Skip to main content
PMC home page
Cellular & Molecular Biology Letters logoLink to Cellular & Molecular Biology Letters
. 2014 Feb 22;19(1):98–115. doi: 10.2478/s11658-014-0183-7

Novel estradiol analogue induces apoptosis and autophagy in esophageal carcinoma cells

Elize Wolmarans 1, Thandi V Mqoco 1, Andre Stander 1, Sandra D Nkandeu 1, Katherine Sippel 2, Robert McKenna 3, Annie Joubert 1,✉
PMCID: PMC6275608  PMID: 24563014

Abstract

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.

Keywords: Antiproliferative; 2-ethyl-3-O-sulfamoyl-estra-1,3,5(10)16-tetraene; ESE-16; Esophageal carcinoma; In vitro; Apoptosis; Autophagy

Full Text

The Full Text of this article is available as a PDF (2.2 MB).

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-Osulfamoylestra-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

2-ethyl-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

References

  • 1.Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J. Clin. Oncol. 2006;24:2137–2150. doi: 10.1200/JCO.2005.05.2308. [DOI] [PubMed] [Google Scholar]
  • 2.Kamangar F, Chow WH, Abnet CC, Dawsey SM. Environmental causes of esophageal cancer. Gastroenterol. Clin. North Am. 2009;38:27–57. doi: 10.1016/j.gtc.2009.01.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Du B, Zhao Z, Sun H, Ma S, Jin J, Zhang Z. Effects of 2-methoxyestradiol on proliferation, apoptosis and gene expression of cyclin B1 and c-Myc in esophageal carcinoma EC9706 cells. Cell. Biochem. Funct. 2012;30:158–165. doi: 10.1002/cbf.1830. [DOI] [PubMed] [Google Scholar]
  • 4.Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global Cancer Statistics. CA Cancer J. Clin. 2011;61:69–90. doi: 10.3322/caac.20107. [DOI] [PubMed] [Google Scholar]
  • 5.Chu FS, Li GY. Simultaneous occurrence of fumonisin B1 and other mycotoxins in moldy corn collected from the People’s Republic of China in regions with high incidences of esophageal cancer. Appl. Environ. Microbiol. 1994;60:847–852. doi: 10.1128/aem.60.3.847-852.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Myburg RB, Dutton MF, Chuturgoon AA. Cytotoxicity of fumonisin B1, diethylnitrosamine, and catechol on the SNO esophageal cancer cell line. Environ. Health Perspect. 2002;110:813–815. doi: 10.1289/ehp.02110813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Zhou J, Giannakakou P. Targeting microtubules for cancer chemotherapy. Curr. Med. Chem. Anticancer Agents. 2005;5:65–71. doi: 10.2174/1568011053352569. [DOI] [PubMed] [Google Scholar]
  • 8.Purohit A, Hejaz HA, Walden L, MacCarthy-Morrogh L, Packham G, Potter BV, Reed MJ. The effect of 2-methoxyoestrone-3-O-sulfamate on the growth of breast cancer cells and induced mammary tumors. Int. J. Cancer. 2000;85:584–589. [PubMed] [Google Scholar]
  • 9.Chua YS, Chua YL, Hagen T. Structure activity analysis of 2-methoxyestradiol analogues reveals targeting of microtubules as the major mechanism of antiproliferative and proapoptotic activity. Mol. Cancer Ther. 2010;9:224–235. doi: 10.1158/1535-7163.MCT-09-1003. [DOI] [PubMed] [Google Scholar]
  • 10.Stander A, Joubert F, Joubert A. Docking, synthesis, and in vitro evaluation of antimitotic estrone analogs. Chem. Biol. Drug Des. 2011;77:173–181. doi: 10.1111/j.1747-0285.2010.01064.x. [DOI] [PubMed] [Google Scholar]
  • 11.Choi HJ, Zhu BT. Critical role of cyclin B1/Cdc2 up-regulation in the induction of mitotic prometaphase arrest in human breast cancer cells treated with 2-methoxyestradiol. Biochim. Biophys. Acta. 2012;1823:1306–1315. doi: 10.1016/j.bbamcr.2012.05.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Visagie M, Mqoco T, Joubert A. Sulphamoylated estradiol analogue induces antiproliferative activity and apoptosis in breast cell lines. Cell. Mol. Biol. Lett. 2012;17:549–558. doi: 10.2478/s11658-012-0030-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Mooberry SL. Mechanism of action of 2-methoxyestradiol: new developments. Drug Resist. Updat. 2003;6:355–361. doi: 10.1016/j.drup.2003.10.001. [DOI] [PubMed] [Google Scholar]
  • 14.Zhu BT, Conney AH. Is 2-methoxyestradiol an endogenous estrogen metabolite that inhibits mammary carcinogenesis? Cancer Res. 1998;58:2269–2277. [PubMed] [Google Scholar]
  • 15.Mabjeesh NJ, Escuin D, LaVallee TM, Pribluda VS, Swartz GM, Johnson MS, Willard MT, Zhong H, Simons JW, Giannakakou P. 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. Cancer Cell. 2003;3:363–375. doi: 10.1016/s1535-6108(03)00077-1. [DOI] [PubMed] [Google Scholar]
  • 16.Thaver V, Lottering M, van Papendorp D, Joubert A. In vitro effects of 2-methoxyestradiol on cell numbers, morphology, cell cycle progression, and apoptosis induction in oesophagealcarcinoma cells. Cell Biochem. Funct. 2009;27:205–210. doi: 10.1002/cbf.1557. [DOI] [PubMed] [Google Scholar]
  • 17.Van Zijl C, Lottering ML, Steffens F, Joubert A. In vitro effects of 2-methoxyestradiol on MCF-12A and MCF-7 cell growth, morphology and mitotic spindle formation. Cell Biochem. Funct. 2008;26:632–642. doi: 10.1002/cbf.1489. [DOI] [PubMed] [Google Scholar]
  • 18.Voster CJJ, Joubert AM. In vitro effects of 2-methoxyestradiol-bissulfamate on the non-tumorigenic MCF-12A cell line. Cell Biochem. Funct. 2010;28:412–419. doi: 10.1002/cbf.1671. [DOI] [PubMed] [Google Scholar]
  • 19.Bruce JY, Eickhoff J, Pili R, Logan T, Carducci M, Arnott J, Treston A, Wilding G, Liu G. A phase II study of 2-methoxyestradiol nanocrystal colloidal dispersion alone and in combination with sunitinib malate in patients with metastatic renal cell carcinoma progressing on sunitinib malate. Invest. New Drugs. 2012;30:794–802. doi: 10.1007/s10637-010-9618-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Harrison MR, Hahn NM, Pili R, Oh WK, Hammers H, Sweeney C, Kim K, Perlman S, Arnott J, Sidor C, Wilding G, Liu G. A phase II study of 2-methoxyestradiol (2ME2) NanoCrystal dispersion (NCD) in patients with taxane-refractory, metastatic castrate-resistant prostate cancer (CRPC) Invest. New Drugs. 2011;29:1465–1474. doi: 10.1007/s10637-010-9455-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Tevaarwerk AJ, Holen KD, Alberti DB, Sidor C, Arnott J, Quon C, Wilding G, Liu G. Phase I trial of 2-methoxyestradiol NanoCrystal dispersion in advanced solid malignancies. Clin Cancer Res. 2009;15:1460–1465. doi: 10.1158/1078-0432.CCR-08-1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Newman SP, Ireson CR, Tutill HJ, Day JM, Parsons MF, Leese MP, Potter BVL, Reed MJ, Purohit A. The role of 17beta-hydroxysteroid dehydrogenases in modulating the activity of 2-methoxyestradiol in breast cancer cells. Cancer Res. 2006;66:324–330. doi: 10.1158/0008-5472.CAN-05-2391. [DOI] [PubMed] [Google Scholar]
  • 23.Liu Q, Jin W, Zhu Y, Zhou J, Lu M, Zhang Q. Synthesis of 3′-methoxy-E-diethylstilbestrol and its analogs as tumor angiogenesis inhibitors. Steroids. 2012;77:419–423. doi: 10.1016/j.steroids.2011.12.024. [DOI] [PubMed] [Google Scholar]
  • 24.Chiche J, Ilc K, Laferriere J, Trottier E, Dayan F, Mazure NM, Brahimi-Horn MC, Pouysségur J. Hypoxia-inducible carbonic anhydrase IX and XII promote tumor cell growth by counteracting acidosis through the regulation of the intracellular pH. Cancer Res. 2009;69:358–368. doi: 10.1158/0008-5472.CAN-08-2470. [DOI] [PubMed] [Google Scholar]
  • 25.Visagie MH, Joubert AM. In vitro effects of 2-methoxyestradiol-bissulfamate on reactive oxygen species and possible apoptosis induction in a breast adenocarcinoma cell line. Cancer Cell Int. 2011;11:43–49. doi: 10.1186/1475-2867-11-43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Supuran CT, Scozzafava A. Carbonic anhydrases as targets for medicinal chemistry. Bioorg. Med. Chem. 2007;15:4336–4350. doi: 10.1016/j.bmc.2007.04.020. [DOI] [PubMed] [Google Scholar]
  • 27.Genis C, Sippel KH, Case N, Cao W, Avvaru BS, Tartaglia LJ, Govindasamy L, Tu C, Agbandje-McKenna M, Silverman DN, Rosser CJ, McKenna R. Design of a carbonic anhydrase IX active-site mimic to screen inhibitors for possible anticancer properties. Biochemistry. 2009;48:1322–1331. doi: 10.1021/bi802035f. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Stander BA, Joubert F, Tu C, Sippel KH, McKenna R, Joubert AM. In vitro evaluation of ESE-15-ol, an estradiol analogue with nanomolar antimitotic and carbonic anhydrase inhibitory activity. PLoS One. 2012;7:e52205–e52215. doi: 10.1371/journal.pone.0052205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Leese MP, Leblond B, Newman SP, Purohit A, Reed MJ, Potter BV. Anti-cancer activities of novel D-ring modified 2-substituted estrogen-3-Osulfamates. J. Steroid Biochem. Mol. Biol. 2005;94:239–251. doi: 10.1016/j.jsbmb.2005.01.005. [DOI] [PubMed] [Google Scholar]
  • 30.Chander SK, Foster PA, Leese MP, Newman SP, Potter BV, Purohit A, Reed MJ. In vivo inhibition of angiogenesis by sulfamoylated derivatives of 2-methoxyoestradiol. Br. J. Cancer. 2007;96:1368–1376. doi: 10.1038/sj.bjc.6603727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Visagie MH, Joubert AM. The in vitro effects of 2-methoxyestradiolbis-sulfamate on cell numbers, membrane integrity and cell morphology, and the possible induction of apoptosis and autophagy in a non-tumorigenic breast epithelial cell line. Cell. Mol. Biol. Lett. 2010;15:564–581. doi: 10.2478/s11658-010-0030-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Stander BA, Joubert F, Tu C, Sippel KH, McKenna R, Joubert AM. Signaling pathways of ESE-16, an antimitotic and anticarbonic anhydrase estradiol analog, in breast cancer cells. PLoS One. 2013;8:e53853–e53871. doi: 10.1371/journal.pone.0053853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Mqoco T, Marais S, Joubert A. Influence of estradiol analogue on cell growth, morphology and death in esophageal carcinoma cells. Biocell. 2010;34:113–120. [PubMed] [Google Scholar]
  • 34.Stander XX, Stander BA, Joubert AM. In vitro effects of an in silicomodelled 17-beta-estradiol derivative in combination with dichloroacetic acid on MCF-7 and MCF-12A cells. Cell. Prolif. 2011;44:567–581. doi: 10.1111/j.1365-2184.2011.00789.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Stander BA, Marais S, Vorster CJ, Joubert AM. In vitro effects of 2-methoxyestradiol on morphology, cell cycle progression, cell death and gene expression changes in the tumorigenic MCF-7 breast epithelial cell line. J. Steroid Biochem. Mol. Biol. 2010;119:149–160. doi: 10.1016/j.jsbmb.2010.02.019. [DOI] [PubMed] [Google Scholar]
  • 36.Kanzawa T, Kondo Y, Ito H, Kondo S, Germano I. Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res. 2003;63:2103–2108. [PubMed] [Google Scholar]
  • 37.Knizhnik AV, Roos WP, Nikolova T, Quiros S, Tomaszowski KH, Christmann M, Kaina B. Survival and death strategies in glioma cells: autophagy, senescence and apoptosis triggered by a single type of temozolomide-induced DNA damage. PLoS One. 2013;8:e55665–e55676. doi: 10.1371/journal.pone.0055665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Moriya S, Che XF, Komatsu S, Abe A, Kawaguchi T, Gotoh A, Inazu M, Tomoda A, Miyazawa K. Macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress-mediated CHOP induction in myeloma cells. Int. J. Oncol. 2013;42:1541–1550. doi: 10.3892/ijo.2013.1870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Taylor JP, Tanaka F, Robitschek J, Sandoval CM, Taye A, Markovic-Plese S, Fischbeck H. Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein. Hum. Mol. Genet. 2003;12:749–757. doi: 10.1093/hmg/ddg074. [DOI] [PubMed] [Google Scholar]
  • 40.Garcia-Mata R, Gao YS, Sztul E. Hassles with taking out the garbage: aggravating aggresomes. Traffic. 2002;3:388–396. doi: 10.1034/j.1600-0854.2002.30602.x. [DOI] [PubMed] [Google Scholar]
  • 41.Simms-Waldrip T, Rodriguez-Gonzalez A, Lin T, Ikeda AK, Fu C, Sakamoto KM. The aggresome pathway as a target for therapy in hematologic malignancies. Mol. Genet. Metab. 2008;94:283–286. doi: 10.1016/j.ymgme.2008.03.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Nkandeu DS, Mqoco TV, Visagie MH, Stander BA, Wolmarans E, Cronje MJ, Joubert AM. In vitro changes in mitochondrial potential, aggresome formation and caspase activity by a novel 17-beta-estradiol analogue in breast adenocarcinoma cells. Cell. Biochem. Funct. 2013;31:566–574. doi: 10.1002/cbf.2937. [DOI] [PubMed] [Google Scholar]
  • 43.Bialik S, Zalckvar E, Ber Y, Rubinstein AD, Kimchi A. Systems biology analysis of programmed cell death. Trends Biochem. Sci. 2010;35:556–564. doi: 10.1016/j.tibs.2010.04.008. [DOI] [PubMed] [Google Scholar]
  • 44.Pradelli LA, Beneteau M, Ricci JE. Mitochondrial control of caspase-dependent and -independent cell death. Cell. Mol. Life. Sci. 2010;67:1589–1597. doi: 10.1007/s00018-010-0285-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Wang C, Klionsky DJ. The Molecular Mechanism of Autophagy. Mol. Med. 2003;9:65–76. [PMC free article] [PubMed] [Google Scholar]
  • 46.Tanida I, Ueno T, Kominami E. LC3 and Autophagy. Methods Mol. Biol. 2008;445:77–88. doi: 10.1007/978-1-59745-157-4_4. [DOI] [PubMed] [Google Scholar]
  • 47.Zaarur N, Meriin AB, Gabai VL, Sherman MY. Triggering aggresome formation. Dissecting aggresome-targeting and aggregation signals in synphilin 1. J. Biol. Chem. 2008;283:27575–27584. doi: 10.1074/jbc.M802216200. [DOI] [PubMed] [Google Scholar]
  • 48.Hsieh YC, Athar M, Chaudry IH. When apoptosis meets autophagy: deciding cell fate after trauma and sepsis. Trends Mol. Med. 2009;15:129–138. doi: 10.1016/j.molmed.2009.01.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Thorburn A. Apoptosis and autophagy: regulatory connections between two supposedly different processes. Apoptosis. 2008;13:1–9. doi: 10.1007/s10495-007-0154-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and Self-killing: crosstalk between autophagy and apoptosis. Mol. Cell Biol. 2007;8:741–752. doi: 10.1038/nrm2239. [DOI] [PubMed] [Google Scholar]

Articles from Cellular & Molecular Biology Letters are provided here courtesy of BMC

RESOURCES