Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR INDUCING APOPTOSIS AND
INHIBITING PROLIFERATION IN CANCER CELLS
FIELD OF THE INVENTION
The subject invention is directed generally to
methods for decreasing proliferation of cancer cells, or
of inducing apoptosis of cancer cells, or of inducing
differentiation of cancer cells into non-cancerous cells
and to methods for treating adenocarcinoma in a subject.
BACKGROUND OF THE INVENTION
Pancreatic cancer is one of the most enigmat_c
and aggressive malignant diseases facing oncologists
(Parker et al., "Cancer Statistics. 1996," CA Cancer J.
Clin.., 46:5-27 (1996) ("Parker") ). it is now the fourth
leading cause of cancer death in both men and women in
the United States, and the incidence of this disease has
significantly increased over the past 20 vears (Parker;
=rede et al., "Survival Afte~.~ Pancreaticoduodenectomy:
118 Consecutive Resections Without ar_ Operative
Mortality," Ann. Sura., 211:4S7-458 (1990); Cameron et
al., "One Hundred and Forty-five Consecutive Pancreatico-
duodenectomies Without Mortality," Ann. Surg., 217:430-
438 (1993) ; Horward, "Pancreatic Adenocarcinoma, Curr.
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Prob. in Cancer, 20:286-293 (1996) ("Horward"); Poston
et al., Gut. Biology of Pancreatic Cancer, 32:800-812
(1991) ("Poston"); and Black et al., "Treatment of
Pancreatic Cancer: Current Limitations, Future
Possibilities," Oncolociy, 10:301-307 (1996) ("Black")).
Pancreatic cancer is responsible for 27,000 deaths per
year in the United States. Because of lack of early
diagnosis and poor therapeutic responsiveness of
pancreatic cancer, less than 2% of patients survive
beyond five years, and the median expectation of life
after diagnosis of pancreatic cancer is less than 6
months (Horward; Poston; and Black).
Colonic cancer is the second most common form
of cancer in the United States (Doll et al., "Mortality
in Relation to Smoking: 20 Years' Observations on Male
British Doctors," BMJ, 2:1525-1536 (1976); Hruban et
al., "Molecular Diagnosis of Cancer and Micrometastases,"
Adv. Anat. Pathol., 5:175-178 (1998) ("Hruban");
Figueredo et al., "Adjuvant Therapy for Stage II Colon
Cancer After Complete Resection. Provincial
Gastrointestinal Disease Site Group," Cancer Prev.
Control, 1:379-92 (1997) ("Figueredo"); Ness et al.,
"Outcome States of Colorectal Cancer: Identification and
Description Using Patient Focus Groups," Am. J.
Gastroenterol., 93:1491-7 (1998) ("Ness"); Trehu et al.,
"Cost of Screening for Colorectal Cancer: Results of a
Community Mass Screening Program and Review of
Literature," South Med. J., 85:248-253 (1992); and Wingo
et al., "Cancer Statistics," CA Cancer J. Clin., 45:8-30
(1995) ("Wi.ngo")). Colonic cancer occurs in more than
138,000 patients and is responsible for more than 55,000
deaths in the United States each year (Wingo). Up to 70
% of patients with colonic cancer develop hepatic
metastasises by the time of death, indicating that non-
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detectable micro-metastases are present at the time of
surgery (Hruban; Figueredo; and Ness). Furthermore,
metastatic cancer is often not responsive to standard
chemotherapeutic regimens, resulting in treatment failure
(Figueredo and Ness). The overall response of advanced
or non-resectable colorectal cancer patients to
chemotherapeutic agents varies from 26 to 44 percent.
For example, less than one third of colorectal cancer
patients with liver metastases respond to treatment with
agents such as 5-FU and leucovorin (Id.).
Breast cancer has the highest incidence of any
cancer in women with the diagnosis being made in more
than 275,000 per year in the USA (Richards et al.,
"Influence of Delay on Survival in Patients with Breast
Cancer: A Systematic Review," Lancet, 353:1119-26 (1999);
Norton, "Adjuvant Breast Cancer Therapy: Current Status
and Future Strategies -- Growth Kinetics and the Improved
Drug Therapy of Breast Cancer," Semin. Oncol., 26:1-4
(1999); Morrow et al., "Current Controversies in Breast
Cancer Management," Curr. Probl. Surg., 36:163-216
(1999); and Ruppert et al., "Gene Therapy Strategies for
Carcinoma of the Breast," Breast Cancer Res. Treatment,
44:93-114 (1997)). Even though five year survival has
increased to more than 80%, more than 77,000 women still
die from this disease each year (Id.).
Thus, another dimension in chemotherapeutic
agents for pancreatic, colonic, and breast cancer would
be extremely beneficial, especially to control metastatic
and unresectable disease.
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SUNIMARY OF THE INVENTION
The present invention relates to a method of
decreasing proliferation of adenocarcinoma cells, or of
inducing apoptosis of adenocarcinoma cells, or of
inducing differentiation of adenocarcinoma cells into
non-cancerous cells. The method includes contacting a
sample comprising adenocarcinoma cells with a compound
having the formula:
CH3 -( CH2 ) n- CH=CH- ( CH2 ) m- COOH
wherein n is an integer from 0 to 15, m is an integer
from about 1 to 16, and the sum of m and n is an integer
from 6 to 16, or a pharmaceutically acceptable ester,
salt, amide, solvate, or metabolite thereof.
The present invention also relates to a method
of treating adenocarcinoma in a subject. The method
includes administering to the subject an effective amount
of a compound having the formula:
CH3 -( CH2) n- CH=CH- ( CH2 ) R,- COOH
wherein n is an integer from 0 to 15, m is an integer
from about 1 to 16, and the sum of m and n is an integer
from 6 to 16, or a pharmaceutically acceptable ester,
salt, amide, solvate, or metabolite thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A and 1B are bar graphs showing the
effects of myristoleic acid.obtained from Sigma Chemicals
(St. Louis, Missouri) (Figure lA) and myristoleic acid
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obtained from Matreya, Inc. (State College, Pennsylvania)
(Figure 1B) on the proliferation of AsPC-1 human
pancreatic cancer cells.
Figures 2A and 2B are bar graphs showing the
effects of impure cetyl myristoleic acid (Figure 2A) and
pure palmitoleic acid (Figure 2B) on the proliferation of
AsPC-1 human pancreatic cancer cells.
Figures 3A and 3B are bar graphs showing the
effects of myristoleic acid on the proliferation of AsPC-
1 (Figure 3A) and PANC-1 (Figure 3B) human pancreatic
cancer cells.
Figure 4 is a set of four fluorescence
micrograph images showing the effect of myristoleic acid
on annexin V binding.
Figures 5A and 5B are dot plots showing TUNEL
assay results of AsPC-1 cells pancreatic cancer cells
treated with 10 g/ml of myristoleic acid for 24 hours
(Figure 5B) as compared to control (Figure 5A).
Figure 6 is an image of human pancreatic cancer
xenografts in athymic mice which were treated with
250mg/kg/day of myristoleic acid (labeled "myristoleic
acid") or control solution (labeled "control").
Figures 7A and 7B are graphs showing the
effects of myristoleic acid on tumor volume of
subcutaneous xenografts of AsPC-1 (Figure 7A) and HPAC
(Figure 7B) human pancreatic cancer cells in athymic mice
as a function of time.
Figures 8A and 8B are bar graphs showing the
effects of myristoleic acid on tumor weight of
subcutaneous xenografts of AsPC-1 (Figure 8A) and HPAC
(Figure 8B) human pancreatic cancer cells in athymic mice
at the end of the experiment.
Figures 9A-9D are images produced with an in
situ TUNEL assay conducted on sections of AsPC-1
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pancreatic cancer cell xenografts harvested from athymic
mice that were treated with control solution (vehicle
only) (Figures 9A and 9B) or myristoleic acid (Figures 9C
and 9D).
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of
decreasing proliferation of adenocarcinoma cells, or of
inducing apoptosis of adenocarcinoma cells, or of
inducing differentiation of adenocarcinoma cells into
non-cancerous cells. The method includes contacting a
sample comprising adenocarcinoma cells with a compound
having the formula ("Formula I")
CH3 -( CH2 ) n- CH= CH -( CH2 ) R, - COOH
wherein n is an integer from 0 to 15, m is an integer
from about 1 to 16, and the sum of m and n is an integer
from 6 to 16, or a pharmaceutically acceptable ester,
salt, amide, solvate, or metabolite thereof.
Examples suitable compounds having Formula I
include those in which m is 1 and n is 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15; in which m is 2 and n is 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, or 14; in which m is 3 and n
is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13; in which in
which m is 4 and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or
12; in which m is 5 and n is 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, or 11; m is 6 and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10; m is 7 and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; m
is 8 and n is 0, 1, 2, 3, 4, 5, 6, 7, or 8; m is 9 and n
is 0, 1, 2, 3, 4, 5, 6, or 7; m is 10 and n is 0, 1, 2,
3, 4, 5, or 6; m is 11 and n is 0, 1, 2, 3, 4, or 5; m is
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12 and n is 0, 1, 2, 3, or 4; m is 13 and n is 0, 1, 2,
or 3; m is 14 and n is 0, 1, or 2; m is 15 and n is 0 or
1; and m is 16 and n is 0. Preferably, when m is 7, n is
0, 1, 2, 3, 4, 5, 6, 8, or 9.
As indicated above, pharmaceutically acceptable
base addition salts of the compounds of Formula I can
also be used. Such salts include those derived from
inorganic bases, such as ammonium and alkali and alkaline
earth metal hydroxides, carbonates, bicarbonates, and the
like, as well as salts derived from basic organic amines,
such as aliphatic and aromatic amines, aliphatic
diamines, hydroxy alkylamines, and the like. Such bases
useful in preparing the salts useful in the practice of
the present invention thus include ammonium hydroxide,
potassium carbonate, sodium bicarbonate, calcium
hydroxide, methyl amine, diethyl amine, ethylene diamine,
cyclohexylamine, ethanolamine, and the like.
Pharmaceutically acceptable esters and amides
of the compound of Formula I can also be employed in the
method of the present invention. Examples of suitable
esters include alkyl, aryl, and aralkyl esters, such as
methyl esters, ethyl esters, propyl esters, dodecyl
esters, benzyl esters, and the like. Examples of
suitable amides include, unsubstituted amides,
monosubstituted amides, and disubstituted amides, such as
methyl amide, dimethyl aminde, methyl ethyl amide, and
the like.
In addition, the method of the present
invention can be practiced using solvate forms of the
compounds of Formula I or salts, esters, amides, and/or
metabolies thereof, such as ethanol solvates, hydrates,
and the like.
It is recognized that the compounds of Formula
I can be in the cis or trans configuration. The method
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of the present invention car: be practiced with pure cis
isomer, pure trans isomer, a racemic mixture of cis and
trar_s isomers, or any other mixture of cis and trans
isomers.
Illustrative compounds which can be used in the
practice of the method of the present invention include
mvristoleic acid er a pharmaceutically acceptable salt,
ester (e.g., a cetyl ester), amide, metabolite, and/or
solvate thereof.
Compounds of Formula I can be synthesized bv
established methods, such as those set forth in Beilstein
2(2) 423.
As explained above, the above-identified
compounds can be used to decrease proliferation of
adenocarcinoma cells, and/or induce apoptosis of
adenocarcinoma cells, and/or induce differentiation of
adenocarcinoma cells into non-cancerous cells. The
meaning of the terms "prolzferation", "apoptosis", and
"differentiation are readily understood in the art.
Illustrative methods for assa_ving for proliferation,
apoptosis, or differentiation are nrovided in the
examples which follow.
"Adenocarcinoma cells", as used herein, are
meant to include cancerous epith.eliai cells, such as
nrostate cancer cells, lung cancer cells, stomach cancer
cells, breast cancer cells, pancreatic cancer cells, and
colon cancer cells. The methods of the present invention
can be practiced in viLro or in vivo.
More particularly, the method of the present
invention can be used in vivo to treat adenocarcinomas,
such as prostate cancer, 1-ung cancer, stomach cancer,
pancreatic cancer, breast cancer, and colon cancer. In
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the case where the method of the present invention is
carried out in vivo, for example, where the
adenocarcinoma cells are present in a human subject,
contacting can be carried out by administering a
therapeutically effective amount of the compound to the
human subject, for example, by directly injecting the
compound into a tumor. Details with regard to
administering compounds in accordance with the method of
the present invention are described below.
The present invention, in another aspect
thereof, relates to a method of treating adenocarcinomas,
such as prostate cancer, lung cancer, stomach cancer,
breast, pancreatic cancer, colon cancer, esophageal
cancer, uterine cancer, ovarian cancer, or other cancers
involving epithelial cells. The method includes
administering, to the subject, a compound of Formula I or
a pharmaceutically acceptable ester, salt, amide,
solvate, or metabolite thereof.
Suitable subjects include, for example mammals,
such as rats, mice, cats, dogs, monkeys, and humans.
Suitable human subjects include, for example, those which
have previously been determined to be at risk of having
prostate cancer, lung cancer, stomach cancer, pancreatic
cancer, colon cancer, and/or breast cancer and those who
have been diagnosed as having prostate cancer, lung
cancer, stomach cancer, pancreatic cancer, colon cancer,
and/or breast cancer. Preferably, the subject suffers
from only one of these types of cancers, for example,
from only pancreatic cancer.
In subjects who are determined to be at risk of
having adenocarcinoma, the above-identified c.ompounds of
Formula I or salts, esters, amides, solvates, and
metabolites thereof are administered to the subject,
preferably under conditions effective to decrease
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proliferation and/or induce apoptosis and/or induce
differentiation of the adenocarcinoma cells in the event
that they develop. Such preventive (which is not used in
the absolute 100% sense) therapy can be useful in high
risk individuals as long as the adverse side effects of
the administration of these compounds are outweighed by
the potential benefit of prevention.
Any of the compounds described above can be
used in the treatment method of the present invention.
For example, compounds may be administered alone or in
combination with compatible carriers as a composition.
Compatible carriers include suitable pharmaceutical
carriers or diluents. The diluent or carrier ingredients
should be selected so that they do not diminish the
therapeutic effects of the compounds used in the present
invention.
The compositions herein may be made up in any
suitable form appropriate for the desired use. Examples
of suitable dosage forms include oral, parenteral, or
topical dosage forms.
Suitable dosage forms for oral use include
tablets, dispersible powders, granules, capsules,
suspensions, syrups, and elixirs. Inert diluents and
carriers for tablets include, for example, calcium
carbonate, sodium carbonate, lactose, and talc. Tablets
may also contain granulating and disintegrating agents,
such as starch and alginic acid; binding agents, such as
starch, gelatin, and acacia; and lubricating agents, such
as magnesium stearate, stearic acid, and talc. Tablets
may be uncoated or may be coated by known techniques to
delay disintegration and absorption. Inert diluents and
carriers which may be used in capsules include, for
example, calcium carbonate, calcium phosphate, and
kaolin. Suspensions, syrups, and elixirs may contain
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conventional excipients, for example, methyl cellulose,
tragacanth, sodium alginate; wetting agents, such as
lecithin and polyoxyethylene stearate; and preservatives,
such as ethyl-p-hydroxybenzoate.
Dosage forms suitable for parenteral
administration include solutions, suspensions,
dispersions, emulsions, and the like. They may also be
manufactured in the form of sterile solid compositions
which can be dissolved or suspended in sterile injectable
medium immediately before use. They may contain
suspending or dispersing agents known in the art.
Examples of parenteral administration are
intraventricular, intracerebral, intramuscular,
intravenous, intraperitoneal, rectal, and subcutaneous
administration.
In addition to the above, generally non-active
components of the above-described formulations, these
formulations can include other active materials,
particularly, actives which have been identified as
useful in the treatment of prostate, lung, stomach,
breast, colon, pancreatic cancers and/or other
adenocarcinomas. These actives can be broad-based anti-
cancer agents, such that they also are useful in treating
other types of cancers (i.e., in addition to
adenocarcinomas) or they may be more specific, for
example, in the case where the other active is useful for
treating adenocarcinomas or particular types of
adenocarcinomas. The other actives can also have non-
anti-cancer pharmacological properties in addition to
their anti-adenocarcinoma properties. For example, the
other actives can have anti-inflammatory properties, or,
alternatively, they can have no such anti-inflammatory
properties.
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It is understood that some of the compounds
described above (i.e., some of the compounds that are
useful in the methods of the present invention) are
naturally occurring. The compositions used in the
treatment method of the present invention can be
substantially free of one or more of the components with
which the compound is typically found when it is
naturally occurring. Alternatively or additionally, the
compositions used in the treatment method of the present
invention can be substantially free of all but one of the
components with which the compound is typically found
when it is naturally occurring. Still alternatively or
additionally, the compositions used in the treatment
method of the present invention can be substantially free
of all of the components with which the compound is
typically found when it is naturally occurring. For the
purposes of the present application, a composition is
considered to be substantially free of component X when
the amount of component X is less than 10% by weight
(such as less than 5% by weight, less than 2% by weight,
and/or less than 1% by weight) relative to the weight of
the composition.
It will be appreciated that the actual
preferred amount of compound to be administered according
to the present invention will vary according to the
particular compound, the particular composition
formulated, and the mode of administration. Many factors
that may modify the action of the compound (e.g., body
weight, sex, diet, time of administration, route of
administration, rate of excretion, condition of the
subject, drug combinations, and reaction sensitivities
and severities) can be taken into account by those
skilled in the art. Administration can be carried out
continuously or periodically within the maximum tolerated
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dose. Optimal administration rates for a given set of
conditions can be ascertained by those skilled in the art
using conventional dosage administration tests.
The present invention is further illustrated
with the following examples.
EXAMPLES
Example 1 -- Effect of Myristoleic Acid on Proliferation
of Human Pancreatic Cancer Cells
Myristoleic acid, obtained from two separate
commercial sources, caused a concentration-dependent
inhibition of proliferation of AsPC-1 human pancreatic
cancer cells as measured by thymidine incorporatiori at 24
hours. This can be seen in Figure lA (results using
myristoleic acid obtained from Sigma Chemicals (St.
Louis, Missouri)) and Figure 1B (results using
myristoleic acid obtained from Matreya, Inc. (State
College, Pennsylvania)).
Example 2 -- Effect of Cetyl Myristoleic Acids and
Palmitoleic Acid on Proliferation of Human Pancreatic
Cancer Cells
Impure cetyl myristoleic acid (approximately 5%
pure) and pure palmitoleic acid also caused a
concentration-dependent inhibition of proliferation of
AsPC-1 human pancreatic cancer cells as measured by
thymidine incorporation. The results are presented in
Figure 2A (cetyl myristoleic acid) and Figure 2B
(palmitoleic acid). As Figures 2A and 2B show, the
effects of impure cetyl myristoleic acid and palmitoleic
acid on AsPC-1 human pancreatic cancer cells
proliferation are similar to, though less potent than,
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the effects of myristoleic acid (shown in Figures 1A and
1B). Irrespective of whether myristoleic acid, cetyl
myristoleic acid, or palmitoleic acid was used, the
inhibition of proliferation was accompanied by
morphological changes associated with apoptosis,
including membrane blebbing, cellular rounding, and
detachment from the culture plates (data not shown).
Example 3 -- Effect of Myristoleic Acid on Proliferation
of AsPC-1 and PANC-1 Human Pancreatic Cancer Cells
The thymidine incorporation experiments
described in Example 1 were repeated in triplicate on
each of two malignant human pancreatic cancer cell lines,
AsPC-1 and PANC-l. The results are set forth in Figure
3A (AsPC-1) and Figure 3B (PANC-1). In each case,
concentration-dependent effects of myristoleic acid on
pancreatic cancer cell proliferation were observed. From
these experiments it was determined that myristoleic acid
has potent anti-proliferative activity in AsPC-1 and
PANC-1 pancreatic cancer cells with effects in the low
micromolar range.
Example 4 -- Effect of Myristoleic Acid on Annexin V
Binding in AsPC-1 Human Pancreatic Cancer Cells
The effect of myristoleic acid (10 g/ml)
("MA") on annexin V binding in AsPC-1 cells at 3 or 5
hours after beginning treatment is shown in Figure 4.
Marked membrane fluorescence indicates early apoptosis in
these cells. This specific test utilizes the
translocation of phophatidyl serine from the inner to
outer plasma membrane, which is a feature of early
apoptosis. The results set forth in Figure 4 are
representative of three separate experiments.
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Example 5 -- Effect of Myristoleic Acid on DNA
Fragmentation by TUNEL Assay
The effect of myristoleic acid (10 g/ml)
("MA") on terminal deoxynucleotidyl transferase-mediated
dUTP nick end labeling ("TUNEL") assay at 24 hours in
AsPC-1 cells was investigated. The results are presented
in Figures 5A (control) and 5B (10 g/ml myristoleic
acid). As Figures 5A and 5B demonstrate, 10 g/ml
myristoleic acid caused a marked increase in the number
of apoptotic cells, shown in the upper right panel
compared with control in the upper left. The results set
forth in Figures 5A and 5B are representative of three
separate experiments.
Example 6 -- Effect of Myristoleic Acid on Growth of
Human Pancreatic Cancer Xenografts in Athymic Mice
The effects of daily intraperitoneal injection
of myristoleic acid (250mg/kg/day) on the growth of AsPC-
1 and HPAC xenografts in athymic mice were investigated.
The results are presented in Figure 6. Animals were
injected daily with myristoleic acid (250mg/kg/day)
(labeled "myristoleic acid" in Figure 6) or with a
control solution (labeled "control" in Figure 6) once
visible tumors were established (about five days after
implantation). As the sizes of the tumors in Figure 6
demonstrate, myristoleic acid caused a marked reduction
in tumor size throughout the experimental period. The
experiment was repeated twice with AsPC-1 cells and also
with HPAC cells. The results set forth in Figure 6 are
representative of these experiments.
Tumor volumes of the above-described athymic
mice were measured as a function of time, and the results
are presented in Figure 7A (AsPC-1 cancer cells) and
Figure 7B (HPAC cancer cells). Figures 7A and 7B
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demonstrate that myristoleic acid caused a marked
reduction in both AspC-1 and HPAC tumor volume throughout
the experimental period.
At the end of the experiment, the AsPC-1 and
HPAC xenografts were harvested from the athymic mice and
the weights of the tumors were measured. The results,
presented in Figure 8A (AspC-1 cancer cells) and Figure
8B (HPAC cancer cells), show that myristoleic acid caused
a marked reduction in AspC-1 and in HPAC tumor weight in
all three experiments.
Example 7 -- In Situ TUNEL Assay of Human Pancreatic
Cancer Xenografts in Athymic Mice
To determine whether the effects observed in
the athymic mouse experiments described in Example 7
might be due to apoptosis, in situ TUNEL assays were
carried out on the AsPC-1 xenografts that were harvested
from the athymic mice at the end of the experiment.
Images of xenograft sections from mice treated with the
control solution (vehicle only) (Figures 9A and 9B) show
minimal staining. In contrast, images of xenograft
sections from mice treated with the myristoleic acid
(Figures 9C and 9D) show many apoptotic cells (visualized
by the dark brown staining). Thus, Figures 9A-9D
demonstrate that cells in the myristoleic acid-treated
tumor are undergoing apoptosis (programmed cell death).
Although preferred embodiments have been
depicted and described in detail herein, it will be
apparent to those skilled in the relevant art that
30' various modifications, additions, substitutions and the
like can be made without departing from the spirit of the
invention and these are therefore considered to be within
the scope of the invention as defined in the claims which
follow.
