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.