Cytosolic and mitochondrial ROS production resulted in apoptosis induction in breast cancer cells treated with Crocin: The role of FOXO3a, PTEN and AKT signaling.

Different groups have reported the Crocin anticancer activity. We previously showed Crocin-induced apoptosis in rat model of breast and gastric cancers, through the increased Bax/Bcl-2 ratio and caspases activity, as well as the cell cycle arrest in a p53-dependent manner. Since Crocin antioxidant activity has been shown under different conditions, it is interesting to elucidate its apoptotic mechanism. Here, we treated two breast cancer cell lines, MCF-7 and MDA-MB-231, with Crocin. MTT and ROS assays, cell cycle arrest, Bax/Bcl-2 ratio and caspase3 activity were determined. PARP cleavage and expression of some proteins were studied using Western blotting and immunofluorescence. The results indicated stepwise ROS generation in cytosol and mitochondria after Crocin treatment. Attenuating the early ROS level, using diphenyleneiodonium, diminished the sequent mitochondrial damage (decreasing Δψ) and downstream apoptotic signaling. Crocin induced ROS production, FOXO3a expression and nuclear translocation, and then, elevation of the expression of FOXO3a target genes (Bim and PTEN) and caspase-3 activation. Application of N-acetylcysteine blocked AKT/FOXO3a/Bim signaling. FOXO3a knockdown resulted in a decrease of Bim, PTEN and caspase 3, after Crocin treatment. PTEN knockdown caused a decrease in FOXO3a, Bim and caspase 3, in addition to an increase in p-AKT and p-FOXO3a, after Crocin treatment. In conclusion, Crocin induced apoptosis in MCF-7 and MDA-MB-231 human breast cancer cells. The ROS-activated FOXO3a cascade plays a central role in this process. FOXO3a-mediated upregulation of PTEN exerted a further inhibition of the AKT survival pathway. These data provide a new insight into applications of Crocin for cancer therapy.