXAMPLE 2
MATERIALS AND METHODS
Reagents.
As203 and Trolox, were purchased from Sigma Chemical (St Louis, MO).
As203 was dissolved in 0.4N NaOH and then diluted in phosphate-buffered
saline (PBS). Trolox was resuspended in dimethylsulfoxide (DMSO) at a stock
solution of 0.1 mol/L. The final DMSO concentration in the medium was not
greater than 0.1 %.
Cell lines
P388 was kindly provided by Dr. Dai Jing, Department of Medicine, Mount
Sinai Medical Center, New York, and was maintained in DMEM (Life
Technologies,Bethesda, MD) supplemented with 10% fetal bovine calf serum
(FBS). Cells were grown in a humidified chamber at 37°C with a 5% C02
environment.
Growth Assays
P388 cells were seeded at 1x105 cells/ml in 24-well plates. Cells were treated
with various concentrations of As203 alone or in combination with 100~M
trolox for six days. Viable cells were counted by trypan blue exclusion on day
1, 3 and 6. All cells were maintained at a density lower than 1 x1 O6 cells/ml
through dilution as required, and media +/- treatment was replaced every third
day.
Annexin V I Propidium Iodide staining
Cells were stained with Annexin-V-FITC and Propidium Iodide in binding
buffer according to the manufactures recommendations (BD Pharmigen, San
Diego, CA). The fluorescent signals of FITC and PI were detected by FL1 at
518 nm and FL2 at 620 nm respectively on a FACScan (Becton Dickson, San
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Jose, CA). Apoptotic cells (Annexin V positive/ PI negative) were quantified
using the CeIIQUEST software.
DNA Fragmentation Analysis
DNA fragmentation assay, a qualitative index of apoptotic cell death, was
performed using agarose gel electrophoresis. Cells (2x106) were fixed with
70% ethanol, stored at -20°C for 24 h and collected by centrifugation.
Degraded oligonucleosomal DNA was extracted with 40 IuL of phosphate-citric
acid buffer at room temperature for 1 h and vacuum dried for 15 min. The
powder was resuspended in 3 pL of 0.25% Nonidet P-40 and 3 pL of 1 mg/mL
RNase and then incubated at 37°C for 30 min. Three pL of 1 mg/mL
proteinase K was added to the solution and incubated at 37°C for
another 30
min. The mixture, together with 12 pL of loading buffer, was loaded on a 0.8%
agarose gel containing 0.5 mg/mL ethidium bromide and electrophoresed at 2
V/cm overnight. The DNA laddering was recorded with a Chemilmager 4000
image analyser (Alpha Innntech Corporation, San Leandro, CA, USA).
Detection of the Mitochondria) Membrane Potential (04~m)
Changes in ~4~m were determined with the J-aggregate-forming lipophilic
cationic fluorochrome 5,5',6,6'-tetrachloro-1,1',3,3'-
tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Molecular Probes,
Eugene, OR). JC-1 loading into mitochondria is detected by a shift in
fluorescence from green, which is characteristic of its monomeric form, to
orange, which reflects its aggregation in mitochondria. Cells were incubated
with 2.5 mg/mL JC-1 (dissolved in DMSO) for 15 min at room temperature in
darkness. After centrifugation for 5 min at 1200rpm, cells were washed twice
with PBS at 4°C, resuspended in 0.5 mL PBS, and analyzed on a
FACSCalibur flow cytometer. Data were analyzed with the computer software
CELLQuest (Becton Dickinson) to quantify the percentage of red (polarized)
and green (depolarized) fluorescence. The ratio of mean florescence intensity
(MFI) between FL1 and FL2 was also calculated for each tested sample. For
a positive control in the assay, calcium ionophore was used to induce
mitochondria) depolarization to nearly 100 percent.
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Preparation of S-100 Fractions and Assessment of Cytochrome c
Release.
P388 cells were harvested after drug treatment by centrifugation at 1200rpm
for 10 min at 4°C. The cytosolic S-100 fraction was prepared as
described
previously, with minor modifications. Cell pellets were washed once with ice
cold phosphate-buffered saline (PBS) and resuspended in 5 volumes of buffer
(75mM NaCI, 8mM Na2HP04, 1 mM NaH2P04, 1 mM EDTA, 350ug/ml
digitonin, 1 mM dithiothreotol, 0.1 mM phenylmethylsulfonyl fluoride, 10~mo1/L
aprotinin and 10~,mol/L leupeptin). After chilling for 30 minutes on ice, the
cells were disrupted by 15 strokes of a glass homogenizer. The homogenate
was centrifuged twice to remove unbroken cells and nuclei (750g, 10 min,
4°C). S-100 fractions (supernatants) were then obtained by
centrifugation at
100,OOOg, 60 min at 4°C. All steps were performed on ice or at
4°C.
Cytochrome c release into the S-100 fraction for each condition was assessed
by Western blot analysis of the resulting fractions as detailed below.
Western Blotting
Cells were washed with cold PBS and resuspended in 0.1 ml lysis buffer
(5mM NaH2P04, 1 mM DTT, 10% glycerol, 1 mM PMSF, 1 Olug/ml each
aprotinin and leupeptin, pH 7.4) at 4°C. Extracts were centrifuged at
14,000
rpm at 4°C, and supernatants were transferred to fresh tubes. Protein
concentration was determined with the Bio-Rad protein assay (Bio-Rad,
Mississauga, Ontario, Canada). Livers from treated mice were disrupted by 2-
4 sec bursts of a Polytron homogenizer. Cell debris was removed by
centrifugation at 700g for 15 min, followed by centrifugation of the
supernatant
twice at 14,400g for 15 min and finally at 100,OOOg for 1 h at 4 °C.
Proteins
were separated and probed as described previously (Diaz et al. Blood.
February 1, 2005, Vol. 105, Number 3). The source and dilution of antibodies
were as follows: cytochrome c, 1:500, Clontech; HO-1, 1:1000, Stressgen;
Actin 1:10,000, Sigma. Following three washes with PBS and 0.5% Triton X
100, blots were incubated with a horseradish peroxidase-conjugated
secondary antibody (1:10,000; PharMingen) for one hour at room
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temperature. Bands were visualized by enhanced chemiluminescence
(Amersham Pharmacia Biotech, Baie d'Urfe, Quebec, Canada).
Immunostaining for (3-actin was used to confirm equal protein loading.
In vivo toxicity experiments.
BDF1 mice were obtained from Charles River Laboratories (Wilmington, MA).
All procedures confirmed to the Canadian Institute for Health Research
guidelines for the care and use of laboratory animals. Mice were randomly
divided into eight groups each with six mice. Each group received Trolox (2.5,
10, 20 and 50 mg/kg) and As203 (7.5 mg/kg) alone or in combination
intraperitoneally every other day for fourteen times. Animals were weighted
every other day. One day after the last dose of arsenic, blood was collected
by cardiac puncture. Serum was separated after allowing the blood to
coagulate at room temperature for 2 h. Total protein levels, glucose content,
serum activities of alanine aminotransferase (ALT), aspartate
aminotransferase (AST) and alkaline phosphatase (AKP) were assayed using
commercially available kits. Mice were sacrificed by cervical dislocation,
followed by decapitation. Liver and kidney were extracted and washed in ice-
cold isotonic saline solution to remove debris and blood. Livers were weighted
using an analytic balance. A representative portion of all the extracted
organs
were fixed in 10% neutral-buffered formalin and embedded in paraffin for
histological analysis. The rest was frozen in liquid nitrogen for biochemical
assays.
In vivo anti-tumor experiments.
Transplantable P388 cells were injected intraperitoneally in DBA/2 mice
(Charles River Laboratories, Wilmington, MA) and allowed to growth for fifteen
days. Cells were collected from the peritoneum, washed and resuspended in
PBS. For experiments, 0.1 mL containing 2x106 cells obtained from the
ascites was inoculated intraperitoneally in BDF1 mice. Mice were randomly
divided into six groups each with eight mice. After 24 hours, each group was
given saline, As203 (7.5 or 10 mg/kg), and trolox (50 mg/kg) alone or in
combination intraperitoneally every other day for thirty times. The percentage
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increase in lifespan over control (ILS) was calculated as follows: ILS% =(T-
C)/C x 100, where T is the test mean survival time, and C is the control mean
survival time.
Statistical Analysis
The significance of data was determined using Prism version 3.0 (GraphPad
software, San Diego, CA). Analysis of variance followed by Newman-Keuls
post-tests were used to determine if treatments produced significant changes.
RESULTS
Trolox increases the growth inhibitory effects of Asz03 in murine
lymphoma P388 cells.
Our laboratory has previously documented that Trolox increases As203-
mediated growth inhibition in a variety of cell lines. In the present study we
investigated whether this combination have similar effects in lymphoma cells.
As Fig 8 illustrates, As203 was able to induce a dose dependent growth
inhibition in the murine lymphoma cell line P388. This cell line is less
sensitive
to As203 than some leukemic cell lines where 0.5uM and 1 uM As203 are
sufficient to induce a similar effect. However, when Trolox was combined with
2 or 4uM of As203, 25 and 47% growth inhibition were observed. These
results indicate that this combination is also effective in this cell line.
Trolox enhances Asz03 -induced apoptosis in P388 cells.
To further study the growth inhibitory effect observed when this combination
was used we used conventional techniques to establish the ability of this cell
line to undergo apoptosis.
We use a combined staining with Annexin V and Propidium Iodide to
discriminate between viable (Annexin V-negative/PI-negative), apoptotic
(Annexin V-positive/PI-negative), and necrotic (Annexin V-positive/PI-
positive)
cells. As depicted in Fig 9A approximately 25% of P388 cells were apoptotic.
This effect was increased to 45% when Trolox was added to the culture
media. Similar effects were observed at 4uM. Consistent with these results,
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we observed an increase in DNA fragmentation when the combination was
used at the two doses studied (Fig 9B.)
It is generally accepted that the apoptotic pathway is related to a breach in
the
mitochondria) integrity such that the usually impermeable inner mitochondria)
membrane becomes permeable to the nonspecific passage of ions and small
molecules, causing complete loss (depolarization) of the transmembrane
potential. We employed the fluorescent dye JC-1, which exhibits potential
dependent accumulation in mitochondria, to determine the effect of As203 and
Trolox on mitochondria permeability in P388 cells. As depicted in Fig 9C., a
decrease in the ratio of red to green fluorescence was observed in the As203-
treated cells in a dose dependent manner compared to control which
indicatives mitochondria) depolarization. One of the small molecules that are
released when the membrane potential is compromised is cytochrome c. As
shown in Fig 9D, an increase in cytoplasmic cytochrome c content was
observed when Trolox and arsenic were used in combination at all the doses
studied.
Thus, using a variety of complementary techniques, our data indicate that
trolox increases As203-induced apoptosis in P388 cells.
Trolox decreases As203-mediated toxicity in BDF1 mice.
In our previous work we demonstrated that trolox does not enhance
cytotoxicity of As203 in colony forming assays using human hematopoietic
peripheral blood mononuclear cells and in mouse embryo fibroblasts. This
suggests that cytotoxic enhancement accruing from trolox exposure could be
specific to tumor cells.
We have now extended these in vitro results to in vivo studies. Toxicological
studies were conducted to define the maximum tolerable dose of trolox in
BDF1 mice and the toxicity associated with the combination of As203 and
trolox. The selected arsenic dose (7.5mg/kg) had been previously used in rat
and other mouse strains and identified in the literature as well tolerated and
efficacious.
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Considering the low toxicity of trolox, and the lack of synergy we observed
between this drug and As20s in non-malignant cells, we asked whether trolox
could decrease As203-associated liver toxicity in vivo. Mice were randomly
divided into six groups each with six mice. Each group received trolox (10 or
20mg/kg) and As203 7.5 mg/kg alone or in combination (Fig 10A). None of the
animals exhibited discomfort or obvious distress throughout the duration of
the experiment. No differences in weight were observed at the endpoint of the
experiment in any of the treated groups compared to control.
The liver has been reported to be susceptible to arsenic-induced damage,
marked by tissue necrosis and other histological as well as biochemical
changes in different animal models. Liver damage as a consequence of
arsenic poisoning has also been reported in human subjects. Consistent with
this literature, we observed arsenic-induces liver toxicity in this
experiment.
As depicted in Figure 11A, moderate hepatomegaly was observed in the
As203-treated group. However, in the groups treated with the combination of
As203 and trolox, the hepatomegaly was completely reduced.
Aspartate aminotrasferase (AST) and alanine aminotransferase (ALT) levels
in blood are common means of detecting liver damage, the enzymes being
raised several fold in the first 24 hours after damage. AST and ALT activities
were increased in the As203-treated group by 4.2 and 3.5 fold compared to
the control group respectively. However in the animals treated with the
combination of As20s and either 10 or 20 mg/kg Trolox, a significant reduction
of AST and ALT activities were observed (p<0.05). The activity of alkaline
phosphatase, an indication of cholestasis, was not significantly affected by
any of the treatments (data not shown), which suggests that As203 can induce
a direct injury to the hepatocytes without blocking bile excretion. We did not
observe any change in glucose or total protein levels in any of the groups.
To explore the mechanism by which Trolox significantly decreases markers
for liver toxicity and hepatocellular death or necrosis, we analyzed the
expression of heme oxygenase-1 (HO-1 ), which is widely accepted as a
sensitive and reliable marker of cellular oxidative stress. As depicted in Fig
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11 D, As20s increases oxidative stress in the liver of the treated mice. The
addition of trolox induced a significant decrease in HO-1 protein expression
suggesting restoration of the hepatocellular redox homeostasis.
Thus, we have shown that arsenic treatment induces toxicity in mice, as
evidenced from the induction of hepatomegaly and alterations in the
enzymatic activities of AST and ALT. The addition of trolox reduces these
arsenic toxicities.
Trolox increases As203-mediated antitumor effects in BDF1 mice
bearing lymphoma P388 cells.
On the basis of the in vitro potency and favorable in vivo toxicity, As203 and
trolox were evaluated for an in vivo antitumor efficacy in mice bearing P388
murine lymphoma tumors. P388 cells were injected intraperitoneally in BDF1
mice. Animals were randomly divided in six groups and injected with saline
solution, As203, alone or in combination with trolox (Fig10B.) Based on low
preliminary toxicity at 7.5mg/kg dose, arsenic was given at 7.5 and 10mg/kg
and trolox at 50mg/kg, which was not toxic but approached the maximum
solubility. As shown in Figure 12A, As203 treatment prolonged survival, with
median survival times of 20 and 18 for As203 7.5mg/kg and 10mg/kg
respectively (p<0.001). The median survival time when the animals were
treated with the combination of As203 and trolox was further prolonged to 24.5
and 22 (p<0.001).
As shown in Fig 12B the combination treatment improved survival time, with
an increase in life span (ILS) of 46.4% when animals were treated with As20s
7.5mg/kg. When the same dose was combined with trolox we observed an
ILS of 73.5%. The significantly prolonged survival was without additive
toxicity
as compared with As203 or trolox treatment alone. The use of 10mg/kg of
As203 increased the life span of the treated mice only by 28.6%. Previous
results suggested that this dose is toxic in BDF1 mice. However, the addition
of trolox more than doubled the ILS at this dose of As203 (Fig 12B).
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Example 3
Chronic myelogenous leukemia (CML) is a haematological malignancy that
affects the myeloid lineage. In most cases of CML, the leukemic cells share a
chromosome abnormality: a reciprocal translocation between one
chromosome 9 and one chromosome 22 resulting in the fusion of two proteins
Bcr and Abl. The selective Abl kinase inhibitor, STI-571 (a small molecule Abl
inhibitor developed by Novartis), is toxic to CML cells in culture, causes
regression of CML tumors in nude mice, and is currently used to treat CML
patients.
In an attempt to analyze the efficacy of a combined theraphy using As203,
Trolox and the new dual Src/Abl kinase inhibitor (SK1606), animal experiments
were conducted as follows: CML cells (BaF stable transfected with mutated
Bcr-Abl tyrosine kinase) were implanted subcutaneous in nude mice. After 24
hours, animals were divided in groups and injected intraperitoneally with
saline solution, As203 (5mg/kg), and Trolox 50mg/kg alone or in combination.
SKI (75mg/kg) was injected intravenous in all the animals except controls.
Animals were treated every day for 11 days. After 8 days the non-treated
animals (Controls) developed tumors. In the animals treated with
As203+SKI606 tumors were developed after 10 days and the group treated
with As203+Trolox+SKI606 was tumor free after 29 days, when the animals
were sacrificed.
While the invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of further
modifications and this application is intended to cover any variations, uses,
or
adaptations of the invention following, in general, the principles of the
invention and including such departures from the present disclosure as come
within known or customary practice within the art to which the invention
pertains and as may be applied to the essential features hereinbefore set
forth, and as follows in the scope of the appended claims.
