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 muM) 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 (SKOV-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 As2O3 treatment resulted in a time-dependent accumulation of cells in the G(2)/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 beta-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 beta-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 G(2)/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.
