Abstract

Arsenic trioxide (As₂O₃) has been found to induce apoptosis in leukemia cell lines and clinical remissions in patients with acute promyelocytic leukemia. In this study, we investigated the cytotoxic effect and mechanisms of action of As₂O₃ in human tumor cell lines. As₂O₃ caused inhibition of cell growth (IC₅₀ range, 3–14 μM) in a variety of human solid tumor cell lines, including four human non–small-cell lung cancer cell lines (H460, H322, H520, H661), two ovarian cancer cell lines (SK-OV-03, A2780), cervical cancer HeLa, and breast carcinoma MCF-7, as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry analysis showed that As₂O₃treatment resulted in a time-dependent accumulation of cells in the G₂/M phase. We observed, using Wright-Giemsa and 4′,6-diamidine-2-phenylindole-dihydrochloride staining, that As₂O₃ blocked the cell cycle in mitosis. In vitro examination revealed that As₂O₃ markedly promoted tubulin polymerization without affecting GTP binding to β-tubulin. Immunocytochemical and EM studies of treated MCF-7 cells showed that As₂O₃ treatment caused changes in the cellular microtubule network and formation of polymerized microtubules. Similar to most anti-tubulin agents, As₂O₃ treatment induced up-regulation of the cyclin B1 levels and activation of p34^cdc2/cyclinB1 kinase, as well as Bcl-2 phosphorylation. Furthermore, activation of caspase-3 and -7 and cleavage of poly(ADP-ribose) polymerase and β-catenin occurred only in As₂O₃-induced mitotic cells, not in interphase cells, suggesting that As₂O₃-induced mitotic arrest may be a requirement for the activation of apoptotic pathways. In addition, As₂O₃ exhibited similar inhibitory effects against parental MCF-7, P-glycoprotein–overexpressing MCF-7/doxorubicin cells, and multidrug resistance protein (MRP)-expressing MCF-7/etoposide cells (resistance indices, 2.3 and 1.9, respectively). Similarly, As₂O₃ had similar inhibitory effect against parental ovarian carcinoma A2780 cells and tubulin mutation paclitaxel-resistant cell lines PTx10 and PTx22 (resistance indices, 0.86 and 0.93, respectively), suggesting that its effect on tubulin polymerization and G₂/M phase arrest is distinct from that of paclitaxel. Taken together, our data demonstrate that As₂O₃ has a paclitaxel-like effect, markedly promotes tubulin polymerization, arrests cell cycle at mitosis, and induces apoptosis. In addition, As₂O₃ is a poor substrate for transport by P-glycoprotein and MRP, and non–cross-resistant with paclitaxel resistant cell lines due to tubulin mutation, suggesting that As₂O₃ may be useful for treatment of human solid tumors, particularly in patients with paclitaxel resistance.