Evaluating the cytotoxic effect of crocin on MDA-MB-468 cell line based on apoptosis induction, ER stress, and autophagy markers

Crocin, an important saffron ingredient, showed anticancer activity in a variety of cancer types, particularly breast cancer. However, little information is available on the mechanism of its action. Previous studies indicate apoptosis induction by crocin in some cancer cells. This study aims to investigate the effect of crocin on the MDA-MB-468 breast cancer cell line in order to investigate its effect on caspase 9 (Cas9) and cleaved-Cas9, expression and splicing of XBP1, and accumulation of LC3-II.
We used the MTT assay to investigate the cytotoxic effect of crocin on MDA-MB-468 breast cancer cells. Next, Cas9 and cleaved-Cas9 levels were evaluated by Western blot analysis. Splicing of XBP1 mRNA and expression of the spliced protein (XBP1s) was investigated by RT-PCR and Western blot, respectively. The accumulation of LC3-II was also evaluated by Western blot. The obtained results were analyzed and reported by Image J software.
The results showed a time and dose-dependent cytotoxic effect of crocin in MDA-MB-468 cells. The expression of Cas9 and its cleavage, therefore, the ratio of cleaved-Cas9/Cas9 significantly increased. Crocin treatment led to a noticeable increase in splicing of XBP1 mRNA, expression of XBP1s, accumulation of LC3-II, and increased the LC3-II/LC3-I ratio in these cells.
The data have shown induction of apoptosis in these breast cancer cell lines after crocin treatment. Because of the observed changes in UPR markers and autophagy, it seems that these pathways are possibly involved in this process and in intracellular regulations.