Abstract
Crocus sativus and its major constituent crocin are well established to have anti-cancer properties in breast cancer cells (MCF-7). However the role of C. sativus extract (CSE) and crocin on caspase signaling mediated MCF-7 cell death at molecular level is remains unclear. In this study, we tried to unravel role of CSE and crocin on caspase mediated MCF-7 cells death and their in vivo preclinical toxicity profiling and immune stimulatory effect. CSE extract was fractionated by HPLC and crocin was isolated and characterized by NMR, IR, and MS. MCF-7 cells were treated with both CSE and crocin and expression of Bcl-2 and Bax was assessed after 24 and 36 hours. Furthermore, caspase 3, caspase 8 and caspase 9 expression was determined by Western blotting after 24 hours of treatment. DNA fragmentation analysis was performed for genotoxicity of CSE and crocin in MCF-7 cells. The in vivo toxicity profile of CSE (300 mg/kg of b.wt) was investigated in normal Swiss albino mice. In addition, peritoneal macrophages were collected from crocin (1, 1.5 and 2 mg/kg body weight) treated mice and analyzed for ex vivo yeast phagocytosis. Immunoblot analysis revealed that there was time dependent decline in anti-apoptotic Bcl-2 with simultaneous upregulation of Bax in CSE and crocin treated MCF-7 cells. Further CSE and crocin treatment downregulated caspase 8 and 9 and cleaved the caspase 3 after 24 hours. Both CSE and crocin elicited considerable DNA damage in MCF-7 cells at each concentration tested. In vivo toxicity profile by histological studies revealed no observable histopathologic differences in the liver, kidney, spleen, lungs and heart in CSE treated and untreated groups. Crocin treatment elicited significant dose and time dependent ex vivo yeast phagocytosis by peritoneal macrophages. Our study delineated involvement of pro-apoptotic and caspase mediated MCF-7 cell death by CSE and crocin at the molecular level accompanied with extensive DNA damage. Further we found that normal swiss albino mice can tolerate the maximum dose of CSE. Crocin enhanced ex vivo macrophage yeast phagocytic ability.
Highlights
Breast cancer is the most common malignancy in women throughout the world, and it accounts for 18% of all female cancers and there are approximately 600,000 annual deaths worldwide (Kumar et al, 2011)
Taken together these results suggest that C. sativus extract (CSE) and crocin were capable of inducing apoptosis in MCF-7 cells with extensive DNA damage
Ex vivo immune stimulatory effect of purified crocin crocin leads to up regulation of caspase 3, 8 and 9 in time dependent manner (Figure 7). These findings suggest that the CSE and crocin induced apoptosis in MCF-7 cells via the Bcl-2 down regulation and caspase 3 dependent pathways
Summary
Breast cancer is the most common malignancy in women throughout the world, and it accounts for 18% of all female cancers and there are approximately 600,000 annual deaths worldwide (Kumar et al, 2011). Crocin is major active component of saffron coated TLC plates mesh size 60-120 was obtained from and it is reported by our group and elsewhere We intended to elaborate the diluted with 10 mmol/l phosphate buffered saline (PBS, molecular mechanism of caspase mediated MCF-7 pH=7.4), and the concentration of crocin was adjusted to cell death induced by saffron. Different concentration of CSE (25, 50 and 75 μg/ml) the precise molecular mechanism of apoptosis induced was freshly prepared by reconstituting them in cell culture by saffron and its active constituent crocin in MCF-7 medium. Further In vivo toxicity and macrophage activation μg/ml) and crocin (10 μg/ml) treated MCF-7 cells of saffron were investigated in normal Swiss albino mice.
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