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An estrogen analogue and promising anticancer agent refrains from inducing morphological damage and reactive oxygen species generation in erythrocytes, fibrin and platelets: a pilot study
Cancer Cell International
An estrogen analogue and promising anticancer agent refrains from inducing morphological damage and reactive oxygen species generation in erythrocytes, fibrin and platelets: a pilot study
Lisa Repsold 0
Etheresia Pretorius 0
Annie Margaretha Joubert 0
0 Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria , Pretoria , South Africa
Background: 2-Methoxyestradiol is known to have antitumour and antiproliferative action in vitro and in vivo. However, when 2-methoxyestradiol is orally administered, it is rapidly oxidized by the enzyme 17β-hydroxysteriod dehydrogenase in the gastrointestinal tract. Therefore, 2-methoxyestradiol never reaches high enough concentrations in the tissue to be able to exert these antitumour properties. This resulted in the in silico-design of 2-methoxyestradiol analogues in collaboration with the Bioinformatics and Computational Biology Unit (UP) and subsequent synthesis by iThemba Pharmaceuticals (Pty) Ltd (Modderfontein, Midrand, South Africa). One such a novelty-designed analogue is 2-ethyl-3-O-sulphamoyl-estra-1, 3, 5(10)16-tetraene (ESE-16). Methods: This pilot study aimed to determine the morphological effect and possible generation of reactive oxygen species by ESE-16 on erythrocytes and platelet samples (with and without added thrombin) by means of scanning electron microscopy, transmission electron microscopy and flow cytometry. Results: Erythrocytes and platelets were exposed to ESE-16 at a concentration of 180nM for 24 hours. Scanning- and transmission electron microscopy indicated that ESE-16 did not cause changes to erythrocytes, platelets or fibrin networks. Flow cytometry measurements of hydrogen peroxide and superoxide indicated that ESE-16 does not cause an increase in the generation of reactive oxygen species in these blood samples. Conclusion: Further in vivo research is warranted to determine whether this novel in silico-designed analogue may impact on development of future chemotherapeutic agents and whether it could be considered as an antitumour agent.
2-Methoxyestradiol analogues; 2-Ethyl-3-O-sulphamoyl-estra-1; 3; 5(10)16-tetraene; Reactive oxygen species; Cancer
Introduction
Cancer is currently one of the leading causes of mortality
across the world’s population [
1
]. Research aimed at
finding possible compounds to treat or cure cancer is
therefore of extreme importance. 2-Methoxyestradiol (2ME)
(Figure 1) is a metabolite of 17β-estradiol which has been
tested for its antitumour and antiangiogenic properties
in vitro and in vivo [
2,3
].
2ME however, has poor bioavailability limiting the
action in vivo as indicated in earlier phase I studies where
2ME was orally administered to cancer patients in the
form of a capsule [
5
]. The rate at which 2ME becomes
available to the site of drug action is low since the rate
of oxidation of 2ME is higher than absorption [
5
]. 2ME
is rapidly oxidized in the gastrointestinal tract when
orally administered by the enzyme 17β-hydroxysteriod
dehydrogenase [
6,7
]. This leads to decreased levels of
2ME, thus being too low to exert significant antitumour
effects [8]. The development of a 2ME NanoCrystal®
colloidal dispersion (NCD) was subsequently formulated.
The latter increased bioavailability, however, the
antitumour effects were not optimal in patients [
9
]. This led
to the development and synthesis of analogues of 2ME
to test for a compound with increased antitumour and
antiangiogenic properties with increased bioavailability
[
10,11
]. Such analogues of 2ME have been in
silicodesigned in our laboratory (Figure 2). One novel in
silicodesigned analogue is 2-ethyl-3-O-sulphamoyl-estra-1, 3, 5
(10)16-tetraene (ESE-16) (Figure 2).
2ME exerts its anticancer signaling by disrupting
mitochondrial function, generating reactive oxygen species
(ROS) and by targeting microtubule dynamics in vitro
and in vivo [
13
]. 2ME results in apoptosis involving both
the extrinsic and intrinsic pathways [
14
]. 2ME activates
apoptosis via the extrinsic pathway by means of death
receptors (DRs) such as death receptor 5 (DR5) and
activates caspases known to promote the extrinsic pathway
[
14
]. The extrinsic pathway of apoptosis comprises of
the instigation of caspase 8, caspase 3, and upregulation
of DR5 with activation of caspase 9 present in the
intrinsic pathway through crosstalk with caspase 8 [
14,15
].
Production of ROS in 2ME- and analogue-treated cells
is likely to be a result of the inhibition of the
mitochondrial electron transport complex I [
13
] leading to
autophagy by means of beclin-1 upregulation and autophagy
protein 4 (Atg4) inactivation [
16
]. Generation of ROS
upregulates beclin-1 which results in the increased
incidence of autophagy, while hydrogen peroxide causes
autophagy and the inactivation of Atg4 responsible for the
induction of autophagy [
17
]. 2ME also induces apoptosis
in cancer cells as a result of the upreg (...truncated)