Abstract 1349: Hydrogen sulfide-releasing aspirin suppresses NF-κB signaling in estrogen receptor negative breast cancer cells in vitro and in vivo
Authors: Barsegian Arpine , Chattopadhyay Mitali , Kodela Ravinder , Boring Daniel , Crowell James A. , Kashfi Khosrow 
Published: June 2012

Abstract
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. Hydrogen sulfide-releasing aspirin (HS-ASA) is a safer ASA consisting of an ASA molecule to which an H2S-releasing moiety (5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH) has been covalently attached. We have observed that HS-ASA is very potent in inhibiting the growth of ER(−) breast cancer cells an effect which is modulated by NF-κB.
Methods: HS-ASA was synthesized and purified at our lab with 1H-NMR verification. Cell growth: MTT; Cell cycle phase distribution: Flow cytometry; Apoptosis: subdiploid (sub-G0/G1) peak in DNA content histograms; Proliferation: PCNA; NF-κB: enzyme-linked immunosorbent assay (ELISA). Xenografts: Male athymic SKID mice were implanted s.c. in the right flank with MDA-MB-231 (3 × 106) cells suspended in 50% Matrigel, after 5 days the animals were randomly divided into 2 groups (N = 6/group) and gavaged daily with HS-ASA (100 mg/kg body weight) or vehicle. Tumor volume and animal weight were recorded every 2 days. After 4 weeks of treatment, mice were sacrificed, tumors excised, weighed, and fixed in 10% buffered formalin for immunohistochemical studies.
Results: HS-ASA inhibited the growth of MDA-MB-231 cells, an ER(−) human breast cancer cell line; at 24h the IC50 being 3.6 ± 0.5 µM whereas for ASA the IC50 was &gt;5,000 µM. The ratio ASA/HS-ASA is &gt;1000, meaning that HS-ASA is at least 1000 times more potent than traditional ASA. In cultured ER(−) breast cancer cells, HS-ASA treatment for 24 hrs showed the following: 1. There was a combined inhibition of cell proliferation, induction of apoptosis and a cell cycle block at G0/G1; PCNA expression was reduced dose dependently, at 0.5xIC50, 1xIC50, and 2xIC50 the reduction was 79 ± 2 %, 47 ± 3 % and 36 ± 2 % respectively; % of apoptotic cells at 0.5xIC50, 1xIC50, and 2xIC50 were 15 ± 2%, 43 ± 3%, and 64 ± 5% respectively; 2. Activation of NF-κB was inhibited as demonstrated by ELISA, at 0.5xIC50, 1xIC50, and 2xIC50 the reduction was 10 ± 2%, 31 ± 3%, and 50 ± 3% respectively. Xenografts: 1. HS-ASA had no effect on the weight of the mice; 2. HS-ASA reduced tumor mass by 55 ± 7% and tumor volume as a function of time; 3. HS-ASA inhibited growth of ER(−) breast cancer cells as a result of reduced proliferation (decreased PCNA expression), and induction apoptosis (increased number of TUNEL positive cells); 4. NF-κB, activated in control xenografts was significantly inhibited by HS-ASA.
Conclusions: HS-ASA inhibits NF-κB which is central in inflammatory cascades. This suggests that HS-ASA may have a role in chemoprevention, in particular in ER(−) breast cancer.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1349. doi:10.1158/1538-7445.AM2011-1349