Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~ CA 02223489 1998-01-07 ~ 9 6 / ~ ~ 7 3 6
i ~)~JS ~ APR t99
~O~ FOR TREATING CANCER
USING AHPN
FIELD OF THE INVENTION
The present invention relates to the use of a reti-
noid having unique properties for the treatment or pre-
vention of breast cancer or leukemia. More specifical-
~, ly, the present invention relates to the use o~ 6-[3-[1-
adamantyl]-4-hydroxyphenyl]-2-naphthalene carboxylic
acid (AHPN) to treat or prevent breast cancer or
leukemia.
BACKGROUND OF THE INVENTION
Retinoids are de~ined as substances that can elicit
speci~ic biological responses by binding to and activat-
, ing a specific receptor or set o~ receptors. Retinoids
are known to play a ~nn~m~ntal role in normal cell
growth and differentlation. (Roberts, A.B. et al, in
"The Retinoids," ed. by M.B. Sporn, A.B. Roberts and D.
S. Goodman, Vol. 2, pp. 209-256, Academic Press, Orlan-
do, Fla., (1984); Sporn, M.B. et al, ~. Amer. Acad.
20 Dermatol., 15:756-764 (1986)). Multiple retinoic acid
nuclear receptors (RAR~, ~ and ~) and retinoid X recep-
tors (RXR~, ~ and ~) have been identi~ied (Evans, R.M.,
Science, 240:889-895 (1988); O'Malley, B.W., Mol.
Endocrin., 4:363-364 (1990); Gudas, L.J. Cell Growth
A~EN~0 $~
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Differ, 3:655-662 (1992)); Lohnes et al, Cell Sci., 16
(Suppl): 69-76 (1992). Moreover, numerous isoforms of
the various nuclear receptors exist as a result of
alternative splicing (Gudas L.J., ~. Biol. Chem.,
269:15399-15402 (1994)).
Retinoic acid receptors mediate gene transcription
through a variety of mechanisms. These nuclear recep-
tors can bind to specific DNA consensus sequences termed
retinoid receptor response elements (RAREs or RXREs)
which are located in the regulatory regions of the reti-
noid target genes (Gudas, L. J., Cell Growth Differ.,
3:655-662 (1992); Lohnes et al, Cell Sci., 16
(Suppl.):69-76 (1992)). Nuclear receptor binding to
these response elements preferably occurs through
heterodimer formation between the RAR and RXR, although
homodimer binding and subsequent gene activation has
also been found (Hermann et al, Mol. Endocrinol.,
6:1153-1162 (1992); Leid et al, Cell, 68:377-395 (1992);
Zhang X, Nature, 355:441-446 (1992)). The RXRs can
mediate gene transcription via heterodimer formation
with the RARs, with the vitamin D, thyroid hormone (Yu
et al, Cell, 67:1251-1266 (1991); Hermann et al, Mol.
Endocrinol., 6:1153-1162 (1992); Kliewer et al, Nature,
355:446-449 (1992); Leid et al, Cell, 68:377-395 (1992);
Zhang et al, Nature, 355:441-446 (1992)), and a number
of orphan receptors. (Apfel et al, Mol. Cell Biol.,
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.
--3--
14:7025-7035 (1994); Song et al, Proc. Natl. Acad. Sci.,
USA 91:10809-10813 (1994)). These orphan receptors can,
in turn, inhibit the activity of RARs and thyroid nucle-
ar receptors (TRs) (Kliewer et al, Proc. Natl. Acad.
Sci. , USA, 89:1448-1452 (1992); Tran et al, Mol. Cell
Biol., 12:4666-4676 (1992); Apfel et al, Mol. Cell
Biol., 14:7025-7035 (1994), Casanova et al, Mol. Cell
Biol., 14:5756-5765 (1991); Song et al, Proc. Natl.
Acad. Sci., USA, 91:10809-10813 (1994)).
The retinoid receptor response elements usually
consist of direct repeats (DRs) in which the half-sites
are separated by a number of base pair spacers. Selec-
tivity for binding appears to be determined by the num-
ber of base pairs utilized as spacers, as well as by the
sequence of the response element itself (Kim et al, Mol.
Endocrinol., 6:1489-1501 (1992), Mader et al, J. Biol.
Chem., 268:591-600 (1993)).
RAR and RXR inhibition of AP-1-mediated gene tran-
scription that does not require RAR or RXR binding to
DNA has also been observed (Pfahl, Endocrin. Reviews,
14:651-658 (1993), and references cited therein); RAR
and RXR when complexed to their ligands have been shown
to inhibit c-Jun/c-Fos binding to the AP-l consensus
sequence and subsequent gene activation (Pfahl, Endo-
crine Reviews, 14:651-658 (1993), and references cited
therein). Negative regulation of transcription by RA
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can apparently also occur by means that do not involve
RAR binding to the promoter region but by inhibiting
enhancer activity (Gudas, J. Biol . Chem., 269:15399-
15402 (1994)). In addition, negative regulation of RAR-
mediated, as well as TR-mediated, gene transcription
occurs by the competitive binding o~ the orphan receptor
coup and v-ErbA to RARE and TREs (Tran et al, Mol. Cell.
Biol ., 12:4666-4676 (1992), Hermann et al, Oncogene,
8:55-65 (1993)).
Most cell types express more than one RAR and RXR
receptor. RAR homologous recombination studies have
suggested that RAR functional redundancy exists among
the different RARs (Li et al, Proc. Natl. Acad. Sci.,
USA, 90:1590-1594 (1993), Lohnes et al, Cell, 73: 643-
658 (1993), Lufkin et al, Proc. Natl. Acad. Sci., USA,
90:7225-7229 (1993)). However, other studies have indi-
cated that the various receptor subtypes possess dis-
tinct functions and may indeed modulate the activity of
distinct genes (Nagpal et al, Cell, 70:1007-1019 (1992);
Boylan et al, Mol. Cell Biol., 15:843-851 (1995)).
Evidence also suggests a unique role for each of the
receptor subtypes: (1) receptor selectivity towards
specific transactivating response elements has been
demonstrated (Nagpal et al, Cell, 70:1007-1019 (1992));
and (2) specific cell types have become re~ractory to
the antiproli~erative and dif~erentiating ef~ects o~ RA
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with the loss of one receptor subtype, despite the pres-
ence o~ other RAR subtypes (Sheikh et al, J. Cell
Biochem., 53:393-403 ~1993); Moasser et al, Oncogene,
9:833-840 (1994)).
The RARs bind both RA and its isomer 9-cis-RA,
while the RXRs only bind 9-cis-RA (Allenby et al, J.
Biol. Chem., 269:16689-16695 (1995), and references
cited therein). To further document a unique function
for each receptor subtype, conformationally restricted
retinoids have been synthesized that selectively bind to
and enhance transcriptional activation by selective RAR
and RXR subtypes (Graupner et al, Biochem. Biophys. Res.
Commun., 179:1554-1561 (1991); L~hm~nn et al, Cancer
Res., 61:4804-4809 (1991), B~hm~nn et al, Science,
258:1944-1946 (1992); Dawson et al, in "Retinoids: New
Treatments in Research and Clinical Applications".
Livrea MA and Packer L., (eds) Marcel Dekker: NY pp 205-
221 (1992); Davies et al, Amer. Ass'n of Cancer Res.
Conf., Banff,=Alberta, Canada, March 15-20 (1993) Abst.
B-28; Jong et al, J. Med. Chem., 36:2605-2613 (1993);
Reichert et al, ~rom "Mol. Biol. to Therapeutics: Phar-
macology of the Skin", Vol. 5, Bernard BA and Shroot B
(eds), Karger: B.2d pp 117-127 (1993); Beard et al,
Bioorg. Med. Chemical, 4:1447-1452 (1994); and Boehm et
al, J. Med. Chem., 37:2936-2941 (1994)).
-
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These synthetic receptor-selective retinoids have
further con~irmed the uniqueness o~ specific RAR sub-
types in modulating RA responses in various cell types
(Rudd et al, Cancer Letter, 73:41-49 (1993); Sheikh et
al, ~. Biol. Chem., 269:21440-21447 (1994)). Recently,
a series o~ synthetic retinoids has been described that
selectively transactivate RAR~ (Bernard et al, Biochem.
Biophys. Res. Comm., 186(2):977-983 (1992)).
Because o~ the ability o~ retinoids to a~ect cell
growth and di~erentiation, these compounds have been
disclosed to be use~ul ~or the treatment or prevention
o~ diseases and conditions involving abnormal cell
proli~eration and di~erentiation. For example, the
usage o~ retinoids as e~icient therapeutics ~or the
treatment o~ various skin diseases and neoplasms has
been reported (Roberts, A. B. and Sporn, M.B., in "The
Retinoids", Sporn et al, pp 209-286, Academic Press,
Orlando, Fla; Bollag et al, Ann. Oncol., 3:513-526
(1992); Smith et al, ~. Clin. Oncol., 10:839-864
(1992)).
To date, the best results o~ retinoid therapy have
typically been achieved with a regimen which combines
retinoid administration with the administration o~ other
di~erentiation or cytotoxic agents. Besides retinol
and retinoic acid, isotretoin (13-cis-retinoic acid) and
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- etretinate have been used, as well as 9-cis retinoic
acid and N-(9-hydroxyphenyl)retinoid.
The most convincing results have been documented in
the ~ield of dermological disorders, where topical
application can circumvent the toxic effects sometimes
observed during systemic administration of retinoids.
For example, retinoids have been reported to be useful
for the treatment of a variety of dermatoses including
psoriasis, cystic acne, cutaneous disorders of keratina-
zation, among others.
Besides dermatological disorders, retinoids haveimportant potential as anti-cancer agents. For example,
retinoid compounds have been disclosed to have potential
for the prevention of skin cancer, for the treatment of
acute myeloid leukemia (AML), acute promyelocytic leuke-
mia (APL) ~or the treatment o~ other hematopoietic
malignancies such as myelodysplastic syndrome, juvenile
chronic myelogenous leukemia, Sézary syndrome, squamous
cell carcinomas o~ the upper aerodigestive tract, non-
small lung cancer, and human head and neck carcinomas.(See Pfahl et al, Vit;~m;n.q and Hormones, 49 :327-382
(1993) at 363-366, which reviews the usage of retinoids
as therapeutics).
In the specific case of breast cancer, the growth
o~ some breast cancer cell lines has been reported to be
inhibited by retinoids (La Croix et al, J. Clin.
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Invest., 65:586-591 (1980)). Also, etretinate has been
reported to prevent the growth of xenotransplanted
breast carcinoma cells in athymic mice (Halter et al,
Cancer Res ., 48:3733-3736 (1988)). Further, it has been
reported that N-(4-hydroxyphenyl) retinamide (4-HPR)
induces apoptosis and the differentiation of breast
cancer cell lines, assertedly independent of the status
of estrogen receptor (ER) and RAR expression (Pellegrini
et al, Cell Growth Differ., 6(7):863-869 (1995)).
Also, a recent patent issued to Curley et al, U.S.
Patent No. 5,516,792, assigned to Ohio State
Research Foundation, teaches the use of retinoyl beta-
glucuronide N-glycoside analogs for the treatment, pre-
vention and study of cancers, including breast cancer.
Further, N-(4-hydroxyphenyl) retinamide (4-HPR), a
derivative of trans-retinoic acid, is currently in clin-
ical trials as a chemopreventive agent for breast
cancer.
Additionally, retinoic acid in combination with
RAR-~ receptors has been reported to promote apoptosis
o~ estrogen receptor-positive (ER+) human breast cancer
cell lines (Liu et al, Mol. Cell Biol. 16(3):1138-1149
(1996)).
Further, the use of retinolc acid in combination
with interferon, speci~ically alpha inter~eron or gamma
inter~eron, has been reported to inhibit the proli~era-
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tion of some breast cancer cell lines (Widschwendter et
al, Anticancer Res., 16(1):369-374 (1996); Wi.dschwendter
et al, Cancer Res., 55(10):2135-2139 (1995)).
Also, 9-cis retinoic acid has been reported to
inhibit the growth of breast cancer cells and to down-
regulate estrogen receptor RNA and protein (Rubin et al,
Cancer Res ., 54(24):6549-6556 (1994)).
Still further, the use of (4-HPR) in combination
with the anti-estrogen tamoxifen as a potential
synergistic combination for breast cancer
chemoprevention has been reported (Costa, A., Eur . J.
Cancer, 29A(4):589-592 (1993)).
However, while some retinoids have been reported to
have potential as anticancer agents, and specifically
for the treatment or prevention of breast cancer and
leukemia, the identification of retinoids having
improved therapeutic properties which are suitable for
the treatment or prevention of such cancers would be
highly beneficial. In particular, the identification of
retinoids which are cytotoxic to either estrogen
receptor positive or estrogen receptor negative breast
cancers would be highly beneficial.
OBJECTS AND BRIEF DESCRIPTION OF THE INVENTION
It is an object of the invention to provide a novel
method for the treatment or prevention of breast cancer
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--10--
or leukemia, involving the administration o~ a retinoid
which induces Go/G1 arrest and apoptosis.
It is a more speci~ic object o~ the invention to
provide a novel method ~or treating or preventing breast
cancer or leukemia involving the administration o~ a
therapeutically or prophylactically e~ective amount o~
6-[3-adamantyl-4-hydroxypropyl]-2-naphthalene carboxylic
acid to a subject in need o~ such treatment or
prevention.
It is a more specific object of the invention to
provide a novel method ~or treating or preventing breast
cancer involving the administration o~ a therapeutically
or prophylactically e~ective amount of 6-[3-adamantyl-
4-hydroxypropyl]-2-naphthalene carboxylic acid to a
subject in need o~ such treatment or prevention.
It is another object o~ the invention to provide a
novel composition adopted ~or the treatment or
prevention o~ breast cancer or leukemia which comprises
a therapeutically or prophylactically e~ective amount
o~ 6-t3-adamantyl-4-hydroxyphenyl]-2-naphthalene
carboxylic acid.
Thus, the subject invention essentially relates to
the usage o~ a speci~ic retinoid, 6-[3-adamantyl-4-
hydroxyphenyl]-2-naphthalene carboxylic acid which
induces Go/Gl arrest and apoptosis ~or the treatment or
prevention o~ breast cancer or leukemia, as well as
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- pharmaceutical compositions adopted for the treatment or
prevention of breast cancer or leukemia which contain an
effective amount of 6-[3-adamantyl-4-hydroxyphenyl]-2-
naphthalene carboxylic acid.
- DETAILED DESCRIPTION OF THE FIGURES
Figure 1 shows the inhibition of breast carcinoma
cell proliferation by AHPN. The tested breast carcinoma
cell lines were seeded at a cell concentration o~ 3 x 10
or 1 x 104 cells per well in DMEM:F-12 medium
supplemented with 5~ FBS. The cells were then incubated
overnight at which time AHPN was added to a final
concentration of l~m. The data in the figure represents
the mean +- SEM o~ three different experiments.
Figure 2 shows the effects o~ varying the
concentration o~ AHPN on MCF-7 and MDA-MB-231
proliferation. MCF-7 cells and MDA-MB-231 cells were
seeded in DMEM:F-12 supplemented with 5~ FBS in 24-well
plates at a cell concentration of 3 x 104 and 1 x 104
cells, respectively, per well. The cells were incubated
for 24 hours at which time varying concentrations of
AHPN were added. Control cells were treated with
vehicle alone. The medium and A~PN were changed every 2
days and cells in triplicate wells were counted after a
6 day incubation period. The data represent the mean -
SEM o~ three independent experiments.
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-12-
Figures 3A and 3B, respectively, show Gl arrest of
MCF-7 and MDA-MB-231 cells by AHPN. AHPN was added to a
final concentration of l~M to MCF-7 and MDA-MB-231 cells
logarithmically growing in DMEM:F-12 supplemented with
5~ FBS. Cells were harvested at various times, the DNA
stained with propidium iodide and the cell cycle phase
distribution then determined.
Figures 4A and 4B compare AHPN- and RA-mediated
RARE and RXRE transactivation and anti-AP-1 activity in
MCF-7 and MDA-MB-231 cells. MDA-MB-231 and MCF-7 cells
logarithmically growing in 10 cm plates (approximately 3
x 106 cells per plate) in regular medium were transiently
transfected with the indicated plasmid and treated with
either l~M RA or l~M AHPN for 48 hours. CAT assays were
then effected to measure gene expression. Also, 14C-
activity was quantified by laser densitometry.
Activation is expressed as the ratio of converted 14C
activity in the diacetate and monoacetate forms to the
total 14C activity. Figure 4A shows the results of a
representative CAT assay and Figure 4B is the
quantification of two different experiments. The values
are expressed relative to respective controls, which
were given an arbitrary value of 1 The error bars
represent the standard errors.
Figure 5 shows AHPN-mediated inhibition of HL-60R
proliferation. HL-60R cells were seeded at cell
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-13-
- concentrations of 3 x 104 cells per well in DMEM:F-12
supplemented with 5~ FBS. AHPN or RA was added to a
final concentration of l~M and medium and supplements
were changed every 2 days. Cell counts were per~ormed
utilizing a hemocytometer. The results represent the
mean +- SEM of three independent experiments.
Figure 6 shows DNA fragmentation induced by AHPN in
MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells
were seeded in DMEM:F-12 medium supplemented with 5~ FBS
and incubated overnight. AHPN or RA were added to final
concentrations of l~M and the cells incubated for
various times. DNA was extracted and fractionated by
gel electrophoresis.
Figures 7A-7E show the effect of varying
concentrations of AHPN on AHPN-mediated apoptosis in
MDA-MB-231 cells. MDA-MB-231 cells were seeded in
DMEM:F-12 medium supplemented with 5~ FBS, incubated
overnight and exposed to vehicle alone (Figure 7A), or
30nM (Figure 7B), lOOnM (Figure 7C), or 500nM (Figure
7D) of AHPN for 72 hours (magnification x 100). Medium
and supplement were changed every 18 hours.
Immunoperoxidase staining of cells was ef~ected and the
apoptotic index quantified for the different AHPN
dosages. These results are contained in Figure 7E.
Figures 8A-8E show AHPN-mediated apoptosis in MDA-
MB-231 cells. MDA-MB-231 cells were seeded in DMEM:F-12
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-14-
medium supplemented with 5~ FBS, incubated overnight and
expose~ to vehicle alone (Figure 8A), or l~M AHPN ~or 24
hours (Figure 8B), 48 hours (Figure 8C) or 72 hours
(Figure 8D). Immunoperoxidase staining for apoptotic
cells was e~ected (magni~ication x 100). The percent
o~ apoptotic cells was then quantified as a ~unction o~
length o~ exposure to AHPN. These results are in Figure
8E.
Figures 9A-9B show AHPN induction o~ WAFl /CIPl mRNA
expression in MCF-7 and MDA-MB-231 cells. MCF-7 and
MDA-MB-231 cells were incubated in DMEM:F-12 (l:l)
supplemented with 5~ FBS and AHPN (l~M) was added at
various times during a total incubation period o~ 72
hours. All cultures were harvested at the end o~ the 72
hour incubation period. The zero-time culture that was
incubated over the 72 hour period in the absence o~ AHPN
served as the control ~or those cells incubated ~or
various times with AHPN. Total RNA was extracted and
Northern blots per~ormed. Figure 9A shows a
representative Northern blot assay. Figure 9B
quanti~ies the results of two independent experiments.
The values are expressed relative to respective
controls, which were again given an arbitrary value o~
l. The error bars represent the standard errors.
Figure lO shows AHPN-enhanced WAFl /CIPl
transcription in MDA-MB-231 and MCF-7 cells. MCF-7 and
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MDA-MB-231 cells logarithmically growing in 10 cm plates
(3 x 106 cells/plate) in DMEM:F-12 (1:1) supplemented
with 5~ FBS were transiently transfected with a
WAFl/CIPl luciferase reporter construct, and luciferase
activity then determined.
Figures llA and llB show AHPN modulation of bc1-2
and bax mRNA levels. MCF-7 cells seeded in DMEM:F-12
supplemented with 5~ FBS were grown in the presence and
absence of l~M AHPN for varying amounts of time. Total
RNA was extracted and Northern blots performed. Figure
llA shows a representative Northern blot assay. Figure
llB quantifies two independent experiments. The values
are expressed relative to respective controls, which
were again given an arbitrary value of 1. The error
bars represent the standard errors.
Figures 12A and 12B show AHPN modulation of cyclin
A, B1, D1 and E on RNA levels. MCF-7 cells seeded in
DMEM:F-12 supplemented with 5~ FBS were grown in the
presence and absence o~ l~M AHPN for varying amounts o~
time. Total RNA was extracted and Northern blots
performed. Figure 12A shows a representative Northern
blot assay. Figure 12B is a quantification o~ two
independent experiments. The values are expressed
relative to respective controls, which were again given
an arbitrary value o~ 1. The error bars represent the
standard errors.
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DETAILED DESCRIPTION OF THE I~v~NL~lON
The present invention is based on the
identification of a synthetic retinoid 6-[3-adamantyl-4-
hydroxyphenyl]-2-naphthalene carboxylic acid (APHN)
which has been discovered to possess novel properties
which render it well suited as an anti-cancer agent, in
particular for the treatment or prevention of breast
cancer or leukemia. Specifically, it has been
discovered that this retinoid mediates Go/Gl arrest and
apoptosis of breast cancer and leukemia cell lines.
Thus, 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene
carboxylic acid (AHPN) provides for the programmed death
of cancer cells. This is surprising, because previous
retinoids having anticancer activity have typically been
cytostatic in their inhibition o~ cancer cell growth.
Also surprising is the fact that while AHPN
selectively transactivates RAR~ (Bernard et al, Biochem.
Biophys. ~es. Commun., 186:977-983 (1992)), this
compound apparently mediates Go/G1 arrest and apoptosis
via a mechanism which operates independently o~ retinoic
acid receptor (RAR) and retinoid X receptor (RXR)
expression. This is substantiated by the discovery that
AHPN induces Go/Gl arrest and apoptosis in both retinoic
acid-sensitive (RA-sensitive) and retinoic acid-
resistant (RA-resistant) cancer cell lines.
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- Further surprising is the fact that AHPN induces
Go/G1 arrest and apoptosis of both estrogen receptor
positive (ER+) and estrogen receptor negative (ER-) cell
lines. By contrast, other compounds such as retinoic
acid and tamoxifen have been reported only to be active
against ER+ breast cancers. This is therapeutically
significant, because this allows AHPN to be used for the
treatment or prevention of both ER+ and ER- breast
cancers.
As noted, the therapeutic activity of AHPN occurs
by Go/G1, arrest and apoptosis. It is known that
apoptosis of cancer cells can be triggered through a
number of different pathways. (Isaacs et al, Curr.
Opin. Oncol., 6:82-89 (1994)). Moreover, a number of
different cellular proteins have been reported to
enhance or inhibit G1 arrest or programmed cell death.
For example, the cellular proteins bc1-2 (Vaux, R.L.
Proc. Natl. Acad. Sci., USA, 90:786-789 (1993)) bclXL
~Boise et al, Cell, 79:597-608 (1993), Ich-Is (Wang et
al, Cell, 78:739-750 (1994)) promote cell survival,
while WAFI/CIPl induces G, arrest. (Hunter et al, Cell,
79:573-582 (1994)), and bcX (Oltvai et al, Cell, 74:609-
619 (1993)), Ichm (Wang et al, Cell, 78:731-750 (1994)),
and TGF/~ (Polyak et al, Ge~es Dev., 8:9-22 (1994)),
enhance cell death.
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-18-
Based on the results of gene expression studies
which are discussed in greater detail in the examples
which follow, it appears that AHPN induces Go/G1 arrest
and apoptosis via a unique pathway which apparently
involves the activation of downstream effectors of p53,
but which operates in a p53-independent manner. This is
supported by the fact that AHPN has been discovered to
markedly enhance WAFl /CIPl mRNA levels in several breast
cancer cell lines (MCF-7 and MDA-MB-231), while
significantly decreasing bc1-2 mRNA levels in another
breast cancer cell line (MCF-7). Also, AHPN was found
to significantly enhance bax mRNA levels by a breast
cancer cell line (MCF-7). By contrast, it was found
that AHPN apparently does not modulate either TGF-B1
mRNA or protein levels.
Also, it has been discovered that exposure of
cultured breast cancer cell lines to AHPN results in
significant decreases in mRNA levels of some cyclins,
e.g., cyclin D1, cyclin A and cyclin B1. However, when
the same cell lines were exposed to AHPN, no effects on
cyclin E mRNA levels were observed. This further
substantiates the apoptosis inducing activity o~ AHPN,
because cyclins are well known to be important mediators
o~ cell cycle progression (van der Hevvel et al,
Science, 262:2050-2054 (1993)).
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~ That AHPN induces apoptosis is also substantiated
by morphological changes which were found to occur to
cancer cell lines which were exposed to AHPN in culture.
These changes include marked nuclear ~ragmentation and
chromosome condensation. Moreover, that AHPN induced
these morphological changes is substantiated by the fact
that such morphological changes were observed to
progressively increase as AHPN concentration was
increased.
The effects of AHPN on cancer cells, and in
particular on breast cancer cells and leukemia cells
indicate that this compound may be used for the
treatment or prevention of breast cancer or leukemia in
subjects in need of such treatment.
In particular, AHPN can be used to treat persons
who have been diagnosed to have breast cancer or
leukemia, or to prevent cancer in persons who are at
elevated risk of developing breast cancer or leukemia.
The latter group includes persons with a family history
o~ breast cancer or leukemia, or who have been shown to
express markers, e.g., proteins, the expression o~ which
is correlated with an increased incidence o~ breast
cancer or leukemia.
Treatment or prevention of breast cancer or
leukemia according to the invention is effected by
administering a therapeutically or prophylactically
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-20-
effective amount of AHPN to a subject in need of such
treatment or prevention. A therapeutically or
prophylactically effective dosage of AHPN will range
from about .001 mg/kg to 10 mg/kg by body weight, more
preferably from .01 to 5 mg/kg by body weight and most
preferably the dosage will approximate that which is
typical for the administration of retinoic acid, which
typically ranges from about 2 ~g/kg/day to 2 mg/kg/day.
Such administration may be effected in a single or
multiple dosages, which typically will be administered
daily. However, the dosage regimen may be varied
dependent upon the condition of the subject treated, and
other factors, such as whether AHPN is administered in
conjunction with any other anti-cancer agents or
treatments such as radiation therapy. Treatment will
typically be effected over a prolonged time period.
The administration of AHPN for the treatment or
prevention of breast cancer or leukemia can be effected
by any pharmaceutically acceptable route, e.g., orally,
intraocularly, parenterally, topically, or via
inhalation.
Parenteral administration according to the present
invention includes intravenous, intramuscular,
subcutaneous, rectal, surgical and intraperitoneal
routes of administration as well as the administration
of slow or sustained release compositions. Of these,
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intravenous, intramuscular and subcutaneous routes of
administration are generally preferred.
AHPN will be provided in the form of a
pharmaceutically acceptable formulation by the addition
of one or more acceptable carrier(s) or excipients and
optionally by the addition of other active agents, e.g.,
other anti-cancer agents. The carrier(s) or
excipient(s) are acceptable in the sense of being
compatible with the other ingredients in the formulation
and being safe for pharmaceutical usage. (See, e.g.,
Remingtons Pharmaceutical Sciences, by E. W. Martin
(Mack Publ. Co., Eastern PA), for a listing o~ typical
pharmaceutically acceptable carriers and excipient and
conventional methods of preparing pharmaceutically
acceptable formulations.)
Depending on the intended mode of administration,
the pharmaceutical compositions may be in the form of
solid, semi-solid or liquid dosage forms, such as, for
example, tablets, suppositories, pills, capsules,
powders, liquids, suspensions, lotions, creams, gels, or
the like, preferably in unit dosage form suitable for
single administration of a precise dosage.
Essentially, these compositions will include, as
noted above, a therapeutically or prophylactically
effective amount of AHPN in combination with a
pharmaceutically acceptable carrier and, in addition,
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may include other active agents, e.g., other anti-cancer
agents, pharmaceutical agents, carriers, adjuvants,
diluents, etc.
For solid compositions, conventional nontoxic solid
carriers include, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium
saccharin, talc, cellulose, glucose, sucrose, magnesium
carbonate, and the like. Liquid pharmaceutically
administrable compositions can, for example, be prepared
by dissolving, dispersing, etc., AHPN and optionally
pharmaceutical adjuvants in an excipient, such as, for
example, water, saline, aqueous dextrose, glycerol,
ethanol, or the like, to form a solution or suspension.
If desired, the pharmaceutical composition may also
contain minor amounts of nontoxic auxiliary substances
such as wetting or emulsifying agents, pH buffering
agents and the like, for example, sodium acetate,
sorbitan monolaurate, triethanolamine sodium acetate,
triethanolamine oleate, etc. Suitable methods for
preparing such dosage forms are well known in the art.
(See, e.g., Remington's Pharmaceutical Sciences,
referenced above.)
For oral administration, fine powders or granules
may contain diluting, dispersing and/or surface active
agents, and may be presented in water or in a syrup, in
capsules or sachets in the dry state, or in a nonaqueous
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solution or suspension wherein suspending agents may be
included, in tablets wherein binders and lubricants may
be included, or in a suspension in water or a syrup.
Where desirable or necessary, flavoring, preserving,
suspending, thickening, or emulsifying agents may be
included. Tablets and granules are preferred oral
administration forms, and these may be coated.
Parenteral ~m;n; stration, if used, is preferably
effected by injection. Injectables can be prepared in
conventional forms, either as li~uid solutions or
suspensions, solid forms suitable for solution or
suspension in liquid prior to injection, or as
emulsions. Also parenteral administration includes use
of a slow release or sustained release system, such that
a constant level of dosage is maintained. See e.g.,
U.S. Patent No. 3,710,795, which is incorporated by
reference herein.
As noted, the subject AHPN formulations may contain
other active agents. Such active agents include any
compound suitable for the prevention or treatment of
cancer, or more specifically the treatment or prevention
of breast cancer or leukemia.
For example, the subject AHPN containing
formulations may include other retinoids reported to
possess anti-cancer activity, e.g., retinoic acid, N-(4-
hydroxyphenyl) retinamide (4-HPR), 9-cis-retinoic acid,
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retinoyl-glucuronide N-glycoside analogs (see, U. S.
Patent No. 5,516,792), or other anti-cancer agents, e.g.
methotrexate, anti-estrogens, such as tamoxi~en and
toremifene, doxorubicin, daunorubicin, adriamyicin, etc.
Other active agents which may be incorporated in the
subject AHPN formulations include cytokines, e.g.,
interferons such as gamma, beta or alpha interferon,
alpha or beta tumor necrosis ~actors, interleukins,
colony stimulating factors, among others. The
incorporation o~ an interferon in the subject AHPN
formulation is potentially advantageous because it has
been previously reported that some retinoids exhibit
synergistic anti-cancer activity when used in
conjunction with an inter~eron, speci~ically alpha
interferon or gamma inter~eron.
Similarly, the incorporation o~ anti-estrogens,
such as tamoxifen or toremi~ene, in the subject AHPN
containing ~ormulations is potentially advantageous
because it has been reported that tamoxi~en exhibits
synergistic breast cancer chemopreventive effects when
combined with the retinoid ~enretinimide (4-HPR) (Costa,
A. Eur. ~. ~ancer, 29A(4):589-592 (1993)).
The ~ollowing examples illustrate the preparation
and use of specific embodiments o~ the invention, but
are in no way intended to limit the scope of the
invention.
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EXAMPLES
The following materials and methods were used in
the examples:
Retinoid Materials
All- trans-RA was obtained ~rom Sigma (St. Louis,
MO) and 4-[1,5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-
naphthalenyl) cyclopropyl]benzoic acid (SR11246) was
prepared as described by Dawson et al (In Retinoids: New
Trends in Research and Clinical Applications, Livrea,
M.A. and Packer, L. (eds.), Marcel Dekker: New York, pp.
205-221 (1992)). The synthesis o~ 6-[3-[1-adamantyl]-4-
hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) is
described in U.S. Patent No. 4,717,720 (see Example 13).
Supplies
Dulbecco's modi~ied Eagle's medium, Ham's F-12
medium and ~etal bovine serum (FBS) were obtained ~rom
Gibco-BRL (Grand Island, NY). Dulbecco's modi~ied
Eagle's medium (Phenol red-~ree) was purchased ~rom
Bio~luids (Rockville, MD). HEPES, tamoxifen, and
charcoal limpet sul~atase, were obtained ~rom Sigma
Chemicals (St. Louis, MO). [l~C]chloramphenicol (53 mCi
mmol~1) and [32p] ~dCTP (3000 Ci mmol~1) was supplied by
Amersham (Arlington Heights, IL).
=:
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Cell lines and cul ture
The MCF-7, T47D and MDA-MB-231 cell lines were a
kind gi~t o~ Dr. Marc Lippman (Lombardi Cancer Center,
Washington, D.C.). The MDA-MB-468 cells were provided
by Dr. Anne Hamburger (University o~ Maryland Cancer
Center, Baltimore, MD). Cells were maintained in
Dulbecco's modi~ied Eagle's (DMEM): Ham's F-12 medium,
supplemented with 5~ FBS as previously reported
(Fontana, J.A., Exp. Cell Res., 55:136-144 (1987)).
Growth e~periments
Cells were plated in DMEM:F-12 (1:1) medium
supplemented with FBS ~or 24 h. MCF-7 cells were plated
at an initial cell concentration o~ 3 x 104 cells per
well, while T47D, MDA-MB-231 and MDA-MB-468 were plated
at a cell concentration o~ 1 x 104 cells per well. The
treatment with RA or AHPN was ~or 3, 6, or 9 days in the
same medium. The medium and retinoids were changed
every 2 days. The retinoids were dissolved in dimethyl
sul~oxide (DMSO). The ~inal concentration o~ DMSO in
all the cultures was 0.1~; control cells were incubated
with DMSO at the same ~inal concentration.
Plasmid constructs, transient transfection and CAT assay
The $2-RARE-tk-CAT and the CRBP II-tk CAT and APO-l-
tk CAT reporter plasmids, which were kindly provided by
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- Dr. X-k Zhang of the La Jolla Cancer Center (La Jolla,
CA), carry the RARE and RXRE, respectively, upstream of
the thymidine kinase (tk) promoter containing the CAT
gene. Variations in transfection efficiencies were
adjusted by using the plasmid pRSV2 (MacGregor et al,
Somat. Cell Mol. Genet., 13:253-265 (1987)), which
carries an Escherichia coli lac Z gene under the control
of a Rous sarcoma virus long terminal repeat and encodes
the ~-galactosidase enzyme. The (AP-1) 3- tk-CAT reporter
construct was obtained ~rom Dr. Ronald Evans (La Jolla,
CA). WWP-Luc carries a 2.4 kb 5'-proximal region of the
WAF1/CIP1 gene ~used to a promoterless luci~erase
reporter gene (El-Deiry et al, Cell, 75:817-825 (1993)).
cDNA probes
The ~ull-length human WAF1 cDNA probe was kindly
provided by Drs. Kinzler and Vogelstein (Johns Hopkins
University, Baltimore, MD). The human cyclin D1 and E
cDNA probes (Xiong et al, Cell, 65:691-699 (1991), and
Xiong et al, Genomics, 13:575-584 (1992)) were obtained
~rom Dr. David Beach (Howard Hughes, Cold Spring Harbor
Laboratory, NY). The human cyclin A and Bl cDNA probes
(Pines et al, Cell, 58:833-846 (1989); and Pines et al,
Nature, 346:760-763 (1990)), were a gi~t o~ Dr. Tony
Hunter (The Salk Institute, San Diego, CA). Probes were
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labelled according to the random primer method o~ -
Feinberg et al, (Anal. Biochem., 132:6-13 (1983)).
Transient transfections, CAT and luciferase assays
Transient trans~ections were per~ormed as
previously described (Sheikh et al, J. Cell. Biochem.,
53:393-404 (1993)). Cells were plated in a 10 cm plate
at a density o~ 3 x lo6 cells per plate. The medium was
changed 2 h be~ore trans~ection. The cells were co-
trans~ected with various plasmids and the total amount
o~ DNA was corrected to 20 ~g per plate by adding
plasmid pUCl9. MDA-MB-231 and MCF-7 cells were shocked
with 20~ glycerol ~or 7 min and 2 min, respectively, 6 h
~ollowing the addition o~ DNA. Fresh medium was added
following washing the cells with 1 x PBS. The cells
were harvested 48 h a~ter the trans~ection. For CAT
assays, the cells were trypsinized and resuspended in
100 ~l 0.25 M Tris-HCl, pH 8Ø A~ter three cycles o~
~reezing and thawing, cell lysates were collected and
CAT assays per~ormed as previously described (Sheikh et
al, J. Cell. Biochem., 53 :393-404 (1993)).
For luci~erase assays, the cells were washed with
PBS, and 300-400 ~l o~ lysis bu~er (25mM glycylglycine
pH 7.8, 1.5 mM MgSO4 a, 4 mM EGTA, 100 mM DTT and 1~
Triton X-100) added to the cells and the cell harvested.
The lysates were centri~uged ~or 5 min in a
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- microcentrifuge. The supernatants were supplemented
with 12 mM K2HPO4 and 1.6 mM ATP and assayed ~or
luciferase activity by measuring light units in a
standard luminometer for 10 s. Relative light units
were corrected with respect to ~-galactosidase activity.
Northern blots
Total RNA was extracted and 20 ~g of total RNA was
loaded in agarose gel and Northern blot analysis were
performed essentially as previously described (Sheikh et
al, Biophys. Res. Comml7n., 183:1003-1010 (1992)).
Analysis o~ cell cycle phase distribution
Flow cytometric analysis DNA content was per~ormed
to assess the cell cycle phase distribution as described
previously (Sheikh et al, Anticancer Res., 13:1387-1392
(1993)). AHPN (1 ~M) was added to logarithmically
growing MCF-7 or MDA-MB-231 cells. Cells were harvested
by trypsinization at various times and stained for DNA
content using propidium iodide fluorescence as
previously described (Sheikh et al, Anticancer Res.,
13:1387-1392 (1993)). The computer program Multicycle
~rom Phenix Flow Systems (San Diego, CA) was used to
generate histograms, which were used to determine the
cell cycle phase distribution.
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Apoptosi~ A~~ay
Apoptosis was detected by labelling the 3'OH ends
o~ DNA utilizing digoxigenin incorporation by terminal
deoxynucleootidyltransferase. Antidigoxigenin
antibodies and immunoperoxidase staining were utilized
to demonstrate digoxigenin-nucleotide incorporation by
utilizing the Apotag detection system (Oncor,
Gaithersburg, MD). In brief, cells were spun onto
microscope slides, rinsed with PBS and finally incubated
in a reaction mixture containing terminal transferase
and digoxigenin dUTP at 37~C for 1 h. The specimens
were then washed, antidigoxigenin antibody coupled to
horseradish peroxidase was then added, and the cells
were incubated for 30 min at room temperature.
Following the rinsing with PBS, diaminobenzidine
tetrachloride (DAKO, Carpenteria, CA) was added and the
cells incubated for 10 min. The percent peroxidase
positive cells were determined by counting 200 cells in
random fields in two separate experiments.
EXAMPLE 1
AHPN inhibition o~ breast carcinoma proliferation
Numerous investigators have demonstrated that
estrogen receptor (ER)-positive breast carcinoma cells
are sensitive to the antiproliferative effects of RA
while ER-negative cells are refractory (Roman et al,
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Cancer Res., 53:5940-5945 (1993); Sheikh et al, ~. Cell .
Biochem., 53:393-404 (1993); and van der Burg et al,
Mol. Cell. Endo., 91:149-157 (1993)). Therefore, in
order to determine whether AHPN displays the same
spectrum of antiproliferative activity against breast
carcinoma cells as RA, the ability of AHPN to inhibit
the proliferation o~ both ER-positive and ER-negative
breast carcinoma cells was investigated. The results in
Figure 1 show that AHPN significantly inhibited the
proliferation of both the RA-sensitive, ER-positive MCF-
7 and T47D, as well as the RA-refractory, ER-negative,
MDA-MB-231 and MDA-MB-468 human breast carcinoma cell
lines (HBC). In addition, AHPN displayed signi~icantly
greater antiproliferative activity against MCF-7 and
T47D cells than that noted with RA (data not shown).
The concentration of AHPN required ~or 50~ inhibition of
growth (ICso) was 300 nM and 150nM for MCF-7 and MDA-MB-
231 cells, respectively (see Figure 2). By contrast, RA
did not inhibit the growth of MDA-MB-231 or MDA-MB-468
cells and displayed an ICso of 100 nM when tested against
MCF-7 and T47D cells (Sheikh et al, ~. Biol . Chem.,
269:21~40-21447 (1994)). Also, it was discovered that
AHPN arrested MCF-7 and MDA-MB-231 cells in the Go/Gl
phase (Figure 3A and 3B). Exposure of both these cell
lines to AHPN resulted in a significant increase in
cells in Go/G1/ accompanied by a decrease in the percent
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of cells in G2+M and S phase. RA had no e~ect on the
cell cycle phase distribution o~ MDA-MB-231 cells (data
not shown).
EXAMPLE 2
AEPN hin~;ng and activation of endogenous retinoid
receptors
AHPN has been reported as RAR~-selective in both
retinoid receptor binding and transcriptional activation
assays (Bernard et al, Biochem. Biophys. Res. Commun.,
186:977-983 (1992)). Tritiated 6-(5,6,7,8-tetrahydro-
5,5,8,8-tetramethyl-2-anthracenyl)benzoic acid gave Kd
values ~or RAR~ and RAR~ that were higher by 32-fold and
84-~old, respectively, than ~or the RAR~.
Transcriptional activation assays in HeLa cells using
expression vectors for the receptors and the (TRE)3-tk-
CAT reporter plasmid required AHPN concentrations ~or
50~ m~;m~l activation that were 19-~old and 3.9-~old
higher for RAR~ and RAR~, respectively than ~or RARr
(Bernard et al, Biochem. Biophys. Res. Commun., 186:977-
983 (1992)).
The present inventors theorized that i~ AHPN-
mediated inhibition o~ breast carcinoma proliferation
occurred through activation o~ RAR~, similar or greater
transactivation o~ a transfected RARE-mediated reporter
gene with AHPN than that ~ound with RA, in MDA-MB-231
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- cells, which only possesses RAR~ and are refractory to
the antiproli~erative e~ects o~ RA (Roman et al, Cancer
Res., 53:5940-5945 (1993); Sheikh et al, J. Cell.
Biochem., 53:393-404 (1993); and van der Burg et al,
Mol . Cell . Endo., 91:149-157 (1993)) would be expected
to be seen. There~ore, the ability o~ AHPN to activate
the RARE pathway in MCF-7 and MDA-MB-231 cells was
compared utilizing a $2-RARE-controlled CAT reporter
gene. The $2-RARE is predominantly activated by RAR/RXR
heterodimers and to a lesser extent by RXR homodimers
and has a direct repeat 5 (DR5)-type response element
located in the promoter region o~ the RAR$2 gene (Zhang,
Nature, 355:441-446 (1992); and Zhang et al, Mol . Cell .
.Biol ., 14:4311-4323 (1994)). As shown in Figure 4, AHPN
displayed signi~icantly decreased potency than RA in its
ability to transactivate the endogenous retinoid
receptors in both MCF-7 and MDA-MB-231 cells.
Based on these results, it was concluded that AHPN
apparently does not mediate its antiproli~erative
ef~ects through the RAR pathway since AHPN is a much
more potent inhibitor o~ MCF-7 and MDA-MB-231 growth
than RA but displays markedly less transactivation o~
the $2RARE response element than RA in both cell types.
HL-6OR cells do not possess a ~unctional RAR and their
growth is not inhibited by RA (Robertson et al, Bl ood,
80:1885-1889 (1982)). The e~fect o~ AHPN on HL-60R
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growth was also studied to ~urther assess the role of
the RAR pathway on AHPN-mediated ihhibition of growth.
AHPN was found to markedly inhibit the proliferation of
HL-60R leukemia cells while RA had no e~ect (Figure 5).
These results further substantiate that the RAR pathway
is not involved in AHPN-mediated growth inhibition.
In order to investigate whether AXPN functions
through the RXR homodimer selective pathway, the ability
of AHPN to transactivate two naturally occurring RXREs,
i.e. APO-AI and CRBPII which were transfected into MCF-7
and MDA-MB-231 was compared. The APO-AI, which is
activated by RXR/RXR homodimers and to a lesser extent
by RAR/RXR heterodimers, is a DR-2 type response element
located in the promoter region of the APO-AI gene (Zhang
et al, Nature, 358:587-597 (1992); and Zhang et al, Mol.
Cell. Biol., 14 :4311-4323 (1994)). The CRBPII RXRE is
activated by RXR/RXR homodimers and is a DR-1-type
retinoid response element. SR11246 which induces RXR
homodimer formation, was a good activator of the RXRE
pathway, while minimal activation o~ the RXRE was noted
in the presence o~ AHPN (Figure 4).
EXAMPLE 3
A~PN modulation of AP-l activity
Activation o~ AP-1 mediated gene transcription is
closely associated with cellular proli~eration (Pardee,
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.
-35-
Science, 246:603-606 (1989)). RA through its RAR and
RXR nuclear receptors can antagonize c-Fos/c-Jun-
mediated activation of the AP-1 consensus se~uence
(Schule et al, Proc. Natl. Acad. Sci. USA, 88:6092-6096
(1991); Busam et al, ~. Biol. Chem. , 267:19971-19977
(1992); Jaf~ey et al, Cancer Res., 52:2384-2388 (1992);
and Salbert et al, Mol . Endocrinol ., 7:1347-1356
(1993)). Therefore, the effect of AHPN on AP-1-mediated
CAT activity was studied in both MCF-7 and MDA-MB-231
cells. As shown in Figure 4, l~M RA significantly
inhibited AP-l-mediated CAT activity in MCF-7 cells,
whereas AHPN had no e~fect. Neither RA nor AHPN
modulated AP-1-mediated CAT activity in MDA-MB-231
cells.
EXAMPLE 4
AHPN-media ted apoptosis
The decrease in MDA-MB-231 and MCF-7 cell counts
(Figure 1), and change in cell appearance following
exposure to AHPN, but not to RA, suggested that exposure
to APHN resulted in programmed cell death, i.e.,
apoptosis. To further investigate this possibility,
cell morphology was ~ml ned and DNA electrophoretic
analysis was performed on MDA-MB-231 and MCF-7 cells
~ollowing exposure to l~M RA or l~M AHPN. Following
exposure to AHPN, MCF-7 cells demonstrated morphologic
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changes associated with the apoptotic process (Isaacs,
J.T., Curr. Opinion in Oncol., 6:82-89 (1994)). The
cells had marked nuclear fragmentation and chromatin
condensation with the nuclear and cytoplasmic membranes
rem~;n;ng intact (data not shown). No such changes were
found in MCF-7 cells following exposure to RA.
Incubation of MDA-MB-231 or MCF-7 cells with 1 ~M AHPN
resulted in internucleosomal cleavage and laddering of
the DNA on gel electrophoresis (Figure 6), indicative of
apoptosis (Isaacs, J.T., Curr. Opinion in Oncol ., 6:82-
89 (1994)). Internucleosomal cleavage and DNA laddering
was not seen following exposure of MCF-7 or MDA-MB-231
cells to RA (Figure 6).
AHPN-mediated apoptosis was also evaluated in MDA-
MB-231 cells utilizing the Apotag assay in which 3'0H
ends o~ the DNA breaks associated with apoptosis are
labeled with digoxigenin-coupled dUTP using terminal
deoxynucleotidyl trans~erase and digoxigenin detected
using immunoperoxidase staining. As shown in Figure 7,
increasing AHPN concentrations resulted in a progressive
increase in the percent o~ apoptotic cells as indicated
by ~ragmentation of the DNA and nuclear immunoperoxidase
staining. Also, exposure to 1 ~M AHPN for varying
periods o~ time (0, 24, 48 o~ 72 h) resulted in a
progressive increase in the percent o~ apoptotic cells
(Figure 8). Exposure to 1 ~M RA ~or 72 h resulted in 8
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o~ MCF-7 cells displaying digoxigenin UTP incorporation
while no staining was seen in MDA-~B-231 cells (data not
shown).
EXAMPLE 5
AHPN induction of WAFl/CIPl
Apoptosis can be triggered in cells through a
variety o~ pathways (Isaacs, J.T., Curr. Opinion in
Oncol ., 6:82-89 (1994)). A number o~ cellular proteins
either enhance or inhibit G1 arrest or programmed cell
death. The cellular proteins bc1-2 (Vaux, Proc. Natl.
Acad. Sci. USA, 90:786-789 (1993)), bclXL (Boise et al,
Cell, 74:597-608 (1993), Ich-ls (Wang et al, Cell,
78:739-750 (1994)) promote cell survival, while
WAF1/CIP1 induces Gl arrest (Hunter et al, Cel l, 79:573-
582 (1994)), and bax (Oltvai et al, Cell, 74:609-619
(1993)), IchL (Wang et al, Cell , 78:739-750 (1994), and
TGF-$1 (Polyak et al, Genes Dev., 8: 9-22 (1994)) enhance
cell death. There~ore, the ability o~ AHPN to modulate
WA1/CIP1, bax, TGF-$1 and bcl-2 mRNA levels was
evaluated.
As shown in Figure 9, 1 ~m AHPN markedly enhanced
WAF1/CIP1 mRNA levels in both MCF-7 cells and MDA-MB-231
cells. AHPN elevated WAF1/CIP1 mRNA levels as early as
1 h a~ter addition to MDA-MB-231 cells, and within 6 h
a~ter addition to MCF-7 cells (Figure 9). There was a
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six-~old and 35-fold increase in WAFl/CIPl mRNA levels
a~ter 72 h incubation with AHPN in MCF-7 and MDA-MB-231
cells respectively. MDA-MB-231 cells expressed
signi~icantly lower WAFl/CIPl mRNA levels than MCF-7
cells due to the presence o~ a mutated (non~unctional)
p53, as previously demonstrated (Sheikh et al, Oncogene,
9:3407-3415 (1994)). RA did not modulate WAFl/CIPl mRNA
levels (data not shown) in either cell type. That AHPN
enhanced WAFl/CIPl gene transcription may be seen ~rom
the results contained in Figure lO. A six-~old and two-
~old increase in luci~erase activity in MDA-MB-231 and
MCF-7 cells, respectively, was observed when a WAF-luc
reporter construct was trans~ected into the cells in the
presence of AHPN.
EXAMPLE 6
A~PN modulation of bc1-2 and bax m~2NA expression
Negative and positive regulators o~ apoptosis have
been identi~ied. Bc1-2 expression enhances cell
survival and blocks TGF$1 induced cell death while bax
promotes cell death and may enhance p53-mediated
apoptosis (Selvakumaran et al, Oncogene, 9:1791-1798
(1994)). Accordingly, the e~ect o~ AHPN on bc1-2 and
bax mRNA expression was ~m; ned These results
indicated that while AHPN signi~icantly enhanced
WAFl/CIPl mRNA levels in MCF-7 cells within 6 h, it
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~ signi~icantly decreased bc1-2 mRNA levels (Figure 11).
Bc1-2 mRNA expression could not be detected in MDA-MB-
231 cells in the absence or presence o~ AHPN.
Similarly, AHPN (1 ~M) signi~icantly elevated bax mRNA
levels within 6 h in MCF-7 cells, while bax mRNA
expression could not be detected in MDA-MB-231 cells.
Also, it was found that AHPN (1 ~M) did not modulate
TGF~1 mRNA or protein levels in either MCF-7 or MDA-MB-
231 cells (data not shown).
EXAMPLE 7
A~PN modulation of cyclin expression
Cyclins are important mediators o~ cell cycle
progression (van der Hevvel et al, Science, 262:2050-
2054 (1994)). In addition, numerous investigators have
suggested that cyclin D1 and cyclin E are rate-limiting
~or progression through Gl (Jiang et al, Oncogene,
8:3447-3457 (1993); Quelle et al, Genes Dev., 7:1559-
1571 (1993); and Baldin et al, Genes Dev., 7:812-821
(1994)). Since exposure to AHPN resulted in Go/Gl
arrest, the e~ect o~ AHPN treatment on cyclin E, D1, A
and B1 expression in MCF-7 cells was ~m; ned As shown
in Figure 12, cyclin D1 mRNA levels were signi~icantly
decreased within 6 h o~ exposure to AHPN. Decreases in
cyclin A and cyclin B1 mRNA were also noted within 6 h
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following the addition of AHPN, while there was no
modulation of cyclin E mRNA levels (Figure 12).
CONCLUSIONS
Based on these results, it was concluded that while
5 AHPN displays enhanced binding to and transactivation
of the RAR~y receptor, AHPN apparently also functions
through a RAR and RXR-independent mechanism. Also, AHPN
was found to significantly inhibit the proliferation of
both ER-positive MCF-7 and T47D cells, as well as ER-
negative MDA-MB-231 and MDA-MB-468 cells, whereas RA
inhibited only ER-positive breast carcinoma cell
proliferation. Moreover, in MDA-MB-231 cells, AHPN was
found to display significantly less transactivation than
RA of the RAR receptor on the RARi32 R~RE as well as the
15 APO-1 RXRE, which can also be activated by RAR-RXR
heterodimers. These results indicate there to be no
correlation between AHPN transactivation of the RARr
receptor and its ability to inhibit breast carcinoma
cell proliferation, even in MDA-MB-231 cells which only
20 possess a functional RARy receptor. Also, the results
indicate that AHPN does not activate the RXRE pathway
(as demonstrated by the lack of CAT activity on
transfection of MCF-7 and MDA-MB-231 cells with the
CRBPII and APO-1 RXRE-tk-CAT reporter constructs in the
25 presence of AHPN).
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Further, AHPN was ~ound to inhibit the
proliferation of the RA-resistant HL-60R cells which
possess a defective RAR~ due to a point mutation in the
RAR~ gene resulting in its truncation and inability to
bind RA. (Robertson et al, Blood, 80:1885-1889 (1982)).
HL-60R cells do not possess a RAR~ or RAR~ receptor
(Nervi et al, Proc. Natl . Acad . Sci . USA, 86:5854-5858
(1989)). Accordingly, these results indicate that
growth inhibition by AHPN is not mediated via the RAR
pathway in these cells.
As opposed to the cytostatic effect noted with RA
in breast cancer cells (Fontana, J.A., Exp . Cell Res .,
55:136-144 (1987)), these results indicate that exposure
to AHPN results in apoptosis. That AHPN induces
apoptosis was demonstrated by several lines of evidence.
For example, AHPN treatment of MCF-7 and MDA-MB-231
cells resulted in chromatin condensation and DNA
fragmentation while the nuclear and plasma membranes
remained intact. These ~indings are consistent with the
process of apoptosis, as opposed to cellular necrosis
where there is increased plasma membrane permeability
accompanied by cellular edema and osmotic lysis o~ the
cell (Isaacs, J.T., ~urr. Opinion in Oncol ., 6: 82-89
(1994)).
-
Also, AHPN arrested MCF-7 and MDA-MB-231 cells in
the Go/G1 phase o~ the cell cycle. This arrest in Go/G
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was preceded by the marked increased transcription o~
WAFl /CIPl mRNA. WAF1/CIPl inhibits the function o~ a
number o~ cyclin/cyclin-dependent protein kinase (CDK)
complexes including cyclin A-CDK2, cyclin E-CDK2 and
cyclin D-CDK- resulting in the arrest o~ the cells in G
(Hunter et al, Cell, 79:573-582 (1994)). p53-mediated G
arrest in response to DNA damage appears to require the
elevation o~ WAFl /CIPl levels (Hunter et al, Cel l,
79:573-582 (1994)).
The WAFl/CIPl promoter has a p53 consensus sequence
and thus the post-transcriptional elevation o~ p53
levels following DNA damage results in enhanced
WAFl /CIPl gene transcription and subsequent Gl arrest.
It has previously been shown that MCF-7 cells possess a
wild-type (~unctional) p53 while MDA-MB-231 cells
possess a mutant (non~unctional) p53 (Sheikh et al,
Oncogene, 9:3407-3415 (1994)). Niewolik et al
(Oncogene, 10:881-890 (1995)) have also reported that
p53 derived ~rom MDA-MB-231 cells does not bind to the
p53 consensus sequence. Thus, the AHPN-mediated
increase in WAFl/CIPl transcription in MDA-MB-231 and
presumably MCF-7 cells must occur through a p53-
independent mechanism. It has previously been reported
that DNA-damaging agents, as well as serum starvation-
induced growth arrest, did not increase p53 levels butdid increase WAFl /CIPl mRNA levels in cells carrying
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W O 97/03682 PCTAUS96/11736
mutant p53 (Sheikh et al, Oncogene, 9:3407-3415 (1994)).
Incubation with AHPN did not result in elevated p53
levels in either MCF-7 or MDA-MB-231 cells (data not
shown). Several investigators have demonstrated that
RA-mediated differentiation of HL-60 cells is associated
with a p53 independent elevation of WAFl/CIP1 levels
(Jiang et al, Oncogene, 9:3397-3406 (1994); and Steinman
et al, Oncogene, 9:3389-3396 (1994)). Michiel et al
(Cancer Res., 54:3391-3395 (1994)) have hypothesized the
existence of two separate pathways for the induction of
WAFl/CIPl, a mitogen-activated p53-independent mechanism
and a DNA damage-activated, p53-dependent mechanism. In
addition to elevating WAFl/CIP1 levels and inhibiting
cyclin/cyclin-dependent protein kinase activity, AHPN
significantly decreased cyclin D1, A and B mRNA levels,
perhaps also contributing to G1 arrest.
The bc1-2 oncoprotein is found in the inner
mitochondrial membrane, as well as other subcellular
compartments, i.e., the endoplasmic reticulum and the
nuclear membrane (Oltvai et al, Cell, 74:609-619
(1993)), where its overexpression results in
prolongation of cell survival (~ockenberry et al, Proc.
Natl. Acad. Sci. USA, 88:6961-6965 ~1991)). Bc1-2 is
reported to both homodimerize and heterodimerize with a
number of other proteins, including bax, bcl XL~ bcl Xs~
and Mcl-1 (Sato et al, Proc. Natl. Acad. Sci. USA,
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W O 97/03682 PCTAJS96/11736
-44-
91:9238-9242 (1994)). Heterodimer formation with bcl Xs
or bax, as well as homodimer formation by bax,
inactivates bcl-2. Selvakumaran et al, Oncogene,
9:1791-1i98 (1994) and Miyashita et al, Oncogene,
9:1799-1805 (1994) have reported that the elevation in
p53 leads to the upregulation of bax and the
downregulation of bc1-2 levels. These observations
suggest that p53 markedly inhibits bc1-2 mRNA
expression, while elevating the levels of bax mRNA
levels. Interestingly, bc1-2 is also down-regulated by
mutant p53 (Haldar et al, Cancer Res., 54:2095-2097
(1994) and Zhan et al, Oncogene, 9:3743-3751 (1994))
have speculated that elevation of bax and down
regulation o~ bc1-2 play major roles in irradiation-
induced apoptosis. AHPN-mediated elevation of bax mRNA
levels and downregulation o~ bc1-2 mRNA occurred in MCF-
7 cells, which possess a wild-type p53. Bcl-2 or bax
mRNA expression in MDA-MB-231 cells were not detected in
the previously described experiments. By contrast,
Haldar et al, Cancer Res. , 54:2095-2097 (1994), detected
low bc1-2 protein levels in MDA-MB-231 cells. This
discrepancy may be attributable to different strains o~
MDA-MB-231. The results also indicate that MDA-MB-231
cells are exquisitely sensitive to AHPN-mediated Go/Gl
arrest and programmed-cell death. It is possible that
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-45-
the low levels o~ bc1-2 may enhance the sensitivity of
MDA-M3-231 to this retinoid.
Moreover, the foregoing results show that AHPN
displays a wide spectrum o~ action by inhibiting the
growth o~ both ER-positive and ER-negative breast
carcinoma cell lines. In addition, as opposed to other
retinoids, exposure to AHPN results in programmed death
o~ breast carcinoma and leukemia cells. Thus, based on
these results, AHPN is well suited ~or the treatment or
prevention o~ breast cancer or leukemia.
While the invention has been described and
illustrated with respect to speci~ic embodiments and
features, it will be appreciated that various changes
and modi~ications, can be made without departing ~rom
the invention.
