Novel estradiol analogue induces apoptosis and autophagy in esophageal carcinoma cells

Cellular & Molecular Biology Letters, Mar 2014

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-O-sulfamoylestra-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-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.

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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 - 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)


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Elize Wolmarans, Thandi V. Mqoco, Andre Stander, Sandra D. Nkandeu, Katherine Sippel, Robert McKenna, Annie Joubert. Novel estradiol analogue induces apoptosis and autophagy in esophageal carcinoma cells, Cellular & Molecular Biology Letters, 2014, pp. 98-115, Volume 19, Issue 1, DOI: 10.2478/s11658-014-0183-7