RTA-408 induces JNK-dependent apoptosis and autophagy in breast cancer cells.

RTA-408 is a synthetic activator of the nuclear factor erythroid 2-related factor 2 pathway with antioxidant and anti-inflammatory act2ivity, but its antitumor effects in breast cancer (BC) have not fully been defined. The present study evaluated the antiproliferative efficacy and underlying mechanisms of RTA-408 in BC cells. Estrogen receptor-positive MCF-7 and triple-negative MDA-MB-231 BC cells were exposed to 0-1,000 nM RTA-408 for 72 h in standard serum-containing culture medium. Cell viability was quantified using a tetrazolium-based colorimetric assay and apoptosis was evaluated by the Muse® Annexin V & Dead Cell assay based on Annexin V and 7-amino-actinomycin D staining; the expression levels of JNK, p38, ERK, beclin-1, microtubule-associated protein 1 light chain 3B (LC3B), p62/sequestosome 1 (SQSTM1) and poly (ADP-ribose) polymerase (PARP) were assessed by western blotting. Pathway dependence was examined using the JNK inhibitor SP600125. Results showed that RTA-408 reduced cell viability in a concentration-dependent manner, with an IC50 of ~400 nM, decreasing survival to 28% in MCF-7 cells and 22% in MDA-MB-231 cells at 1,000 nM. RTA-408 increased annexin V+ apoptotic fractions, enhanced PARP cleavage and increased beclin-1, LC3B and p62/SQSTM1 levels. RTA-408 markedly enhanced JNK phosphorylation, with more modest activation of p38 and ERK. Pharmacological inhibition with SP600125 attenuated JNK phosphorylation, reduced apoptotic responses and diminished autophagy-associated marker accumulation, supporting the notion that JNK signaling contributes, at least in part, to these effects. These findings indicate that RTA-408 exerts nanomolar antiproliferative activity in both hormone receptor-positive and triple-negative BC cells through a JNK-dependent mechanism that simultaneously engages apoptosis and autophagy, supporting further in vivo and translational investigation.