title: 2-methoxyestradiol induces mitotic arrest, apoptosis, and synergistic cytotoxicity with arsenic trioxide in human urothelial carcinoma cells.
abstract: 2-Methoxyestradiol (2-ME), an endogenous derivative of 17β-estradiol, has been reported to elicit antiproliferative responses in various tumors. In this study, we investigated the effects of 2-ME on cell viability, proliferation, cell cycle, and apoptosis in human urothelial carcinoma (UC) cell lines. We used two high-grade human bladder UC cell lines (NTUB1 and T24). After treatment with 2-ME, the cell viability and apoptosis were measured by MTT assay and flow cytometry (fluorescence-activated cell sorting), with annexin V-FITC staining and propidium iodide (PI) labeling. DNA fragmentation was analyzed by agarose gel electrophoresis. Flow cytometry with PI labeling was used for the cell cycle analyses. The protein levels of caspase activations, poly (ADP-ribose) polymerase (PARP) cleavage, phospho-histone H2A.X, phospho-Bad, and cell cycle regulatory molecules were measured by Western blot. The effects of the drug combinations were analyzed using the computer software, CalcuSyn. We demonstrated that 2-ME effectively induces dose-dependent cytotoxicity and apoptosis in human UC cells after 24 h exposure. DNA fragmentation, PARP cleavage, and caspase-3, 7, 8, 9 activations can be observed with 2-ME-induced apoptosis. The decreased phospho-Bad (Ser136 and Ser155) and mitotic arrest of the cell cycle in the process of apoptosis after 2-ME treatment was remarkable. In response to mitotic arrest, the mitotic forms of cdc25C, phospho-cdc2, cyclin B1, and phospho-histone H3 (Ser10) were activated. In combination with arsenic trioxide (As2O3), 2-ME elicited synergistic cytotoxicity (combination index <1) in UC cells. We concluded that 2-ME significantly induces apoptosis through decreased phospho-Bad and arrests bladder UC cells at the mitotic phase. The synergistic antitumor effect with As2O3 provides a novel implication in clinical treatment of UC.
authors: Department of Urology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors: Kuan-Lin
authors: KL
authors: Kuo
authors: Wei-Chou
authors: WC
authors: Lin
authors: I-Lin
authors: IL
authors: Ho
authors: Hong-Chiang
authors: HC
authors: Chang
authors: Ping-Yi
authors: PY
authors: Lee
authors: Yuan-Ting
authors: YT
authors: Chung
authors: Ju-Ton
authors: JT
authors: Hsieh
authors: Yeong-Shiau
authors: YS
authors: Pu
authors: Chung-Sheng
authors: CS
authors: Shi
authors: Kuo-How
authors: KH
authors: Huang
mesh_terms: D000077584:2-Methoxyestradiol; D000368:Aged; D017209:Apoptosis / Q000187:drug effects*; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000633:toxicity; D005260:Female; D006657:Histones / Q000378:metabolism; D006801:Humans; D008938:Mitosis / Q000187:drug effects; D010087:Oxides / Q000494:pharmacology* / Q000633:toxicity; D010766:Phosphorylation / Q000187:drug effects; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D014571:Urologic Neoplasms / Q000378:metabolism*; D051025:bcl-Associated Death Protein / Q000378:metabolism
chemical_list: D001152:Arsenicals; D006657:Histones; D010087:Oxides; D051025:bcl-Associated Death Protein; D004958:Estradiol; D000077584:2-Methoxyestradiol; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D000077237:Arsenic Trioxide
references: Cancer. 2001 Aug 1;92(3):500-9
references: 11505393
references: Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2009 Apr;17(2):335-9
references: 19379562
references: J Steroid Biochem Mol Biol. 2010 Apr;119(3-5):149-60
references: 20193762
references: EMBO J. 2002 Jun 17;21(12):3000-8
references: 12065413
references: Urology. 2010 Feb;75(2):334-9
references: 19963252
references: Urology. 2002 Aug;60(2):346-50
references: 12137851
references: J Cell Mol Med. 2003 Jul-Sep;7(3):249-57
references: 14594549
references: J Biol Chem. 2005 Apr 15;280(15):14773-9
references: 15708859
references: CA Cancer J Clin. 2010 Sep-Oct;60(5):277-300
references: 20610543
references: J Clin Oncol. 2000 Sep;18(17):3068-77
references: 11001674
references: PLoS One. 2012;7(3):e33615
references: 22438966
references: Cancer Cell. 2003 Apr;3(4):363-75
references: 12726862
references: Int J Cancer. 1973 May;11(3):765-73
references: 4133950
references: Cell. 1999 Apr 2;97(1):99-109
references: 10199406
references: Cell Death Differ. 2002 Nov;9(11):1240-7
references: 12404123
references: J Clin Oncol. 2001 Feb 15;19(4):1229-31
references: 11181690
references: Cancer Lett. 2002 Dec 10;187(1-2):77-86
references: 12359354
references: Carcinogenesis. 1998 Jan;19(1):1-27
references: 9472688
references: Carcinogenesis. 2003 Feb;24(2):209-16
references: 12584169
references: Biochim Biophys Acta. 2012 Aug;1823(8):1306-15
references: 22580043
references: Biochem Biophys Res Commun. 2008 Jun 6;370(3):483-8
references: 18384749
references: Eur J Gynaecol Oncol. 2009;30(3):275-80
references: 19697620
references: J Pharmacol Exp Ther. 2010 Oct;335(1):114-23
references: 20605902
references: Mol Pharm. 2010 Dec 6;7(6):2030-9
references: 20831190
references: Nature. 1995 Mar 9;374(6518):131-4
references: 7877684
references: Adv Enzyme Regul. 1984;22:27-55
references: 6382953
references: J Org Chem. 2009 Aug 21;74(16):6362-4
references: 19610601
references: Blood. 2003 May 15;101(10):4098-104
references: 12531810
references: Pharmacotherapy. 2003 Feb;23(2):165-72
references: 12587805
references: Anticancer Res. 2004 Mar-Apr;24(2B):873-80
references: 15161040
references: Cancer Lett. 2008 Sep 18;268(2):295-307
references: 18492602
references: Steroids. 2010 Oct;75(10):625-31
references: 20214913
references: Int J Oncol. 1999 Oct;15(4):639-46
references: 10493943
references: Anticancer Res. 2006 Jan-Feb;26(1A):375-8
references: 16475721
references: Cancer Res. 1987 Nov 15;47(22):6066-73
references: 2822239
references: Anticancer Agents Med Chem. 2010 Jul;10(6):504-10
references: 20812901