百里醌诱导膀胱癌细胞凋亡作用机制的研究

目的 探讨百里醌对人膀胱癌细胞株BIU-87细胞凋亡的影响及对其诱导凋亡的潜在作用机制。方法 应用CCK8法检测细胞增殖活性, Hoechst33258染色法检测细胞凋亡, 荧光定量PCR检测基因Bcl-2、Bax、caspase-3、c-myc mRNA的表达水平, Wsetern blot法检测不同浓度百里醌作用后Bcl-2、Bax、c-myc蛋白表达水平的变化。结果 细胞增殖抑制曲线结果显示百里醌能明显抑制BIU-87细胞增殖, 其24、48、72h半数抑制浓度(IC₅₀)分别是38.75±0.58、34.33±1.01和32.43±0.71μmol/ L。百里醌能以剂量依赖性方式诱导膀胱癌细胞凋亡。百里醌作用于BIU-87细胞后, Bcl-2、c-myc mRNA及Bcl-2、c-myc蛋白的表达量呈浓度依赖性降低, 而caspase-3、Bax mRNA及caspase-3、Bax蛋白表达水平呈浓度依赖性递增。结论 百里醌能抑制BIU-87细胞的增殖, 且能诱导BIU-87细胞的凋亡, 其诱导凋亡机制可能与Bcl-2、c-myc表达水平降低及caspase-3、Bax表达水平上调有关。     

Thymoquinone: An edible redox-active quinone for the pharmacotherapy of neurodegenerative conditions and glial brain tumors. A short review

There exist few efficient agents in the neurological and neurosurgical armamentarium for treatment of neurotrauma, refractory seizures and high grade glial tumors. Pathophysiological conditions of diverse neural injuries have converging common pathways including oxidative stress and apoptosis. Targeted therapies have been throughly investigated, but limited success has been achieved until now. Phytochemical drugs may provide easily achievable and cheap adjunctive sources. Thymoquinone is an edible quinone obtained from Nigella sativa seed oil and exerts powerful antiinflammatory, antioxidant and antitumor activities in experimental models. Recently emerging studies conducted with animal models suggest that thymoquinone – bearing a very simple molecular structure – significantly crosses the blood brain barrier and exerts neuromodulatory activities. Indeed, in animal studies, the following actions of thymoquinone were demonstrated: 1—Protection against ischemic brain damage. 2—Reduction of epileptic seizures and associated cerebral oxidative injury. 3—Reduction of morphine tolerance and associated oxidative brain damage. 4—Anxiolytic effects and reduction of immobility stress-associated cerebral oxidative injury. 5—Reduction of diabetes-induced cerebral oxidative stress, 6—Reduction of cerebral oxidative injuries induced by noxious exposures including toluene, lead and ionizing radiation. Substantial in vitro data suggest that thymoquinone may be beneficial in treatment of glial tumors. However, there is no clinical study investigating its antitumor effects. In fact, thymoquinone suppresses growth and invasion, and induces apoptosis of glial tumor cells via degrading tubulins and inhibiting 20S proteasome, telomerase, autophagy, FAK and metalloproteinases. A simple and easily available agent may be a promising adjunctive treatment option in neurological and neurosurgical practice.     

The Th1/Th2 paradigm in lambda cyhalothrin-induced spleen toxicity: The role of thymoquinone

This study investigates the retrofitted role of thymoquinone (TQ) in the Th1/Th2 paradigm imbalance in lambda-cyhalothrin (LCT) treated rats. Four groups of male Wistar rats were formed: Group I served as control. Group II received 5 mg TQ/(kg bw) daily. Group III received 0.6 mg LCT/(kg bw). Group IV was treated with TQ and LCT. All treatments were given orally for 10 weeks. The LCT-treated group elicited a significant increase in MDA and NO levels with up-regulation of NF-κB/p65 and pro-inflammatory genes expression and their levels. Meanwhile, GSH and immunoglobulins concentrations were markedly decreased concomitant with lessening the activities of antioxidant enzymes and anti-inflammatory cytokine genes mRNA levels. The co-administration of TQ and LCT improved the altered antioxidant enzymes activities and concentration of cytokines with attenuation of NF-κB/p65 mRNA. These data support the antioxidant role of TQ in the Th1/Th2 imbalance paradigm during LCT toxicity.     

Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1

The salvage anti-tumoral pathway which implicates the p53-related p73 gene is not yet fully characterized. We therefore attempted to identify the up- and down-stream events involved in the activation of the p73-dependent pro-apoptotic pathway, by focusing on the anti-apoptotic and epigenetic integrator UHRF1 which is essential for cell cycle progression. For this purpose, we analyzed the effects of a known anti-neoplastic drug, thymoquinone (TQ), on the p53-deficient acute lymphoblastic leukemia (ALL) Jurkat cell line. Our results showed that TQ inhibits the proliferation of Jurkat cells and induces G1 cell cycle arrest in a dose-dependent manner. Moreover, TQ treatment triggers programmed cell death, production of reactive oxygen species (ROS) and alteration of the mitochondrial membrane potential (ΔΨm). TQ-induced apoptosis, confirmed by the presence of hypodiploid G0/G1 cells, is associated with a rapid and sharp re-expression of p73 and dose-dependent changes of the levels of caspase-3 cleaved subunits. These modifications are accompanied by a dramatic down-regulation of UHRF1 and two of its main partners, namely DNMT1 and HDAC1, which are all involved in the epigenetic code regulation. Knockdown of p73 expression restores UHRF1 expression, reactivates cell cycle progression and inhibits TQ-induced apoptosis. Altogether our results showed that TQ mediates its growth inhibitory effects on ALL p53-mutated cells via the activation of a p73-dependent mitochondrial and cell cycle checkpoint signaling pathway which subsequently targets UHRF1.     

百里醌对体内外乳腺癌细胞生长和凋亡的作用

目的 研究百里醌在体内外对人乳腺癌细胞株MCF-7生长和凋亡的影响,并探讨其作用机制.方法 采用四甲基偶氮唑蓝(MTT)法检测细胞增殖;Annexin V-FITC/PI双染法检测细胞凋亡;Western blot检测survivin以及半胱天冬酶(pro-caspase 3、pro-caspase 8、pro-caspase 9)蛋白的表达,并利用活力检测试剂盒检测caspase 3、caspase 8、caspase 9的活力;建立裸鼠乳腺癌皮下移植瘤模型,原位末端标记(TUNEL)法检测肿瘤组织细胞的凋亡情况;采用免疫组化方法检测肿瘤组织中ki-67、survivin和caspase 3的表达.结果 百里醌对体外MCF-7细胞有明显的增殖抑制和凋亡诱导作用;百里醌可下调MCF-7细胞中survivin、pro-caspase 3和pro-caspase 8的表达,但对pro-caspase 9的表达无明显影响;活力测定结果进一步显示,百里醌作用MCF-7细胞后,caspase 3和caspase 8被激活,而对caspase 9无明显影响;在体内试验中,百里醌可抑制裸鼠皮下移植瘤生长,同时诱导体内乳腺癌细胞凋亡,另外百里醌还可分别下调和上调肿瘤组织细胞中survivin和caspase 3的表达.结论 百里醌对体内外乳腺癌细胞有凋亡诱导作用,死亡配体途径可能是百里醌诱导乳腺癌细胞凋亡的主要机制之一.     

Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apoptosis with down-regulation of Bcl-2 protein

Thymoquinone (TQ), the active constituent of Nigella sativa or black cumin exhibited cytotoxic effects in several cancer cell lines. In this study, the cytotoxicity of TQ in human cervical squamous carcinoma cells (SiHa) was investigated. TQ was cytotoxic towards SiHa cells with IC50 values of 10.67 ± 0.12 and 9.33 ± 0.19 μg/mL as determined by MTT assay and trypan blue dye exclusion test, respectively, after 72 h of incubation. TQ was more cytotoxic towards SiHa cells compared to cisplatin. Interestingly, TQ was less cytotoxic towards the normal cells (3T3-L1 and Vero). Cell cycle analysis performed by flowcytometer showed a significant increase in the accumulation of TQ-treated cells at sub-G1 phase, indicating induction of apoptosis by the compound. Apoptosis induction by TQ was further confirmed by Annexin V/PI and AO/PI staining. Significant elevation of p53 and down-regulation of the anti-apoptotic Bcl-2 protein was found in the treated cells, without any changes in the expression of the pro-apoptotic Bax protein. In conclusion, thymoquinone from N. sativa was more potent than cisplatin in elimination of SiHa cells via apoptosis with down-regulation of Bcl-2 protein.     

Thymoquinone induces heme oxygenase-1 expression in HaCaT cells via Nrf2/ARE activation: Akt and AMPKα as upstream targets

Thymoquinone (TQ), an active constituent of Nigella sativa, possesses anti-inflammatory and anticancer properties. Multiple lines of evidence suggest that the induction of heme oxygenase-1 (HO-1) suppresses inflammation and carcinogenesis. In the present study, we examined the effect of TQ on HO-1 expression in human keratinocytes (HaCaT) and elucidated its underlying molecular mechanisms. TQ induced the expression of HO-1 in HaCaT cells in a concentration- and time-dependent manner. Treatment with TQ increased the localization of nuclear factor (NF)-erythroid2-(E2)-related factor-2 (Nrf2) in the nucleus and elevated the antioxidant response element (ARE)-reporter gene activity. Knockdown of Nrf2 abrogated TQ-induced HO-1 expression and the ARE luciferase activity. TQ induced the phosphorylation of extracellular signal-regulated kinase (ERK), Akt and cyclic AMP-activated protein kinase-α (AMPKα). Pharmacological inhibition of Akt or AMPKα, but not that of ERK, abrogated TQ-induced nuclear localization of Nrf2, the ARE-luciferase activity and the expression of HO-1. TQ also generated reactive oxygen species (ROS) and pretreatment with N-acetyl cysteine (NAC) abrogated TQ-induced ROS accumulation, Akt and AMPKα activation, Nrf2 nuclear localization, the ARE-luciferase activity, and HO-1 expression in HaCaT cells. Taken together, TQ induces HO-1 expression in HaCaT cells by activating Nrf2 through ROS-mediated phosphorylation of Akt and AMPKα.     

Macrophage-derived cytokine and nitric oxide profiles in type I and type II diabetes mellitus: effect of thymoquinone

Abstract Comparing macrophage-derived cytokine and nitric oxide (NO) profiles in type I and type II diabetes mellitus (DM); and determining whether thymoquinone (TQ) has any modulatory effect were the main objectives of the present study. Peritoneal macrophages have been collected from Otsuka Long-Evans Tokushima Fatty (OLETF) as a model for type II DM and its control Long-Evans Tokushima Otsuka (LETO) rats, as well as from streptozotocin (STZ)-injected LETO ones as a model for type I DM. The cells were cultured and incubated with or without TQ (10 µM) in the absence or presence of lipopolysaccharide (LPS; 1 µg/ml). The same parameters have been also assessed in sera of the used animals with or without TQ treatment (3 mg/kg) under both LPS-stimulated (10 mg/kg) and unstimulated conditions. Nitrite, IL-1 and TNF- were significantly higher in macrophage supernatants and sera of the acutely affected STZ-LETO rats either with or without LPS stimulation compared to corresponding controls. On the other hand, chronically diabetic OLETF rats macrophage supernatants showed significant decreases of IL-1 and TNF- levels upon LPS stimulation or even without stimulation (IL-1); and insignificant increase in nitrite concentration, which turned significant upon LPS stimulation. Sera of these animals, however, showed significant increase in TNF- level. TQ normalised the elevated nitrite and cytokine profiles both in vitro and in vivo, yet had no significant effect on the already decreased parameters in chronically affected OLETF rats. These data suggest that there is a tendency for macrophage inflammatory products to increase in acute type I and to decrease in chronic type II DM; and that TQ has the potential to normalise the elevated levels of these macrophage-derived inflammatory mediators.     

Prevention of doxorubicin-induced experimental cardiotoxicity by Nigella sativa in rats

IntroductionDoxorubicin (DOX) is an anthracycline cytotoxic chemotherapeutic drug that is commonly used in cancer treatment. A major side effect limiting the clinical use of DOX is cardiotoxicity due to oxidative injury. Nigella sativa (NS) is an annual flowering plant with antioxidant properties. Its seeds contain several bioactive constituents such as saturated and unsaturated fatty acids, thymoquinone, dithymoquinone, thymohydroquinone, and thymol. In this study, we investigated the effect of NS extract on DOX-induced cardiotoxicity.MethodsThe experimental study animals consisted of 28 male Sprague Dawley rats weighing between 300 and 400 g. Four study groups each of seven rats were defined: controls; NS extract; DOX; and DOX+NS. Control and DOX rats received standard food, while each rat in the NS and DOX+NS groups also received 100 mg/kg NS extract orally. At day 28 of follow-up, rats in the DOX groups were administered a single 10 mg/kg intraperitoneal dose of DOX, while rats in the control and NS groups received a single 10 mg/kg dose of physiological saline solution. All animals were monitored for 35 days. On day 35, the rats were decapitated and serum and cardiac tissue samples were obtained. Troponin and NT-proBNP levels were measured in blood sera, while malondialdehyde (MDA), nitric oxide, total antioxidant capacity (TAC), and total oxidative stress (TOS) levels were quantified in sera and tissue samples. Histological alterations that were assessed in cardiac tissue included myocyte disarray, small vessel disease, myocyte hypertrophy, and fibrosis.ResultsThe DOX group had significantly higher NT-proBNP, TOS, and MDA, with greater histopathological derangement. TAC was significantly elevated in the DOX+NS group, which also exhibited significantly lower troponin, TOS, and MDA, as well as significantly higher TAC compared to the DOX group. Histopathological examination showed that the significant structural derangement observed in DOX rats was markedly and significantly reduced in DOX+NS rats.ConclusionOur results suggest that NS extract may prevent DOX-induced cardiotoxicity and thus represents a promising cardioprotective agent.