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Biochemistry and Biophysics |
Department of Pharmaceutics and Pharmacodynamics, Center for Pharmaceutical Biotechnology [S. M., V. H., A-N. T. K.] College of Pharmacy and Department of Surgical Oncology, College of Medicine [K. C.], University of Illinois at Chicago, Chicago, Illinois 60612
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, which have been extensively
studied (2, 3, 4, 5, 6, 7)
. The biotransformation in humans is
primarily attributable to the action of CYP 3A4 (8)
and
CYP 2D6 (9)
. In humans as well as in rodents, OHT and DMT
are shown to be the major metabolites, present in high levels in both
the plasma and the tumor tissues (2
, 4
, 10
, 11)
. It has
been reported that the metabolites OHT and DMT possess several hundred
times more or equal affinity, respectively, toward the ER
as
compared with the parent TAM (12
, 13)
. Thus, OHT and DMT
are considered to be more active than or equally active as TAM in
retarding the estrogen-dependent cell growth and proliferation of
ER-positive mammary tumors.
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(25)
, and others. The cleavage of the
various substrates contributes to the typical morphological and
biochemical features observed in the apoptosis of cells. Induction of apoptosis can be considered as one of the pharmacodynamic end points in the measurement of the efficacy of chemotherapeutic agents (26) . It has been shown that daily administration of TAM (1 mg/kg) resulted in the regression of NMU-induced rat mammary tumors, and that this has been associated with the inhibition of cell proliferation and induction of apoptosis (27 , 28) . NMU-induced mammary tumors are ER-positive and have been used as a preclinical in vivo model for most of the breast cancer chemoprevention and therapy studies, because they are very similar in morphology and origin to human breast cancer (29) . Because the antitumor mechanism of TAM action is yet to be completely understood and its metabolites have been shown to possess growth inhibitory activity, and because caspase-dependent apoptosis may be a determinant of the cytotoxicity of anticancer agents both in vitro and in vivo, we evaluated the role of TAM metabolism in terms of caspase activation and induction of apoptosis in rat mammary tumors as well as in human breast cancer cells. In contrast to most previous studies, here we assessed the early (1–4 days) cellular responses of mammary tumors to TAM and its metabolites.
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MATERIALS AND METHODS |
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-tocopherol), DAPI were all obtained from Sigma (St. Louis, MO).
The hydrochloride of DMT was generously provided by Dr. M. Matilde
Marques (Instituto Superior Tecnico, Lisbon, Portugal). The fluorogenic
tetrapeptide substrates of caspase-3 (Ac-DEVD-MCA), and caspase-1
(Ac-YVAD-MCA) were obtained from Peptides International (Louisville,
KY), and substrates for caspase-8 (Ac-IETD-MCA), caspase-9
(Ac-LEHD-FCA), and the pan-caspase inhibitor, z-VAD-fmk were purchased
from Calbiochem (San Diego, CA). For treatment with chemicals, the
medium was removed when cells were about 80% confluent and was
replaced with serum-free MEM. For pretreatment, the cells were
incubated with the respective inhibitors for 1 h followed by
treatment with chemicals.
Animals and Treatment.
The protocol for the animal experiment was approved by the Animal Care
Committee of University of Illinois. Adult female Sprague Dawley rats
(average weight, 250 g) were housed three per cage and fed 4%
Purina diet ad libitum. The rats received two i.p.
injections of NMU (Sigma; 50 mg/kg of body weight in acidified saline)
at the age of 50 and 57 days. Three weeks after NMU injection, the
animals were palpated weekly to monitor the appearance of mammary
tumors. When the palpable tumors reached 
10 mm in diameter, TAM
(emulsified in corn oil at 1 mg/ml; injection volume, 0.25 ml)
treatment was initiated. All of the animals except the controls
received two s.c. injections of TAM, 1 mg/kg of body weight, 12 h
apart (8 p.m. and 8 a.m.). After several repeats of this schedule,
the rat mammary tumors showed significant regression
(30)
. Therefore, we sought to investigate the early
signaling pathway leading to this response, i.e., induction
of caspase-dependent apoptosis in mammary tumors. The rats were divided
into groups of five animals each; one group was left untreated
(control) and others were killed at 1.5, 3.5, 5, 7, 18, 24, 48, 72, and
96 h after the second TAM administration (no samples were taken in
between the two TAM doses; and for the sake of convenience, the data
are reported by considering the time of the second TAM administration,
which was 12 h after the first dose as zero time point). After
light ether anesthesia, the animals were killed by exsanguination via
the abdominal aorta. Blood was collected, allowed to clot for 30 min at
4°C, and centrifuged at 12,000 rpm to separate the serum. The serum
was stored at -70°C for subsequent HPLC analyses of TAM and
metabolites concentration as described below. Mammary tumors were
dissected out and cut into small portions, a portion of the sample was
stored in formalin for histochemical analysis, and others were frozen
at -70°C for subsequent determination of caspase activity.
HPLC Assay for TAM and Metabolites.
The parent drug TAM and its metabolites (OHT, DMT), together with the
internal standard TPP (which was added to 1 ml of serum at a final
concentration of 100 ng/ml), were extracted from rat serum by
solid-phase extraction using Oasis HLB extraction cartridges
according to the manufacturer’s instructions. The final methanol
extract was evaporated to dryness in a stream of helium. The samples
were reconstituted in 150 µl of methanol, and 100 µl was injected
onto the Beckman ODS reverse phase C18
HPLC column (4.6 x 150 mm, 5-µm particle size; Varian
Inc.). The compounds were separated using a Shimadzu LC-10ADvp HPLC
system, with a mobile phase of methanol (A):ammonium acetate [50
mM (pH 7.0); B] in a gradient elution as follows: 75%
A:25% B for the first 10 min followed by 95% A:5% B for next 15 min,
at a solvent flow rate of 1 ml/min. Detection of compounds was
performed using a UV-VIS detector at 260 and 280 nm. The peak area
ratios of TAM:TPP, OHT:TPP, or DMT:TPP after integration were used to
calculate the concentration of TAM and its metabolites in various serum
samples, using extracted standard curves of pure compounds prepared in
blank rat serum (Hilltop Laboratories, NJ).
Caspase Activity Assay.
The tumors (about 100 mg) were first homogenized in lysis buffer [50
mM Tris (pH 7.4), 50 mM ß-glycerophosphate,
15 mM MgCl2, 15 mM EDTA,
100 µM phenylmethylsulfonyl fluoride, 1 mM
DTT, and 150 µg/ml digitonin] by grinding with a pestle in a
Potter-Elvehjem tissue homogenizer (Wheaton). The lysates were further
sonicated twice for 15 s at 50 W using a tissue dismembrator
(Fisher Scientific) and were kept on ice for 30 min. The lysates were
clarified by centrifugation at 12,500 RPM for 20 min at 4°C. Caspase
activity in the supernatant was determined as described previously
(31)
. Briefly, 100 µg of total protein, as determined by
the Bio-Rad protein assay (Bio-Rad, Richmond, VA), was incubated with
200 µM fluorogenic peptide substrates Ac-YVAD-MCA
(caspase-1), Ac-DEVD-MCA (caspase-3), Ac-VEID-MCA (caspase-6),
Ac-IETD-MCA (caspase-8) or Ac-LEHD-MCA (caspase-9) in 50 µl of
caspase assay buffer {100 mM HEPES (pH 7.5), 10%
sucrose, 10 mM DTT, and 0.1%
3-[3-(chloramidopropyl) dimethylamino]-1-propanesulfonate}.
After incubation at 37°C for 2 h, the release of AMC was
measured with a spectrofluorometer (PerSeptive Biosystems, Inc.,
Framingham, MA) at an excitation wavelength of 360 nm and an emission
wavelength of 460 nm. The release of AMC was monitored at 360/530 nm.
When estimating the caspase activity in human breast cancer cell lines
treated with TAM metabolites, the same protocol was followed except
that the cells were lysed by passing through a 23-gauge needle
several times.
Apoptosis Assay by TUNEL or DAPI Staining.
The ApopTag in situ hybridization detection kit (Oncor,
Gaithersburg, MD) was used to identify the apoptotic cells within rat
mammary tumors as described previously (30
, 32
, 33)
.
Negative controls included the top sections on each slide that were
incubated without digoxigenin-dUTP. Positive controls were sections
(provided by manufacturer) from mammary glands of rats 6 days after
ovariectomy. In addition to the brown staining of apoptotic
cells by TUNEL method, the following cytomorphological criteria were
examined: condensation of chromatin alongside of the nuclear membrane,
condensation of cytoplasm around the nucleus, and presence of apoptotic
bodies in the tumor cells. The slides were counterstained with
hematoxylin or methyl green for assessment of tumor morphology
(30
, 32, 33, 34)
. For DAPI staining, human breast cancer cells
treated with TAM metabolites were harvested by trypsinization and fixed
in methanol:acetic acid (3:1) solution for 30 min on ice as described
previously (31)
. The fixed cells were placed on slides and
stained with 1 µg/ml aqueous solution of DAPI for 15 min. The nuclei
were observed under Zeiss fluorescence microscope. A portion of breast
cancer cells harvested as above was stained with 1% trypan blue and
the percentage of necrotic cells was counted with a hemocytometer.
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RESULTS |
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and Table 1
) to BDL after 5 h. One of the active metabolites, OHT, presumably
metabolized by CYP 3A1 (rat), was formed rapidly after TAM
administration and attained a Cmax of
385 ± 132 ng/ml at 5 h, and subsequently declined
to BDL after 7 h (Fig. 2B
;Table 1
). A second
metabolite, DMT, presumably biotransformed by CYP 2D1 (rat) and also
3A1, appeared relatively late as compared with OHT (Fig. 2C)
. It reached a Cmax of
2136 ± 1516 ng/ml at 48 h, subsequently declined
slowly, and was detected even at 96 h. The total exposure in rat
serum of OHT and DMT was considerably higher than that of the parent
TAM with regard to the area under the serum concentration
versus time curve (AUC). The estimated
AUC were 0.57, 2.66, and 14.9 µg·h/ml for TAM, OHT, and
DMT, respectively (Table 1)
. Thus, after TAM administration, high
levels of metabolites were found in the plasma of female rats.
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. The elevation in caspase activity was first seen at
18 h after the second TAM dose and reached the peak value at
24 h. The high caspase activity was sustained until 48 h,
gradually decreasing at 72 h and returning to basal level at
96 h. Caspase-8 and -9 were also potently activated, although no
induction in caspase-1 activity was detected under the experimental
conditions. Apoptosis in mammary tumors was determined by TUNEL
staining, apoptotic cells being recognized by brown staining of the
nucleus (Fig. 3B)
. In control tumors, apoptotic cells were
rare in peripheral proliferating areas but frequent in the areas close
to necrosis (30
, 32, 33, 34, 35)
. In TAM-treated animals, the
number of apoptotic cells started to increase 18 h after the
second TAM administration (Fig. 3C)
, and the number of
apoptotic cells continued to rise at subsequent sampling points, up to
96 h. The highest number of apoptotic cells were found in the
animals killed at 48–72 h after the second TAM administration. Thus,
kinetically, the activation of caspases (peak at 24 h) preceded
the induction of apoptosis (peak at 48 h) in the rat mammary
tumors. Because the plasma concentrations of TAM and OHT preceded, and
those of DMT paralleled, the caspase kinetics, we next determined
whether these metabolites of TAM were able to induce caspase-dependent
apoptosis.
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, as well as previous findings
(2
, 4)
, suggest that high levels of TAM metabolites are
formed after the administration of TAM to rats. Therefore, we wanted to
determine whether these metabolites of TAM were capable of inducing
caspase activity and/or apoptosis in human breast cancer cell lines. We
recently reported that TAM (5 µM) induces the activity of
caspase-3-, -8-, and -9-like proteases prior to inducing apoptosis in
both ER-positive and ER-negative breast cancer cells (14)
.
In the present study, 5 µM of OHT or DMT was added to
MCF-7 (ER-positive) or BT-20 and MDA-MB 231 (both ER-negative) human
breast cancer cell lines, and induction of caspase activities and
apoptosis were investigated over a period of 48 h. As shown in
Fig. 4A
, both metabolites caused a time-dependent increase in the
activity of caspase-3-like protease in the ER-negative MDA MB-231
cells. The caspase-3 activity appeared at 15 h and peaked at
24 h after treatment. Whereas OHT also potently activated
caspase-9, DMT did so to a lesser extent. Both of the metabolites also
increased the activity of caspase-8, but neither of them activated
caspase-1. Similar results were obtained in ER-negative BT-20 cells. In
contrast, no activation of caspase-3 activity was observed in MCF-7
cells (data not shown), consistent with what was reported in the
literature (36)
. The effect of the metabolites on cell
death was studied using two methods as described previously (31
, 37)
: (a) DAPI staining to detect nuclear changes such
as fragmentation and condensation; and (b) trypan blue
exclusion to detect cell death attributable to membrane damage.
Induction of cell death by both compounds occurred at least after
24 h of exposure. The increase in condensed nuclei paralleled that
of trypan blue-positively stained cells, which suggested that the
cytotoxicity induced by the metabolites was attributable to both
necrotic and apoptotic death (Fig. 4B)
. To further define
the role of caspases in cell death induced by TAM metabolites, we
pretreated the breast cancer cell lines with the broad-specificity
caspase inhibitor, z-VAD-fmk, as described previously (31
, 37)
. Pretreatment of the cells with z-VAD-fmk completely
inhibited both OHT- and DMT-induced cell death (Fig. 4C)
, as
detected by both DAPI and trypan blue staining. This suggests that the
cell death induced by the metabolites of TAM was caspase-dependent. We
had also reported that the lipid-soluble antioxidant V-E inhibited
TAM-induced caspase activity and apoptosis in breast cancer cells
(14)
. Similarly, V-E also inhibited OHT-induced, but not
DMT-induced, caspase activity and cell death.
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DISCUSSION |
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The HPLC analysis of rat serum after s.c. TAM administration indicated
that the parent compound was metabolized to OHT and DMT. OHT was formed
rapidly and disappeared rapidly, whereas peak levels of DMT did not
occur until 48 h and were detected at 96 h. This observation
is consistent with a previous report (4)
in which rats
were given 200 mg/kg TAM p.o. This pharmacokinetic difference between
OHT and DMT suggests differential substrate specificity and reaction
rate constants for the two cytochrome P-450 isoforms, presumably CYP
2D1 and 3A1 (CYP 2D6 and CYP 3A4 in humans in the formation of these
metabolites from TAM. It is possible that the early conversion of TAM
to DMT and a prolonged elimination phase for the parent as well as for
the metabolites may have been undetected because of the limited
sensitivity of UV-detection in our HPLC assay as compared with
fluorescence or mass-spectrometric methods used by others. It remains
to be determined whether CYP inhibition led to decreased clearance and,
therefore, the accumulation of DMT. Importantly, both OHT and DMT were
found in concentrations much higher than that of the parent drug. In
addition, previous reports have shown that the concentration of TAM and
its metabolites in mammary tumors are much higher than their plasma
levels (2
, 11
, 38
, 39)
. This, together with the facts that
both metabolites are active ER-binding moieties and effective in
inducing growth arrest of breast cancer cells, prompted us to
investigate whether the metabolites themselves can induce
caspase-dependent apoptosis. Whereas treatment with 1 mg/kg TAM
resulted in high circulating levels of metabolites in rat serum (Fig. 2)
, no metabolites were detected in culture on treatment of human
breast cancer cell lines with TAM at 5 µM for up to
48 h (data not shown), presumably because of low CYP activity.
Therefore, to determine their individual activity, each of the TAM
metabolites was added to human breast cancer cell lines. Both OHT and
DMT induced caspase activation with similar kinetics comparable with
that induced by TAM (Fig. 4A
, Ref. 14
),
although OHT was somewhat more potent. Importantly, the general
inhibitor of caspases, z-VAD-fmk, completely blocked cell death induced
by all three of the compounds, which suggested that caspases may play a
pivotal role in cell death (Fig. 4C)
induced by these
compounds. Interestingly, the lipid-soluble antioxidant V-E also
completely inhibited TAM- and OHT-induced cell death but had little
effect on DMT-induced cell death (Fig. 4C)
. The reasons for
these differences are not clear but may be related to oxidative stress
or cell membrane partitioning. Thus, it is conceivable that the delayed
and sustained caspases activation and apoptosis in rat mammary tumors
after TAM administration may be attributable to the conversion of TAM
to its active metabolites OHT and DMT—because both metabolites are
found in high concentrations in the rat mammary tumors (2
, 38)
—and/or could be attributable to the indirect effect of TAM
that requires signaling intermediates. A confirmatory experiment to
demonstrate the in vivo activity of the metabolites of TAM
will be to administer the metabolites directly to mammary tumor-bearing
rats and measure caspase activation and apoptosis in the tumors.
Caspase-3 is the downstream effector caspase that cleaves various
cytoplasmic and nuclear substrates, eventually giving rise to the
typical morphological characteristics of apoptosis. A time-dependent
induction in caspase-3 activity by TAM metabolites prior to the
induction of apoptosis suggests that the caspase cascade may be
responsible for the metabolite-induced cell death in human breast
cancer cell lines and possibly also in rat mammary tumors. The more
upstream caspases, caspase-8 and -9, were also activated by the
metabolites. Caspase-8 belongs to the DED family, and is
activated on binding of tumor necrosis factor (TNF-)-related ligands
to their respective cell surface receptors. Caspase-9, on the other
hand, is activated upon release of the mitochondrial cytochrome
c, which binds to Apaf-1 and triggers its own activation and
subsequent caspase-3. Both the upstream caspases have been shown to be
involved in apoptosis induced by some anticancer drugs, such as
camptothecin and Adriamycin, either through the induction of FasL
expression (40, 41, 42)
, or through the
mitochondrial-cytochrome c-Apaf-1 pathway
(43, 44, 45)
. Thus, TAM and its metabolites may initiate
activation of the caspase cascade through different pathways, either by
inducing expression of TNF- family ligands and/or through the
mitochondrial-cytochrome c-Apaf-1 pathway, possibly through
generation of oxidative stress. Indeed, recently, we have found that
the phenolic antioxidant butylated hydroxyanisole (BHA) induces loss of
mitochondrial membrane potential (
m),
release of cytochrome c, and activation of caspase-3, -8,
and -9 (46)
. Future studies will explore the second
messengers and mediators of TAM- and metaboliteinduced signaling
events leading to caspase activation and apoptosis.
In summary, we have provided the first evidence that caspases are activated in rat mammary tumors early, within 18–24 h after TAM administration, and that the OHT and DMT metabolites of TAM can induce caspase-dependent apoptosis in various human breast cancer cells. This caspase-dependent apoptosis may contribute to the regression of rat mammary tumors after TAM treatment and may add to the effectiveness of TAM in breast cancer patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH Grant R01-CA73674 (to
A-N. T. K.). 
2 Present Address: DuPont Pharmaceuticals Company,
1094 Elkton Road, P. O. Box 30, Newark, DE 19711.
3 To whom requests for reprints should be
addressed, at Department of Pharmaceutics and Pharmacodynamics, Center
for Pharmaceutical Biotechnology, M/C 865, College of Pharmacy,
University of Illinois at Chicago, 833 South Wood Street, Chicago, IL
60612. Phone: (312) 413-9646; Fax: (312) 413-9303; E-mail: KongT{at}uic.edu
4 The abbreviations used are: TAM,
tamoxifen; OHT, 4-hydroxy TAM; DMT, N-desmethyl TAM;
NMU, N-nitroso N-methylurea; ER, estrogen
receptor; z-VAD-fmk, benzyloxy Val-Ala-Asp-fluoromethyl ketone; AMC,
7-amino-4-methylcoumarin; TUNEL, terminal deoxynucleotidyl
transferase-mediated dUTP nick-end labeling; DAPI, diamidino
phenylindole; V-E, vitamin E; BDL, below detection limit; CYP,
cytochrome P-450; Apaf-1, apoptotic protease activating factor 1; HPLC,
high-performance liquid chromatography; TPP, 2,4,6-triphenyl phenol;
AUC, area under the (serum) concentration versus time
curve.
Received 4/12/00. Accepted 9/29/00.
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REFERENCES |
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in induction of apoptosis. J. Biol. Chem., 272: 20317-20320, 1997.This article has been cited by other articles:
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  J. Cheng, D. V. Yu, J.-H. Zhou, and D. J. Shapiro Tamoxifen Induction of CCAAT Enhancer-binding Protein {alpha} Is Required for Tamoxifen-induced Apoptosis J. Biol. Chem., October 19, 2007; 282(42): 30535 - 30543. [Abstract] [Full Text] [PDF]
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 Y. M. Shah, M. Al-Dhaheri, Y. Dong, C. Ip, F. E. Jones, and B. G. Rowan Selenium disrupts estrogen receptor {alpha} signaling and potentiates tamoxifen antagonism in endometrial cancer cells and tamoxifen-resistant breast cancer cells Mol. Cancer Ther., August 1, 2005; 4(8): 1239 - 1249. [Abstract] [Full Text] [PDF]
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 J. Gauduchon, F. Gouilleux, S. Maillard, V. Marsaud, J.-M. Renoir, and B. Sola 4-Hydroxytamoxifen Inhibits Proliferation of Multiple Myeloma Cells In vitro through Down-Regulation of c-Myc, Up-Regulation of p27Kip1, and Modulation of Bcl-2 Family Members Clin. Cancer Res., March 15, 2005; 11(6): 2345 - 2354. [Abstract] [Full Text] [PDF]
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 K. B. Bouker, T. C. Skaar, D. R. Fernandez, K. A. O'Brien, R. B. Riggins, D. Cao, and R. Clarke Interferon Regulatory Factor-1 Mediates the Proapoptotic but Not Cell Cycle Arrest Effects of the Steroidal Antiestrogen ICI 182,780 (Faslodex, Fulvestrant) Cancer Res., June 1, 2004; 64(11): 4030 - 4039. [Abstract] [Full Text] [PDF]
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H. J. Thompson, Z. Zhu, and W. Jiang Identification of the Apoptosis Activation Cascade Induced in Mammary Carcinomas by Energy Restriction Cancer Res., February 15, 2004; 64(4): 1541 - 1545. [Abstract] [Full Text] [PDF]
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M. Cuendet, K. Christov, D. D. Lantvit, Y. Deng, S. Hedayat, L. Helson, J. D. McChesney, and J. M. Pezzuto Multiple Myeloma Regression Mediated by Bruceantin Clin. Cancer Res., February 1, 2004; 10(3): 1170 - 1179. [Abstract] [Full Text] [PDF]
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M. Obrero, D. V. Yu, and D. J. Shapiro Estrogen Receptor-dependent and Estrogen Receptor-independent Pathways for Tamoxifen and 4-Hydroxytamoxifen-induced Programmed Cell Death J. Biol. Chem., November 15, 2002; 277(47): 45695 - 45703. [Abstract] [Full Text] [PDF]
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