Induction of cytotoxicity and cell cycle arrest by As₂O₃. (A) To show the inhibitory effects of As₂O₃ on the cell proliferation, exponentially growing cells were treated with the indicated concentrations of As₂O₃ for 24 hour and 48 hours, and cell proliferation was assessed by using the MTS reagent (see Materials and Methods). The cytotoxic effect of As₂O₃ was increased in a concentration- and/or time-dependent manner. These data represent the mean±SD of independent experiments. (B) To observe the apoptotic effects of As₂O₃, NB4 cells were treated with either a vehicle (control) or each concentration of As₂O₃ and paclitaxel for 24 hours. PI-stained cells were analyzed by flow cytometry as described in the Materials and Methods section, and the percentage of cells in each phase of the cell cycle was analyzed using flow cytometry (Table 1). Flow cytometry analysis revealed a significant increased apoptotic effect that resulted in a concentration-dependent accumulation of NB4 cells in the G₂/M phase on the As₂O₃-treated cells.

Alterations in tubulin polymerization induced by As₂O₃. (A) To demonstrate that the fraction of depolymerized tubulin is increased after treatment with As₂O₃, NB4 cells were treated with drugs or without drugs as a control for 6 hours at the indicated concentrations. Cell lysates were separated into polymerized (P) or soluble (S) fractions. Aliquots of equal volume were separated on an SDS-PAGE gel, and evaluated by western blotting with an anti α-tubulin antibody. Compared to the baseline proportion of α-tubulin in the soluble fraction, the depolymerized proportion observed after treatment of As₂O₃ was approximately 52-72% and the tubulin shift to the "S" fraction was noted. (B) To show the effects of a combination of drugs, NB4 cells were treated with 5 µM As₂O₃, 10 µM As₂O₃ and 40 nM paclitaxel at alone or in combination, and treated for 6 hours. Cell lysates were separated into "P" or "S" fractions as described above. Aliquots of equal volume were loaded onto SDS-PAGE gels, and the blots probed with antibody against α-tubulin. The results indicate that As₂O₃ and paclitaxel did not synergistically influence microtubule assembly. The intensity of each band was quantified by densitometry and the percentage of soluble tubulin (%S) was calculated by multiplying the fraction of tubulin in the soluble fraction [S/(S+P)] by 100 for each "S-P" pair.

Effects of As₂O₃ on microtubule stabilization. NB4 cells were treated with drugs or no drugs as a control, for 6 hours and 24 hours at the indicated concentration. Lysates were separated into polymerized (P) or soluble (S) fractions. The blots probed with anti-acetylated α-tubulin. The amount of acetylated α-tubulin in the P fraction of paclitaxel treatment was increased, compared with that of the control, but the proportion in P fraction of As₂O₃ treatment was decreased in a concentration-dependent manner. This result indicated that As₂O₃ interfered with important modifications necessary for the stability of microtubules. The intensity of each band was quantified by densitometry and the blots were stripped and reprobed with GAPDH-specific antibody as a loading control.

Effects of As₂O₃ on microtubule polymerization in vitro. Purified tubulin from bovine brain tissue (Cytoskeleton) was incubated at 37℃ in reaction mixtures containing 1 mM GTP, 10 nM paclitaxel (Taxol), 10 nM vincristine, 5 µM As₂O₃ and the mock-treated solution as a control. Tubulin polymerization was determined by measuring absorbance at 340 nm. This revealed that As₂O₃ directly influences tubulin and induces tubulin depolymerization.

Effects of As₂O₃ on the organization of cellular microtubule network. NB4 cells were treated with 5 µM As₂O₃, 40 nM paclitaxel, and 20 nM vincristine. Mock-treated cells were used as a control. After a 6-hour incubation, cells were harvested and fixed with formaldehyde. Cells were incubated with monoclonal anti-α-tubulin antibody at room temperature for 30 minutes. After incubation with FITC-conjugated secondary antibody, the cellular microtubules were imaged using an Olympus IX-81 fluorescence microscope. The normal organization of microtubule network was seen in control cells, increased density of polymerized microtubules were found in paclitaxel-treated cells, and a degraded microtubule network in cytoplasm was observed in As₂O₃-and vicristine-treated cells.