Nitric oxide-donating aspirin induces G2/M phase cell cycle arrest in human cancer cells by regulating phase transition proteins

Abstract

NO-aspirin (NO-ASA), consisting of aspirin and a nitric oxide-releasing group, is safer than aspirin and effective in colon cancer prevention. Here, we examined the mechanism of action of NO-ASA by focusing primarily on its effects on the cell cycle. NO-ASA reduced the growth of several cell lines from colon, pancreas, skin, cervix and breast cancer much more potently than aspirin, with 24-h IC(50) values of 133-268 µM, while those of ASA were >1,000 µM. NO-ASA elevated the intracellular levels of reactive oxygen species, generating a state of oxidative stress. In all cell lines examined, NO-ASA induced cell cycle arrest in the G(2)/M phase transition accompanied by altered expression of G(2)/M transition-related proteins. In SW480 colon cancer cells NO-ASA modulated proteins controlling this transition. Thus, it markedly increased the levels of cyclin B1, decreased the expression of cyclin D1 and Cdc25C, and increased the Thr14/Tyr15-phosphorylation of Cdk1 while leaving unchanged its protein levels. These changes, including the G2/M arrest, were prevented by pretreating the cells with the anti-oxidant N-acetyl-cysteine, indicating that redox signaling is likely responsible for the cell cycle changes, a conclusion consistent with the known redox regulation of these proteins. Collectively, these results confirm the profound cytokinetic effect of NO-ASA and provide strong evidence that it regulates cell cycle transitions through its ability to induce oxidative stress, which activates redox signaling in the target cell.

Figure 1

The chemical structure of NO-ASA. NO-ASA consists of aspirin, a spacer moiety and a nitric oxide-releasing moiety (−ONO₂). This is the meta-isomer of NO-ASA in terms of the position of the −ONO₂ moiety with respect to its closest carboxylic ester.

Figure 2

Effect of NO-ASA on proteins regulating the G₂/M transition in SW480 cells. SW480 cells were treated with 200 μM NO-ASA at the indicated times (A) and with various concentrations of NO-ASA for 24 h (B). Cellular proteins were extracted and the levels of those indicated were assayed by immunoblotting. Loading control: actin and α-tubulin.

Figure 3

Effect of NO-ASA on proteins regulating G₂/M transition in HT-29 cells. HT-29 cells were treated with 200 μM NO-ASA for the indicated periods of time. Cellular proteins were extracted and assayed by immunoblotting. Loading control: α-tubulin.

Figure 4

Effect of NO-ASA-induced ROS on G₂/M arrest in S480 cells. (A), SW480 cells were pre-loaded with H₂DCFDA or DHE for 1 h followed by 200 μM NO-ASA for 4 h. Fluorescence was measured by flow cytometry in single-cell suspensions of the attached cells. (B), SW480 cells were treated with or without 2.5 mM NAC for 1 h before the start of treatment with 200 μM NO-ASA for 24 h. Cells were stained with PI and analyzed by flow cytometry. The table presents the corresponding cell cycle results from triplicate experiments. Values: mean ± SEM.

Figure 5

NO-ASA-induced apoptosis in SW480 cells. (A), After SW480 cells were treated with either 200 μM NO-ASA or vehicle for the indicated periods of time, they were stained with PI and analyzed by flow cytometry. (B), SW480 cells were treated with 200 μM NO-ASA for the indicated periods of time, when proteins were extracted and analyzed by immunoblotting. Loading control: α-tubulin.

Figure 6

The mechanism of G₂/M arrest by NO-ASA. NO-ASA increases intracellular ROS levels, inducing a state of oxidative stress. Redox signaling is then activated, altering the expression of proteins that control the G₂/M transition, as indicated here. The end result is cell cycle arrest that contributes to the anticancer effect of NO-ASA.