Antiestrogenic and antiproliferative potency of secoisolariciresinol diglucoside derivatives on MCF-7 breast cancer cells

Article abstract of DOI:10.1080/14786419.2020.1826479

Secoisolariciresinol diglucoside (SDG) is isolated from Linum usitatissimum seeds. The antiproliferative effects of SDG (1) and its derivatives secoisolariciresinol (2) and secoisolariciresinol-4′, 4″-diacetate (3) have been evaluated on MCF-7 breast cancer cells and normal breast epithelial line MCF-10A. Lignan 1 has not shown cytotoxic effects on MCF-7 cells, while derivatives 2 and 3 have inhibited cell growth with IC₅₀ values of 25 and 11 μM, respectively. Estrogen receptor alpha is a key growth driver in MCF-7 cells. Compound 1 did not affect the activity of ERα, while derivatives 2 and 3 showed significant antiestrogenic effects. Compounds 2 and 3 caused apoptosis in the MCF-7 line, determined by the cleavage of PARP. SDG derivative 3 enhanced the effect of doxorubicin. SDG derivatives can be considered as promising agents that exhibit a combined antiestrogen and proapoptotic effect in hormone-dependent breast cancer cells.

Full text of DOI:10.1080/14786419.2020.1826479

Natural Product Research
Formerly Natural Product Letters
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Antiestrogenic and antiproliferative potency of
secoisolariciresinol diglucoside derivatives on
MCF-7 breast cancer cells
Alexander M. Scherbakov , Olga V. Stasevich , Diana I. Salnikova , Olga E.
Andreeva & Ekaterina I. Mikhaevich
To cite this article: Alexander M. Scherbakov , Olga V. Stasevich , Diana I. Salnikova , Olga
E. Andreeva & Ekaterina I. Mikhaevich (2020): Antiestrogenic and antiproliferative potency
of secoisolariciresinol diglucoside derivatives on MCF-7 breast cancer cells, Natural Product
Research, DOI: 10.1080/14786419.2020.1826479
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1826479
BRIEF REPORT
Antiestrogenic and antiproliferative potency of
secoisolariciresinol diglucoside derivatives on MCF-7
breast cancer cells
Alexander M. Scherbakova , Olga V. Stasevichb, Diana I. Salnikovaa,c, Olga E.
Andreevaa
and Ekaterina I. Mikhaevicha
aDepartment of Experimental Tumor Biology, Blokhin N.N. National Medical Research Center of
b
Oncology, Moscow, Russia; Department of Physical-Chemical Methods for Products Certification,
c
Belarusian State Technological University, Minsk, Belarus; Faculty of Medicine, Lomonosov Moscow
State University, Moscow, Russia
ABSTRACT
ARTICLE HISTORY
Received 21 April 2020
Accepted 4 September 2020
Secoisolariciresinol diglucoside (SDG) is isolated from Linum usita-
tissimum seeds. The antiproliferative effects of SDG (1) and its
derivatives secoisolariciresinol (2) and secoisolariciresinol-40, 400-
diacetate (3) have been evaluated on MCF-7 breast cancer cells
and normal breast epithelial line MCF-10A. Lignan 1 has not
shown cytotoxic effects on MCF-7 cells, while derivatives 2 and 3
have inhibited cell growth with IC50 values of 25 and 11 mM,
respectively. Estrogen receptor alpha is a key growth driver in
MCF-7 cells. Compound 1 did not affect the activity of ERa, while
derivatives 2 and 3 showed significant antiestrogenic effects.
Compounds 2 and 3 caused apoptosis in the MCF-7 line, deter-
mined by the cleavage of PARP. SDG derivative 3 enhanced the
effect of doxorubicin. SDG derivatives can be considered as prom-
ising agents that exhibit a combined antiestrogen and proapop-
totic effect in hormone-dependent breast cancer cells.
KEYWORDS
Lignans; secoisolariciresinol
diglucoside; derivatives;
secoisolariciresinol; secoiso-
lariciresinol-40, 400-diacetate;
breast cancer cells;
apoptosis; cytotoxicity;
estrogen receptor
CONTACT Alexander M. Scherbakov
alex.scherbakov@gmail.com; a.sherbakov@ronc.ru
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1826479.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

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A. M. SCHERBAKOV ET AL.
1. Introduction
Lignans are natural biologically active compounds that consist of two phenylpropane
ꢀ ꢁꢀ
units joined by a b-b-bond (Gigliarelli et al. 2018; Zalesak et al. 2019). Lignans are an
available chemical class for drug development. The antitumor and antioxidant proper-
ties of lignans and their derivatives have been previously described (Gigliarelli et al.
ꢀ ꢁꢀ
2018; Zalesak et al. 2019; Teodor et al. 2020; Zhu et al. 2020a, 2020b). However, to
date, the exact molecular mechanisms of action of this chemical class have not been
fully studied.
The evaluation of antiproliferative effects of natural lignan SDG and its structural
analogues is essential for understanding the biochemical basis and mechanism of anti-
carcinogenic and antitumor effects of this class of lignans, also for study of structure-
activity relationships. Considering that the presence of glucose moiety makes the SDG
molecule more hydrophilic and this factor influenced greatly on the penetration of
this molecule through the lipophilic membrane of the cells, we reduced this part, as it
happens firstly during the metabolism of this lignan (Kitts et al. 1999). Further lipophi-
lization of the molecule of secoisolariciresinol could contribute to its faster penetration
through the cell membrane. Also, we expect the increase in antiestrogenic effect due
to this chemical modification, because the structures of secoisolariciresinol and secoi-
solariciresinol-40,400-diacetate are more similar to estrogens than the structure of SDG.
It was partly confirmed by SwissTargetPrediction tool for estimation of the most prob-
able macromolecular targets (Daina et al. 2019).
Lignan secoisolariciresinol diglucoside 1 was isolated from Linum usitatissimum seeds
by extraction with 50% aqueous ethanol, followed by chromatographic purification on
Diaion HP-20 ion-exchange sorbent and C18 reversed-phase silica gel (Stasevich et al.
2009). The synthesis of secoisolariciresinol (2) was performed by acid hydrolysis of 1, as
described previously by us (Stasevich et al. 2009). Secoisolariciresinol-40, 400-diacetate 3
was obtained in the reaction between 2 and acetyl chloride (Stasevich et al. 2009)
(Scheme S1).
Considering high activity of various natural compounds against the luminal type of
breast cancers (Salehi et al. 2020; Zhu et al. 2020a) and their potent antitumor effects
described by us in (Stasevich et al. 2010), we have analyzed the effect of the com-
pounds on MCF-7 breast cancer cells and identified signaling pathways involved in
the response to SDG derivatives 2 and 3.
2. Results and discussion
Cytotoxic effects on MCF-7 breast cancer cells and MCF-10A epithelial cells
MCF-7 cells were treated with SDG 1 and its derivatives 2 and 3 for 72hours. As shown
in Figure S1A, SDG did not exert a cytotoxic effect on MCF-7 cells, while its derivatives
caused inhibition of cell growth. The IC50 value of SDG derivative 2 on MCF-7 cells was
25.1 2.9 mM, whereas the IC50 value of compound 3 was 11.5 1.8 mM.
Evaluation of the potential toxicity of new compounds is important for in vitro stud-
ies. The MCF-10A cell line is an in vitro model of normal breast epithelium (Graminha

NATURAL PRODUCT RESEARCH
3
et al. 2020; Waleka et al. 2020). SDG and its derivatives did not exert a cytotoxic effect
on MCF-10A cells at concentrations lower than 50 mM. Cytotoxicity of 2 and 3 was
demonstrated only at elevated concentrations (Figure S1B) with the IC50 values of
88.6 6.9 mM and 93.5 8.5 mM, respectively. Thus, the treatment with compounds 2
and 3 unveiled their effective and selective action towards MCF-7 breast cancer cells.
Since SDG 1 did not show cytotoxic effects on tumor cells, further studies were per-
formed with compounds 2 and 3 demonstrating high activities.
The effect of glycosylation could be the reason for the absence of cytotoxic effect
of natural lignan SDG in comparison with secoisolariciresinol and secoisolariciresinol-
40,400-diacetate against MCF-10A cell line. The analogous results were presented by us
earlier against B-cell lymphoblastoid human cancerous cell line Raji (Stasevich et al.
2010) and in the research with flavonoid quercetin. It was shown that quercetin dem-
onstrated significant antiproliferative effect, while its glucoside was inactive in concen-
trations below 463,4 mM (Kwak et al. 2009; Razavi et al. 2009). Acetylation, therefore,
was demonstrated as a powerful tool to increase the cytotoxicity of secoisolariciresinol.
The same results were presented earlier with the acetylated form of quercetin
(Dell’Albani et al. 2017). This effect may be due to increased lipophilicity in the acety-
lated molecules.
The alternative explanation of the antiproliferative effects of secoisolariciresinol and
secoisolariciresinol-40,400-diacetate arises from their structural similarity with estrogen. It
was reported that estrogen significantly inhibits cell growth of the ERa-negative
human monoblastic leukemia cell line U937 via cell cycle arrest (Shim et al. 2003).
Antiestrogen activity of SDG derivatives and apoptosis
Estrogen receptor alpha (ERa) is expressed in about 70% of breast cancers. This makes
hormone therapy possible in most breast cancer patients. Considering that ERa is
expressed at a high level in MCF-7 cells and is a key proliferation driver, we have ana-
lyzed the effects of 1, 2, and 3 on the activity of this factor. For ERE-Luc assay, cells
are transfected with the plasmids containing the luciferase reporter gene controlled
by the promoter with estrogen-responsive elements (ERE-Luc). Cells are treated with a
physiological receptor ligand, 17b-estradiol, and novel compounds. Figure S2 shows
that compounds 2 and 3 inhibit the 17b-estradiol-induced luciferase activity. In the
assay, both SDG derivatives were less active than the antiestrogen fulvestrant. Thus,
tested SDG derivatives showed antiestrogenic potency. The explanation of effects for
secoisolariciresinol and secoisolariciresinol-40,400-diacetate arises from their structural
similarity with estrogens, this fact is partly confirmed by SwissTargetPrediction tool for
estimation of the most probable macromolecular targets (Daina et al. 2019).
The current classification of estrogen receptor regulators includes two classes: SERM
(Selective estrogen receptor modulators) and SERD (Selective estrogen receptor
degraders). The “gold standard” for hormone therapy, tamoxifen, belongs to the SERM
group. Tamoxifen can inhibit the activity of the estrogen receptor, but in some tissues,
it exhibits agonistic activity. Despite the undoubted success of this drug in clinical
practice over the past 40 years, its agonistic activity and other side effects are limiting
factors. The SERD group, which includes a fulvestrant, does not have agonistic activity

4
A. M. SCHERBAKOV ET AL.
and its action is focused on ERa degradation. By immunoblotting, we have evaluated
the effect of compounds 2 and 3 on the ERa expression level. It turned out that the
treatment with 2 and 3 leads to a significant reduction of ERa expression in MCF-7
cells. Considering the nature of the effect on the level of ERa, compounds 2 and 3
can be assigned to SERDs.
The effect of 2 and 3 on the apoptosis marker, PARP cleavage, was analyzed using
immunoblotting. Figure S3 shows that compounds 2 and 3 cause dose-dependent
PARP cleavage. Thus, apoptosis is the main type of cell death induced by compounds
2 and 3.
Combination with doxorubicin
One of the important problems in chemotherapy is the occurrence of toxicity when
using high doses of drugs. One of the possible approaches to overcome this problem
is the search for potential chemo-sensitizers. We used a combination of doxorubicin
and compound 3 in low doses to treat MCF-7 cells. Figure S4 shows a significant
increase in the effect of doxorubicin combined with compound 3 (CI < 1). Thus, the
development of strategies of combining 3 with chemotherapeutics is promising. New
intriguing data published by Arken (Arken 2019) indicate the great potential of lignans
to overcome the resistance of tumors to doxorubicin. In Arken’s work, the multidrug
resistance reverse effect of schizandrol A, a lignan from Schisandra chinensis, was dem-
onstrated with P-gp overexpressing drug-resistant K562/A02 cells. Similar trends are
identified in (Laiolo et al. 2018) for analogues of the lignan pinoresinol.
Apoptosis plays a pivotal role in the pathogenesis of cancers and is linked to many
cell signaling pathways. Lignan-mediated enhancement of apoptosis has been shown
in several studies; it can occur through many mechanisms. For example, the data
acquired by Li-Hua Chen et al. (Chen et al. 2007) show the inhibitory effect of entero-
lactone on LNCaP cell (human prostate carcinoma) growth. Treatment of LNCaP cells
with 25 to 100 mM of enterolactone for 24 h resulted in the disruption of mitochondrial
membrane potential and induction of mitochondrial-mediated cell death.
Enterolactone also caused a dose-dependent release of cytochrome c and an increase
in the cleavage of caspase-3 and PARP, events that are considered molecular hallmarks
of apoptosis. Enterolactone-induced apoptosis may also be mediated through the
inactivation of the Akt signaling pathway, as suggested by the reduced phosphoryl-
ation of Akt and its downstream targets (GSK3 and MDM2), and enhanced p53 expres-
sion. The similar trends were seen in the study (Buckner et al. 2019), where treatment
of B16-BL6 murine melanoma and MCF-7 breast cancer cells with flaxseed oil induced
apoptosis as determined by changes in cell morphology (increase in the sub-G1 popu-
lation in treated cells), annexin V staining (associated with phosphatidylserine
“flipping” to the outer leaflet of the plasma membrane), DNA fragmentation and/or
caspase activation (activation of caspase-9 is consistent with activation of the intrinsic
apoptotic pathway and results in activation of caspase-3; activation of caspase-8 is
associated with the extrinsic apoptosis pathway that results from a receptor-mediated
activation of an apoptotic signal in the plasma membrane).
As it was shown, lignans demonstrate weak binding properties to estrogen receptor
a (ERa) and ERb. The results of Stefan O. Mueller et al (Mueller et al. 2004) study show

NATURAL PRODUCT RESEARCH
5
that enterolactone and 6-OH-enterolactone were partial agonists/antagonists of ERa
or ERb.
Yunyun Di et al. (Di et al. 2018) have gathered evidence that flaxseed lignans, secoi-
solariciresinol and enterolactone, could enhance the anticancer activity of therapeutic
agents against breast cancer cell lines. Secoisolariciresinol selectively enhanced the
cytotoxicity of docetaxel in SKBR3 and MDA-MB-231 cells. Exposure with 50 mM enter-
olactone plus docetaxel gave the most stable combination effect and consistently
resulted in very high DRI values of up to 10-fold in SKBR3 and MDA-MB-231 cells.
Furthermore, enterolactone significantly increased the cytotoxicity of therapeutic con-
centrations of metformin in MDA-MB-231 cells in a concentration-dependent manner,
at 50 mM increased the sensitivity of MDA-MB-231 cells to docetaxel by 10-fold and
metformin by 2.3-fold. These data support the possibility of using flaxseed lignan sup-
plementation as an adjuvant therapy to decrease cytotoxic chemotherapeutic drug
dosage requirements while enhancing their effectiveness.
Lignans and other natural compounds can be used to overcome resistance to
chemotherapy (Li et al. 2018; Morsy et al. 2018). In the 2020 work (Morsy et al. 2020),
secoisolariciresinol alone showed dose-dependent cytotoxicity, where 25 or 50 mM of
secoisolariciresinol caused significantly less viability of doxorubicin-resistant cells com-
pared to control. When 50 mM secoisolariciresinol was added to doxorubicin, secoiso-
lariciresinol significantly enhanced doxorubicin-induced cytotoxicity compared to
doxorubicin alone. Morsy’s et al results suggest secoisolariciresinol as a novel P-gp
inhibitor that can sensitize cancer cells during doxorubicin therapy (Morsy et al. 2020).
There are various natural inhibitors of P-gp to overcome doxorubicin-resistance in the
resistant cell line (Morsy et al. 2018). In the 2018 work of the same research group,
both in silico and in vitro studies confirm that piperine is an inhibitor of P-gp (Morsy
et al. 2018).
In general, the effect of new derivatives of SDG on breast cancer cells and normal
epithelium was analyzed. The tested compounds induced apoptosis in the MCF-7 line,
determined by the PARP cleavage, and demonstrated selectivity for malignant cells.
Antiproliferative effects of compound 3 are associated with inhibition of ERa, the main
0
driver of growth of luminal breast cancer. Moreover, the secoisolariciresinol-4 , 4
00-diacetate (derivative 3) enhanced the effect of doxorubicin. New derivatives of SDG
can be considered for further development, including for new approaches to therapy
in combination with doxorubicin and other chemotherapeutic drugs. The combination
of compound 3 with doxorubicin may be of interest for preclinical studies to lower
the drug doses.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The study was financially supported by the Russian Foundation for Basic Research, project 18-
29-09017 (ERE-Luc assays and experiments with MCF-10A cells). Special thanks to Dr. Irina
Stasevich for the inspiring discussion at the 8th EACR-OECI Joint Course: Molecular Pathology

6
A. M. SCHERBAKOV ET AL.
Approach to Cancer held at De Rode Hoed near beautiful canals of Amsterdam. The densitom-
etry protocol for immunoblotting “Using ImageJ to quantify blots” was provided by The
University of Queensland (https://di.uq.edu.au/community-and-alumni/sparq-ed/sparq-ed-serv-
ices/using-imagej-quantify-blots). The authors thank Aglaya Shcherbakova for her kind help in
the design of graphical abstract. Graphical abstract was created using Servier Medical Art tem-
plates. Original templates are licensed under a Creative Commons Attribution 3.0 Unported
License (https://smart.servier.com/).
ORCID
Alexander M. Scherbakov
http://orcid.org/0000-0002-2974-9555
Olga E. Andreeva http://orcid.org/0000-0002-6015-6619
Ekaterina I. Mikhaevich
http://orcid.org/0000-0002-1299-9080
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