Abstract
A91
Introduction: Estrogen receptor negative [ER(-)] breast cancer is very aggressive, responds poorly to current treatments and has a poor prognosis. The NF-κB signaling pathway is strongly implicated in ER(-) tumorigenesis. Epidemiological studies show a chemopreventive effect for aspirin (ASA) against ER(+) but not for ER(-) breast cancers. Nitric oxide-releasing aspirin (NO-ASA) is a safer ASA consisting of an ASA molecule and a NO-releasing moiety linked via an aromatic spacer. We have observed that NO-ASA is very potent in inhibiting the growth of ER(-) breast cancer cells an effect which is modulated by NF-κB through induction of reactive oxygen species. Methods: Cell growth: MTT; Cell cycle phase distribution: Flow cytometry; Apoptosis:subdiploid (sub-G0/G1) peak in DNA content histograms; NF-κB: electrophoretic mobility shift assays (EMSA); Reactive oxygen species (ROS): 2',7'-dichlorodihydrofluorescein (H2DCFDA, probe for peroxides) or dihydroethidium (DHE, probe for superoxide anion) measured fluorescence by flow cytometry; Xenografts: Female athymic nude mice, 8-weeks-old, 2 groups (n = 10/group) gavaged daily with NO-ASA (100 mg/kg body weight) or vehicle. After one week, animals in both groups were implanted s.c. in the right flank with MDA-MB-231cells suspended in 50% Matrigel. After 4 weeks of treatment, mice were sacrificed, tumors excised, fixed in 10% buffered formalin for immunohistochemical studies. Results: NO-ASA inhibited the growth of MDA-MB-231 cells, an ER(-) human breast cancer cell line; at 24 and 48 hrs the respective IC50s were 4.5 ± 0.5 µM and 3.2 ± 0.3 µM, whereas for ASA was >2,000 µM at both time points. The ratio ASA/NO-ASA is >444, meaning that NO-ASA is at least 444 times more potent than traditional ASA. In cultured ER(-) breast cancer cells, NO-ASA treatment for 24 hrs showed the following: 1. There was a combined inhibition of cell proliferation, induction of apoptosis and induction of a G2/M cell cycle block, PCNA expression was reduced dose dependently, at 5 µM the reduction was 81 ± 2 % and at 15 µM it was 42 ± 5 %; 2. Activation of NF-κB was inhibited as demonstrated by three independent approaches: a) EMSA, b) translocation from the cytoplasm into the nucleus, c) effect on transfected reporter constructs; 3. Induction of oxidative stress as evidenced by increases in both H2DCFDA and DHE-derived fluorescence representing increases in intracellular levels of H2O2 and superoxide anion respectively. Xenografts: NO-ASA inhibits proliferation of ER(-) breast cancer cells in the nude mice xenografts (reduced PCNA expression), and induces apoptosis (increased number of TUNEL positive cells). NF-κB, activated in control xenografts is significantly inhibited by NO-ASA. Conclusions: NO-ASA inhibits NF-κB in vitro and in vivo ER(-) breast cancer xenografts, suggesting merit for further evaluation.
[Fifth AACR International Conference on Frontiers in Cancer Prevention Research, Nov 12-15, 2006]