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.