Correction: Frankincense essential oil suppresses melanoma cancer through down regulation of Bcl-2/Bax cascade signaling and ameliorates heptotoxicity via phase I and II drug metabolizing enzymes.

[This corrects the article DOI: 10.18632/oncotarget.26930.].

Frankincense essential oil suppresses melanoma cancer through down regulation of Bcl-2/Bax cascade signaling and ameliorates heptotoxicity via phase I and II drug metabolizing enzymes.

Melanoma is a deadly form of malignancy and according to the World Health Organization 132,000 new cases of melanoma are diagnosed worldwide each year. Surgical resection and chemo/drug treatments opted for early and late stage of melanoma respectively, however detrimental post surgical and chemotherapy consequences are inevitable. Noticeably melanoma drug treatments are associated with liver injuries such as hepatitis and cholestasis which are very common. Alleviation of these clinical manifestations with better treatment options would enhance prognosis status and patients survival. Natural products which induce cytotoxicity with minimum side effects are of interest to achieve high therapeutic efficiency. In this study we investigated anti-melanoma and hepatoprotective activities of frankincense essential oil (FEO) in both in vitro and in vivo models. Pretreatment with FEO induce a significant (p < 0.05) dose-dependent reduction in the cell viability of mouse (B16-F10) and human melanoma (FM94) but not in the normal human epithelial melanocytes (HNEM). Immunoblot analysis showed that FEO induces down regulation of Bcl-2 and up regulation of BAX in B16-F10 cells whereas in FM94 cells FEO induced dose-dependent cleavage of caspase 3, caspase 9 and PARP. Furthermore, FEO (10 µg/ml) treatment down regulated MCL1 in a time-dependent manner in FM94 cells. In vivo toxicity analysis reveals that weekly single dose of FEO (1200 mg/kg body weight) did not elicit detrimental effect on body weight during four weeks of experimental period. Histology of tissue sections also indicated that there were no observable histopathologic differences in the brain, heart, liver, and kidney compare to control groups. FEO (300 and 600 mg/kg body weight) treatments significantly reduced the tumor burden in C57BL/6 mice melanoma model. Acetaminophen (750 mg/kg body weight) was used to induce hepatic injury in Swiss albino mice. Pre treatment with FEO (250 and 500 mg/kg body weight) for seven days retained hematology (complete blood count), biochemical parameters (AST, ALT, ALK, total bilirubin, total protein, glucose, albumin/globulin ratio, cholesterol and triglyceride), and the level of phase I and II drug metabolizing enzymes (cytochrome P450, cytochromeb5, glutathione-S-transferase) which were obstructed by the administration of acetaminophen. Further liver histology showed that FEO treatments reversed the damages (central vein dilation, hemorrhage, and nuclei condensation) caused by acetaminophen. In conclusion, FEO elicited marked anti-melanoma in both in vitro and in vivo with a significant heptoprotection.

Dietary Crocin Reverses Melanoma Metastasis.

Crocus sativus and its bioactive constituent crocin are well known for anti-tumor potential in different models. However, the efficacy of crocin on in-vivo melanoma metastasis is not yet reported. In this study, melanoma metastatic model was developed by tail vein injection of B16F-10 cells in to C57BL/6 mice. Metastatic mice treated with two different doses of crocin (250 and 500 µg/kg of bodyweight) for 10 days and parameters such as lung metastasis inhibition, mean survival time, lung hydroxyproline, uronic acid and hexosamine levels were analyzed after 21 days of treatment. Then blood was collected and serum gamma glutamyl transpeptidase (g-GGT), sialic acid, tumor necrosis factor alpha (TNF-a), interleukin 10 (IL-10), IL-6, IL-2, and TIMP-1 levels were measured. Further, a lung histological examination was done in crocin treated metastatic mice. Subsequently hallmark metastatic parameters such as matrix metalloproteases (MMPs), extracellular regulated kinase 2 (ERK2), vascular endothelial growth factor (VEGF), and K-ras gene expression were investigated in the lungs of crocin treated metastatic mice. Further, in-vitro adhesion, invasion and migration of B16F-10 cells were examined after 24 h of crocin (5 and 10 µg/mL) treatment. Administration of crocin to tumor bearing C57BL/6 mice reduced the lung metastasis by 85%. Elevated levels of hydroxyproline, uronic acid, hexosamine, serum sialic acid andg-GGT in metastatic control were found to be significantly reduced in crocin treated mice. Crocin also inhibited expression of MMP-2, MMP-9, ERK-2, K-ras, and VEGF. Crocin reduced the ability of B16F-10 cells invasion (p<0.05), migration (p<0.05) and adhesion by upregulating E-cadherin expression. In conclusion, crocin elicited marked anti-metastatic potential by regulating the metastasis induced biomarkers.

Role of Dietary Crocin in In Vivo Melanoma Tumor Remission

Background: Melanoma is a deadly form of malignancy. Early diagnosis might pave the way to cure but its aggressive nature leads to rapid dissemination and colonization of distant organs. Dietary herbs may play a significant role in prevention of cancer. In this study, we tested anti-tumor efficacy of the Crocus sativus derived active constituent crocin, it is well established to have anti-cancer properties in different cancer models by our group and other groups. Notably, crocin is reported to exert anti-proliferative effect on melanoma cells (B16F10) in vitro. However, roles of crocin on in vivo melanoma tumor remission have not yet been reported to our knowledge. Materials and Methods: Melanoma tumor model was established by transplanting B16F10 (5 X 105) cells into C57BL/6 mice, which were then observed for tumor development and once the tumor volume reached 6 mm, mice were divided into (Group I: tumor-bearing animals treated with normal saline and Group II: counterparts treated with crocin at 2 mg/kg body weight for 21 days). . Tumor remission and tumor growth related parameters such as tumor silent period (TSP), tumor volume doubling time (VDT), growth delay (GD), and mean survival time (MST) were determined. In addition, serum protein profiles were analyzed. Results: The 21 days crocin treatment significantly reduced the tumor burden in mice, extending the mean survival time significantly as compared to control. Crocin treatment also significantly increased the TGD and TSP and decreased VDT. Furthermore, while serum proteins such as albumin and globulin (alpha1, alpha2, beta, and gamma) were altered due to tumor burden, crocin treatment resulted in their levels near to normal at the end of the experimental period. Conclusion: Our study provided clear evidence that crocin may exhibit significant melanoma tumor remission properties by positively modulating tumor growth related parameters. In future, the molecular mechanisms of crocin action should be studied extensively in melanoma models before defining crocin-based melanoma drug formulations.

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

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.

DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by crocin from kashmiri saffron in a human pancreatic cancer cell line.

BACKGROUND: Apoptosis, a widely important mechanism that contributes to cell growth reduction, is reported to be induced by Crocus sativus in different cancer types. The present study was designed to elucidate apoptosis induction by crocin, a main component of Crocus sativus in a human pancreatic cancer cell line (BxPC-3). METHODS: Cell viability was measured by MTT assay, Hoechest33258 staining was used to detect the chromatin condensation characteristic of apoptosis, and DNA fragmentation was assessed by gel electrophoresis and cell cycle analysis by flow cytometry. RESULTS: Crocin induced apoptosis and G1-phase cell cycle arrest of BxPC-3 cells, while decreasing cell viability in a dose dependent and time dependent manner. Cells treated with 10µg/L crocin exhibited apoptotic morphology (brightly blue-fluorescent condensed nuclei on Hoechst 33258 staining) and reduction of volume. DNA analysis revealed typical ladders as early as 12 hours after treatment indicative of apoptosis. CONCLUSION: Our preclinical study demonstrated a pancreatic cancer cell line to be highly sensitive to crocin-mediated growth inhibition and apoptotic cell death. Although the molecular mechanisms of crocin action are not yet clearly understood, it appears to have potential as a therapeutic agent.

Crocin from Kashmiri saffron (Crocus sativus) induces in vitro and in vivo xenograft growth inhibition of Dalton's lymphoma (DLA) in mice.

In this study we investigated in vitro and in vivo xenograft growth inhibition by crocin isolated from Kashmiri saffron (Crocus sativus). It was found that crocin decreased cell viability in DLA cells, in a concentration- and time-dependent manner. Significant increase in the lifespan of Dalton's lymphoma bearing animals was noted by 37% and 44%, respectively. Furthermore, animals given treatment before induction of cancer showed 58% increase in lifespan and there was 95.6% reduction of solid tumor in crocin treated animals on the 31st day after tumor inoculation. Crocin also showed significant impact on hematological parameters, like the hemoglobin count and numbers of lymphocytes. These findings support the conclusion that crocin from Crocus sativus has significant anti-tumor activity.