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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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Bioconjugate Chemistry

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 Kumar Singh, ‡ Anil K. Mishra,*† Anupama Datta*†

†

Institute of Nuclear Medicine & Allied Sciences, DRDO, Brig. SK Mazumdar Marg, Delhi110054, India ‡

§

Department of Chemistry, Indian Institute of Technology, Delhi-110016, India

Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow-226031, India

ACS Paragon Plus Environment

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ABSTRACT

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 radiolabeled complex,

99m

Tc. The

Tc-(EST)2DT was obtained in >99% radiochemical purity and 20‒48

99m

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. 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 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 with ER(+) tumors. The clinical outcomes of 18F-FES are encouraging, but the availability and 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. 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,

99m

Tc 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. 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

99m

Tc-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-6Hcyclopenta[α]phenanthren-17-yl)-1H-1,2,3-triazol-1-yl)-2-(2-(2-(4-(3,17-dihydroxy-13-methyl7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[α]phenanthren-17-yl)-1H-1,2,3-triazol-1yl)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

99m

Tc. 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 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, 17αethinylestradiol (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 MCF7 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 2azidoethanamine (1) through copper catalysed click reaction to give compound 2, amino estradiol derivative in 88% yield. The appearance of triazole proton peak at 7.76 ppm in 1H and two triazole carbon peaks at 120.0, 152.3 ppm in

13

C 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). NaN3, H2O,

. NH2 HBr

Br

NH2 N3

90°C

1

OH

N

HO

N

N

. CuSO4 5H2O, Sodium ascorbate, 2:1 tBuOH:H2O, RT

OH HO

H2N

2 COOH O

O O

N

N

N

DMF, Et3N, 60°C

O O

O

1:1

2:1

O

N HO

N N

N H

O

O OH N

HO

OH HO

OH N

O N

O O

O

N H HO

N

N N

O

O

N OH HO

N

N N H

N N N

O HO

HO

3 (EST)2DT HO

4 ESTDT HO

Scheme 1. Synthetic scheme for estradiol based bivalent ligand, (EST)2DT.


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Radiolabeling and Stability. (EST)2DT was radiolabeled with

99m

Tc 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 amount of free

TcO4‒. The purification with C-18 sep-pak cartridge efficiently removed the

99m

free ligand. Radiolabeling efficiency was in the range of 92‒97% as determined by ITLC. The radiochemical purity of

99m

Tc-(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

99m

Tc-(EST)2DT and

99m

Tc-ESTDT was assessed in human serum, and was

also challenged with the potential competitors, histidine and cysteine. The stability of

99m

Tc-

(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. 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

99m

Tc-(EST)2DT and

99m

Tc-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.

99m

Tc-(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 MDAMB-231 cells was very less and did not display any significant difference.

99m

Tc-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

99m

Tc-(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 after 2 h incubation (Figure S17). The blocked uptake of

99m

Tc-(EST)2DT by ~60%

99m

Tc-(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,

99m

Tc-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 99m

uptake of both the tracers in MDA-MB-231 was very low and

Tc-ESTDT was not

internalized in MCF-7 cells significantly therefore, further in vivo evaluation was performed with 99m

Tc-(EST)2DT in MCF-7 xenografts.

In Vivo Scintigraphy and Biodistribution Studies. The ability of

99m

Tc-(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,

99m

Tc-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

99m

Tc-DTPA may be attributed to the

introduction of two EE2 moieties that metabolize in liver. Estrogen mediated tumor uptake of 99m

Tc-(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

99m

Tc-(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

99m

Tc-(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,

99m

Tc-(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). Table 1. Anti-proliferative activity of (EST)2DT in terms of IC50 (Mean± SE, in µM)

Activity in terms of IC50 (Mean± SE, in µM) MCF-7 MDA-MB-231 HEK-293 MCF-7 + E2 (EST)2DT 14.3 ± 4.5 35.3 ± 3.9 74.1 ± 4.1 46.3 ± 3.2 Tamoxifen 9.6 ± 1.5 12.2±1.6 23.6±1.0 9.9 ± 1.2 As the biological activity of the ligand is not reflected by its RBA value, the cytotoxicity of the Compound

(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. 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 antiproliferative activity by 3.2 orders of magnitude. The antiestrogenic activity thus appeared to be ER-mediated and was not non-specific in nature. 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.

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).

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 0.05 versus control.

In transient transfection assay, transcriptional activation of ERα decreased significantly (p

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