Spectroscopic evaluation of synthesized 5β-dihydrocortisol and 5β-dihydrocortisol acetate binding mechanism with human serum albumin and their role in anticancer activity

Article abstract of DOI:10.1080/07391102.2018.1433554

Our study focus on the biological importance of synthesized 5β-dihydrocortisol (Dhc) and 5β-dihydrocortisol acetate (DhcA) molecules, the cytotoxic study was performed on breast cancer cell line (MCF-7) normal human embryonic kidney cell line (HEK293), th

Full text of DOI:10.1080/07391102.2018.1433554

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Spectroscopic evaluation of synthesized 5β-
dihydrocortisol and 5β-dihydrocortisol acetate
binding mechanism with human serum albumin
and their role in anticancer activity
Monika Kallubai, Srinivasa P Reddy, Shreya Dubey, Dhevalapally B
Ramachary & Rajagopal Subramanyam
To cite this article: Monika Kallubai, Srinivasa P Reddy, Shreya Dubey, Dhevalapally B
Ramachary & Rajagopal Subramanyam (2018): Spectroscopic evaluation of synthesized 5β-
dihydrocortisol and 5β-dihydrocortisol acetate binding mechanism with human serum albumin
and their role in anticancer activity, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2018.1433554
To link to this article: https://doi.org/10.1080/07391102.2018.1433554
Accepted author version posted online: 29
Jan 2018.
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Publisher: Taylor & Francis
Journal: Journal of Biomolecular Structure and Dynamics
DOI: http://doi.org/10.1080/07391102.2018.1433554
Spectroscopic evaluation of synthesized 5β-dihydrocortisol and 5β-
dihydrocortisol acetate binding mechanism with human serum
albumin and their role in anticancer activity
Monika Kallubaia , Srinivasa P Reddyb , Shreya Dubeya , Dhevalapally B
Ramacharyb & Rajagopal Subramanyama
a Department of Plant Sciences, School of Life Sciences, University of Hyderabad,
Hyderabad 500046, India
b Catalysis Laboratory, School of Chemistry, University of Hyderabad, Hyderabad
500046, India
*Corresponding author
Rajagopal Subramanyam
Department of Plant Sciences
1

School of Life Sciences
University of Hyderabad 500 046 India
Tel: +91-40-23134572
Fax: +91-40-23010120
Email:srgsl@uohyd.ernet.in
Abstract
Our study focus on the biological importance of synthesized 5β-dihydrocortisol (Dhc)
and 5β-dihydrocortisol acetate (DhcA) molecules, the cytotoxic study was performed on breast
cancer cell line (MCF-7) normal human embryonic kidney cell line (HEK293), the IC50 values
for MCF-7 cells were 28 and 25 µM, respectively, whereas no toxicity in terms of cell viability
was observed with HEK293 cell line. Further experiment proved that Dhc and DhcA induced
35.6% and 37.7% early apoptotic cells and 2.5%, 2.9% late apoptotic cells respectively,
morphological observation of cell death through TUNEL assay revealed that Dhc and DhcA
induced apoptosis in MCF-7 cells. The complexes of HSA–Dhc and HSA–DhcA were observed
as static quenching, and the binding constants (K) was 4.7±0.03×104 M-1 and 3.9±0.05×104 M-1
,
and their binding free energies were found to be -6.4 and -6.16 kcal/mol, respectively. The
displacement studies confirmed that lidocaine 1.4±0.05×104 M-1 replaced Dhc, and
phenylbutazone 1.5±0.05×104 M-1 replaced by DhcA, which explains domain I and domain II are
the binding sites for Dhc and DhcA. Further, FT-IR, synchronous spectroscopy, and CD results
revealed that the secondary structure of HSA was altered in the presence of Dhc and DhcA.
Furthermore, the atomic force microscopy and transmission electron microscopy showed that the
dimensions like height and molecular size of the HSA–Dhc and HSA–DhcA complex were
2

larger compared to HSA alone. Detailed analysis through molecular dynamics simulations also
supported greater stability of HSA–Dhc and HSA–DhcA complexes, and root-mean-square-
fluctuation interpreted the binding site of Dhc as domain IB and domain IIA for DhcA. This
information is valuable for further development of steroid derivative with improved
pharmacological significance as novel anti-cancer drugs.
Keywords: 5β-dihydrocortisol; Apoptosis; Fluorescence quenching; Molecular docking;
Molecular dynamics simulations; Protein conformations
Introduction
Cortisol is a steroid hormone secreted by the adrenal cortex and is an important
glucocorticoid in humans. More than 80% of cortisol binds to cortisol-binding globulin (CBG) in
normal intact plasma (Perogamvros, Aarons, Miller, Trainer & Ray 2011). However, when CBG
is inactivated by heat or absent, then the majority of cortisol associate with albumin. It has been
determined in vitro that at 37 °C the dissociation constant (Kd) for cortisol binding to albumin in
human serum was about 330 and 810 µM (Mendel, Miller, Siiteri & Murai 1990). Cortisol is
usually metabolized in the liver to dihydrocortisol, then to tetrahydrocortisol and finally
conjugated to glucuronic acid, which enters the circulation and excreted in the urine. Cortisol
metabolites, α-dihydrocortisol and β-dihydrocortisol are known to have affinities for both
mineralocorticoid and glucocorticoid receptors. Mineralocorticoid hormones regulate electrolyte
balance, in mammals, they increase K+ and H+ and decrease Na+ in the urine. The levels of
unconjugated dihydrocortisols in urine give the information of either mineralocorticoid act as
agonist or antagonist to the renal aldosterone receptor (Marver & Edelman 1978).
Glucocorticoids have multi-systemic effects that are essential for survival in times of stress,
influencing the regulation of blood pressure, salt and water balance, immune function and
3

cellular energy metabolism. The consequences of the failure of these responses cause several
syndromes leading to morbidity and mortality. The physiological effects of these hormones
result from their specific binding to an intracellular receptor protein. Mineralocorticoid receptor
and glucocorticoid receptor, in the absence of hormone, are primarily in the cell cytoplasm, once
binding the receptor sheds its associated proteins (serum proteins), translocate to the nucleus for
transcription of its target genes. Cortisol has a 21-carbon skeleton, modifications to this skeleton
and other corticosteroids selectively alter the metabolic consequences varying the degree and
duration of activity, and also protein-binding affinity of the resultant compound. In such
modifications, the additional fluorination at the C-9 position enhanced both glucocorticoid and
mineralocorticoid activity, as in 9α-fluorocortisol, while fludrocortisone acetate is 125-fold more
potent than cortisol for mineralocorticoid effect and only 10-fold more for glucocorticoid effect
(Genard & Palem-Vliers 1980). In addition to the tri-hydroxyl groups, 16-acetoxy 10-aldehyde
groups in drugs showed enhanced binding affinity with human serum albumin (HSA) (Zhou et
al. 2015).
Human serum albumin (HSA) plays a fundamental role in the transport of metabolites,
drugs and various endogenous ligands, such as fatty acids. It is synthesized in the liver and
exported as a single non-glycosylated chain, reaching blood concentration of about 7.0×10-4 M
(Fasano et al. 2005; Matsuda et al. 2014; Lambrinidis, Vallianatou & Tsantili-Kakoulidou 2015).
The unfolding/refolding strategies of HSA with their exogenous/endogenous ligand binding
preference are of immense use in therapeutics, as it is the marvelous binder of therapeutic drugs
(Varshney et al. 2010). The X-ray crystallographic analysis of HSA reveals its primary structure
containing three homologous domains I, II, and III. Further, each domain is constituted by two
subdomains A and B, connected by random coils. In a crystal, all six subdomains assemble to
4

form a heart-shaped molecule (Carter & Ho 1994). Terminal regions of sequential domains
contribute to the formation of inter-domain helices linking domain IB to IIA, and IIB to IIIA,
respectively (Curry 2002). Most of the aromatic and heterocyclic ligands are bound within two
hydrophobic pockets in subdomains IIA and IIIA, which are referred as sites I and II (Sudlow,
Birkett & Wade 1975). In our previous work, we have reported that beta-sitosterol, piperine
binds in IIA and L-3,4-dihydroxyphenylalanide binds in IIIA domain of HSA (Sudhamalla,
Gokara, Ahalawat, Amooru & Subramanyam 2010; Yeggoni & Subramanyam 2014; Yeggoni,
Rachamallu, Kallubai & Subramanyam 2015). It can also be able to bind seven equivalents of
long-chain fatty acids (FA1-7) in sub-domains IB, IIIA, IIIB, and on the sub-domain interfaces
(Fasano et al. 2005). The principle binding region in HSA is located at IIA region of domain II,
which play vital role to transport and deliver various drugs to multiple organs of body. Recent
reports showed that various compounds like inhibitor STAT3 for abnormally expressed proteins
in cancer cells, an organophosphorous herbicide diazinon, anticancer drug [Pt(5,5′-
dmbpy)Cl4 (5,5′-dmbpy is 5,5′-dimethyl-2,2′-bipyridine) complex, and an alkaloid 5-hydroxy-1-
methylpiperidin-2-one were interacted in the IIA region of HSA with good binding affinity
(Affandi et al. 2016; Jafari et al. 2017; Shahraki, Somaye, Saeidifar, Shiri & Heidari 2017;
Yadav, Yeggoni, Devadasu & Subramanyam 2017). The structure of bufadienolides showed
strong variations in their binding with HSA, especially 11-OH and 16-OAC groups improved
serum albumin binding contributing to their development as anti-tumor agents (Zhou et al.
2015). Even we reported synthesized nefopam analogues and Androstenedione derivatives
binding in the domain IB, IIA and domain IIIA regions (Gokara et al. 2017; Nerusu, Reddy,
Ramachary & Subramanyam 2017).
Thus, our current study deals with in vitro approach of structure-affinity relationship of
5

synthesized 5β-dihydrocortisol (Dhc) and 5β-dihydrocortisol acetate (DhcA) with HSA, as it is
an important determinant to understand the binding mechanism of these compounds. However,
the mode of interaction of these compounds with HSA and its role in disease control was not
previously reported. Thus our investigation on Dhc and DhcA binding with HSA through cell-
based assay, multiple spectroscopic and molecular docking and dynamics simulation studies
emphasized the biological importance, nature of the binding sites, the strength and variations in
these interactions, as well as the possible conformational changes.
Materials and methods
Synthesis of 5β-Dihydrocortisol and 5β-Dihydrocortisol acetate
General procedure for palladium/charcoal catalyzed hydrogenation of cortisol
The mixture of 5% Pd/C (5 mol%), Hantzsch ester (152 mg, 0.9 mmol) and the cortisol
(0.3 mmol) in EtOH (3.0 mL, 0.1 M) was heated to 80 °C for the 6 h. Crude mixture was then
passed through a pad of celite. Pure hydrogenated product 5β-dihydrocortisol was obtained by
column chromatography (silica gel, mixture of hexane/ethyl acetate) in 86% yield with 72% de.
General procedure for organocatalytic hydrogenation of cortisol acetate
(S)-(+)-1-(2-Pyrrolidinylmethyl)pyrrolidine (12 mg, 0.075 mmol) and D-camphor
sulphonic acid (17 mg, 0.075 mmol) in dry CH3CN (3.0 mL, 0.1 M) were stirred at rt for 5 min,
then added cortisol acetate (0.3 mmol) and stirring was continued at the same temperature for
another 5 min. To this, added Hantzsch ester (152 mg, 0.6 mmol) and refluxed for the 72 h. The
crude reaction mixture was purified with aqueous work-up followed by column chromatography
(silica gel, mixture of hexane/ethyl acetate) in 52% yield with 66% de (Ramachary, Sakthidevi &
Reddy 2013).
Preparation of stock solutions
6

Fatty acid-free with above 95% purity HSA was purchased from Sigma-Aldrich, USA.
The stock solution (1.5 mM) of HSA was prepared in 0.1 M phosphate buffer at pH 7.4.
Dilutions of the HSA stock solutions was made to a final concentration (0.001 mM) in phosphate
buffer of pH 7.4 were prepared immediately before use. The molecular weight of 5β-
dihydrocortisol and 5β- dihydrocortisol acetate are 364.47 and 406.51 Da, and the purity of
molecules are more than 97%, their stock solutions were prepared by dissolving them in small
amount of ethanol and then diluting them to (1.0 × 103 M-1) solution. Site-specific markers
phenylbutazone, lidocaine, and ibuprofen solutions were also prepared at concentrations 0.001
mM. All other chemicals used for the experiments in our study are of analytical grade purchased
from Sigma-Aldrich.
Schematic representation of 5β-dihydrocortisol and 5β-dihydrocortisol acetate
Cell viability assay (MTT assay)
The MCF-7 cells and HEK 293 were procured from the cell repository, National Centre
for Cell Science, NCCS complex, Pune. The cytotoxic activity of Dhc and DhcA on MCF-7 and
HEK 293 were determined by MTT (3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium
bromide) assay. Cells (1×105/well) were seeded in 96-well plates and treated with Dhc and DhcA
7

in increasing concentrations of 20, 40, 60, 80 and 100 µM for 48 h in a final volume of 100 µL.
At the end of treatment, 20 µL of MTT (5 mg MTT/ml in PBS) was added, and the cells were
incubated for 3-4 h, control was maintained without the drug. About 100 µL of DMSO
(solubilizing reagent) was added to each well and mixed well by micropipette. Presence of viable
cells was visualized by the development of purple color due to the formation of formazan
crystals. Relative cell response was evaluated by measuring the optical density at 570 nm on a
microplate reader (μ Quant Bio-tek Instruments, Inc.). Three independent experiments were
performed, measurements were performed, and the concentration required for a 50% inhibition
of viability (IC50) was determined through standard graph plotted by taking a concentration of
the drug in X-axis and percentage inhibition in Y-axis.
Apoptosis assay
The Annexin/PI assay is a widely used approach for studying apoptotic cells. Propidium
iodide in conjunction with Annexin V significantly determines the viable, apoptotic and necrotic
cells through differences in plasma membrane integrity and permeability. The MCF-7 cells (2
mL) at a density of 5 × 103 were grown in 6 well plate and allowed to attach for 24 h after which
cells were treated with half the IC50 values of Dhc and DhcA 14 µM, 12.5 µM for 24 h. Then
cells were washed twice with 1% phosphate buffer saline, resuspended in Annexin V binding
buffer. The cells were centrifuged for 10ꢀmin, and supernatants were discarded, again cells were
suspended in 100ꢀμL Annexin V binding buffer, incubated in the dark for 15 min. Propidium
Iodide (4ꢀμL) diluted in 1x Annexin V binding buffer (1ꢀ:ꢀ10) was added and allowed to incubate
for 15ꢀmin in the dark at room temperature. The experiment were done using the Annexin V-
FITC detection kit (Sigma, cat. no. APOAF) and the results were analyzed by flow cytometry.
Tunel assay
8

TUNEL assay uses (terminal deoxynucleotidyl transferase-TdT mediated dUTP nick end-
labeling) to label 3'-OH ends of DNA fragments that are generated during the process of
apoptosis. The DNA fragmentation was assessed by a commercial kit (In situ Apoptosis
Detection Kit #MK500 from TaKaRa), in accordance with the manufacturer's instructions. MCF-
7 cells (1×106 cells/ml) were exposed to half the IC50 values of Dhc and DhcA obtained from
cell viability assay, i.e. 14 and 12.5 µM, a control was maintained without test compounds for 24
h. The cells were fixed with 4% (v/v) methanol-free formaldehyde/PBS solution at 4○C for 25
min and rinsed again with PBS. Subsequently, the cells were permeabilised by immersion in
0.2% Triton X-100 in PBS (pH 7.4) for 5 min, rinsed in PBS and equilibrated with the
equilibration buffer for 10 min. Cells were labelled by incubating at 37○C with the TdT (terminal
deoxynucleotidyl transferase) incubation buffer for 60 min. The reaction was stopped by
immersing the slides in saline sodium citrate for 15 min. Finally, slides were washed with PBS
and treated with propidium iodide (10 μg/mL in PBS) for 15 min in dark conditions.
Observations were made by using confocal fluorescence scanning microscope (Carl-Zeiss,
Germany). The green fluorescent cells is considered as an apoptotic hallmark.
Fluorescence quenching
Fluorescence spectroscopy is a convenient and simple method to study the binding of
HSA by small ligands. The fluorescence emission spectra were carried out on a LS-55
spectrofluorimeter (PerkinElmer corporate, USA). The excitation and emission slit widths were
set at 5 nm, and the excitation wavelength was 285 nm while emission wavelengths were
recorded between 300-500 nm. The HSA concentration (0.001 mM) was kept constant
throughout the study, although the Dhc and DhcA varied from 0.001 to 0.01 mM. The incubation
time for each concentration of both Dhc and DhcA with HSA was 10 minutes. The experiment
9

was done for three times and each time identical spectra were observed.
The fluorescence intensity was measured through quenching, where a variety of
molecular interactions can occur. Such excited state reactions, molecular rearrangements, energy
transfer, ground-state complex formation and collisional quenching is described by the modified
Stern-Volmer equation (Liang, Tajmir-Riahi & Subirade 2008).
Molecular displacement
Displacement studies between HSA and Dhc and DhcA in the presence of site-specific
markers like lidocaine, phenylbutazone, and ibuprofen for the site I, II and III respectively
(Yeggoni et al. 2014), were executed using the fluorescence spectroscopy. The concentration of
HSA and the site-specific markers were kept constant (0.001 mM), while the concentration of
Dhc and DhcA increased from 0.001 to 0.01 mM. An excitation wavelength of 285 nm was
chosen and the fluorescence emission acquired from 300-500 nm. The fluorescence spectra were
recorded, and the binding constants of the HSA-site marker-ligand were evaluated.
Synchronous fluorescence spectroscopy
To exploit the induced structural changes in HSA with the addition of Dhc and DhcA,
synchronous fluorescence analysis of HSA has been performed. The fixed concentration of HSA
0.001 mM was titrated with increased concentrations of Dhc and DhcA (0.001-0.01 mM). The
spectra were recorded from 300 to 450 nm by setting the excitation and emission wavelength
interval (Δλ) at 15 and 60 nm.
Atomic force microscopy
Atomic force microscopy was carried out to observe the topological changes of free HSA and
HSA–Dhc and HSA–DhcA by NT-MDT Solver scanning probe microscopy. The cantilever
(0.3mm) have an Au high reflectivity coating, tip height 14-16μm, force constant (5, 5 N/m) and
10

the typical imaging resonance frequency was 140 kHz (Yeggoni, Rachamallu & Subramanyam
2016). Samples were prepared as follows: (1) free HSA with 50 휇L of 2 휇M HSA was added to a
glass slide and incubated for 20 min at room temperature before washing with water and (2)
Dhc–HSA and DhcA–HSA complexes with free HSA samples were prepared as described in
step 1: prior to adding 50 휇L of a 10 휇M Dhc and DhcA solutions, incubating for 20 min and
washed with water then dried under N2 for 5 min, and the samples were imaged from AFM in
noncontact mode (Zhang, Gu & Zhang 2014)
Transmission electron microscopy (TEM)
The size and external morphology of HSA and HSA–Dhc and HSA–DhcA complexes in
aqueous solution at pH 7.4 were determined by transmission electron microscopy (TEM) using
FE1 Tecnai G2S-Twin-200kv. The samples (HSA (0.001mM), HSA (10 µM) + Dhc (50 µM)
solution, HSA (0.001M) + DhcA (50 µM) solution) were prepared freshly by placing one
preparation drop (5–10 μL) on a carbon coated copper grids, and the sample was dried
completely, a piece of filter paper is used to absorb any excess liquid present on the grid. Finally
at room temperature, a drop of 2% uranyl acetate negative stain was added before drying and
further observed the structure of HSA and HSA–Dhc and HSA–DhcA complexes under TEM
(Yeggoni, Rachamallu & Subramanyam 2016).
Molecular docking
Docking experiment was performed using Discovery Studio Modeling 2.1 (Accelrys Inc.)
and Autodock 4.2.1. The small molecules Dhc and DhcA (ligands) were constructed using
Discovery Studio Modeling 2.1, hydrogens are added, and the ligands were energy minimized
before docking runs. The crystal structure of HSA (receptor) PDB ID: 1AO6 was extracted from
Protein Data Bank (http://www.rcsb.org/pdb) deleting water (HET atoms) molecules (Sugio,
11

Kashima, Mochizuki, Noda & Kobayashi 1999). Docking calculations were performed using the
standard procedure for rigid protein and a flexible ligand using Autodock 4.2.1 (Morris et al.
2009). The Lamarckian genetic algorithm (LGA) was applied in the docking calculations. All of
the torsional bonds of the ligands were free to rotate while HSA was held rigid. Then, the polar
hydrogen atoms were added for HSA using the AutoDock tools, and Kollman united partial atom
charges (Morris, Goodsell, Huey & Olson 1996), were assigned. A total of 50 independent runs
were carried out with a maximum of energy evaluations to 25,000,000 and a population size to
150. A grid box (126×126×126) with a spacing of 0.534 Å was created. Energy grid maps for all
possible ligand atom types were generated using Autogrid 4.2.1 before performing the docking.
During the docking process, a maximum of 50 conformers was generated in 50 docking runs,
and the conformation with the lowest energy was used for further analysis.
Molecular dynamic simulations
The lowest docked conformer of HSA-ligand complexes with binding energy near to the
experimental value was selected for MD simulations using GROMACS 4.5.5, with a Gromacs96
54a7 force field (Sudhamalla et al. 2010; Pronk et al. 2013). The topologies of HSA were
generated using the Gromacs package directly, whereas ligands (Dhc and DhcA) using the
PRODRG2.5 Server (Schuttelkopf & van Aalten 2004), these topologies for HSA-drug were
standardized in our previous work (Sudhamalla et al. 2010; Yeggoni et al. 2014; Kallubai,
Rachamallu, Yeggoni & Subramanyam 2015). A box was defined which was filled with the
simple point charges, Na+ ions (15 in number) were added to make the entire system neutral.
Energy minimisation was performed using 100 steps of steepest descent to avoid any kind of bad
contacts generated while solvating the system. Subsequently, the temperature was increased
gradually from 0 K to 300 K over a 50 ps time span in the constant-NVT ensemble with a time
12

step of 0.5 fs. This thermalized configuration was utilized to perform long simulation runs in the
constant-NPT ensemble. A Nos´e Hoover thermostat with a coupling constant of 0.1 ps was
employed to maintain the temperature of the system at 300 K, while a Parrinello–Rahman
barostat with a coupling constant of 2 ps was utilized to maintain the system at 1 bar pressure.
The LINCS algorithm was utilized to keep all the covalent bonds involving hydrogen atoms (C–
H, N–H, and O–H) rigid. A time step of 1 fs was used to integrate the equations of motion for all
the production runs. However, simulations to obtain the free energy profiles were carried out
with a time step of 2 fs constraining all covalent bond lengths. Partial mesh Ewald algorithm was
employed for measuring electrostatic, and van der Waals interactions; the short-range neighbor
list cut-off, short-range electrostatic cut-off, and short-range van der Waals cut-off were fixed at
1 nm. The trajectories were generated up to 10-ns. All the simulations were performed on the
Linux cluster with 36 nodes (dual Xeon processor) at the Bioinformatics facility of the
University of Hyderabad.
Results and discussion
Cytotoxic studies of Dhc and DhcA
Steroidal and nonsteroidal endocrine agents are important in the therapy and control of
advanced breast and prostate cancers. For breast cancer, in particular, the presence of specific
estrogen binding in fresh biopsy specimens determines which tumors respond to endocrine
therapy. Previous results explained that β-sitosterol and dihydrocortisol showed anti-cancer
activity in breast cancer cell line (MCF-7) (Zava, Chamness, Horwitz & McGuire 1977; Pradhan
et al. 2017). Thus, we selected human breast carcinoma cells (MCF-7) and normal human
embryonic kidney cell line (HEK 293) to evaluate the cytotoxicity of synthesized compounds of
5β-dihydrocortisol and 5β-dihydrocortisol acetate using MTT assay. Inhibition of cell viability
13

was very less in HEK 293 cells, hence both synthesized Dhc and DhcA did not showed any
effect on cell cytotoxicity in a concentration-dependent manner (Figure 1(B)). Whereas, MCF-7
cells induced cytotoxicity and the IC50 values for Dhc and DhcA were 28 µM and 25 µM
respectively (Figure 1(A)), which are significant value. In another report, acetoxycoumarin drugs
showed strong anti-proliferative activity and induced apoptosis in various cancer cell lines such
as A549 (lung), ACHN (renal), H727 (lung), MCF-7 (breast) and HL-60 (leukemia). 4-(7-
(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate had the highest cytotoxic
activity of 48.1 µM (Musa et al. 2011). Thus, our results explain that due to the addition of
acetate group in 5β-dihydrocortisol acetate could be more toxic to cancer cells. Thus, Dhc and
DhcA are could be the potential therapeutic agents to cure breast cancer.
Apoptosis assay
Apoptosis plays an important role in controlling the cell number and proliferation. Now a
days the development of novel drugs able to restore the normal apoptotic pathway represents a
new strategy in cancer treatment (Cordeu et al. 2007). In the present work we investigated using
half the concentrations of IC50 values of Dhc 14 µM and DhcA 12.5 µM obtained from cell
viability assay to induce apoptosis in MCF-7 cells. The apoptotic effect was evaluated using
Annexin-V-FITC/PI dual staining assay, which can examine phosphatidylserine translocation to
the cell surface precedes nuclear breakdown, DNA fragmentation, and the appearance of most
apoptosis-associated molecules making Annexin V binding a marker of early stage apoptosis.
The data obtained in this study are presented in (Figure 2(A)), as a percentage of early apoptotic
cells, late apoptotic cells, and necrotic cells in quadrants. The results showed that 12.5 µM
concentration of DhcA induced 37.7%, 2.9% of early and late apoptosis, whereas dhc showed
35.6% and 2.5%. Our results suggest that synthesized molecules Dhc and DhcA are potent
14

antitumor agents and cause cell apoptosis, 5β-dihydrocortisol acetate induced more percentage of
apoptotic cells. Even previous reports showed that other molecules like 1'-Acetoxychavicol
acetate induced apoptosis in myeloma cells via different pathways (Ito et al. 2005). Hence, our
results suggest that Dhc and DhcA are a strong inhibitor of MCF--7 cells and hence these
molecules can be used for further in vivo studies for treating breast cancer.
Tunel assay
The pro-apoptotic efficacy of Dhc and DhcA were further verified on MCF-7 cancer cell
line by Tunel assay. Fragment DNA has a free 3′-OH group which is a substrate for binding of
fluorescein-dUTP provide a better evaluation of DNA damage in cell. As expected, the TUNEL-
positive cells were increased after treatment with 14 and 12.5 µM of Dhc and DhcA after 48 h
(Figure 2(B) and Figure 2(D)). The significant increase in the green fluorescent cells were
observed in the treated sample compared to untreated sample, indicates the apoptotic cells with
fragmented DNA and the percentage of TUNEL-positive cells correlated closely with the
annexin V-positive cells in Figure 2(B) and 2(D). Thus, the data from both annexin V-PI double
staining and TUNEL assay consistently showed that Dhc and DhcA predominantly induced
apoptosis in cultured MCF-7 cells.
Analysis of fluorescence emission studies
The intrinsic fluorescence of proteins is derived mainly from tryptophan and tyrosine
residues, while the quantum yield of the phenylalanine residue is very low. Due to a variety of
molecular interactions, there is a decrease in the fluorescence intensity from the fluorophore
which is referred to as fluorescence quenching. The molecular interactions include excited-state
reactions, molecular rearrangements, energy transfer, and ground-state complex formation. To
understand the binding mechanism, the HSA excitation was fixed at 285 nm and titrated with
15

different concentrations of both Dhc and DhcA. Fluorescence emission spectra exhibit the
fluorescence intensity of HSA which quenched by the addition of Dhc and DhcA in a
concentration-dependent manner (0.001 mM-0.010 mM) (Figure 3). The fluorescence intensity is
decreased due to the presence of Trp214 in HSA, which is sensitive to the ligand binding.
Moreover, the Dhc and DhcA did not interfere with the determination of intrinsic fluorescence as
no fluorescence emission observed in the range of 300-500 nm. Therefore, Dhc and DhcA could
quench the intrinsic fluorescence of HSA with a maximum emission peak at 360 nm, and there
was no shift in fluorescence peak indicates that there could be conjugation or complex formation
between ligand and HSA.
Determination of binding constant and binding sites
To understand the fluorescence quenching mode of HSA–Dhc and HSA–DhcA
complexes, the quenching data were analyzed using the following equation:
F0 / F = 1 + Kqt0[Q] = 1 + KD [Q] - (1)
Where F and Fo are the fluorescence intensities in the presence and absence of quencher, [Q] is
the quencher concentration, and KD is the Stern-Volmer quenching constant (Kq), which can be
written as KD = kqt0; where kq is the biomolecular quenching rate constant and t0 is the lifetime
of the fluorophore in the absence of quencher is 5.6 ns (Liang et al. 2008). From the above
equation, the quenching constant was calculated to be 5.0 × 1013 M−1 s−1 for Dhc and 5.4 × 1013
M−1 s−1 for DhcA. Hence, the quenching mechanism is static, as these values are much greater
than the maximum collisional quenching constant 2.0 × 1010 M−1 s−1 (Agudelo et al. 2012). Thus,
Dhc and DhcA binding to HSA is a static process. The binding constant (K) and the number of
bound complexes to HSA (n) were determined by plotting the fluorescence data using modified
Stern-Volmer equation
16

log[(F0 – F)/F] = logKs + nlog[Q]
- (2)
Where n is the number of ligands binding to the protein, Ks is the binding constant, and [Q] is the
drug concentration. The dF is (F0 – F), where F0 is the initial fluorescence of free HSA and F is
fluorescence with different concentrations of Dhc and DhcA. We have plotted a graph log (dF/F)
against log[Q]. A graph was plotted with the slope and the intercept that can be correlated with
the binding constant and the number of binding sites, respectively. The number of binding sites
for Dhc and DhcA is 0.85 and 0.96 indicates that binding of these compounds to HSA would be
one to one interaction (S. Figure 1). Also, the binding constant (Figure 3(A) and 3(B)) found to
be KDhc = 4.7±0.03×104 M-1 and KDhcA = 3.9±0.05 x 104 M-1 indicates the strong binding of Dhc
and DhcA with HSA. The binding constants are ranging from 103 to 105 which is regarded as an
appropriate scope for association and dissociation of the drug-protein system (Schiel & Hage
2009).
Free energy changes upon binding of Dhc and DhcA to the protein
Usually, there are four types of non-covalent forces such as hydrogen bonding, van der
Waals, hydrophobic and electrostatic interactions which stabilize their binding. In order to
understand the binding interactions, the free energy change (∆G°) can be calculated from the
following equation:
∆G° = -RTlnK
-(3)
Where, ∆G° is the free energy change, R is the gas constant at room temperature, and K is the
binding constant. From the above equation, the free energy change for Dhc and DhcA HSA been
calculated and found to be -6.8±0.012 and -5.9±0.035 Kcal/mol at 25°C. Similar results were
found in molecular docking of Dhc and DhcA -6.44 and -6.16 Kcal/mol with HSA. Therefore,
17

these data suggests that the binding between protein and ligand can be stabilized by hydrophobic
interaction and hydrogen bond formation. Our previous results published recently were also
found similar kind of free energies with, 7‑hydroxycoumarin, lupeol and lupeol derivative and
coumarin derivatives (Garg et al. 2013; Yeggoni et al. 2014; Kallubai et al. 2015).
Molecular displacement studies
The displacement experiments were conducted with several site-specific drugs such as
lidocaine, ibuprofen and phenylbutazone reported to be bind with HSA and are specific in their
locations at domain IB, (Hein et al. 2010) site I, (Ghuman et al. 2005) and site II (Yamasaki et al.
2000). The fluorescence intensity was measured after addition of 0.01 mM of site-specific
marker to 0.001 mM HSA, later the quenching was measured at the different concentrations of
Dhc and DhcA, respectively. The experiment was repeated for all the three site-specific markers,
and the binding constants were calculated. Figure 3(C) and 3(D) shows decrease in the value of
binding constant for lidocaine Kld+Dhc = 1.4±0.02×104 in the presence of Dhc compared to HSA–
Dhc (KDhc = 4.7±0.03×104) alone, whereas, phenylbutazone showed decreased binding constant
value of Kpb+DhcA = 1.5±0.03×104 against HSA–DhcA (KDhc = 3.9±0.05×104). However, there are
no observed changes in their binding constants with other site-specific markers of HSA.
Therefore, the binding site of Dhc is domain I and for DhcA is domain II. Even molecular
docking studies showed Dhc binding in the IB region and DhcA in the domain IIA region. These
two molecules bind to different binding pocket because these molecules have different functional
groups like –OH in Dhc and acetate in DhcA.
Change in secondary structure observation through synchronous spectroscopy,
FT-IR and circular dichroism
Synchronous spectroscopy
18

The synchronous fluorescence spectroscopy (SFS) technique was used to study the
conformational change of protein. It involves simultaneous scanning of the excitation and
emission monochromators of a fluorimeter while maintaining a constant wavelength interval
(Δλ) between them. The shift in the position of maximum emission reflects the changes in
polarity around the chromophore molecule (Poulli, Chantzos, Mousdis & Georgiou 2009). When
Δλ is fixed at 60 nm and 15 nm, the synchronous fluorescence of HSA is characteristic of
tryptophan and tyrosine residues. The concentration of HSA was kept constant 0.001 mM and
was progressively titrated with Dhc and DhcA by gradually increasing their concentrations as
stated in the fluorescence spectroscopy experiment. This data shows small shifts in the spectra at
15 nm, which express a slight change in conformation of HSA upon binding with Dhc (S. Figure
2(A) and 2(C)). In contrast at 60 nm, the influence of tryptophan residues was not observed.
While (S. Figure 2(B)) suggests slight change at 15 nm upon binding of DhcA with HSA
indicating a change in microenvironment in the vicinity of tyrosine residues. At 60 nm the
maximum emission peak shifted towards higher wavelength, which indicates the stretch of HSA
molecule after binding DhcA, thus the polarity around the tryptophan residues decreased,
increasing the hydrophobicity (S. Figure 2(D)). Similar results were shown that the
microenvironment around the trptophan residues of HSA, became more hydrophobic, after
addition of norfloxacin at Δλ=60 nm (Azimi, Emami, Salari & Chamani 2011). The results
reveal that the polarity around the tryptophan residues after binding Dhc had no remarkable
change, while the fluorescent aromatic residues buried in nonpolar hydrophobic cavities are
moved to a more hydrophilic environment in the binding process of DhcA, similar results were
showed with Nordihydroguaiaretic acid binding with Human serum albumin and Nintedanib
binding with Bovine serum albumin (Nusrat et al. 2016; Abdelhameed et al. 2017).
19

Fourier transform infrared spectroscopy (FT-IR)
IR spectroscopy is used as a powerful tool for investigating the secondary structure of
proteins. Infrared spectra of a protein exhibit a number of so-called amide bands, which are
sensitive to the secondary structure of proteins. The amides I and II peak occur in the region of
1600–1700 cm−1 (mainly C=O stretch) and 1500–1600 cm−1 (C-N stretch coupled with N-H
bending mode) have been widely used, and both are related to the secondary structure of the
protein (Rahmelow & Hubner 1996). The FT–IR spectrum of free HSA and the difference in the
spectra after binding with Dhc and DhcA were shown in (S. Figure 3). The spectral changes
showing the amide I band shifted from 1649±0.5 to 1634±0.6 cm−1 and 1648±0.2, while the
amide II peak was shifted from 1544±0.7 to 1529±0.9 cm−1 and 1514±0.5 binding with Dhc and
DhcA to HSA (S. Figure 3(A) and 3(B)). These results indicated that Dhc interacted both with
the C=O and C-N groups in the protein polypeptides. The earlier report shows that both
hydrophilic and hydrophobic interactions occur for Chol and DOPE-HSA adducts (Charbonneau,
Beauregard & Tajmir-Riahi 2009). The spectra indicates that when DhcA enters the hydrophobic
binding cavities (subdomain IIA or IIIA) via hydrophobic interactions, the oxygen atom and
hydroxyl group of DhcA can form hydrogen bonding with the C=O and C–N groups of the main
polypeptide in HSA, resulting in rearrangement of the polypeptide carbonyl hydrogen-bonding
network and reduction in the α-helical structure of the protein (Pu, Jiang, Chen & Wang 2014).
Thus HSA–Dhc and HSA–DhcA complex formation showed considerable changes in the rigidity
of protein molecule, which affects secondary structure of HSA and this result supports with the
molecular dynamics simulations data.
Protein conformational changes observed from Circular dichroism
Further to get more information on the structural changes of HSA upon addition of Dhc
20

and DhcA, the CD spectroscopy is one of the best methods for examining the conformational
changes of protein. The CD spectra of HSA exhibit two negative bands in the far-ultraviolet
region at 208 and 220 nm, these peaks attributed to n→π* transition for the peptide bond of α-
helix (Greenfield 1996).
The CD spectra of free HSA (0.001 mM) and in the presence of Dhc and DhcA are given
in (Figure 4(A) and 4(B)). The decrease in intensity of HSA at 208 and 220 nm as the
concentration increases of Dhc and DhcA. This indicated the considerable changes in the protein
secondary structure upon binding of Dhc and DhcA. Specifically, the loss of α-helical stability,
which might be the result of the formation of a complex between the protein and Dhc, DhcA.
Here, CDDN web-based software was used to calculate the secondary structural elements. In free
HSA, the secondary structure consisted of 58.4±0.5% of α-helices, 22.8±1.2% of β-sheets and
18.8±1.5% random coil, respectively. However, with an increase of 0.001 and 0.002 mM of
Dhc, there was a significant change in α-helices i.e. 58±0.3% and 56.3±0.5%, and the percentage
change in secondary structural elements is given in Table 1. The change in protein conformation
could be a partial unfolding of the protein. After addition of 0.001 and 0.002 mM of DhcA, the
α-helical stability was decreased to 56.2±0.8% and 53±0.3% respectively, and this data is in
agreement with synchronous fluorescence. We also checked for the thermal stability of Dhc and
DhcA bound HSA, and the stability of protein was up to 65° C (data not shown), and the results
were similar to the HSA-lupeol complex (Kallubai et al. 2015), indicates that HSA-Dhc/DhcA
complexes are stable. Therefore, Dhc and DhcA were bound to the amino acid residues of HSA
by non-covalent interactions, which destroyed the hydrogen bonding network, causing the partial
unfolding of the polypeptide of HSA in turn change in secondary structure of HSA (Kang et al.
2004). Hence, the HSA–Dhc/DhcA complexes are stable.
21

Atomic force microscopy
AFM experiment was performed to understand the topographical changes of free HSA
upon addition of Dhc and DhcA. Figure 5, displays the results for the free HSA and its HSA–
Dhc, HSA–DhcA complexes. Figure 5(A) and 5(D) shows the even distribution of free HSA on
the glass slide with the mean height of the individual HSA of 16.00±3.0 nm. After addition of 10
µM of Dhc, the HSA molecule became swollen reaching the mean height of molecule to
20.0±5.0 nm (Figure 5(B) and 5(E)) depicts the formation of HSA–Dhc complex. The same
concentration of DhcA added to HSA showed the mean height of 30.0±2.0 nm (Figure 5(C) and
5(F)) and this could be explained that upon binding DhcA perturbs more structural conformation
than Dhc. The different shapes and size distributions point towards distinctly different forms of
the morphology of free HSA, HSA–Dhc and HSA–DhcA complex and also differential
conformation. Thus, the shape of HSA–Dhc complex is different and varied from HSA–DhcA
complex, even the mean height also conclude that HSA–DhcA complex is more than HSA–Dhc
complex. These results explain that after addition of DhcA, the HSA molecule reduced its
surface area of contact with water by molecular aggregation, which further reveal the
microenvironment surrounding the HSA became more hydrophobic after interacting with DhcA.
Similar results were obtained for HSA-corilagin, HSA–S4TdR and HSA–patulin complexes
(Yuqin et al. 2014; Zhang et al. 2014; Yeggoni et al. 2016). The increase in size of the protein is
an indication of formation complexes of HSA–Dhc/DhcA.
Transmission electron microscopy
Further, we also confirmed the binding of th molecule with HSA using an electron
microscope and can provide information about the size and morphological changes to understand
the dynamic properties of molecules. Hence, the electron microscopy is one of the best tools to
22

see the protein topology upon binding of Dhc and DhcA with HSA. Figure 6(A) shows the
evenly distributed spherical shape particles of free HSA with mean particle diameter of 48.3±2.5
nm. Figure 6(B) and 6(C) shows the HSA–Dhc complex with a diameter of 51.2±3.6 nm, while
HSA–DhcA complex with 57.4±4 nm diameter. The results of TEM images explain that the
morphology or shape of free HSA, HSA–Dhc and HSA–DhcA are different. HSA–DhcA
complex showed large variation in diameter compared to HSA–Dhc complex; this may be
probably due to conformational changes within the globular structure of HSA after binding with
DhcA. The morphological difference of HSA upon addition of Dhc are attributed to slight
structural changes. While more structural changes would have happened upon DhcA
complexation. Formation of large aggregates is chracterized by a decrease of helical structure
and slight increase in the β-sheet. Hydrogen bonding from those polar groups probably takes
place in the aqueous environment. The TEM results are very well supported by the AFM data
where we can see a broad aggregate for HSA–DhcA complex. The AFM and TEM data (more
surface changes on protein) are in agreement with cell line data that DhcA is showing better
cytotoxicity activity where it act again breast cancer cell lines.
Molecular docking studies
Molecular docking gives the detailed picture of the binding site of drugs, its position, and
orientations of the protein. This information is crucial as it explains the relationship between
molecular properties of complexes. In our previous studies, we analyzed docking studies on
different PDB structures of HSA with PDB ID: 1AO6, 2BXC, 1BM0 and obtained similar
docking energies (Kallubai et al. 2015). Assuming that docking on different crystal structures of
HSA gives no difference in binding sites as we selected PDB ID: 1AO6 as a template structure to
identify the possible binding sites of Dhc and DhcA. Out of 50 docked conformations, the
23

highest affinity site regarding docked energy for Dhc and DhcA is domain IB and domain IIA.
The docked energies are -6.44 k.cal/mol and -6.18 K.cal/mol, which correlates with the free
energies obtained from experimental data of fluorescence emission. Molecular displacement
studies revealing that the Dhc replaced the lidocaine binding site domain IB and DhcA with
phenylbutazone of domain IIA (Figure 3(C) and 3(D)). The binding site of Dhc obtained from
docking results showed four hydrogen bonds, three OH- groups interacting with Pro110, Leu112
and Leu115, while carbonyl oxygen with His146 with bond distances 2.116, 2.206, 1.869 and
2.145 Å, all these residues lie in domain IB (Figure 7(C)). Another reports supports our results
that tamoxifen an antitumor drug for the treatment of breast cancer and its metabolite 4-
hydroxytamoxifen also showed stronger interaction in the vicinity of domain I region of HSA via
hydrophobic and hydrogen-bonds formation (Bourassa, Thomas & Tajmir Riahi 2016). DhcA
was located between subdomain IIA and IIIA of HSA (Figure 7(D) and 7(E)), during the
interaction with the residues two hydrogen bonds were established between C=O of acetate
group with the residues Trp214, Arg218 with bond distance 1.973 and 1.995 Å, while one –OH
group showed hydrogen bond with polar amino acid residue Cys448 present in domain IIIA with
bond distance 1.82 Å (Figure 7(F)). The same type of results was observed with HSA–phloretin
complex, which showed binding site in between domain IIA and IIIA (Barreca et al. 2016).
There is no surprise that Dhc and DhcA binding to two different domains and previously similar
type of hormone, ie., thyroid hormone thyroxine also showed primary binding site as the site I
and thyroxine di iodophenol ring accommodated in site II (Petersen, Ha, Harohalli, Park &
Bhagavan 1997). This explains from our results that these two molecules are binding in different
binding pockets because DhcA exhibits more conformational change, increase of topology of
electron micrographs and increase size in AFM picture. Bufotalin and cinobufagin are the
24

compounds belongs to bufadienolides had 16-OAc and 21-O groups, 16-acetyl group of
bufotalin showed a stable H-bond interaction in the site I of HSA and also 16-OAc of
cinobufagin via H-bond interacted with polar residue of Arg222 in the site I of HSA (Zhou et al.
2015). The addition of different groups like acetylation at C21 position of aldosterone, 9α-
fluorocortisol and dihydrocortisol showed an increase of ΔG, negativity, indicating a weaker
hydrogen bonding to the receptor of the acetylated side chain (Genard & Palem-Vliers 1983). By
the above observations, we can conclude that the interaction between DhcA and HSA is not
exclusively hydrophobic in nature but also involves polar interactions. Thus the hydrophobic and
hydrogen bond interactions play an important role in stabilizing the HSA–Dhc/DhcA complexes.
The results obtained by synchronous fluorescence spectroscopy, and FT-IR also provide
experimental evidence for the interactions obtained from molecular docking studies.
Molecular dynamics simulation studies
Molecular dynamics simulations (MDS) is useful for filling in the details, where
experimental methods cannot, especially identification of cryptic or allosteric binding sites and
protein folding. A complete understanding of atomic energies and molecular mechanism of
binding is unattainable using experimental techniques as macromolecular motions after ligand
binding are microscopic events that take place in mere millionths of a second. To evaluate the
structural stability of the HSA–Dhc and HSA–DhcA complexes, the properties were examined
by means of the radius of gyration (Rg), root mean square deviation (RMSD) and root mean
square fluctuation (RMSF) of the protein. The radius of gyration (Rg) of a protein is a measure
of its compactness during the simulation. As in Figure 8(A), the HSA alone showed some
fluctuations at 2000 ps, later achieved equilibration around 4000 ps, in our previous report we
25

have showed free HSA became stable after 2000 ps (Sudhamalla et al. 2010), whereas HSA–Dhc
complex showed some disturbance in compactness of 2.55 to 2.62 nm at 4000ps but during the
simulations, especially after 8000 ps, there is an overlap. Whereas the HSA–DhcA complex
compactness is 2.54+0.3 nm lower (Figure 9(A)) comparative to HSA–Dhc complex 2.58+0.1
nm, we may assume that upon binding of Dhc and DhcA molecules the protein-ligand undergo
more conformational changes. The variations of RMSD values regarding HSA alone and HSA–
Dhc and HSA–DhcA complex were plotted from 0 to 10 ns and are shown in Figure 8(B) and
9(B). The analysis of RMSD indicated both systems (HSA and HSA–Dhc and HSA–DhcA
complex) reached stability and balance after 3 ns simulation time till the end of 10 ns MD
simulation time, indicating that the molecular systems were well behaved thereafter. The
calculated average RMSD values of HSA, HSA–Dhc and HSA–DhcA complex were 0.4± 0.5
nm and 0.35± 0.2 nm, 0.32+0.70 nm respectively, at which point they achieved an equilibrium
state. In addition, the RMSF of all amino acid residues of HSA and in HSA–Dhc, HSA–DhcA
complex were examined to reveal the flexibility of residues of HSA. Figure 8(C) shows a plot of
RMSF of HSA and HSA–Dhc complex against residue number. These results indicated that
residues 100-150 (Pro110, Leu112, and Leu115) located in the domain I seem to be more rigid
due to the HSA–Dhc complex formation. Whereas, in Figure 9(C) the HSA–DhcA complex is
showing rigidity of residues 200-230 (Trp214, Arg218, Cys448), which explains that the DhcA
is binding in domain IIA. Thus, the fluctuations of the residues all through 10 ns at the binding
sites clearly illustrated that Dhc bound to HSA in domain I, and DhcA bound in domain II. Thus
the Dhc and DhcA are stable in the domain IB and IIA, which corroborates with all the
spectroscopic results explained above. Hence, MDS results shows that HSA–Dhc and HSA–
DhcA are formed stable complexes. Further, these compounds could be used for further studies
26

to understand the potent anti-cancer activity particularly for breast cancer.
Conclusions
The synthesized steroid compounds of Dhc and DhcA interaction with HSA was studied here.
The results obtained from the fluorescence spectroscopy suggested that Dhc and DhcA can bind
to HSA through static quenching mechanism and the binding constant of the HSA–Dhc and
HSA–DhcA complexes was calculated to be 4.7±0.03×104 M-1 and 3.9±0.05×104 M-1,
respectively. Additionally, molecular displacement experiments proved that the probable binding
site for Dhc is domain I, while for DhcA is site I. Even synchronous fluorescence data showed
spectral changes at λ15 nm for Dhc, a clear indication of involving tyrosine residues around
binding site of Dhc, and spectral changes at λ60 nm for DhcA proves that tryptophan residue is
among the one in the binding cavity. From the FT-IR spectroscopy and CD showed results
emphasized the conformational changes occurred in the HSA. Further, AFM and TEM results
showed that Dhc and DhcA after binding to HSA noticed that there is an increase in the height of
surface topology and molecular size. Furthermore, the binding mode was also characterized by a
molecular docking and molecular dynamics simulations, which indicated that Dhc could bind in
the domain IB via hydrophobic interaction, while DhcA binds in the site I i.e domain IIA via
hydrophobic and hydrophilic interactions. All of the experimental and computational data reveal
that Dhc and DhcA, through binding to HSA, can be effectively transported and distributed in
the blood. Additionally, the cytotoxic studies of Dhc and DhcA reveal that the DhcA showed
better inhibitory activity against human breast cancer cells (MCF-7 cancer cell line) than Dhc
indicates these compounds are acting against breast cancer. In addition, it was found that Dhc
and DhcA induced apoptosis in MCF-7, particularly DhcA exhibited strong anticancer activity in
27

inhibiting the cells at lower concentration and also increasing the percentage of early and late
apoptotic cells. Hence, these results are pivotal in understanding of protein structure-function
relationships which in turn gives an important information in drug discovery.
Acknowledgements
We thank BIF, for Bioinformatics facilities and also acknowledge the AFM facility provided by
School of Chemistry, TEM and Flow cytometer facility provided under DST-Centre for
Nanotechnology, University of Hyderabad.
Funding
This work was supported by Science and Engineering Research Board (SB/EMEQ-064/2014
dated 14-07-2015 and DST-FIST), UGC-SAP and UPE-2 University of Hyderabad. MK
acknowledge UGC (Dr. D. S. Kothari Fellowship) for providing financial support.
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