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.

Figure 1. 2-ME reduces cell viability and induces apoptosis as well as DNA fragmentation in UC cells, but not in SV-HUC cells.

(A) Cells were treated with various concentrations of 2-ME for 24 h. Cell viability was assessed by MTT assay. (B) Cells were exposed to mock (DMSO) and 2-ME (2 µM) for 24 h. Apoptotic cells were analyzed by FACS flow cytometry with propidium iodide (PI) and annexin V-FITC staining. The lower-right panel presented annexin V-positive cells (early apoptotic cells); the upper-right panel presented late apoptotic cells with membranes permeable to PI and annexin V staining. Quantitative analyses of total apoptosis (early and late) population following 2-ME (2 µM) and mock (DMSO) treatments were presented. In (A) and (C), data are presented as means ± SD of three independents experiments. * p<0.05 is interpreted to be significant as compared with control. (C). Agarose gel electrophoresis of DNA extracted from UC cells treated with 2-ME (2 µM) for 24 h. Left lane: DNA marker; Middle lane: treatment with mock (DMSO); Right lane: treatment with 2-ME (2 µM) (D). The total cell lysates were harvested and analyzed by Western blot with specific antibodies against phospho-H2AX.

Figure 2. 2-ME induces caspase activations and PARP cleavage in UC cells.

(A) NTUB1 and (B) T24 cells were treated by 2-ME (2 µM) at different time point. The total cell lysates were harvested and analyzed by Western blot with specific antibodies against caspase-8, 9, cleaved caspase-3, 7 and PARP. CF is the abbreviation of cleaved form. Results shown are representative of at least three independent experiments.

Figure 3. 2-ME induces downregulation of phospho-BAD at Ser136 and Ser155.

(A) NTUB1 (B) T24 cells were exposed to 2-ME (2 µM) for 4 to 24 h. Cell lysates were analyzed by Western blot with anti-phospho-BAD antibodies at Ser136 and 155.

Figure 4. The effect of 2-ME on cell proliferation and cell cycle in UC cells.

(A) NTUB1 and T24 cells were treated with 2-ME (2 µM) or DMSO (as non-treated control) for 24 h, cell proliferation were measured by BrdU incorporation assay. (B) NTUB1 and T24 cells were treated with mock (DMSO) or 2-ME (2 µM) for 12 h and 24 h, and cell cycle analyses were measured by flow cytometry. Quantitative data are presented as means ± SD of three independents experiments. * p<0.05 is interpreted to be significant as compared with control.

Figure 5. The effects of 2-ME on cell cycle regulatory proteins.

NTUB1 and T24 cells were treated with 2ME (2 µM) for 12 and 24 h. The total cell lysates were analyzed for the levels of cell cycle regulatory proteins by Western blot using specific antibodies. (A) and (B) shows the levels of cdc25C, phospho-cdc25C (Thr48 and Ser216), phospho-cdc2 (Tyr15), cyclin B1, phospho-histone H3 (Ser10), cdc2, histone H3. Similarly, (C) and (D) shows the level of p27, cyclin D3, cyclin D1, CDK4, cyclin A2, cyclin E2, CDK2 proteins in NTUB1 and T24 cells after 2-ME treatment. Results shown are representative of at least three independent experiments.

Figure 6. The combinative effects of 2-ME and As₂O₃ on cell viability and the level of cleaved caspase-3 and 7 in UC cells.

(A) NTUB1 and (B) T24 cells were incubated in the presence of 2-ME (0.5 and 1 µM) and different concentration of As₂O₃ (0.75 to 10 µM) individually or in combination. Cell viability was measured by MTT assay. Quantitative analysis of cell viability are presented as means ± SD of three independents experiments. * p<0.05 is interpreted to be significant as compared with As₂O₃ treatment alone. The total cell lysates were harvested and analyzed by Western blot with specific antibodies against cleaved caspase-3 and 7 after treatment of 2-ME (1 µM), As₂O₃ (1 µM) and in combination. Results shown are representative of at least three independent experiments.

Figure 7. The combination with 2-ME and As₂O₃ at the ratio of 1∶ 10 induces synergistic inhibition on the viability of NTUB1 and T24 cells.

UC cells were incubated in the presence of 2-ME, As₂O₃, and in combination at the concentration ratio of 1∶ 10. Cell viability was measured by MTT assay. Cell viability was measured by MTT after 24 h exposure. (A) The dose-effect plot reflects the dose-effect relationships for As₂O₃, 2-ME and the combination. (B) Combination index and fractions affected was plotted in combination with As₂O₃ and 2-ME.