Novel estradiol analogue induces apoptosis and autophagy in esophageal carcinoma cells
Volume
0 McKnight Institute, University of Florida , Gainesville, Florida , USA
1 Department of Biochemistry and Molecular Biology, Baylor College of Medicine , Houston, Texas , USA
2 Department of Physiology, University of Pretoria , South Africa
Cancer is the second leading cause of death in South Africa. The critical role that microtubules play in cell division makes them an ideal target for the development of chemotherapeutic drugs that prevent the hyperproliferation of cancer cells. The new in silico-designed estradiol analogue 2-ethyl-3-Osulfamoyl-estra-1,3,5(10)16-tetraene (ESE-16) was investigated in terms of its in vitro antiproliferative effects on the esophageal carcinoma SNO cell line at a concentration of 0.18 µM and an exposure time of 24 h. Polarization-optical differential interference contrast and triple fluorescent staining (propidium iodide, Hoechst 33342 and acridine orange) revealed a decrease in cell density, metaphase arrest, and the occurrence of apoptotic bodies in the ESE-16-treated cells when compared to relevant controls. Treated cells also showed an increase in the presence of acidic vacuoles and lysosomes, suggesting the occurrence of autophagic processes. Cell death via autophagy was confirmed using the Cyto-
* Author for correspondence; Email; annie; joubert@up; ac; za; phone; +27 12 319 2246; fax; +27 12 321 1679
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Abbreviations used: 2ME – 2-methoxyestradiol; 2-MeOE2bisMATE –
2-methoxyestradiolbis-sulfamate; AAF – aggresome activity factor; AIF – apoptosis inducing factor;
AO – acridine orange; Apaf-1 – apoptosis protease-activating factor; C9 –
2-ethyl-3-Osulfamoyl-estra-1,3,5(10)-tetraen-3-ol-17-one; CAII – carbonic anhydrase II;
CAIX – carbonic anhydrase IX; DMEM – Dulbecco’s modified Eagle’s medium;
DMSO – dimethyl sulfoxide; EC – esophageal cancer; ER – estrogen receptor; ESE-16 –
2ethyl-3-O-sulfamoyl-estra-1,3,5(10)16-tetraene; FACS – fluorescence-activated cell sorting;
FB1 – fumonisin B1; HO – Hoechst 33342; MFI – mean fluorescent intensity;
MOMP – mitochondrial outer membrane permeabilization; PBS – phosphate buffer saline;
PE – phosphatidylethanolamine; PI – propidium iodide; PlasDIC – polarization-optical
transmitted light differential interference contrast microscopy; STS – steroid sulfatase
ID autophagy detection kit and the aggresome detection assay. Results showed
an increase in autophagic vacuole and aggresome formation in ESE-16 treated
cells, confirming the induction of cell death via autophagy. Cell cycle
progression demonstrated an increase in the sub-G1 fraction (indicative of the
presence of apoptosis). In addition, a reduction in mitochondrial membrane
potential was also observed, which suggests the involvement of apoptotic cell
death induced by ESE-16 via the intrinsic apoptotic pathway. In this study, it
was demonstrated that ESE-16 induces cell death via both autophagy and
apoptosis in esophageal carcinoma cells. This study paves the way for future
investigation into the role of ESE-16 in ex vivo and in vivo studies as a possible
anticancer agent.
Esophageal cancer (EC) is the eighth most common incident cancer in the world
and due to its high fatality rate, it ranks sixth among all cancers in terms of
mortality [1–3]. The highest rates of EC are found in Eastern Asia and Eastern
and Southern Africa [4]. One of the main reasons for the high incidence rates in
these areas is believed to be the presence of the mycotoxin fumonisin B1 (FB1).
It occurs in high concentrations in the above-mentioned geographic areas due to
a soil-borne fungus named Fusarium verticillioides, which frequently
contaminates maize and maize products [5, 6]. Studies revealed that FB1 may
act as a promoter or initiator of carcinogenesis in synergy with co-carcinogens
such as the N-nitrosamines found in tobacco [6].
One of the best-known characteristics of cancer cells is their rapid and
uncontrolled division. The critical role that microtubules play in cell division
makes them an ideal target for the development of chemotherapeutic drugs that
prevent the hyperproliferation of cancer cells [7]. Clinical success with several
vinca alkaloids and taxanes on a wide variety of human cancers has proven the
effectiveness of microtubule-targeting drugs and has prompted a worldwide
search for compounds with similar mechanisms [7].
2-methoxyoestradiol (2ME) is an endogenous metabolite derived from 17β-estradiol
(Fig. 1A) [7–12]. Research has shown that this compound has antiproliferative,
antiangiogenic and pro-apoptotic characteristics in vitro and in vivo [3, 7–9, 12–15].
2ME causes abnormal mitotic spindle formation and mitotic accumulation in
estrogen receptor-positive and -negative cells [9, 10, 13–16]. This causes the
activation of the spindle assembly checkpoint leading to metaphase arrest. As
a consequence, cell proliferation is inhibited and cell death is induced [9, 11, 17, 18].
2ME is registered as Panzem by Entremed Inc. and is currently undergoing
Phase I and II clinical (...truncated)