NOSH‐Aspirin (NBS‐1120) Inhibits Estrogen Receptor Negative Breast Cancer in Vitro and in Vivo by Modulating Redox‐Sensitive Signaling Pathways
Authors: Duvalsaint Pascale Louise , Chattopadhyay Mitali , Kodela Ravinder , Kashfi Khosrow 
Published: November 2023

Introduction

Estrogen receptor negative [ER(−)] breast cancer is very aggressive and has a poor prognosis, given the lack of target‐directed therapies. Recent exhaustive meta‐analysis provides evidence that non‐steroidal anti‐inflammatory drugs (NSAIDs) use in general and aspirin in particular are associated with reduced risk for breast cancer. However, long‐term NSAIDs/aspirin use may lead to life‐threatening side effects. In trying to improve the clinical profile of NSAIDs, we developed NOSH‐NSAIDs, which are nitric oxide (NO) and hydrogen sulfide (H2S)‐releasing analogs, the rational being the observations that NO and H2S enhance the local mucosal defense mechanisms. We recently reported on the gastrointestinal safety and pharmacological properties of NOSH‐aspirin. Here we highlight the molecular targets of NOSH‐aspirin using in vitro and in vivo models of ER(−) breast cancer.

Methods

Cell lines: MDA‐MB‐231 and SKBR3 (ER−), HMEpC, normal human mammary epithelial; Cell growth: MTT; Apoptosis and cell cycle phase distribution: Flow cytometry; Proliferation: PCNA; ROS: measured hydrogen peroxide and super oxide by flow cytometry using appropriate dyes. Xenografts: Male athymic nude (NU/NU) mice (N=10) implanted s.c. in the right flank with MDA‐MB 231 cells, after 10 days randomly divided animals and gavaged daily with NOSH‐ASA (100 mg/kg body weight) or vehicle. Tumor volume and animal weight were recorded every 3 days. After 4 weeks of treatment, mice were sacrificed, tumors excised, weighed, and prepared for IHC studies.

Results

In vitro: NOSH‐aspirin preferentially inhibited cancer cell growth the IC50s in μM at 24h being, MDA‐MB‐231=0.09±0.005, SKBR3=0.08±0.007, HMEpC=30±7, aspirin >5,000 in all cell lines. Using MDA‐MB‐231 cells, NOSH‐aspirin dose‐dependently and as a function of time, induced apoptosis; inhibited proliferation; caused a G0/G1 cell cycle block; inhibited NF‐κB and ROS, the latter being blocked by pretreating the cells with the antioxidant N‐acetylcysteine. In vivo xenografts: NOSH‐aspirin had no effect on the weight of the mice and there were no overt signs of toxicity. Tumor volume/growth was reduced as a function of treatment time (99% reduction at sacrifice, P=0.0008) and tumor mass was reduced by 91% (P=0.006). NOSH‐aspirin inhibited growth of these cancer cell xenografts as a result of reduced proliferation (decreased PCNA expression), and induction apoptosis (increased number of TUNEL positive cells), and induction of ROS. Other molecular targets affected were: NF‐κB activated in untreated tumors was reduced by 85±5%; p53 expression was increased by 88±3% while FoxM1 expression was decreased by 78±4% in treated tumors. iNOS expression was also increased in treated tumors while tumor growth decreased questioning the notion that induction of iNOS is a prognostic marker in ER(−) breast and other cancers.

Conclusions

NOSH‐aspirin targets parameters important in determining cellular mass and has a pleotropic mechanism of action affecting multiple pathways that are dis‐regulated in ER(−) breast cancer.

Support or Funding Information

NIH grant R24 DA018055