2-methoxyestradiol induces mitotic arrest, apoptosis, and synergistic cytotoxicity with arsenic trioxide in human urothelial carcinoma cells

PLoS One. 2013 Aug 13;8(8):e68703. doi: 10.1371/journal.pone.0068703. eCollection 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 2-Methoxyestradiol
  • Aged
  • Apoptosis / drug effects*
  • Arsenic Trioxide
  • Arsenicals / pharmacology*
  • Caspases / metabolism
  • Cell Cycle Checkpoints / drug effects*
  • Cell Line, Transformed
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Cell Survival / drug effects
  • DNA Fragmentation / drug effects
  • Dose-Response Relationship, Drug
  • Drug Synergism
  • Enzyme Activation / drug effects
  • Estradiol / analogs & derivatives*
  • Estradiol / pharmacology
  • Estradiol / toxicity
  • Female
  • Histones / metabolism
  • Humans
  • Mitosis / drug effects
  • Oxides / pharmacology*
  • Oxides / toxicity
  • Phosphorylation / drug effects
  • Poly(ADP-ribose) Polymerases / metabolism
  • Urologic Neoplasms / metabolism*
  • bcl-Associated Death Protein / metabolism

Substances

  • Arsenicals
  • Histones
  • Oxides
  • bcl-Associated Death Protein
  • Estradiol
  • 2-Methoxyestradiol
  • Poly(ADP-ribose) Polymerases
  • Caspases
  • Arsenic Trioxide

Grants and funding

This work was supported by grants from Taiwan National Science Council (NSC 98-2314-B-002-056 and 99-2314-B-002-067-MY3). The authors also acknowledge help from the Second and Sixth core laboratories of National Taiwan University Hospital. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.