Bivalent Approach for Homodimeric Estradiol Based Ligand: Synthesis and Evaluation for Targeted Theranosis of ER(+) Breast Carcinomas

Article abstract of DOI:10.1021/acs.bioconjchem.6b00024

The synthesis of estradiol based bivalent ligand [(EST)₂DT] is reported and its potential for targeted imaging and therapy of ER(+) tumors has been evaluated. For the purpose, ethinylestradiol was functionalized with an azidoethylamine moiety via click chemistry. The resultant derivative was reacted in a bivalent mode with DTPA-dianhydride to form the multicoordinate chelating agent, (EST)₂DT which displayed capability to bind ^99mTc. The radiolabeled complex, ^99mTc-(EST)₂DT was obtained in >99% radiochemical purity and 20-48 GBq/μmol of specific activity. RBA assay revealed ～15% binding with estrogen receptor. Evaluation of ligand on ER(+)-cell line (MCF-7) suggested enhanced and ER-mediated uptake. In vivo assays displayed early tracer accumulation in MCF-7 xenografts with tumor to muscle ratio ～6 in 2 h and negligible uptakes in nontargeted organs. MTT assay performed on ER(+) and ER(-) cell lines displayed selective inhibition of ER(+) cancer cell growth with IC₅₀ = 14.3 μM which was comparable to tamoxifen. The anticancer activity of the ligand is possibly due to the increase in ERβ and suppression of ERα protein levels in gene transcription. The studies reveal the potential of (EST)₂DT as diagnostic imaging agent with the additional benefits in therapy. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.bioconjchem.6b00024

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Article
Bivalent Approach for Homodimeric Estradiol based Ligand: Synthesis
and Evaluation for Targeted Theranosis of ER(+) Breast Carcinomas
Kanchan Chauhan, Ashutosh Arun, Saurabh SIngh, Murli Manohar, Krishna Chuttani, Rituraj
Konwar, Anila Dwivedi, Ravi Soni, Ajai K. Singh, Anil Kumar Mishra, and Anupama Datta
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00024 • Publication Date (Web): 21 Mar 2016
Downloaded from http://pubs.acs.org on March 22, 2016
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Bivalent Approach for Homodimeric Estradiol based
Ligand: Synthesis and Evaluation for Targeted
Theranosis of ER(+) Breast Carcinomas
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Kanchan Chauhan, †‡ Ashutosh Arun, § Saurabh Singh, † Murli Manohar, § Krishna Chuttani, † Rituraj
Konwar, § Anila Dwivedi, § Ravi Soni, † Ajai Kumar Singh, ‡ Anil K. Mishra,*† Anupama Datta*†
†Institute of Nuclear Medicine & Allied Sciences, DRDO, Brig. SK Mazumdar Marg, Delhi-
110054, India
‡Department of Chemistry, Indian Institute of Technology, Delhi-110016, India
§Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow-226031, India
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ABSTRACT
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The synthesis of estradiol based bivalent ligand, [(EST)2DT] is reported and its potential for
targeted imaging and therapy of ER(+) tumors has been evaluated. For the purpose
ethinylestradiol was functionalized with an azidoethylamine moiety via click chemistry. The
resultant derivative was reacted in a bivalent mode with DTPA-dianhydride to form the
multicoordinate chelating agent, (EST)2DT which displayed capability to bind 99mTc. The
radiolabeled complex, 99mTc-(EST)2DT was obtained in >99% radiochemical purity and 20‒48
GBq/μmol of specific activity. RBA assay revealed ~15% binding with estrogen receptor.
Evaluation of ligand on ER(+)-cell line (MCF-7) suggested enhanced and ER-mediated uptake.
In vivo assays displayed early tracer accumulation in MCF-7 xenografts with tumor to muscle
ratio ~ 6 in 2 h and negligible uptakes in non-targeted organs. MTT assay performed on ER(+)
and ER(‒) cell lines displayed selective inhibition of ER(+) cancer cell growth with IC50=14.3
μM which was comparable to tamoxifen. The anti-cancer activity of the ligand is possibly due to
the increase in ERβ and suppression of ERα protein levels in gene transcription. The studies
reveal the potential of (EST)2DT as diagnostic imaging agent with the additional benefits in
therapy.
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KEYWORDS. ER(+), MCF-7, Imaging, Therapy
INTRODUCTION
Breast cancer remains the most common malignancy and a foremost cause of mortality in
females worldwide.1 Besides early diagnosis, the understanding of estrogen receptor (ER) levels
in breast tumor is also necessary for the prediction of disease’s prognosis and optimization of the
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appropriate treatment strategy. Estrogens are the major role players in the initiation and
progression of the breast cancer. Their effect is mediated by estrogen receptors that exist in two
forms, ERα and ERβ. The ERα form serves as the best prognostic factor for breast cancers whose
role in the disease is well defined.2 Further, on the basis of ERα expression, the tumor is
subcategorized as estrogen dependent [ER(+)] or independent [ER(‒)]. The ER(+) tumors often
respond to hormonal therapy while ER(‒) tumors need surgical or chemotherapeutic
interventions.3 Immunohistochemical staining is the classical method utilized for the evaluation
of ER status in patients. However, the heterogeneity of ER expression in primary tumors often
results in misleading predictions with false ER status in tumor biopsy. It has been found that
approximately 20% of the assumptions with this golden standard are inaccurate worldwide.4–6
Furthermore, the specimen biopsy procedure is not encouraged by the physicians due to its
invasive nature.7 Consequently, the molecular imaging techniques are being explored to obtain
better information about ER status in breast cancer by non-invasive means. Also, the new
strategies with the blend of non-invasive diagnostic imaging and therapy may serve as an
efficient approach for the quantification of ER expression and appropriate treatment of these
malignant lesions. It thereby, not only allows the understanding of cellular phenotype, tumor
heterogeneity, and disease treatment efficiency but also in certain conditions allows the
simultaneous diagnosis and therapy. These versatile agents have drawn huge attention to serve
the potential applications in cancers.8
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There has been a significant advancement in the development of estrogen based
radiopharmaceuticals for single photon emission computed tomography (SPECT) 9–11 or positron
emission tomography (PET).12,13 The well known PET agent, 16α-18F-17β-estradiol (18F-FES)
has demonstrated promising results in the evaluation of estrogen-dependent tumors. It is
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currently in the phase II trials for the prediction of response to antihormonal therapy in patients
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with ER(+) tumors. The clinical outcomes of F-FES are encouraging, but the availability and
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accessibility of the cyclotron based source (18F) are still an issue in many medical centres.
Therefore, dedicated attempt is being made towards the development of estradiol based tracers
labeled with technitium-99m (99mTc) due to its ideal nuclear decay properties, easy accessibility,
low isotope cost, simple and rich chelating chemistry.3,14–18 However, many of the complexes
displayed suboptimal in vivo target selectivity due to the high lipophilicity or very fast
metabolism. Apparently, there is a pressing requirement of facile chemical strategies that can
provide cost-effective imaging of breast tumor with clinical translation potential.
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Estrogen binding to ER leads to the activation of the intracellular receptor. The unligated
receptor takes up the disordered and mobile globular structure which undergoes various
conformational changes upon activation. The process finally allows the receptor dimerization
and mediates the genomic/non-genomic activity.19,20 The alterations in ER dimeric structure
using suitable bivalent ligands may provide an alternative mechanism to regulate their activity
and consequently, may improve tissue selectivity and uptake. The bifunctional chelating agents
can be used as a simple tool for the conjugation of the biovectors. In this context,
polyaminopolycarboxylic ligands are well suited as they possess functional groups for the
coupling of one or more biovectors as well as the donor atoms for strong complexation with
radiometal ions. These agents may also overcome the limitation of poor water solubility faced in
classical
radiotracers
for
intravenous
administration.
Amongst
them,
diethylenetriaminepentaacetic acid (DTPA) is a conventional example being widely utilized in
nuclear medicine applications. In particular, 99mTc labeled DTPA conjugates have shown
excellent outcomes as radiopharmaceuticals.21–23
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The evidences also demonstrate the induction of programmed cell death (apoptosis) in breast
cancer cells on treatment with estradiol in physiological concentrations. In fact, the synthetic
estrogens like ethinylestradiol or diethylstilbestrol (DES) have been utilized in high doses for the
treatment of breast cancer for the last sixty years.24 Therefore, by exploring the bivalent
approach, design and development of ligands with two estradiol units as biovector may not only
result in the enhanced target specificity required in imaging but can also be tested for therapeutic
efficiency as an additional advantage.
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In this work, it has been hypothesized that the bivalent ligand approach utilized in the
development of homodimeric ligand using ethinylestradiol may provide a system for the dual
application of site-directed imaging and therapy of estrogen-dependent tumors. Here, we report
the synthesis of a bivalent 99mTc-labeled estradiol-DTPA hybrid agent, 5,8-bis(carboxymethyl)-
13-(4-(3,17-dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-
cyclopenta[α]phenanthren-17-yl)-1H-1,2,3-triazol-1-yl)-2-(2-(2-(4-(3,17-dihydroxy-13-methyl-
7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[α]phenanthren-17-yl)-1H-1,2,3-triazol-1-
yl)ethylamino)-2-oxoethyl)-10-oxo-2,5,8,11-tetraazatridecane-1-carboxylic acid [(EST)2DT] and
its evaluation as a potential targeting agent for ER(+) breast cancers. The relative binding affinity
of the ligand towards ER was evaluated and compared with the corresponding monomeric unit.
The tumor localization ability of the ligand was demonstrated through SPECT imaging after
labeling with 99mTc. The therapeutic efficiency of the ligand was checked by evaluating its
anticancer activity in various cancer cell lines. The underlying mechanism by which the ligand
caused cancer cell death was also understood. The ligand is shown to retain both the stability and
specificity throughout the in vitro and in vivo analyses.
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RESULTS & DISCUSSION
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ERα is the key protein overexpressed in the breast cancers and the ligands targeting ERα for
diagnosis or therapy are among the most exploited ones with impressive results in clinics. One of
the approaches in medicinal chemistry research is the development of ligands offering the
combination of imaging and therapeutic functionalities in one scaffold. Towards the same
direction, ER targeting estradiol based ligand has been synthesized for breast cancer imaging and
therapy using bivalent ligand approach. The bivalent approach is often applied to enhance the
biological effects of the biovector. There has been a brief history of utilizing bivalent ligands
using a variety of linkers with linker length in the range of 22‒30 Å for targeting ER.20,25–27 The
key aspect of our strategy was to incorporate a targeting group into the bivalent system using a
multifunctional chelating agent possessing the ability to bind radiometal ions with high stability.
For the purpose, DTPA was the choice of scaffold for creating a long chain alkyl spacer (n= 4)
arm in the bivalent ligand. DTPA not only allows the fast complexation with metal ions but also
enhances the water solubility required in clinical use. It has been vastly exploited with 99mTc as a
radiocomplex with excellent results in medical imaging. Estrogen derivative, 17α-
ethinylestradiol (EE2) was chosen as targeting molecule due to its synthetic accessibility, easy
availability and well-established tolerance for its bioactivity by substitution at 17α-position. The
modified estradiol derivative, (EST)2DT is chemically accessible via simple three step reaction
under mild conditions. The distance between the two estradiol moieties tethered by the
conventional acyclic chelating agent through triazole rings was calculated in the energy
minimized state by using Schrödinger Software; Maestro 9.7 and was observed to be 22.57 Å
(Figure 1). The figure S14 in the supporting information shows the three dimensional structure of
ligand with ERα homodimer.
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Figure 1. Distance between the two E2 moieties in the energy minimized structure of (EST)2DT.
Further, binding affinity has been predicted using radiometric assay with 3H-estradiol. The target
specificity has been evaluated via scintigraphy and tissue distribution studies in orthotopic MCF-
7 tumor bearing nude mice after labeling the ligand with 99mTc. The in vitro studies in cell lines
demonstrate the selectivity of the bioconjugate towards ER(+) breast cancer cells. (EST)2DT
induced ROS generation was dependent on ER protein expression and led to mitochondrial
apoptosis (intrinsic pathway). The developed tracer also exhibits high stability, fast
pharmacokinetics in normal organs.
Ligand Synthesis. The synthesis was facile and involved the bivalent approach where two units
of EE2 were conjugated to DTPA via amide linkage in three simple and straightforward steps
(Scheme 1). The ethinyl group at 17α-position was utilized for conjugation with 2-
azidoethanamine (1) through copper catalysed click reaction to give compound 2, amino
1
estradiol derivative in 88% yield. The appearance of triazole proton peak at 7.76 ppm in H and
two triazole carbon peaks at 120.0, 152.3 ppm in 13C NMR spectroscopy confirmed the
formation of triazole ring. The final reaction of two equivalents of amino derivative (2) with the
diethylenetriaminepentaaceticacid dianhydride gave (EST)2DT (3) in 92% yield. Importantly,
synthetic procedures allowed the reactions to occur in mild conditions with the minimal
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requirement of chromatographic techniques for purification giving products in high yield. The
corresponding monomer, ESTDT (4) was also synthesized using compound 2 in 1:1 ratio under
similar reaction conditions. However, yield of the ligand was very low (< 30%) that it could not
be purified further and used as such in the in vitro assays. All the synthesized products were
characterized by spectrometric methods. As the presence of residual EE2 in the bivalent ligand
may result in the greater biological response, therefore, its presence was analyzed by analytical
HPLC. The final product showed >99% purity (Figure S15).
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,
,
.
a
N N3
H
2O
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NH2
HB
NH2
r
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3
4
E T 2DT
S
(
)
E TDT
S
H
O
H
O
Scheme 1. Synthetic scheme for estradiol based bivalent ligand, (EST)2DT.
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Radiolabeling and Stability. (EST)2DT was radiolabeled with 99mTc for the evaluation of its
diagnostic efficiency in SPECT imaging of ER(+) tumor. The labeling experiments performed
with 90−100 µg of stannous chloride resulted in maximum labeling efficiency with the minimum
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‒
amount of free 99mTcO4 . The purification with C-18 sep-pak cartridge efficiently removed the
free ligand. Radiolabeling efficiency was in the range of 92‒97% as determined by ITLC. The
radiochemical purity of 99mTc-(EST)2DT was estimated chromatographically using HPLC and
was found to be >98%. The specific activity was 20‒48 GBq/μmol. Same procedure was
followed for the radiolabeling of ESTDT and was achieved in >90% efficiency. High stability of
the radio complex is essential before in vivo studies as the dissociation of radiometal from the
chelate may result in non-targeted organs uptake and non-specific toxicities. Therefore, the
kinetic inertness of 99mTc-(EST)2DT and 99mTc-ESTDT was assessed in human serum, and was
also challenged with the potential competitors, histidine and cysteine. The stability of 99mTc-
(EST)2DT in human serum was almost constant under the whole timeframe of 6 h with 95‒99%
of intact tracer at physiological pH (Figure S16). Overall stability of the complex was >85%
when challenged with histidine and >90% with cysteine till 6 h suggested high kinetic inertness
of the radiocomplex. Similarly, the stability of 99mTc-ESTDT was >90% till 6 h (Figure S16).
Biological Evaluation
Relative Binding Affinity. The ER binding affinity of the (EST)2DT was evaluated by
competitive radiometric binding analysis and was compared with the corresponding monomeric
ligand. The binding is calculated as relative binding affinity, relative to the reference β-estradiol
(ES), one of the naturally occurring mammalian estrogenic steroids (RBA = 100%). The RBA
value for (EST)2DT was 15.63 ± 0.14%. Though lower than ES, the RBA of the ligand is
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sufficient enough for it to fall in the category of active binders i.e. the ligands displaying RBA >
10%.28 However, ESTDT displayed only 3.67 ± 0.08% affinity. As the ERα binding pocket is
hydrophobic in nature therefore, lower RBA value of ESTDT can be attributed to its highly polar
nature. The study confirms the enhanced binding efficiency of the dimeric ligand.
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Cellular Uptake Studies. Cellular uptake study is a widely utilized method for the
understanding of the diverse biological activity of compounds at the cellular level. To investigate
the ability of 99mTc-(EST)2DT and 99mTc-ESTDT to penetrate through cancer cells, in vitro
cellular uptake studies were performed on ER(+) MCF-7 and ER(‒) MDA-MB-231 human
breast cancer cell lines at different time intervals. As seen from Figure 2, both the ligands
displayed time dependent cell uptake in MCF-7 cells. 99mTc-(EST)2DT exhibited enough
accumulation with 0.84‒4.5% activity while the extent of uptake was less in the case of
monomer with 0.37‒1.58% uptake in the time interval of 20‒120 min. The lower cell penetration
by 99mTc-ESTDT is possibly due to its lower lipophilicity. However, uptake of tracers in MDA-
MB-231 cells was very less and did not display any significant difference. 99mTc-ESTDT
displayed only 0.09‒1.06% cell uptake while it was 0.18‒1.12% for 99mTc-(EST)2DT.
Figure 2. Binding assay of 99mTc-(EST)2DT with MCF-7 and MDA-MB-231 cells (n=3). The binding is expressed
as percentage radioactivity bound to cell lines.
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The blocking study with the standard ES, was carried out to further assess the mode of binding.
Cells pre-treated with ES led to the decrease in percentage uptake of 99mTc-(EST)2DT by ~60%
after 2 h incubation (Figure S17). The blocked uptake of 99mTc-(EST)2DT due to ES in the
competitive binding study reveal the same binding mode to a significant extent. The results
demonstrated that the binding of the developed ligand was may be through the ER-mediated
mechanism. On the other hand, 99mTc-ESTDT was not significantly blocked by ES thereby
suggesting uptake due to non-ER mediated pathway. As expected in the case of MDA-MB-231,
the presence or absence of ES had no effect on percentage accumulation of the tracers. As the
uptake of both the tracers in MDA-MB-231 was very low and 99mTc-ESTDT was not
internalized in MCF-7 cells significantly therefore, further in vivo evaluation was performed with
99mTc-(EST)2DT in MCF-7 xenografts.
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In Vivo Scintigraphy and Biodistribution Studies. The ability of 99mTc-(EST)2DT to localize
in ER(+) tumor was established by in vivo gamma scintigraphy. The MCF-7 xenografts were
used for imaging at different time periods (0.5‒4 h) (Figure 3). The tumor quantification and
activity accumulation in major organs were realized by determining ROIs encompassing the
whole organ in the image. The percentage uptake is expressed as the average of tracer uptake in
four mice.
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Figure 3. Scintigraphy studies of 99mTc-(EST)2DT in orthotopic tumor-bearing nude mice at 0.5 and 2 h.
The uptake in tumor was seen in early time point of 0.5 h. The ROI analysis of the planar image
demonstrated the tumor to muscle ratio (T/M) was in the range of 1.5‒5.7 in the time interval of
0.5‒2 h. However, the positive control, 99mTc-DTPA, when injected in the tumor bearing nude
mice showed non-significant tumor uptake and renal route of excretion, a characteristic property
of DTPA. The change in the metabolic route in 99mTc-DTPA may be attributed to the
introduction of two EE2 moieties that metabolize in liver. Estrogen mediated tumor uptake of
99mTc-(EST)2DT was evaluated by blocking study performed in the presence of ES which
decreased the tumor localization of the tracer by ~5 fold with T/M ratio = 1.11 ± 0.34 at 2.0 h.
The results validated the receptor mediated uptake of 99mTc-(EST)2DT in ER(+) tumor. As
estrogens are known to vary vascular physiology, expression levels of receptors, and blood flow,
therefore, care must be taken during receptor blocking experiments with high doses of ES.2
To further authenticate the SPECT analysis, tissue distribution study was performed on MCF-7
tumor bearing nude mice after i.v. administration of 99mTc-(EST)2DT. The results were in
concordance with the SPECT study displaying increase in tumor uptake with time. Tumor uptake
was achieved at initial time point of 0.5 h with tumor/muscle ratio (T/M) as 2.04 which reached
to 6.2 at 2 h (Figure 4).
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Figure 4. Biodistribution of 99mTc-(EST)2DT after injection in tumor bearing nude mice (n = 3).
In 2005, Biber et al. reported the synthesis and application of 99mTc-ESTDTPA where one unit of
ES was conjugated to DTPA.29 The ligand was not ER specific in nature and exhibited T/M ratio
as 2.0 at 24 h. The enhanced ER specificity and an apparent improvement in the tumor targeting
efficiency by the use of EE2 (RBA >100%) in the bivalent approach is observed in this study.
Like other steroids, 99mTc-(EST)2DT was mainly eliminated through liver. Also, in the previous
reports, one of the problems encountered with 99mTc-labeled estradiol derivatives was the higher
activity accumulation in liver and relatively lower uptake in tumor than the background.15,30 In
the present study, the elimination of activity from liver was fast, thereby minimizing the
background activity and improving the tumor localization with time. Due to the early tracer
clearance and nearly constant metabolite background, imaging at initial time-point could provide
a good visualization of the tumor even at the location near to the blood pool structure.
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In Vitro Anti-Cancer Activity. The cell viability and proliferation measurements are the bases
for the understanding of the response of cell population to the external factors through in vitro
assays. In this aspect, MTT assay is often used for the screening of various drugs for cell
proliferation or cell death. The toxicity of (EST)2DT was evaluated against human normal cell
line, HEK-293 by MTT assay and compared with the standard anticancer agent, tamoxifen. The
(EST)2DT had an insignificant role in arresting the growth of HEK-293 cells (IC50~74.1µM). Its
IC50 was three order higher in magnitude than tamoxifen suggesting that the ligand was relatively
safer towards the normal cells and thus, suitable for medical purposes (Table 1).
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Table 1. Anti-proliferative activity of (EST)2DT in terms of IC50 (Mean± SE, in µM)
Compound
Activity in terms of IC50 (Mean± SE, in µM)
MCF-7
14.3 ± 4.5
9.6 ± 1.5
MDA-MB-231
35.3 ± 3.9
HEK-293
74.1 ± 4.1
23.6±1.0
MCF-7 + E2
46.3 ± 3.2
9.9 ± 1.2
(EST)2DT
Tamoxifen
12.2±1.6
As the biological activity of the ligand is not reflected by its RBA value, the cytotoxicity of the
(EST)2DT was further assessed on cancer cells by evaluating its growth inhibition effect against
ER(+) MCF-7 and ER(‒) MDA-MB-231 (human breast cancer) cells using MTT assay at
nM‒mM concentration and IC50 was calculated. The ligand did not show proliferative activity in
any of the cell line evaluated. However, anti-proliferative activity was observed in MCF-7 with
IC50 ~ 14.3 µM and MDA-MB-231 with IC50 ~ 35.3 µM. (EST)2DT demonstrated higher potency
towards estrogen-dependent MCF-7 cells in comparison to MDA-MB-231 cells. On the other
hand, tamoxifen displayed similar activity against both the ER(+) (IC50 ~ 9.6 µM) and ER(‒)
(IC50 ~ 12.2 µM) cell lines. The growth inhibition in MCF-7 cells by (EST)2DT was comparable
to that of tamoxifen. Also, ES is reported to inhibit selectively the growth of ER(+) cells,25 this
property was retained in the developed bivalent ligand even after chemical modifications. A
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significant and selective inhibition of MCF-7 cells suggests that (EST)2DT might be a partial ER
antagonist.
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The effect of the presence of ES to reverse the antiestrogenic activity of (EST)2DT in MCF-7
cells was further assessed. The pre-treatment of MCF-7 cells with ES led to the reduction of anti-
proliferative activity by 3.2 orders of magnitude. The antiestrogenic activity thus appeared to be
ER-mediated and was not non-specific in nature.
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Apoptosis Assay. Cancer cell inhibition is frequently associated with induction of apoptosis that
in turn induces morphological changes and nuclear DNA fragmentations in cells. Morphological
study of MCF-7 cells treated with (EST)2DT was performed at the concentration of 14.3 µM
(IC50) and 21µM for 24 h and analyzed with light microscopy. Morphological changes
accompanying the characteristic features of apoptosis including visible rounding, detachment
from the substratum and cell shrinkage in treated groups signified the cell death in the presence
of (EST)2DT (Figure 5).
Figure 5. Morphological alteration of MCF-7 cells by (EST)2DT treatment.
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Apoptosis is favored over other mechanisms for the elimination of cancer cells as it induces cell
death via a sequence of regulated cell events without generating inflammatory effect or harm to
the healthy cells. Conversely, necrosis elicits membrane rupture thereby, triggering inflammatory
response.31 Whether the developed ligand caused cell death through necrosis or apoptosis was
verified by flow cytometry of (EST)2DT treated MCF-7 cells after staining with Annexin-V
FITC and PI (Figure 6). The upper left quadrant of the cytogram is positive only for PI
(necrotic), upper right for both Annexin-V and PI (late apoptotic), lower right for Annexin-V
only (early apoptotic) and lower left negative for both Annexin-V and PI (live). Each quadrant
data presented with the percent of total cell count. A significant decrease in live cell counts and
an increase in early and late apoptotic cell counts at the concentration of 14.3 µM and 21 µM
was observed after 24 h. However, membrane impermeable PI staining which is indicative of
necrotic membrane did not increase with increasing concentration of (EST)2DT suggesting that
(EST)2DT primarily caused apoptosis, but not necrosis.
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Figure 6. Effect of (EST)2DT treatment on apoptosis and necrosis of MCF-7 determined by Annexin-V and PI
staining.
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To further evaluate the intrinsic mechanism behind programmed cell death, cell cycle analysis
was performed by flowcytometry and the histogram of DNA fluorescence was generated for the
cell cycle phases as G0/G1, S and G2/M. The treatment of MCF-7 cells with (EST)2DT at
variable concentration did not show significant alteration in any of the phases of the cell cycle
(Figure S18).
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Cellular ROS Assay. Reactive oxygen species (ROS) generation is important for the functional
role in the immune system. However, its excessive generation causes oxidative stress in cells.
Oxidative stress promotes cell death and ROS are the well established cellular signal that
mediates apoptosis.32 Therefore, impact of (EST)2DT on cellular ROS level was investigated.
Straightforward technique was utilized to directly measure the redox state of the cells by staining
with cell permeable fluorescent dye, 2ʹ,7ʹ–dichlorofluorescein diacetate (DCFDA). Hydrogen
peroxide was used for positive control as it exhibits major contribution in oxidative damage.
An increase in ROS level upon treatment with (EST)2DT with 134.57 and 192.74 counts at 14.3
and 21 μM respectively was observed in comparison to 99.6 counts in the absence of (EST)2DT
(control) (Figure 7). The ROS inducing effect of (EST)2DT was thus, dose-dependent; and
higher concentration of the ligand (21 μM) caused significantly higher production of ROS
compared to H2O2-induced ROS level in MCF-7 cells. Therefore, it was believed that (EST)2DT
induces high ROS level leading to the disturbance in the antioxidant defence system in MCF-7
cells, thereby, causing apoptosis via oxidative pathway.
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Figure 7. Effect of (EST)2DT treatment on cellular ROS level of MCF-7 cells. MCF-7 cells treated with
(EST)2DT for 24 h were harvested, stained with 2′,7′-dichlorofluorescin diacetate (DCFDA), and analyzed using
flowcytometry. H2O2 (30µM) was used for positive control for ROS generation and added 2 h before harvesting for
analysis. Histogram show percent change in cellular ROS level with various treatments. Results were expressed as
mean ± SEM, n= 3. Level of statistical significance presented as p values are ***= p<0.001, **= p < 0.01, c=
p<0.05 and *= p >0.05 versus control.
The process was confirmed by the cells treatment with (EST)2DT in the presence or absence of
ROS scavenger N-acetyl cysteine (NAC). The presence of NAC led to the decrease in early and
late apoptotic cells by 35 and 15% respectively (Figure S19). The apoptosis was thus confirmed
via oxidative pathway.
Mitochondria are both the source and target of ROS which consist of main apoptogenic factors
like apoptosis inducing factor (AIF) and cytochrome c thus playing an intrinsic role in
apoptosis.32,33 Consequently, effect of (EST)2DT concentration on MMP was also evaluated. JC-
1 dye was used for the measurement of mitochondrial membrane potential, where green
fluorescent JC-1 dye changes to red fluorescent JC-1 aggregates when concentrated in energized
mitochondria in response to their higher membrane potential.34 Upon treatment with (EST)2DT,
significant increase in green fluorescence indicative of formation of less JC-1 aggregates
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compared to control was observed suggesting that (EST)2DT is probably causing mitochondrial
depolarization (Figure 8). Effect of (EST)2DT on mitochondria is also supported by earlier
reports suggesting that ROS could induce mitochondrial depolarization or loss of mitochondrial
(intrinsic pathway) membrane potential.
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Figure 8. Effect of (EST)2DT treatment on MMP of MCF-7 cells. MCF-7 cells were grown in DMEM media and
treated with different concentrations of (EST)2DT for 24 h. Cells were harvested and stained with JC-1 dye, and
analyzed using flowcytometry. 10 µM of carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used for positive
control to induce mitochondrial depolarization and added 2 h before harvesting for analysis. Representative
cytogram show cell count scatter with JC-1 monomer (green) and JC-1 aggregates (red) fluorescence intensity.
Gene Transcriptional Activation Assay and Protein Expression Study. (EST)2DT was
initially designed as ER targeting imaging agent; however it also appeared to have significant
anti-proliferative and pro-apoptotic activity. Consequently, its impact on ERα and ERβ through
western blotting was investigated. Upon treatment on MCF-7 cells with 14.3 µM of (EST)2DT
for 24 h, expression of ERα protein decreased with significant concomitant increase in ERβ
protein (Figure 9 A & B).
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Figure 9. A. Effect of (EST)2DT treatment on ERα and ERβ protein. MCF-7 cells were treated with 14.3 µM of
(EST)2DT for 24 h and immunoblotting was performed to detect ERα and ERβ expression in the lysates of MCF-7
cells. The blots were stripped and reprobed with β-actin antibody as a loading control. B. Densitometry of ERα and
ERβ protein in MCF-7 treated with (EST)2DT. The quantity of protein was measured by densitometry,
normalized with β-actin and represented fold change versus control. C. Effect of (EST)2DT at different
concentrations (10 μM, 15 μM and 20 μM) on transcriptional activation of ERα and ERβ in MCF-7 cells. E2
(100 nM) was used as positive control. Results were described as % of normalized relative luciferase unit (RLU).
Results were expressed as mean ± SEM, n = 3. p Values are a = p < 0.001, b = p < 0.01, c = p < 0.05 and d = p >
0.05 versus control.
In transient transfection assay, transcriptional activation of ERα decreased significantly (p<0.05,
p<0.01, p<0.001) at different concentrations (10, 15 and 20 μM respectively) of (EST)2DT in
MCF-7 cells. Whereas, (EST)2DT caused a significant increase (p<0.01, p<0.001) in the
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transcriptional activation of ERβ at 15 and 20 μM. At 100 nM of E2, the activation of both ERα
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and ERβ, were increased significantly (p<0.01) as compared to control (Figure 9 C).
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ERα and ERβ ratio is a very important regulator of cell proliferation and apoptosis in breast
cancer cell. A consistent finding suggests that total ERβ level decreases during breast cancer in
contrast to ERα, supporting the potential role of ERβ in breast tumor suppression.35,36 In general,
elevation of ERβ-like expression is linked with expression of good quality diagnostic probes or
better clinical outcomes in patients usually treated with tamoxifen.37,38 The alteration in ROS
level causing mitochondrial damage and apoptosis is anticipated as due to the activation of ERβ
and alteration in the ratio of ERα/ERβ protein expression by (EST)2DT. It was further proved by
measuring the cellular ROS level generated by (EST)2DT, in the presence of ER antagonist
fulvestrant (ICI 182780). The results displayed the significant reversal of (EST)2DT induced
ROS generation upon treatment with ICI 182780 suggesting (EST)2DT induced ROS is
dependent on ER protein expression (Figure S20). The process also confirms the cellular
internalization of the ligand and ER-mediated apoptosis in ER(+) cells.
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CONCLUSIONS
An estradiol based bivalent ligand (EST)2DT has been designed, synthesized and bioevaluated
for its role in diagnosis and therapy of breast cancer. The two ethinylestradiol units were tethered
with the conventional acyclic chelator and the developed ligand was radiolabeled with 99mTc in
high yield and stability. Early tumor localization of 99mTc-(EST)2DT was viable and feasible
along with the improved tumor-targeting performance by the bivalent approach. The unlabeled
bivalent ligand possessed higher affinity and specificity towards ER. The additional benefit of
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the unlabeled ligand in therapeutics was also evaluated in cancer cell lines. It exhibited
anticancer activity against ER(+) cells with IC50 comparable to tamoxifen. The underlying
mechanism for programmed cell death was the elevation of ERβ and suppression of ERα levels
in gene transcription leading to the ROS-mediated mitochondrial apoptosis (intrinsic pathway).
The results are sufficiently encouraging for further evaluation of the ligand or the related
compounds in possible imaging/therapy of breast cancer. Furthermore, the application of
(EST)2DT can also be extended with other metal radioisotopes including 67/68Ga, 153Gd, thereby
offering the versatility for mapping breast cancer.
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EXPERIMENTAL PROCEDURES
Chemicals. All reagents and solvents were purchased from Sigma-Aldrich, Fischer-Scientific or
Merck, Germany and used without further purification unless otherwise specified. The reaction
progress was monitored by thin-layer chromatography (Silica gel 60, F254, Merck).
Radiocomplexation and the radiochemical purity were checked by ITLC-SG (Paul German,
USA). 99mTc was obtained from Regional Centre for Radiopharmaceuticals (Northern Region),
Board of Radiation and Isotope Technology (BRIT), Department of Atomic Energy, India.
Instrumentation. NMR spectra were recorded on Bruker Avance II spectrometer (1H NMR 400
MHz and 13C NMR 100 MHz). Mass spectral data were obtained by Agilent 6310 ion trap mass
spectrometer. IR studies were performed on Perkin-Elmer FT-IR spectrophotometer and the
values are reported as λmax per centimeter. HPLC trace analyses were performed using Agilent
1200 LC coupled to UV detector (λ = 214 and 254 nm) and C-18 RP Agilent columns (5 µm, 4.6
mm × 250 mm). The mobile phase was 60 % acetonitrile (A) and 40 % water (B) mixtures with
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the flow rate of 0.6 mL/min. Studies with radioisotopes were carried out using γ-scintillation
counter GRS230, ECIL (India). SPECT images were acquired in HAWKEYE γ-Camera (USA).
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Cell Culture. MCF-7 (ER(+) breast cancer), MDA-MB-231 (ER(‒) breast cancer) cell lines
were originally obtained from ATCC, USA and maintained in laboratory. HEK-293 (human
embryonic kidney epithelial) cells were procured from the institutional repository of CSIR-
CDRI. MCF-7 and HEK-293 cells were routinely sub-cultured in DMEM phenol red, MDA-MB-
231 in DMEM without phenol red incubated at 37 ˚C with 5% CO2.
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Animal Models. All animal protocols were according to our institution’s Animal Ethics
Committee (Reg. No. 8/GO/a/99/CPCSEA). New Zealand rabbits (weighing 2–3 kg), athymic
mice were used for blood clearance and scintigraphy studies. Female athymic nude mice were
injected orthotopically with human breast cancer cells, MCF-7 (100 μL, 5 × 107) into the
mammary fat pad and tumor growth was monitored daily. Animals were used for the studies
when tumors size were 3 ‒ 4 mm in diameter (2 ‒ 3 weeks).
Ligand Synthesis
2-Azidoethanamine (1). The synthesis was performed as reported earlier.39 Briefly, sodium
azide (2.39 g, 36.89 mmol) was added to a stirring solution of 2-bromoethylamine hydrobromide
(2.52 g, 12.29 mmol) in water (15 mL). The solution was heated to 80 ˚C for 15 h and cooled
down to 0 ˚C. The potassium hydroxide (10 g) and diethyl ether were then added (10 mL) to the
reaction mixture. The mixture was kept for vigorous stirring for 15 min and the organic phase
was separated. The aqueous layer was extracted with diethyl ether (3 × 25 mL). The organic
layers were combined, dried over sodium sulfate, and carefully evaporated under reduced
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pressure to afford a volatile colorless oil (0.68 g, 65%). 1H NMR (CD3OD, 400 MHz): δ 3.39 (t,
2H, J=6 Hz), 2.77 (t, 2H, J=6 Hz). 13C NMR (CD3OD, 100 MHz): δ 53.61, 40.32.
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17-(1-(2-Aminoethyl)-1H-1,2,3-triazol-4-yl)-13-methyl-7,8,9,11,12,13,14,15,16,17-
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decahydro-6H-cyclopenta[α]phenanthrene-3,17-diol (2). 17α-Ethinylestradiol (250 mg, 0.84
mmol) and 1 (108.80 mg, 1.26 mmol) were dissolved in a 2:1 mixture of tert-butyl alcohol and
water (2 mL). Freshly prepared aqueous sodium ascorbate (33.39 mg, 0.168 mmol) was added to
the reaction mixture followed by the addition of copper (II) sulphate pentahydrate (21.08 mg,
0.08 mmol). The mixture was stirred vigorously and the reaction progress was monitored by
TLC. It was diluted with water and the precipitate was filtered as off-white solid (284.03 mg,
88%). IR (cm‒1, film): 3358, 3290, 3146, 2931, 1609, 1503, 1449, 1288, 1220, 1140, 1056, 1022,
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874, 815. H NMR (CD3OD, 400 MHz): δ 7.76 (s, 1H), 6.91 (d, 1H, J=8.4 Hz), 6.40 (m, 2H),
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4.38 (t, 2H, J=4.8 Hz), 3.05 (t, 2H, J=6 Hz), 2.69‒1.27 (15H), 0.97 (s, 3H). C NMR (CD3OD,
100 MHz): δ 154.50, 154.03, 137.40, 131.07, 125.72, 123.09, 114.62, 112.27, 81.82, 52.18,
48.24, 43.48, 41.28, 39.68, 37.05, 32.93, 29.33, 27.33, 26.13, 23.24, 13.48. MS(ESI) for
C22H30N4O2 + H, calcd 383.2, found 383.5, for C22H30N4O2 + Na, calcd 405.2, found 405.3.
5,8-Bis(carboxymethyl)-13-(4-(3,17-dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-
decahydro-6H-cyclopenta[α]phenanthren-17-yl)-1H-1,2,3-triazol-1-yl)-2-(2-(2-(4-(3,17-
dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[α]phenanthren-
17-yl)-1H-1,2,3-triazol-1-yl)ethylamino)-2-oxoethyl)-10-oxo-2,5,8,11-tetraazatridecane-1-
carboxylic acid, [3, (EST)2DT]. DTPA-dianhydride (93.28 mg, 0.26 mmol) was dissolved in
DMF (5 mL) and a solution of 2 (200 mg, 0.52 mmol) in DMF (10 mL) was added dropwise.
The pH of the reaction mixture was adjusted to 8.0 using triethylamine and heated at 60 ˚C under
nitrogen atmosphere for 48 h. The reaction mixture was brought to room temperature, diethyl
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ether (50 mL) was added and it was left for stirring for another 24 h. The precipitate obtained
was filtered and dried in vacuum to give pale yellow amorphous solid (269.26 mg, 92%). IR
(cm‒1, film): 3320.0, 2931.4, 287.3, 1723.5, 1647.5, 1499.6, 1149.0, 1377.1, 1284.2, 1229.3,
1140.6, 1056.1, 1022.4, 870.31, 819.63, 692.92. 1H NMR [(CD3)2SO, 400 MHz]: δ 8.30 (s, 2H),
7.88 (s, 2H), 6.92 (d, 2H, J=8.4 Hz), 6.40 (m, 4H), 4.42 (t, 4H, J=6 Hz), 3.53‒1.19 (50 H), 0.90
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(s, 6H), 0.56 (t, 2H, J=11.2 Hz). C NMR [(CD3)2SO, 100 MHz]: δ 172.71, 170.05, 169.10,
154.81, 153.95, 137.14, 130.51, 125.86, 122.93, 114.85, 112.63, 81.05, 57.41, 54.92, 52.61,
50.32, 48.57, 47.47, 46.68, 43.00, 37.10, 35.83, 32.51, 30.62, 29.26, 27.16, 26.12, 23.64, 14.24.
MS(ESI) for C58H79N11O12 + 2H, calcd 1123.6, found 1123.0.
2,2'-(1-Carboxy-2-(carboxymethyl)-13-(4-(3,17-dihydroxy-13-methyl-
7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[α]phenanthren-17-yl)-1H-1,2,3-triazol-
1-yl)-10-oxo-2,5,8,11-tetraazatridecane-5,8-diyl)diacetic acid, [4, ESTDT]
The synthetic procedure for ESTDT was similar to (EST)2DT. The only change was the use of
1:1 molar ratio of compound 2 and DTPA dianhydride in the reaction. The product was obtained
as off-white solid (28.73%). 1H NMR (CD3OD, 400 MHz): δ 7.88 (1H), 6.99 (1H), 6.47 (m, 2H),
4.57 (t, 2H, J=6 Hz), 3.86‒1.19 (37 H), 1.049 (s, 3H), 0.67 (1H); 13C NMR (CD3OD, 100 MHz):
δ 173.69, 172.00, 171.72, 169.49, 154.57, 153.98, 137.45, 131.15, 125.84, 123.22, 114.66,
112.31, 81.77, 55.53, 55.44, 55.16, 51.17, 50.71, 49.98, 49.02, 48.25, 47.89, 46.97, 43.38, 39.67,
38.99, 37.11, 32.88, 29.34, 27.33, 26.15, 23.25, 13.53.
Radiolabeling. Freshly eluted Na99mTcO4 (110 MBq) was added to a shielded vial containing
lyophilized sample of (EST)2DT/ESTDT (1 mg) and stannous chloride (SnCl2, 95 μg) in de-
ionized water (500 μL). The pH of the reaction mixture was adjusted to 6.5‒7.0 with 0.1 M
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Na2CO3 and incubated at room temperature for 10 min. The radiocomplex was purified using C-
18 Sep-pak cartridge preconditioned with ethanol (20 mL) and activated with ethanol/water
(30%, v/v). The tracer was loaded onto the cartridge, rinsed with de-ionized water (5 mL)
followed by the elution of radiotracer in 5% (v/v) ethanol/water (2 mL). The radiolabeling
efficiency and purity of the complex formed was assessed by ascending instant thin layer
chromatography on ITLC-SG strips. The mobile phase used was 100% acetone and
pyridine/acetic acid/water (PAW) 3:5:1.5 (v/v/v). The specific activity of 99mTc-(EST)2DT and
99mTc-ESTDT was in the range of 47 - 49.5 GBq/μmol and 26.8 – 28.5 GBq/μmol respectively
after purification.
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Human Serum Stability. The evaluation of 99mTc-(EST)2DT/99mTc-ESTDT stability in human
serum was performed using protocol as reported.40
Cysteine and Histidine Challenge. The kinetic inertness of 99mTc-(EST)2DT/99mTc-ESTDT was
tested by displacement of ligand bound activity with cysteine and histidine. A mixture of the
known amount of each ligand and the 100-fold molar excess of cyteine/histidine was incubated
at 37 ˚C for uto 6 h. The samples were analyzed at 0.5, 1, 3.0 and 6.0 h by HPLC and the
percentage of intact tracer was determined.
Relative Binding Affinity: The ERα competitive binding experiment was performed according
to the previous procedure.16 The reaction mixture containing different concentration of ligands
(50 μL) in the ERα binding buffer [prepared from dithiotheitol (2 mM), glycerol (10%), Tris-HCl
(pH 7.5, 10 mM) and BSA (1 mg/mL) was treated with 3H-Estradiol (3H-ES, 23.8 nM) and ERα
protein solution (0.25 pmol) (Sigma-Aldrich). The non-specific binding was determined by the
addition of ES (50 μM). The mixture was incubated at 4˚C for 18‒24 h. The ER-ligand
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complexes were absorbed on hydroxyapatite (HA) by adding HA slurry [(200 μL, slurry was the
50% (v/v) concentration of HA in Tris-HCl (50 mM, pH 7.4)]. The final mixture was then
incubated and vortexed three times for 15 min. It was washed in triplicates with washing buffer
[containing KCl (100 mM, pH 7.4) and Tris-HCl (40 mM)], centrifuged and supernatant was
removed. The HA pellets were resuspended in cold ethanol (750 μL), vortexed, centrifuged and
supernatants were collected in the scintillation vials for radioactivity measurement in the
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scintillation counter. The data is expressed as percentage of specific binding of H-ES vs log
molar concentration of tested compounds.
Cellular Uptake Studies. To demonstrate the cellular uptake of the radiolabeled complexes,
human breast tumor cell line MCF-7/MDA-MB-231 (105 cells/well in each well of 96-well
microculture plates) was incubated with 99mTc-(EST)2DT or 99mTc-ESTDT (6 μg/well) for 0.5‒4
h. In the blocking assay, cells were pretreated with β-estradiol (54 μg/well) or DMSO (control)
for 30 min. To this, 99mTc-(EST)2DT (6 μg/well) was added and kept for incubation upto 2 h. The
cells were rinsed thrice with cold PBS (0.5 mL) and trypsinized using EDTA (0.2 mL). After 5
min, PBS (0.4 ml) was added, and radioactivity counts in cell lysates were determined using γ-
well counter to calculate the percentage of uptake in cells. The experiment was carried out in
triplicates.
In Vivo Scintigraphy. In vivo scintigraphic images were acquired as the statistic acquisition at
different time intervals (0.5–4 h) after i.v. injection of 99mTc-(EST)2DT or 99mTc-DTPA (3.5‒4.8
MBq) using a gamma camera. In the blocking study, each mouse was pretreated with E2 (n=3,
616 μmol/kg, i.v.) 1 h prior to 99mTc-(EST)2DT injection and imaged at the various time point.
Tumor/background ratio was determined by semiquantitative analysis from the computer-
outlined regions of interest (ROI) through Entegra® software.
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MTT Assay. The antiproliferative activity of the ligands was assessed using MTT, (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, reduction assay.41 MCF-7, MDA-MB-
231 and HEK-293 cells were seeded in 96-well microculture plates (104 cells/200 μL per well)
for 24 h. A stock solution of (EST)2DT was prepared in DMSO and was diluted to the desired
concentrations in culture medium supplemented with 0.5% FBS. Cells cultured in the 96-well
plate were treated with the variable concentration of ligand for 24 h at 37 ˚C. After the treatment,
MTT (20 µl of 5 mg/mL in PBS) was added, and the plate was further incubated for 4 h. The
medium from each well was carefully removed; formazan crystals were dissolved in DMSO (200
µl), and absorbance was recorded at 540 nm. The percentage cell inhibition was calculated by
comparing absorbance of (EST)2DT/ESTDT and untreated control group. The percent cell
inhibition was plotted against concentrations of each compound, and IC50 concentration of
compounds was calculated using Graphpad Prism software.
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The ES competition assay was done with the cells pretreated with ES (50 μM) for 1 h. After the
treatment the ligand addition was done in complete media with 50 μM of ES.42 The cell viability
was then analyzed after 24 h incubation as described above.
Cell Cycle Analysis. The cytotoxicity of (EST)2DT against HEK-293 and MCF-7 cells were
identified and subjected to cell cycle analysis.43 Briefly, MCF-7 cells were treated with
(EST)2DT; the adherent cells were trypsinized after incubation, combined with any floating cells
present and then washed with cold PBS. Cells were fixed in 70% ethanol, treated with RNase A
(200 μg/mL) followed by staining with propidium iodide (PI) (50 μg/ml) and incubated for 30
min. The flow cytometry readings (FACScan, BD Biosciences, USA) were acquired after cell
treatment.
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Morphological Analysis of Cells. The effect of (EST)2DT on breast cancer cells was observed
under a bright field microscope after treatment of the cells with the variable concentrations of
ligand.44 MCF-7 cells were seeded in the 6-well plate (105 cells/well) and incubated for 24 h. The
medium was then carefully replaced, and cells were treated with desired concentration of the
compound for 24 h. For the untreated control, cells were treated with DMSO (0.001%) in the
culture medium. After incubation, random fields of cells from each group were captured by
bright field inverted microscope (Leica, Germany).
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Evaluation of Apoptosis with Flow Cytometry. MCF-7 cells (1×106) were plated in 6-well
plates and allowed to grow overnight. The medium was then replaced and cells were treated with
desired concentration of (EST)2DT for 24 h.45 Cells treated with DMSO (0.001%) in culture
medium were included as controls for all experiments. After 24 hours of treatment, cells from
supernatant and adherent monolayer cells were harvested by trypsinization and washed with PBS
at 300 g. The cells (1×105) were then stained with Annexin V-FITC and propidium iodide using
the Annexin-V-PI apoptosis detection kit (Sigma). Flow cytometry was carried out using an
FACScan (Becton Dickinson) equipped with a single 488 nm argon laser. Annexin V-FITC were
analyzed using excitation and emission settings of 488 nm and 535 nm (FL-1channel) whereas PI
was recorded with 488 nm and 610 nm (FL-2 channel). Debris and clumps were gated out using
forward, and orthogonal light scatter.
Cellular ROS Assay: MCF-7 cells treated with (EST)2DT for 24 h were harvested, stained with
2′,7′-dichlorofluorescin diacetate (DCFDA), and analyzed using flowcytometry. H2O2 (30µM)
was used for positive control for ROS generation and added 2 h before harvesting for analysis.
Results were expressed as mean ± SEM, n= 3. Level of statistical significance presented as p
values are ***= p<0.001, **= p < 0.01, c= p<0.05 and *= p >0.05 versus control.
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