PI3K-AKT Pathway Modulation by Thymoquinone Limits Tumor Growth and Glycolytic Metabolism in Colorectal Cancer.

Colorectal cancer (CRC) is the third leading cause of death in men and the fourth in women worldwide and is characterized by deranged cellular energetics. Thymoquinone, an active component from Nigella sativa, has been extensively studied against cancer, however, its role in affecting deregulated cancer metabolism is largely unknown. Further, the phosphoinositide 3-kinase (PI3K) pathway is one of the most activated pathways in cancer and its activation is central to most deregulated metabolic pathways for supporting the anabolic needs of growing cancer cells. Herein, we provide evidence that thymoquinone inhibits glycolytic metabolism (Warburg effect) in colorectal cancer cell lines. Further, we show that such an abrogation of deranged cell metabolism was due, at least in part, to the inhibition of the rate-limiting glycolytic enzyme, Hexokinase 2 (HK2), via modulating the PI3/AKT axis. While overexpression of HK2 showed that it is essential for fueling glycolytic metabolism as well as sustaining tumorigenicity, its pharmacologic and/or genetic inhibition led to a reduction in the observed effects. The results decipher HK2 mediated inhibitory effects of thymoquinone in modulating its glycolytic metabolism and antitumor effects. In conclusion, we provide evidence of metabolic perturbation by thymoquinone in CRC cells, highlighting its potential to be used/repurposed as an antimetabolite drug, though the latter needs further validation utilizing other suitable cell and/or preclinical animal models.

Nigella sativa callus treated with sodium azide exhibit augmented antioxidant activity and DNA damage inhibition.

Nigella sativa L. (NS) is an herbaceous plant, possessing phytochemicals of therapeutic importance. Thymoquinone is one of the active phytochemicals of NS that confers noteworthy antioxidant properties. Sodium azide, an agent of abiotic stress, can modulates antioxidant system in plants. In the present investigation, sodium azide (0, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM and 200 µM) doses administered to the in vitro NS callus cultures for production/modification of secondary metabolites with augmented activity. 200 µM sodium azide treated NS callus exhibited maximum peroxidase activity (1.286 ± 0.101 nanokatal mg-1 protein) and polyphenol oxidase activity (1.590 ± 0.110 nanokatal mg-1 protein), while 100 µM sodium azide treated NS callus for optimum catalase activity (1.250 ± 0.105 nanokatal mg-1 protein). Further, 200 µM sodium azide treated NS callus obtained significantly the highest phenolics (3.666 ± 0.475 mg g-1 callus fresh weight), 20 µM sodium azide treated NS callus, the highest flavonoids (1.308 ± 0.082 mg g-1 callus fresh weight) and 100 µM sodium azide treated NS callus, the highest carotenes (1.273 ± 0.066 mg g-1 callus fresh weight). However, NS callus exhibited a decrease in thymoquinone yield/content vis-à-vis possible emergence of its analog with 5.3 min retention time and an increase in antioxidant property. Treatment with 200 µM sodium azide registered significantly the lowest percent yield of callus extract (4.6 ± 0.36 mg g-1 callus fresh weight) and thymoquinone yield (16.65 ± 2.52 µg g-1 callus fresh weight) and content (0.36 ± 0.07 mg g-1 callus dry weight) and the highest antioxidant activity (3.873 ± 0.402%), signifying a negative correlation of the former with the latter. DNA damage inhibition (24.3 ± 1.7%) was recorded significantly maximum at 200 µM sodium azide treatment. Sodium azide treated callus also recorded emergence of a new peak at 5.3 min retention time (possibly an analog of thymoquinone with augmented antioxidant activity) whose area exhibits significantly negative correlation with callus extract yield and thymoquinone yield/content and positive correlation with antioxidant activity and in vitro DNA damage inhibition. Thus, sodium azide treatment to NS callus confers possible production of secondary metabolites or thymoquinone analog (s) responsible for elevated antioxidant property and inhibition to DNA damage. The formation of potent antioxidants through sodium azide treatment to NS could be worthy for nutraceutical and pharmaceutical industries.

Dichlorvos Induced Oxidative and Neuronal Responses in Rats: Mitigative Efficacy of Nigella sativa (Black Cumin).

Poisoning from Organophosphates (OPs), especially Dichlorvos (DDVP) has become endemic due to theincreasing use in house hold and agricultural pests control, with most marked effects in the nervous system. However, it isevidenced that natural antioxidants are efficacious against OPs toxicity. Thus, this study investigated the possible antidotalefficacy of Nigella sativa oil (NSO) in Dichlovos (DDVP) induced oxidative and neuronal damages in Wistar rats. DDVPwas administered at sub-chronic daily dosage of 8.8 mg/kg.bw for 7 days and a post-administration of NSO at 1 ml/kg.bwfor the subsequent 7 days. The rats were euthanized on the 15thday, blood sample collected via cardiac puncture, centrifugedand the plasma used for biochemical analysis of total antioxidant capacity (TAC), reduced glutathione (GSH) and totalreactive oxygen species (ROS), while the frontal, occipital and cerebellar cortices and the medulla were removed for histomorphological examinations. The results showed significant (P≤0.05) decrease in plasma TAC and GSH, while a significant(P≤0.05) increase in ROS was recorded, and some vacuolation around the neurons especially in the frontal and cerebellarcortices following DDVP exposure. However, post treatment with NSO was observed to be efficacious in the recovery ofthe oxidative activities and the neuro-architectural integrities. Thus, it can be concluded that the antioxidant capacity of NSOcould be efficacious against OPs induced oxidative damages, especially in dichlorvos accidents.

Effect of nanoparticle treatment on expression of a key gene involved in thymoquinone biosynthetic pathway in Nigella sativa L.

Thymoquinone is the most important secondary metabolite in black Cumin, which has several pharmaceutical applications. In this study, effect of TiO2 and SiO2 nanoparticles as new elicitors, on expression of Geranyl diphosphate synthase gene (GPPS gene), as a key gene involved in thymoquione biosynthesis pathway was investigated in two Iranian accessions. Plants were treatment in the early flowering stage and after 24 h of 50 and 100 mg/L of each nanoparticle, separately. After RNA extraction, GPPS gene expression was analysed by qRT-PCR method. The results showed that the TiO2 and SiO2 nanoparticles, generally stimulates the GPPS expression. The TiO2 nanoparticles were more effective than SiO2 for the induction of GPPS expression. Also, 100 mg/L treatment of nanoparticles raised gene expression more than 50 mg/L concentration. It can be concluded these nanoparticles can be used as robust elicitors to enhance the production of Thymoquinone in black cumin through up-regulation of related metabolic pathway genes.

Nigella Sativa Oil Protects the Rat Ovary from Oxidative Injury Due to Ischemia-Reperfusion.

BACKGROUND The aim of this study was to evaluate the effects of Nigella sativa (N. sativa) oil (NSO) on ovarian oxidative damage following ischemia-reperfusion injury, using a rat model of ovarian torsion. MATERIAL AND METHODS Forty-eight female albino Wistar rats were divided into six groups: (Group 1) laparotomy only; (Group 2) intraperitoneal NSO (2 ml/kg), 1 hour following laparotomy; (Group 3) 3 hours of ovarian ischemia; (Group 4) 3 hours of ovarian ischemia followed by 3 hours of reperfusion; (Group 5) 3 hours of ovarian ischemia and 2 ml/kg of NSO 1 hour before laparotomy; (Group 6) 3 hours of reperfusion after 3 hours of ovarian ischemia and 2 ml/mg of NSO 1 hour before laparotomy. RESULTS The antioxidant status, ceruloplasmin level, native thiol, total thiol, and disulfide levels of the control group (Group 1) were significantly increased compared with the ovarian ischemia-reperfusion group treated with NSO (Group 6) (p=0.003, p=0.002, p=0.006, p=0.001 and p=0.003, respectively); these levels in the ovarian ischemia group (Group 3) and ischemia-reperfusion group (Group 4) were statistically similar to those of the ovarian ischemia + NSO group (Group 5) and ovarian ischemia-reperfusion + NSO group (Group 6). CONCLUSIONS In this preliminary rat study, administration of NSO shortly after the onset of ovarian ischemia-reperfusion injury, did not significantly reduce levels of markers of oxidative injury. Further studies are required to evaluate the ovarian changes at the tissue level, and to determine the optimum dose of NSO.

Methyl jasmonate induced accumulation of kalopanaxsaponin I in Nigella sativa.

Hydroponically cultivated Nigella sativa L. plants treated with methyl jasmonate (MeJA) showed a twelve-fold increase in levels of the monodesmosidic triterpene saponins alpha-hederin and kalopanaxsaponin I (KsI) in the leaves. We will demonstrate that these two saponins accounted for approximately 10% of the dry plant matter, of which 93% was KsI and 7% alpha-hederin. To address the molecular basis of saponin induction by MeJA, we cloned and characterized the beta-amyrin synthase gene (NsbetaAS1) encoding one of the key enzymes in triterpene saponin biosynthesis. As expected, NsbetaAS1 transcription was induced by MeJA and led to the production of beta-amyrin when over-expressed in yeast.