Nitric Oxide-Releasing Aspirin Suppresses NF-κB Signaling in Estrogen Receptor Negative Breast Cancer Cells in Vitro and in Vivo

Estrogen receptor negative (ER(−)) breast cancer is aggressive, responds poorly to current treatments and has a poor prognosis. The NF-κB signaling pathway is implicated in ER(−) tumorigenesis. Aspirin (ASA) is chemopreventive against ER(+) but not for ER(−) breast cancers. Nitric oxide-releasing aspirin (NO-ASA) is a safer ASA where ASA is linked to an NO-releasing moiety through a spacer. In vitro, we investigated anti-proliferation effects of NO-ASA (para- and meta-isomers) against ER(−) breast cancer cells MDA-MB-231 and SK-BR-23, effects on NF-κB signaling, and reactive oxygen species by standard techniques. In vivo, effects of NO-ASA were evaluated in a mouse xenograft model using MDA-MB-231 cells. p-NO-ASA inhibited the growth of MDA-MB-231 and SK-BR-3 cells at 24 h, the respective IC50s were 13 ± 2 and 17 ± 2 μM; ASA had an IC50 of \u3e3000 μM in both cell lines. The IC50s for m-NO-ASA in MDA-MB-231 and SK-BR-3 were 173 ± 15 and 185 ± 12 μM, respectively, therefore, implying p-NO-ASA as a stronger inhibitor of growth p-NO-ASA reduced cell growth by inhibiting proliferation, inducing apoptosis and causing G0/G1 cell cycle block. Activation of NF-κB was inhibited by both isomers as demonstrated by decreases in NF-κB-DNA binding and luciferase activity at 24 h, However, m-NO-ASA produced transient effects at 3 h such as increased NF-κB-DNA-binding, increased levels of nuclear p50, even though both isomers inhibited IκB degradation. Increase in nuclear p50 by m-NO-ASA was associated with translocation of p50 in to the nucleus as observed by immunoflouresence at 3 h. NO-ASA induced reactive oxygen species (ROS) as evidenced by overall increases in both H2DCFDA (2′,7′-dichlorodihydrofluorescein) and DHE (dihydroethidium)-derived fluorescence. Inhibition of ROS by N-acetyl-cysteine reversed the m-NO-ASA-mediated translocation of p50 in to the nucleus. In xenografts, p-NO-ASA inhibited tumor growth by inhibiting proliferation (PCNA and tumor volume), inducing apoptosis (TUNEL positive cells) and reducing NF-κB expression. Both isomers inhibit cancer cells, inhibit NF-κB pathway and induce ROS, and have potential as anticancer compounds

H2S biosynthesis and catabolism: new insights from molecular studies

Hydrogen sulfide (H2S) has profound biological effects within living organisms and is now increasingly being considered alongside other gaseous signalling molecules, such as nitric oxide (NO) and carbon monoxide (CO). Conventional use of pharmacological and molecular approaches has spawned a rapidly growing research field that has identified H2S as playing a functional role in cell-signalling and post-translational modifications. Recently, a number of laboratories have reported the use of siRNA methodologies and genetic mouse models to mimic the loss of function of genes involved in the biosynthesis and degradation of H2S within tissues. Studies utilising these systems are revealing new insights into the biology of H2S within the cardiovascular system, inflammatory disease, and in cell signalling. In light of this work, the current review will describe recent advances in H2S research made possible by the use of molecular approaches and genetic mouse models with perturbed capacities to generate or detoxify physiological levels of H2S gas within tissue