Molecular Mechanism of Crocin Induced Caspase Mediated MCF-7 Cell Death: In Vivo Toxicity Profiling and Ex Vivo Macrophage Activation

Abstract

Background: 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. Materials and Methods: 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. Results: 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. Conclusions: 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.

Crocin, a plant-derived carotenoid, modulates microglial reactivity

“…The exact mechanisms how crocin increases microglial phagocytosis are currently unknown. However, crocin-treated peritoneal mouse macrophages also showed increased yeast phagocytosis, corroborating our data [23] .

Fig.

…”

Clinical and metabolic responses to crocin in patients under methadone maintenance treatment: A randomized clinical trial

“…It has also been demonstrated that crocin reduces DNA damage and cell necrosis and prevents the death of PC‐12 cells through sphingomyelinase‐ceramide signaling by enhancing glutathione synthesis (H. Hosseinzadeh, Abootorabi, & Sadeghnia, ; Ochiai et al, ). In addition, several reports showed that crocin has different properties on clinical and metabolic disturbance including increases in mRNA and brain‐derived neurotrophic factor, protein status of VGF neuropeptide, cAMP response element binding (Dorri et al, ; Razavi et al, ), downregulation of mitogen‐activated protein kinase, mitogen‐activated protein kinase‐activated protein signaling pathway, miRNA‐122 expression (Vahdati Hassani, Mehri, Abnous, Birner‐Gruenberger, & Hosseinzadeh, ), inhibiting apoptosis (Razavi, Hosseinzadeh, Abnous, Khoei, & Imenshahidi, ), decreased TNF‐α and IL‐6 levels in striatum (L. Mohammadzadeh, Hosseinzadeh, Abnous, & Razavi, ), modulates IL‐4/IL‐13 signaling (Yosri, Elkashef, Said, & Gameil, ), inhibiting TAU protein hyperphosphorylation, and antiapoptotic effects (Leila Mohammadzadeh, Abnous, Razavi, & Hosseinzadeh, ), alteration of gene expression profile of T24 (transitional cell carcinoma of bladder) cell (Lv, Luo, Ji, & Zhao, ), and regulate the expression of Bcl‐2 with simultaneous up regulation of pro apoptotic Bax in MCF‐7 cells (Bakshi, Hakkim, & Sam, ).…”

Dietary crocin reverses melanoma metastasis

“…As per the WHO, dietary herbs and their constituents are highly tolerable by the human system compared to synthetic products. Our previous study showed that 300 mg/kg of body weight of C. sativus extract administration to normal mice is safe and there was no adverse histopathologic differences observed in the major organs [ 35 ] . The extent of lung fibrosis during metastasis correlated with lung collagen hydroxyproline content because during lung fibrosis, collagen is deposited massively in the alveolus of the lungs.…”