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.

Photoactivated hypericin increases the expression of SOD-2 and makes MCF-7 cells resistant to photodynamic therapy

“…Indeed, accumulation of ROS was accompanied by upregulation of SOD activity in MCF-7 cells (Fig. 1B) which is known from the literature [21] and from our previous results [20] as the least sensitive cell line to HYP-PDT. On the other hand, we have not observed any significant increase of SOD activity in MDA-MB-231 cells neither at 1 h nor at 3 or 12 h after HYP-PDT (Fig.…”

“…Based on previous results of our group [20] as well as the findings of Vandenbogaerde et al [21] that MCF-7 cells are the least sensitive to HYP-PDT, we decided to shed more light on this issue and take a look on SOD-2 and its role in susceptibility to PDT. In this regard our aim was to monitor the correlation between the ROS and SOD activity after HYP-PDT in MCF-7 and MDA-MB-231 cells.…”

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX–Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model

“…Increase in prolidase activity in these cells was accompanied by an increase in proline levels in cytoplasm. It is well recognized that MOE induces mitotic arrest and apoptosis in carcinoma cells [31]. However, in ovarian carcinoma cells lacking PRODH/POX [32], on the contrary, MOE stimulated cell proliferation [33].…”

Selenium-enriched Spirulina protects INS-1E pancreatic beta cells from human islet amyloid polypeptide-induced apoptosis through suppression of ROS-mediated mitochondrial dysfunction and PI3/AKT pathway

“…Its activation will subsequently cleave some specific substrate, such as PARP, which is a DNA repair enzyme. Furthermore, cleavage of PARP resulted in chromosomal DNA break, finally leading to cell apoptosis [37][38][39]. Our results indicate that Se-SE prevented activation of caspase-3 and cleavage of PARP caused by hIAPP in INS-1E cells, suggesting that the protective effects of Se-SE was mediated through inhibiting the caspase-mediated pathway.…”

“…Excessive ROS may induce damage of mitochondrial enzymes, DNA mutation and lipid peroxidation of membranes. Moreover, ROS are known to affect mitochondrial membrane potential and trigger a series of mitochondria-associated events that initiate apoptosis [37,40]. Previous findings suggested that overproduction of ROS was involved in hIAPP-induced beta cell toxicity [35,41].…”