Antiestrogen binding site and estrogen receptor mediate uptake and distribution of 4-hydroxytamoxifen-targeted doxorubicin-formaldehyde conjugate in breast cancer cells

Article abstract of DOI:10.1021/jm049496b

The anthracycline antitumor drug, doxorubicin (DOX), has long been used as a broad spectrum chemotherapeutic. The literature now documents the role of formaldehyde in the cytotoxic mechanism, and anthracycline-formaldehyde conjugates possess substantially

Full text of DOI:10.1021/jm049496b

J. Med. Chem. 2004, 47, 6509-6518
6509
Antiestrogen Binding Site and Estrogen Receptor Mediate Uptake and
Distribution of 4-Hydroxytamoxifen-Targeted Doxorubicin-Formaldehyde
Conjugate in Breast Cancer Cells
Patrick J. Burke,† Brian T. Kalet, and Tad H. Koch*
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309
Received June 24, 2004
The anthracycline antitumor drug, doxorubicin (DOX), has long been used as a broad spectrum
chemotherapeutic. The literature now documents the role of formaldehyde in the cytotoxic
mechanism, and anthracycline-formaldehyde conjugates possess substantially enhanced
activity in vitro and in vivo. We have recently reported the design, synthesis, and preliminary
evaluation of a doxorubicin-formaldehyde conjugate targeted, via 4-hydroxytamoxifen, to the
estrogen receptor (ER) and antiestrogen binding site (AEBS), which are commonly present in
breast cancer cells. The lead targeted doxorubicin-formaldehyde conjugate, called DOX-TEG-
TAM, was found to possess superior cell growth inhibition characteristics relative to clinical
doxorubicin and an untargeted control conjugate, especially in ER-negative, multidrug resistant
MCF-7/Adr cells. The enhanced activity in the absence of estrogen receptor raised the possibility
that targeting was also mediated via AEBS. Fluorescence microscopy of an ER-negative, AEBS-
positive cell line as a function of time showed initial DOX-TEG-TAM localization in cytosol, in
contrast to initial DOX and untargeted doxorubicin-formaldehyde conjugate localization in
the nucleus. DOX-TEG-TAM was taken up by four AEBS-positive cell lines to a greater extent
than doxorubicin and an untargeted doxorubicin-formaldehyde conjugate. Of the four cell lines,
three were ER negative. DOX-TEG-TAM uptake was inhibited in a dose-dependent manner
by the presence of a competing AEBS ligand. DOX-TEG-TAM retains 60% of the affinity of
4-hydroxytamoxifen for AEBS. DOX-TEG-TAM was also taken up by the AEBS-negative, ER-
positive cancer cell line Rtx-6; with these cells uptake was inhibited in a dose-dependent manner
by the ER ligand, estradiol. The data support the hypothesis that uptake of 4-hydroxytamoxifen
targeted doxorubicin-formaldehyde conjugate is mediated by both the antiestrogen binding
site and estrogen receptor.
Introduction
proteins commonly present in large quantities in breast
cancer cells. The targeting group was 4-hydroxytamox-
ifen (4-OHT), the active metabolite of the antiestrogen
tamoxifen. At least two isoforms of the estrogen receptor
have been identified, designated R and â. They are both
expressed in MCF-7 breast cancer cells,18 bind estradiol
equally, and bind 4-OHT with comparable affinity.19
Photoaffinity labeling experiments indicate that AEBS
in liver microsomes consists of at least four proteins,
three of which have been identified as microsomal
epoxide hydrolase, carboxyesterase (ES10) and liver
fatty acid binding protein (L-FABP), and all are involved
in lipid metabolism.20 Recent experiments indicate that
two of the proteins of AEBS in MCF-7 cells are 3â-
hydroxysterol-∆8-∆7-isomerase and 3â-hydroxysterol-∆7-
reductase and that both are involved in cholesterol
biosynthesis.21
The design of the targeted conjugates is shown in
Figure 1. The formaldehyde function was incorporated
in the form of an N-Mannich base joining the amide of
a salicylamide moiety to the amine of doxorubicin.22 The
salicylamide moiety was used as a time-based chemical
trigger to release the doxorubicin-formaldehyde con-
jugate, the presumed doxorubicin active metabolite,
with a half-life for hydrolysis of about 60 min under
physiological conditions.15,22 The salicylamide trigger
was tethered via ethylene glycol units to 4-hydroxyta-
For several decades the anthracycline antitumor drug,
doxorubicin, has been employed in the treatment of
breast cancer, the second leading cause of cancer-related
death among women behind only lung cancer.1 Doxo-
rubicin exhibits a broad range of antineoplastic activity
that has stimulated an immense research effort toward
the elucidation of the cytotoxic mechanism and the
development of superior doxorubicin derivatives.
While the anthracycline cytotoxic mechanism remains
a matter of debate, DNA has clearly emerged as a
target.2,3 Anthracycline-DNA adducts have been cor-
related with cancer cell death,4 and substantial evidence
indicates that the drug-DNA lesion is mediated by a
formaldehyde linkage.5-10 Additionally, formaldehyde-
releasing prodrugs have been shown to enhance the
activity of doxorubicin.11,12 Finally, anthracycline-
formaldehyde conjugates have been shown to possess
intriguing in vitro13-15 and in vivo16 activity.
We recently reported the design, synthesis, and
preliminary evaluation of a class of doxorubicin-
formaldehyde conjugates17 targeted to the estrogen
receptor (ER) and antiestrogen binding site (AEBS),
* Author to whom correspondence should be addressed. Tel: 303-
492-6193. Fax: 303-492-5894. E-mail: tad.koch@colorado.edu.
† Current address: Wayne State University, Department of Chem-
istry, Detroit, MI 48201.
10.1021/jm049496b CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/18/2004

6510 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Burke et al.
Figure 1. Structures of clinical DOX (1), targeted doxorubicin-formaldehyde conjugates (2a-c), untargeted DOX-saliform (3),
clinical tamoxifen (4), and E/Z-4-hydroxytamoxifen targeting group (5).
Scheme 1. Synthesis of Hydroxytamoxifen-Targeted Doxorubicin-Formaldehyde Conjugatesa
a Reagents and conditions: (a) n-BuLi, KOt-Bu, TMEDA; (b) 4-methoxy-4′-methoxymethylbenzophenone (97%); (c) 6 M HCl (93%); (d)
(n-Bu)4NHSO4, NaOH, 1,2-dibromoethane (90%), (e) BBr3 (57%); (f) excess MeNH2 (91%); (g) DIPEA (55-68%); (h) hydrazine (67-74%);
(i) TFA, EtOH, water (∼50%).
moxifen, the active metabolite of tamoxifen. The target-
ing group was selected on the basis of its high binding
affinity to ER and AEBS. An equimolar mixture of E
and Z geometric isomers of 4-hydroxytamoxifen was
utilized because previous work indicates that p-hydroxy-
substituted triarylbutenes isomerize under cell culture
conditions, compromising the interpretation of results
with pure isomers.23
The synthesis of the targeted conjugates is shown in
Scheme 1. Commercially available n-propylbenzene was
metalated at the R position, and 4-methoxy-4′-meth-
oxymethylbenzophenone was then added. The resulting
carbinol was dehydrated and MOM-deprotected under
acidic conditions. The free phenol was then bromoeth-
ylated via phase transfer conditions. Following removal
of the methyl aryl ether with BBr3, the primary bromide
was aminated with excess methylamine, affording E/Z-
desmethyl-4-hydroxytamoxifen. The resultant secondary
amine was alkylated with norbornyl dicarboximide
protected tethers. The norbornyl dicarboximide protect-
ing group was then removed via hydrazinolysis to reveal
the hydroxylamine ether functionality. Oximation with
DOX-5-formylsalicylamide17,22 proceeded under acidic
conditions, providing 2a-c. A detailed description of the
synthesis has been reported.17

Uptake and Distribution of Tamoxifen-Targeted Doxorubicin
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6511
Table 1. Comparison of Growth Inhibition for Various Breast
Cancer Cell Lines by Targeted and Untargeted Drugs as a
Function of ER, MDR, and AEBS Expressiona
ligand if targeting is mediated by an AEBS binding
interaction.
The reliance on anthracycline fluorescence to quantify
cell uptake is complicated by the effect of local environ-
ments on the anthracycline fluorophore. For example,
drug fluorescence is enhanced in lipid membranes26 and
partially quenched by drug-DNA intercalation.27,28
However, cellular doxorubicin fluorescence has been
shown to increase in a time- and dose-dependent man-
ner;29 as well, cell growth inhibition is directly correlated
with doxorubicin fluorescence.30 Therefore, drug fluo-
rescence provides a reliable indication of the relative
degree of drug uptake and retention.
ER/AEBS/
MDR
DOX-TEG-
TAM
cell line
MCF-7
MCF-7/Adr
Rtx-6
DOX
DOXSF
+/+/-
200 ( 26
70 ( 5
30 ( 5
-/+/+ 10000 ( 1300 2000 ( 320 60 ( 9
+/-/-
200 ( 20
300 ( 33
150 ( 14
61 ( 8
80 ( 9
50 ( 9
67 ( 10
30 ( 5
40 ( 6
MDA-MB-231 -/+/-
MDA-MB-435 -/+/-
a IC50 values are reported in nM and represent the concentration
of drug that inhibits 50% of the cell growth. IC50 values for MCF-
7, MCF-7/Adr, MDA-MB-231, and MDA-MB-435 cells were re-
ported earlier.17 IC50 values for Rtx-6 cells were determined as
described in the Experimental Section.
In addition to flow cytometry, fluorescence microscopy
was utilized to assess the cellular distribution of doxo-
rubicin fluorophore following treatment with targeted
conjugate and untargeted controls. If the targeted
conjugate experiences binding to extranuclear AEBS as
part of the mechanism, the drug should appear cytosolic
following short (5-60 min) treatment times, prior to
trigger hydrolysis.
We now report that DOX-TEG-TAM is taken up and
retained by AEBS-positive MCF-7, MCF-7/Adr, MDA-
MB-231, and MDA-MB-435 cancer cell lines to a greater
extent than clinical DOX and untargeted DOXSF.
Furthermore, DOX-TEG-TAM uptake in MDA-MB-435
cells is reduced in the presence of tamoxifen, as a
competing ligand, in a dose-dependent manner. DOX-
TEG-TAM appears cytosolic by fluorescence microscopy
after short treatment times (5-60 min) in contrast to
DOX and DOXSF, which both appear nuclear. DOX-
TEG-TAM retains 63% of the AEBS binding affinity
relative to the targeting group alone. DOX-TEG-TAM
is also taken up by AEBS-negative, ER-positive, Rtx-6
cells, but with these cells uptake is inhibited by the ER
specific ligand estradiol. These data support a targeting
mechanism mediated by AEBS as well as ER.
Preliminary biological evaluation identified DOX-
TEG-TAM (2c), the conjugate containing the triethylene
glycol derived tether, as the lead compound.17 DOX-
TEG-TAM was more cytotoxic than DOX (1) and un-
targeted control conjugate DOX-saliform (3, DOXSF)
in all four breast cancer cell lines tested, regardless of
ER and multidrug resistance (MDR) expression. Struc-
tures for all compounds are shown in Figure 1. The most
dramatic enhancement in activity for DOX-TEG-TAM
relative to DOX and DOXSF was observed in MCF-7/
Adr cells, an ER-negative breast cancer cell line that
expresses MDR.24 DOX-TEG-TAM was 140-fold and 28-
fold more cytotoxic to MCF-7/Adr cells than DOX and
DOXSF, respectively, as summarized in Table 1. MCF-
7/Adr cells are a doxorubicin (trade name Adriamycin)
resistant variant of MCF-7 cells. In addition to growth
inhibition assays, the targeted conjugates’ ER binding
affinity was investigated. DOX-TEG-TAM retained 2.5%
of the ER binding affinity relative to targeting group
alone.
The dramatic enhancement in growth inhibition of the
targeted doxorubicin-formaldehyde conjugates in MCF-
7/Adr cells relative to doxorubicin and untargeted
DOX-saliform cannot be explained in terms of targeting
the ER; MCF-7/Adr cells are ER negative.25 These
observations raise the possibility that targeting is
occurring, at least in part, through binding interaction
with AEBS. The AEBS targeting hypothesis is as
follows: (1) the targeted conjugates (2a-c) passively
diffuse across the cytoplasmic membrane, (2) the target-
ing group binds to cytosolic AEBS, (3) the AEBS serves
to sequester the conjugate, preventing drug efflux by
the p-glycoprotein drug efflux pump (expressed as part
of the MDR phenotype in MCF-7/Adr cells), and (4) the
trigger fires, releasing the doxorubicin active metabolite,
which then intercalates and alkylates DNA, leading to
cell death. On the other hand, in the case of doxorubicin
and DOXSF, following diffusion across the cell mem-
brane, the p-glycoprotein efflux pump would rapidly
transport them out of the MCF-7/Adr cells.
Results and Discussion
Uptake and Release of DOX-TEG-TAM. The up-
take of 500 nM DOX, DOXSF, and DOX-TEG-TAM
following treatment for various times up to 1 h was
assessed by flow cytometry. Uptake beyond that treat-
ment time was not explored since the half-life for
hydrolysis of the formaldehyde conjugates is 1 h.17 The
release of drugs following a 500 nM treatment for 1 h
was assessed at various times out to 6 h posttreatment.
Resistant MCF-7/Adr cells were first assessed as they
provide a means to evaluate the AEBS targeting hy-
pothesis (above) to explain the dramatic improvement
in tumor cell growth inhibition observed for DOX-TEG-
TAM relative to DOXSF and clinical DOX. The uptake
in MCF-7/Adr cells following treatment for 20, 40, and
60 min with 500 nM DOX, DOXSF, or DOX-TEG-TAM
is shown in Figure 2, panel A. DOX and DOXSF were
taken up by the cells to the extent of one relative
fluorescent unit (RFU), while DOX-TEG-TAM was
taken up almost twice as much. Drug release for all
three compounds was observed at 0.5, 1, 3, and 6 h
following a 1 h treatment with 500 nM of each cytotoxin.
In all three cases the drug fluorescence decreased
dramatically up to 1 h posttreatment, as free drug was
transported from the cell. At 6 h posttreatment DOX-
TEG-TAM maintained a higher level of drug retention
As a means to address the validity of the hypothesis,
we performed a series of experiments to measure the
uptake and retention of the lead compound, DOX-TEG-
TAM (2c), relative to doxorubicin and untargeted DOX-
SF. The cellular accumulation of drug was observed by
tracking the presence of the anthraquinone fluorophore
via flow cytometry. Enhanced accumulation of targeted
conjugate relative to controls would suggest effective
targeting. Furthermore, uptake of the targeted conju-
gate should be reduced in the presence of a competing

Figure 2. (A-D) Cellular uptake and release of drug following treatment with 500 nM DOX, DOXSF, or DOX-TEG-TAM as a function of time, measured by flow cytometric
monitoring of fluorescence from the DOX fluorophore. (E) Relative uptake after 1 h of drug treatment.

Uptake and Distribution of Tamoxifen-Targeted Doxorubicin
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6513
relative to DOX and DOXSF. The enhanced uptake and
retention of DOX-TEG-TAM relative to DOX and DOX-
SF parallels the growth inhibition data shown in Table
117 and provides evidence in favor of the targeting
hypothesis involving interaction with AEBS.
The uptake and release of DOX, DOXSF, and DOX-
TEG-TAM in ER-positive, drug sensitive MCF-7 cells
is shown in Figure 2, panel B. DOX-TEG-TAM was
taken up to a greater extent (25 RFU) than both DOXSF
(16 RFU) and DOX (5 RFU). DOX-TEG-TAM showed
the highest level of doxorubicin fluorophore retention
following drug release after 6 h. Again, the uptake and
retention parallel the growth inhibition data for DOX,
DOXSF, and DOX-TEG-TAM (Table 1).17
Additionally, uptake and release data were obtained
for DOX, DOXSF, and DOX-TEG-TAM in ER-negative
MDA-MB-435 (Figure 2, panel C) and MDA-MB-231
cells (Figure 2, panel D). In both cell lines DOX-TEG-
TAM was also taken up to a much greater extent than
DOX and DOXSF. In MDA-MB-435 cells DOX-TEG-
TAM was taken up 3-fold and 5-fold more than DOXSF
and DOX, respectively. In MDA-MB-231 cells DOX-
TEG-TAM was taken up 2-fold and 6-fold more than
DOXSF and DOX, respectively. In both cell lines doxo-
rubicin fluorophore, following 1 h treatment with 500
nM DOX-TEG-TAM, was retained to a greater extent
than it was following treatment with 500 nM DOXSF
or DOX.
The comparison of the extent of DOX-TEG-TAM
uptake as a function of breast cancer cell type is shown
graphically in Figure 2, panel E. The data illustrate that
MDA-MB-435 cells take up substantially more drug
following a 1 h treatment with 500 nM DOX-TEG-TAM.
MCF-7 and MDA-MB-231 cells take up about 25%
relative to MDA-MB-435 cells; while MCF-7/Adr cells,
consistent with the overexpression of the p-glycoprotein
drug efflux pump, take up a relatively small amount of
drug. The enhanced uptake of DOX-TEG-TAM in MDA-
MB-435 cells relative to MCF-7 and MDA-MB-231 cells
was not anticipated based on growth inhibition data,
as the IC50s for all three cell lines following 4 h DOX-
TEG-TAM treatment are quite similar (30-40 nM)
(Table 1).17
Competitive Inhibition of Drug Uptake. If DOX-
TEG-TAM targeting is AEBS mediated, the presence
of a competitive ligand should inhibit uptake. MDA-MB-
435 cells were utilized for competition experiments as
they take up all three compounds to a greater extent
than the other breast cancer cells evaluated (Figure 2,
panel E). MDA-MB-435 cells were treated with 0.5 µM
DOX-TEG-TAM, DOX, or DOXSF for 1 h in the presence
of various concentrations of tamoxifen. In the presence
of 10 µM tamoxifen competitor, the uptake of DOX and
DOXSF relative to DOX-TEG-TAM decreased by only
8% and 6%, respectively, while in the case of cells
treated with DOX-TEG-TAM, uptake of targeted drug
decreased dramatically in a dose-dependent manner as
shown in Figure 3. At 10 µM tamoxifen, the uptake was
47% of the uptake of DOX-TEG-TAM in the absence of
competitor, supporting the hypothesis that DOX-TEG-
TAM targeting is AEBS mediated.
Figure 3. Drug uptake in MDA-MB-435 cells following 1 h
treatment with 0.5 µM DOX-TEG-TAM in the presence of
increasing concentration of tamoxifen as measured by fluo-
rescence from the DOX fluorophore.
fluorophore following treatment with DOX-TEG-TAM
should be predominantly cytosolic following short treat-
ment times (treatment time less than the half-life for
hydrolysis). Furthermore, once the trigger has fired, the
DOX fluorophore should appear nuclear. DOX was used
as a control since, with DOX treatment, drug fluores-
cence typically appears nuclear as the clinical drug
accumulates in nuclear DNA.29,31
Fluorescence micrographs of MDA-MB-435 cells fol-
lowing treatment with 500 nM DOX or 500 nM DOX-
TEG-TAM as a function of time are shown in Figure 4.
Following treatment for 5 min, almost no fluorescence
was observed for the DOX treated cells (column A,
Figure 4) and very little was observed for DOX-TEG-
TAM cells (column B, Figure 4). However, after 20 min,
DOX fluorophore was observed in the nucleus of the
cells following treatment with DOX, while cytosolic
fluorescence was observed for DOX-TEG-TAM treated
cells. Following 40 min of treatment, the DOX treated
cells were observed to have accumulated more nuclear
fluorophore. However, in the cells treated with DOX-
TEG-TAM for 40 min the fluorescence still appeared
extranuclear; however, the fluorophore appeared to have
localized at a cytosolic site. The observed localization
of DOX-TEG-TAM fluorophore appeared even more
dramatic following treatment for 1 h. Following DOX
treatment for 1 and 3 h, the fluorescence continued to
appear exclusively nuclear. Following DOX-TEG-TAM
treatment for 3 h, the fluorescence appeared predomi-
nantly nuclear. This is consistent with the observation
that the half-life for hydrolysis of the trigger is about
60 min; after three half-lives the majority of the DOX-
TEG-TAM should have hydrolyzed from the targeting
group and the liberated doxorubicin active metabolite
translocated to the nucleus, forming DNA virtual cross-
links.32 In an additional control experiment, untargeted
DOXSF was found to mimic the localization pattern
(exclusively nuclear) that was observed for DOX (data
not shown).
Analysis of Drug Distribution by Fluorescence
Microscopy. If drug targeting is mediated by extra-
nuclear AEBS, the cellular distribution of doxorubicin

6514 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Burke et al.
Figure 5. Binding affinity of DOX-TEG-TAM for AEBS
relative to E/Z-hydroxytamoxifen.
Following incubation, free, unbound tamoxifen was
stripped from solution with 2% dextran-coated charcoal
(DCC) buffered suspension; bound 3H-tamoxifen in
solution was then quantified via scintillation counting.
Representative results are shown in Figure 5. Three
concentrations of 3H-tamoxifen binding in the presence
of 50 nM competitor were subtracted from total 3H-
tamoxifen binding (no competitor); the differences are
plotted versus the concentration of 3H-tamoxifen in
Figure 5. Theoretically, a compound (competitor) with
no AEBS affinity would result in no difference between
total binding and competitor. At all three concentrations
3
of H-tamoxifen, DOX-TEG-TAM binding affinity was
less than that of the targeting group alone. DOX-TEG-
TAM binding affinity was 70%, 72%, and 48% relative
to the targeting group (5) at 0.05 nM, 0.5 nM, and 5
3
nM H-tamoxifen, respectively. Taken as an average,
DOX-TEG-TAM (2c) retains 63% ( 13% of the total
binding affinity of the targeting group, E/Z-4-OHT (5).
Effect of DOX-TEG-TAM on AEBS-Negative Rtx-6
Breast Cancer Cells. Rtx-6 cells, kindly provided by
Dr. Marc Poirot (Toulouse, France), were evaluated as
an AEBS-negative control cell line. Rtx-6 cells, a tamox-
ifen resistant breast cancer cell line, are a clonal variant
of MCF-7 cells.33-35 The Rtx-6 cells were utilized to
determine the effect of the absence of AEBS on the
cytotoxicity and uptake of DOX, DOXSF, and DOX-
TEG-TAM.
Rtx-6 cells, in logarithmic growth, were treated with
DOX, DOXSF, or DOX-TEG-TAM to establish the
concentration that inhibited 50% of cell growth (IC50)
following a 4 h treatment. The IC50s are compared in
Table 1 with those previously determined for MCF-7,
MCF-7/Adr, MDA-MB-231, and MDA-MB-435 cells. The
concentrations inhibiting 50% of cell growth following
4 h treatment with DOX, DOXSF, and DOX-TEG-TAM
were 200 nM, 61 nM, and 67 nM, respectively.
The uptake and release in Rtx-6 cells were also
investigated. Rtx-6 cells were treated with DOX, DOX-
SF, or DOX-TEG-TAM as described above to determine
both the uptake and the release of drug following a 1 h
treatment. The results are shown in Figure 6. DOX-
TEG-TAM was taken up to a greater extent than DOX
(>3-fold) and DOXSF (>2-fold). The enhanced uptake
relative to DOX and DOXSF could be attributed to the
presence of ER and the lipophilicity of DOX-TEG-TAM.
The uptake of DOX-TEG-TAM in Rtx-6 cells is perhaps
best compared to the uptake in the parent MCF-7 cell
Figure 4. Fluorescence micrographs of MDA-MB-435 cells
as a function of time following treatment with 500 nM DOX
(A) and 500 nM DOX-TEG-TAM (B).
DOX-TEG-TAM Binding Affinity to AEBS. The
binding affinity of DOX-TEG-TAM relative to E/Z-4-
OHT was determined using MCF-7 cell lysate as an
AEBS source. The lysate was incubated with 3H-
tamoxifen and cold competitors; 1000 nM estradiol was
added to saturate ERs present in the cell lysate.

Uptake and Distribution of Tamoxifen-Targeted Doxorubicin
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6515
Figure 6. Flow cytometric measurement of cellular uptake and release in Rtx-6 cells following treatment with 500 nM DOX,
DOXSF, and DOX-TEG-TAM as a function of time.
Figure 8. Relative concentration of AEBS in MCF-7, MCF-
7/Adr, and MDA-MB-435 cell lysates.
Figure 7. Drug uptake in Rtx-6 cells following 1 h treatment
with 500 nM DOX-TEG-TAM in the presence of increasing
concentration of estradiol, measured by flow cytometric moni-
toring of fluorescence from the DOX fluorophore.
in breast cancer cells independent of ER expression,
with levels typically higher in ER+ cell lines.36,37
MCF-7/Adr and MDA-MB-435 cell lysates were pre-
pared in the same manner as the MCF-7 lysate utilized
for AEBS binding assays. MCF-7, MCF-7/Adr, and
MDA-MB-435 cell lysates containing 5 nM 3H-tamoxifen
were incubated in the presence and absence of 5000 nM
cold tamoxifen. Additionally, cold estradiol (1000 nM)
was added to every sample to saturate the ER present
in the low speed lysate. Total lysate binding was
measured for each cell lysate in the absence of cold
tamoxifen, while nonspecific binding was measured in
the presence of 1000-fold cold tamoxifen. The difference
between total binding and nonspecific binding is, by
definition, the AEBS specific binding. The ratio of AEBS
specific binding for MCF-7/Adr and MDA-MB-435 ly-
sates relative to the AEBS specific binding for MCF-7
lysate is presented in Figure 8. MCF-7/Adr cell lysate
contained about 53% of the AEBS present in the MCF-7
cell lysate. MDA-MB-435 cell lysate contained 126% of
the AEBS present in the MCF-7 cell lysate.
line. DOX-TEG-TAM was taken up to a greater extent
in MCF-7 cells (25 RFU) relative to Rtx-6 cells (14 RFU).
One possible explanation for this difference is the
presence of both AEBS and ER in MCF-7 cells versus
only ER in Rtx-6 cells. Consistent with this explanation
is the dose-dependent inhibition of uptake of DOX-TEG-
TAM by the ER specific ligand, estradiol shown in
Figure 7. Estradiol at 20 nM inhibited the uptake by
70%. No further inhibition occurs at higher concentra-
tions, possibly because of nonspecific binding of DOX-
TEG-TAM at hydrophobic sites in Rtx-6 cells. In con-
trast, 20 nM estradiol had no effect on the uptake of
DOX-TEG-TAM by ER-negative, MDA-MB-435 cells
(data not shown).
Detection of AEBS in MCF-7/Adr and MDA-MB-
435 Cell Lines. An exhaustive search of the literature
uncovered no indication of the presence or expression
level of AEBS in MCF-7/Adr and MDA-MB-435 cell
lines. There is evidence for the presence of AEBS in
MCF-7 and MDA-MB-231 cell lines, and the expression
level has been reported as 140000 sites/cell and 82000
sites/cell for MCF-7 (ER+) and MDA-MB-231 (ER-),
respectively.36 AEBS have been reported to be present
In conclusion, the enhanced uptake measured by flow
cytometry (Figure 2) and fluorescence microscopy (Fig-
ure 4) experiments support the hypothesis that DOX-
TEG-TAM targeting is mediated through the antiestro-
gen binding site as well as the ER. In addition to DOX-
TEG-TAM retaining roughly 60% of the AEBS binding
affinity of the targeting group for AEBS (Figure 5),

6516 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Burke et al.
units/mL), and streptomycin (100 µg/mL). MDA-MB-435 cells
were maintained in vitro by serial culture in DMEM medium
supplemented with 5% fetal bovine serum, L-glutamine (2
mM), sodium pyruvate (1 mM), and nonessential amino acids
and vitamins for minimum essential media. Rtx-6 cells were
maintained in vitro by serial culture in RPMI 1640 medium
supplemented with 5% FBS, L-glutamine (2 mM), HEPES
buffer (10 mM), penicillin (100 units/mL), streptomycin (100
µg/mL), and 1.00 µM tamoxifen (Sigma, St. Louis, MO).
Tamoxifen was excluded from the media when experiments
were performed. Phenol red free media supplemented with
L-glutamine was obtained from Sigma (St. Louis, MO). Cells
were maintained at 37 °C in a humidified atmosphere of 5%
CO2 and 95% air.
Uptake and Release of DOX, DOXSF, and DOX-TEG-
TAM. The uptake and release of DOX-TEG-TAM in breast
cancer cells relative to DOX and untargeted DOXSF was
performed as described with modifications.29,30 Breast cancer
cells in log phase growth were dissociated with trypsin-EDTA,
counted, resuspended in media at 1.5 × 105 cells/mL, and
plated into six well plates (450000 cells/well) and allowed to
adhere overnight. The cells were treated with 0.5 µM DOX,
DOXSF, or DOX-TEG-TAM for various amounts of time (20,
40, and 60 min). Drug treatment was accomplished by addition
of 30 µL of 100× drug solution (50 µM) to 3 mL of medium
which was mixed and immediately added to the cells. For each
time point, the medium was removed, cells were trypsinized,
and trypsinization was quenched with 3 mL of phenol red free
RPMI 1640 medium (no serum) at 4 °C. Cells were pelleted
by centrifugation at 300g for 5 min at 10 °C. The supernatant
was decanted, and the cells were resuspended in 1 mL of
serum-free and phenol red free RPMI 1640 medium and placed
on ice.
DOX-TEG-TAM is taken up and retained to a signifi-
cantly greater extent (up to 6-fold) than DOX and
DOXSF. When MDA-MB-435 cells are treated with
DOX or DOXSF for various times up to 3 h, anthracy-
cline fluorescence appears nuclear, while DOX-TEG-
TAM remains in the cytosol following treatment for
times less (5-40 min) than the 60 min half-life for
hydrolysis. From 40 to 60 min, DOX-TEG-TAM fluoro-
phore appears to localize at a cytosolic site, and follow-
ing release from the trigger/targeting group, the DOX
fluorophore appears nuclear.
Perhaps the most compelling evidence for the role of
AEBS and ER in the targeting mechanism comes from
the competition experiments (Figures 3 and 7). The
uptake of DOX-TEG-TAM by AEBS-positive, ER-nega-
tive MDA-MB-435 cells was substantially reduced in the
presence of tamoxifen, an AEBS ligand, and by AEBS-
negative, ER-positive Rtx-6 cells, in the presence of
estradiol, a specific ER ligand. The competitive inhibi-
tion was observed to be dose dependent, with DOX-TEG-
TAM uptake reduced by over 50% in the presence of 20-
fold tamoxifen or 0.02-fold estradiol, respectively, to a
level similar to untargeted DOXSF. In summary, the
data support the hypothesis that the DOX-TEG-TAM
targeting mechanism involves an interaction with the
antiestrogen binding site and ER.
Experimental Section
General. The concentrations of test compounds were de-
termined spectrophotometrically by UV/vis absorption with a
Hewlett-Packard 8452A diode array spectrophotometer inter-
faced to an Agilent ChemStation data system as described for
each biological assay. Flow cytometric measurements were
performed using a Becton Dickinson FACScan (Franklin
Lakes, NJ) flow cytometer. Fluorescence microscopy was
performed using a Leica DM IRB fluorescence microscope with
an ebq 100 mercury lamp power source (E Licht Company,
Denver, CO) equipped with a Photometrics Sensys (Tucson,
AZ) digital CCD camera system. Fluorescence images were
processed using IPLab Spectrum software. Centrifugation of
AEBS cell lysates was accomplished with a Beckman J2-21
centrifuge. Cell lysis was performed with a Misonix Sonicator
Ultrasonic processor fitted with a microtip.
For drug retention samples, the cells were treated for 1 h
with 0.5 µM DOX, DOXSF, or DOX-TEG-TAM. Following drug
treatment, the medium was removed and replaced with 3 mL
of fresh, 37 °C, full (containing serum and phenol red indicator)
cell medium and the cells were incubated for various times
(0.5, 1, 3, and 6 h). Following the allotted release times, the
cells were prepared as described above. Drug treatment was
performed such that all cell samples would be prepared within
2 h. Previous work has demonstrated that no loss of fluores-
cence is observed from cells stored on ice for up to 4 h.29
The extent of drug uptake and retention was measured by
flow cytometry on a Becton Dickinson FACScan (Franklin
Lakes, NJ) flow cytometer. Cells were analyzed with excitation
at 488 nm (15 mW Ar ion laser), with emission monitored
between 570 and 600 nm. Instrument settings were optimized
for each cell line and held constant for all experiments; 10000
cells were analyzed for anthracycline fluorescence. The data
are presented as the mean fluorescence for each condition with
the background, drug-free cell fluorescence subtracted.
Tamoxifen Competition Experiments. MDA-MB-435
cells were treated with 0.5 µM DOX, DOXSF, or DOX-TEG-
TAM in the absence and presence of the competitor, tamoxifen.
Following drug treatment for 1 h, both anthracycline and
tamoxifen were removed. The cell samples were prepared and
analyzed by flow cytometry as described above.
Estradiol Competition Experiments. Rtx-6 cells were
treated with 0.5 µM DOX-TEG-TAM in the absence and
presence of the competitor, estradiol. Following drug treatment
for 1 h, both anthracycline and estradiol were removed. The
cell samples were prepared and analyzed by flow cytometry
as described above.
3H-Tamoxifen was obtained from Amersham Pharmacia
Biotech (Buckinghamshire, England). Cold tamoxifen and
estradiol were all obtained from Sigma (St. Louis, MO).
Doxorubicin hydrochloride was purchased from Hande Tech
(Houston, TX), and DOX-saliform and DOX-TEG-TAM were
synthesized as previously described.17,22 Complete-Mini pro-
tease inhibitor cocktail tablets were obtained from Boehringer
Mannheim (Indianapolis, IN). Cell lysate total protein was
measured with a Micro Protein Determination kit from Sigma
Diagnostics (Dorset, England). Liquid scintillation counting
was performed using a Packard (Downers Grove, IL) Tri-Carb
1600 TR liquid scintillation analyzer. Econo-Safe biodegrad-
able liquid scintillation cocktail was from Research Products
International Corporation (Mount Prospect, IL).
All tissue culture materials were obtained from Gibco Life
Technologies (Grand Island, NY) unless otherwise stated.
MCF-7 and MDA-MB-231 cells were obtained from American
Type Culture Collection (Rockville, MD). MCF-7/Adr doxoru-
bicin-resistant cells were a gift from Dr. William W. Wells
(Michigan State University, East Lansing, MI). MDA-MB-435
and Rtx-6 cells were generously provided by Dr. Renata
Pasqualini (MD Anderson Cancer Center, Houston, TX) and
Dr. Marc Poirot (Toulouse, France), respectively. MCF-7, MCF-
7/Adr, and MDA-MB-231 cells were maintained in vitro by
serial culture in RPMI 1640 medium supplemented with 10%
fetal bovine serum (Gemini Bioproducts, Calbassas, CA),
L-glutamine (2 mM), HEPES buffer (10 mM), penicillin (100
Analysis of Drug Distribution by Fluorescence Mi-
croscopy. MDA-MB-435 cells in log phase growth were
dissociated with trypsin-EDTA and counted. Cells were sus-
pended in media at 8.3 × 104 cells/mL, and 3 mL of cell solution
was aliquoted into 6 well plates and allowed to adhere
overnight. The cells were treated with 0.5 µM DOX, DOXSF,
or DOX-TEG-TAM for various amounts of time (5 min, 20 min,
40 min, 1 h, and 3 h). Drug treatment was accomplished by
addition of 30 µL of 100× drug solution (50 µM) to 3 mL of
medium which was mixed and immediately added to the cells.

Uptake and Distribution of Tamoxifen-Targeted Doxorubicin
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6517
Following drug treatment, the medium was removed, cells
were washed once with 3 mL of serum-free and phenol red
free RPMI 1640 at room temperature, and 3 mL of the same
medium was then added back to each well. The cells were then
immediately analyzed by fluorescence microscopy. Each condi-
tion was performed individually to minimize the amount of
time between drug treatment and fluorescence detection.
Microscopic images of the cells were observed at a magni-
fication of 40× and recorded with a Leica DM IRB fluorescence
microscope equipped with a Photometrics Sensys digital CCD
camera system. Cell images were observed with a shutter time
of 0.05 s; fluorescence images were observed with a shutter
time of 1.000 s. Drug fluorescence was observed at wavelengths
above 590 nm with excitation between 515 and 560 nm.
Fluorescence images were processed using IPLab Spectrum
software.
were vortexed and stored on ice for 15 min, with vortexing
every 5 min. DCC was pelleted by centrifugation at 3000g for
10 min at 4 °C; 300 µL of lysate supernatant was transferred
to scintillation vials containing 4 mL of Econosafe biodegrad-
able scintillation cocktail. The vials were then vortexed vigor-
ously, and each sample was counted for 5 repetitions of 3 min
counts. This counting protocol was then repeated to ensure
reproducibility. Scintillation counting background was sub-
tracted from all measurements. The percentage of targeting
group binding for DOX-TEG-TAM relative to targeting group
alone was determined by comparison of the reduction of
tritiated tamoxifen at each concentration. Scintillation count-
ing was performed in triplicate, and each competitor was
assayed in duplicate. Error bars represent one standard
deviation for the percentage DOX-TEG-TAM binding relative
to E/Z-4-OHT at three different tritiated tamoxifen concentra-
tions.
Evaluation of DOX-TEG-TAM Binding Affinity to
AEBS. The binding affinity of DOX-TEG-TAM (2c) relative
to E/Z-4-OHT (5) was measured through competition assay
with tritiated tamoxifen (3H-TAM) through a procedure adapted
from several sources.34,37,38 MCF-7 cells were utilized as the
AEBS source. Cells were cultured in six T-175 flasks to 90%
confluence. To harvest, cells were washed with 10 mL of
Hank’s balanced salt solution and dissociated from the flasks
with 2 mL of trypsin. Trypsinization was quenched with 10
mL of phenol red free RPMI medium supplemented with 10%
dextran-coated charcoal (DCC) stripped fetal calf serum
(“stripped medium”); cells from six T-175 flasks were combined
and pelleted by centrifugation at 300g for 5 min at 25 °C. The
supernatant was decanted, and the cells were resuspended in
50 mL of stripped medium and enumerated with a hemacy-
tometer. The cells were then pelleted again by centrifugation,
as described above. The supernatant was decanted, and the
cells were suspended in pH 7.4 lysis buffer (10% v/v glycerol,
10 mM Tris, 1.5 mM EDTA, 0.5 mM dithiothreitol, 10 mM
Na2MoO4, 1.0 mM phenylmethylsulfonyl fluoride, supple-
mented with Complete-Mini protease inhibitors) at 4 °C such
that the cell density was 38 million cells per mL lysis buffer.
Cells were lysed at 0 °C via sonication with a microtip set at
maximum power for 10 cycles of 6 s on followed by 24 s off.
The AEBS-enriched lysate was obtained by centrifugation of
the homogenate at 12000g for 30 min at 4 °C. The supernatant
was dispensed into 100 µL aliquots and stored at -70 °C. The
lysate protein density was measured with a Sigma Diagnostics
Total Protein kit; for all experiments the stock lysate was
diluted such that the protein density was 2.0 mg/mL.
Growth Inhibition of Rtx-6 Cells. The concentration
inhibiting half the growth (IC50) was determined as previously
described with minor modifications.13 All compounds were
solubilized in dimethyl sulfoxide containing 1% v/v acetic acid.
The concentrations of all 100× DMSO/1% AcOH drug solutions
were determined spectrophotometrically by absorbance at 480
nm (ꢀ ) 11500 L/mol‚cm). Drug treatment lasted 4 h; and cells
were cultured until the control wells had achieved 80%
confluence (typically 4-5 days). Every experiment was per-
formed six times for each drug level and controls. Error bars
represent one standard deviation about the mean for the six
wells per lane measured for each drug concentration.
Detection of AEBS in MCF-7/Adr and MDA-MB-435
Cells. The presence of AEBS was measured through the
binding of 3H-tamoxifen in MCF-7, MCF-7/Adr, and MDA-MB-
435 cell lysates. MCF-7/Adr and MDA-MB-435 cell lysates
were prepared as described above for the AEBS binding assay.
All three cell lysates were diluted to achieve a uniform protein
density of 2.0 mg/mL.
Binding ligands were prepared as 120× solutions in DMSO
containing 1% acetic acid and delivered to cell lysates as
described above. MCF-7, MCF-7/Adr, and MDA-MB-435 cell
3
lysates containing 5 nM H-tamoxifen were incubated in the
presence and absence of 5000 nM cold tamoxifen. Additionally,
cold estradiol (1000 nM) was added to every sample to saturate
the ER present in the low speed lysate; cold estradiol was
prepared as described above. Total lysate binding was mea-
sured for each cell lysate in the absence of cold tamoxifen,
while nonspecific lysate binding was measured in the presence
of cold tamoxifen. The difference between total binding and
nonspecific binding is, by definition, the AEBS specific binding.
Following incubation, the cell lysates were prepared for liquid
scintillation counting as described above. The ratio of AEBS
specific binding for MCF-7/Adr and MDA-MB-435 relative to
the AEBS specific binding for MCF-7 is presented. Error bars
represent one standard deviation from the mean of the
scintillation counting statistics.
Competitive ligands were prepared as 120× solutions (50
nM working concentrations) in DMSO containing 1% acetic
acid. Competitor concentrations were determined for ligands
containing the DOX chromophore by optical density at 480 nm
(ꢀ ) 11500 L/mol‚cm). Three different concentrations (5 nM,
0.5 nM, and 0.05 nM) of tritiated tamoxifen were prepared as
240× solutions in DMSO containing 1% acetic acid. Cold
estradiol (1 µM) was added to every sample to saturate the
ER present in the low speed lysate; cold estradiol was prepared
as a 240 µM (240×) stock solution to provide a 1 µM estradiol
working concentration. Equal volumes of the 240× solutions
of tritiated tamoxifen and cold estradiol were added together
to provide 120× solutions of each ligand in DMSO containing
1% acetic acid. The 120× solutions were then diluted 1:10 in
pH 7.6 TE (10 mM Tris, 1 mM EDTA) buffer to provide 12×
solutions of 3H-TAM/estradiol and competitors. Aliquots of cell
lysate (100 µL) were thawed at 4 °C; 10 µL of 12× competitor
Acknowledgment. The authors would like to thank
the National Cancer Institute of the NIH (CA-92107)
for financial support. We also thank Dr. Renata Pas-
qualini (MD Anderson Cancer Center, Houston, TX), Dr.
Marc Poirot (Toulouse, France), and Dr. William Wells
(Michigan State University, East Lansing, MI) for
generously providing MDA-MB-435, Rtx-6, and MCF-
7/Adr cells, respectively, and Theresa Nahreini for help
with the cell experiments. P.J.B. thanks the Colorado
Institute for Research in Biotechnology (CIRB) and the
U.S. Army Breast Cancer Research Program (DAMD
17-01-1-0213) for predoctoral fellowships. T.H.K. thanks
the University of Colorado Council for Research and
Creative Work for a faculty fellowship.
3
was added, followed by 10 µL of 12× H-TAM/estradiol at each
of the three tritiated tamoxifen concentrations. Total binding
was measured by addition of vehicle (DMSO containing 1%
acetic acid) in the absence of competitor; 3H-TAM binding
inhibition was measured by addition of E/Z-4-OHT or DOX-
TEG-TAM. Reaction lysates were vortexed vigorously and
stored at 4 °C for 18 h.
Following incubation, unbound aromatic organics were
stripped from the lysate by addition of 280 µL of DCC as a 2%
w/v suspension in pH 7.6 TE buffer (10 mM Tris, 1.0 mM
EDTA). Following the addition of the DCC, the reaction lysates

6518 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Burke et al.
(22) Cogan, P. S.; Fowler, C. R.; Post, G. C.; Koch, T. H. Doxsali-
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