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Thymoquinone is the main active compound derived from the essential oil of the Nigella sativa plant seed. While thymoquinone is an antioxidant, it has been reported in several studies that thymoquinone has dose-dependent pro-oxidant activity with the Fenton reaction in the presence of transition elements such as iron and copper. This study aimed to investigate cytotoxic, apoptotic, genotoxic, and reactive oxygen species (ROS) generating effects of thymoquinone treated with copper in colon cancer cells. HT-29 cells were treated with pro-oxidant-acting doses of thymoquinone alone and together with the non-toxic dose of Copper (II) Sulfate for 24 h. Cytotoxic, apoptotic, genotoxic, and ROS production activities were analyzed by MTT viability test, Acridine Orange/Ethidium Bromide (AO/EB) staining, alkaline single cell gel electrophoresis and H2DCF-DA assay, respectively. Viability results showed that thymoquinone and copper synergistically affect cancer cells, and DNA damage was increased with the synergic effect. The intracellular ROS was increased when thymoquinone and copper were applied together. Applying redox-active copper (II) with thymoquinone increases DNA damage, apoptosis, and cell death by increasing the amount of intracellular ROS through pro-oxidant activity. Treatments targeting copper-related pathways may open new therapeutic avenues for cancer treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Natural compounds such as thymoquinone (TQ) have recently gained increasing attention in treating glioblastoma (GBM). However, the effects of TQ in reversing drug resistance are not completely understood. Therefore, we aimed to examine TQ impacts on GBM cells with doxorubicin (DOX) resistance and the involvement of the PI3K/Akt/mTOR pathway. GBM cancer U87 and U87/DOX (resistant cells) cells were exposed to DOX and TQ, and cell proliferation was assessed by the MTT assay. ELISA was applied to evaluate cell apoptosis. The expression of apoptotic mediators such as Caspase-3, Bax, Bcl-2 and PI3K, Akt, mTOR, P-gp, and PTEN was assessed via qRT-PCR and western blot. We found that a combination of TQ and DOX suppressed dose-dependent cell growth capacity in cells and increased the cytotoxic effects of DOX in resistant cells. In addition, TQ treatment increased DOX-mediated apoptosis in U87/DOX cell lines via modulating the pro- and anti-apoptotic markers. A combination of TQ and DOX upregulated PTEN and downregulated PI3K, Akt, and mTOR, suppressing this signal transduction in resistant cells. In conclusion, we showed TQ potentiated doxorubicin-mediated antiproliferative and pro apoptotic function DOX-resistant glioblastoma cells, which is mediated by targeting and suppressing PI3K/Akt/mTOR signal transduction., (© 2024 John Wiley & Sons A/S.)

Background: Studies have concentrated on the therapeutic potential of thymoquinone (TQ), a natural polyphenol, in diverse malignancies, such as colorectal cancer. Nevertheless, the precise mechanisms of TQ-mediated anticancer properties are not yet fully elucidated. Objective: The present study has been designed to scrutinize the impact of TQ on 5-fluorouracil (5-FU)-mediated apoptosis in SW-480 cells. Materials and Methods: SW-480 cells were treated with TQ, 5-FU, and a combination of TQ + 5-FU. MTT assay was employed to assess cell viability. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to evaluate apoptotic markers comprising Bcl-2, Bax, and caspase-9 expression levels. The γ -H2AX protein expression was assessed by western blotting, and Annexin V flow cytometry was implemented to determine the apoptosis rate. Results: 5-FU significantly reversed the cell proliferation in a dose-dependent circumstance. The concurrent administration of TQ and 5-FU led to a substantial inhibition of cell growth in comparison to single treatments ( p < 0.05). TQ also facilitated apoptosis via upregulating Bax and caspase-9 proapoptotic markers and suppressing antiapoptotic mediators, like Bcl-2. In addition, TQ augmented 5-FU-induced apoptosis in SW-480 cells. 5-FU, combined with TQ, increased the protein expression of γ -H2AX in SW-480 cells compared with groups treated with TQ and 5-FU alone. Conclusion: The present study's findings unveil the significance of TQ as a potential therapeutic substance in colorectal cancer, particularly through enhancing 5-FU-induced apoptosis., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Nima Zeinali et al.)

Purpose: In this study, the effect of thymoquinone (TQ) on CP-induced spermatogenesis defects in mice has been investigated., Methods: Sperm parameters, serum testosterone concentration, histology, Bax/Bcl-2 ratio, and expression of autophagy-related biomarkers have been assessed. Total antioxidant capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI) in testicular tissue were examined for the evaluation of oxidative stress levels., Results: CP has induced histological changes and significantly increased the Bax/Bcl-2 ratio, decreased testosterone concentration, testicular weight, and sperm quality. CP induced oxidative stress by elevating OSI in the testicular tissue (p < 0.05). Expression of the autophagy-inducer genes (ATG7, ATG5, and Beclin-1) and ratio of LC3B/LC3A proteins were significantly decreased, while mTOR expression was increased in the CP group. TQ pretreatment dose-dependently decreased the Bax/Bcl-2 ratio and mTOR gene expression while increasing the expression of ATG5 and ATG7 genes, LC3B/LC3A ratio, and Beclin-1 proteins. TQ could also dose-dependently reverse the histology, testosterone level, and sperm quality of the CP-intoxicated mice., Conclusions: These findings show that TQ pretreatment can enhance sperm production by inducing autophagy and reducing apoptosis and oxidative stress in the CP-intoxicated mouse testicles., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Pancreatic cancer (PC) is associated with some of the worst prognoses of all major cancers. Thymoquinone (TQ) has a long history in traditional medical practice and is known for its anti-cancer, anti-inflammatory, anti-fibrosis and antioxidant pharmacological activities. Recent studies on hypoxia-inducible factor-1α (HIF-1α) and PC have shown that HIF-1α affects the occurrence and development of PC in many aspects. In addition, TQ could inhibit the development of renal cancer by decreasing the expression of HIF-1α. Therefore, we speculate whether TQ affects HIF-1α expression in PC cells and explore the mechanism., Aim: To elucidate the effect of TQ in PC cells and the regulatory mechanism of HIF-1α expression., Methods: Cell counting kit-8 assay, Transwell assay and flow cytometry were performed to detect the effects of TQ on the proliferative activity, migration and invasion ability and apoptosis of PANC-1 cells and normal pancreatic duct epithelial (hTERT-HPNE) cells. Quantitative real-time polymerase chain reaction and western blot assay were performed to detect the expression of HIF-1α mRNA and protein in PC cells. The effects of TQ on the HIF-1α protein initial expression pathway and ubiquitination degradation in PANC-1 cells were examined by western blot assay and co-immunoprecipitation., Results: TQ significantly inhibited proliferative activity, migration, and invasion ability and promoted apoptosis of PANC-1 cells; however, no significant effects on hTERT-HPNE cells were observed. TQ significantly reduced the mRNA and protein expression levels of HIF-1α in PANC-1, AsPC-1, and BxPC-3 cells. TQ significantly inhibited the expression of the HIF-1α initial expression pathway (PI3K/AKT/mTOR) related proteins, and promoted the ubiquitination degradation of the HIF-1α protein in PANC-1 cells. TQ had no effect on the hydroxylation and von Hippel Lindau protein mediated ubiquitination degradation of the HIF-1α protein but affected the stability of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90, thus promoting its ubiquitination degradation., Conclusion: The regulatory mechanism of TQ on HIF-1α protein expression in PC cells was mainly to promote the ubiquitination degradation of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90; Secondly, TQ reduced the initial expression of HIF-1α protein by inhibiting the PI3K/AKT/mTOR pathway., Competing Interests: Conflict-of-interest statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Diabetes mellitus (DM) is a complex metabolic condition that causes organ dysfunction. The current experiment sought to determine the effect of thymoquinone (TQ) on hyperglycemia, hyperlipidemia, oxidative/nitrosative stress, inflammation, and apoptosis in diabetic rats prompted by streptozotocin (STZ) (55 mg/kg body weight i/p). The animals were allocated into control, TQ (50 mg/kg B.W. orally administered for 4 succeeding weeks), Diabetic, and Diabetic + TQ groups. This study confirmed that TQ preserves the levels of insulin, fasting blood glucose, HOMA β-cell indices, HbA1c %, body weight, and lipid profile substantially relative to the DC group. Furthermore, hepatic antioxidant (CAT, GSH, and T-SOD) values were reduced. Conversely, the enzymatic activity of liver functions (AST, ALT, ALP, cytochrome P450, and hepatic glucose-6-phosphatase), lipid peroxidation (MDA), pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6), nitric oxide (NO) and inflammatory marker (CRP) enhanced with STZ administration, which is substantially restored after TQ treatment. Relative to the diabetic rats, TQ reestablished the hepatic architectural changes and collagen fibers. Additionally, TQ downregulated the intensity of the immunohistochemical staining of pro-apoptotic marker (caspase-3), p53, and tumor necrosis factor-alpha (TNF-α) proteins in hepatic tissues. Furthermore, TQ displayed abilities to interact and inhibit the binding site of caspase-3, interleukin-6 receptor, interleukin-1 receptor type 1, TNF receptor superfamily member 1A, and TNF receptor superfamily member 1B in rats following the molecular docking modeling. All these data re-establish the liver functions, antioxidant enzymes, anti-inflammatory markers, and anti-apoptotic proteins impacts of TQ in STZ-induced DM rats. Founded on these outcomes, the experiment proposes that TQ is a novel natural supplement with various clinical applications, including managing DM, which in turn is recommended to play a pivotal role in preventing the progression of diabetes mellitus., (© 2024. The Author(s).)

Background: Tongue cancer is the most prevalent type of oral cancer. Recently, natural compounds have been considered important resources for several anticancer drugs. Thymoquinone (TQ) exhibits a potent anti-cancer effect. 5-Fluorouracil (5-FU) is a chemotherapeutic drug that has been utilized in the treatment of cancer. Recently, combination therapy has gained popularity as a treatment option for patients with cancer., Objectives: The present study was carried out to assess the cytotoxic effect of 5-Fluorouracil (5-FU), Thymoquinone (TQ), and their combination on tongue squamous cell carcinoma cell line (HNO-97)., Methods: Tongue carcinoma cell line (HNO-97) was maintained in cultured flasks and the cells were divided into four groups; group Ι: control untreated group, group ΙΙ: HNO-97-treated cells with different concentrations of 5-FU from 0.5 µM/ml to 3µM/ml, group ΙIΙ: HNO-97-treated cells with different concentrations of TQ from 7.25µM/ml to 23.05µM/ml, and group ΙV: HNO-97-treated cells with both 5-FU and TQ in serial concentrations  till (IC50) in a dose of 27.44 µM/ml. Determination of the cytotoxic effect of the tested agents on the HNO-97 cell line was done using methyl thiazole tetrazolium assay, nuclear morphometric analysis, microscopic examination, and annexin-v/ propidium iodide staining assay., Result: The findings revealed that the cytotoxic effect of 5-FU, TQ, and their combination on tongue squamous cell carcinoma cell line (HNO-97) was dose-dependent. The microscopic examination revealed that 5-FU, TQ alone, or their combination induced apoptotic cell death. P-value < 0.05 was statistically significant., Conclusion: The combination of 5-FU and TQ produced a marked cytotoxic effect on HNO-97 cells.

Colon cancer is on the rise in both men and women. In addition to traditional treatment methods, herbal treatments from complementary and alternative medicine are actively followed. Naturally derived from plants, thymoquinone (TQ) has drawn a lot of attention in the field of cancer treatment. MK-801, an N-methyl-D-aspartate agonist, is used to improve memory and plasticity, but it has also lately been explored as a potential cancer treatment. This study aimed to determine the roles of N-Methyl-D-Aspartate agonists and Thymoquinone on mitochondria and apoptosis. HT-29 cells were treated with different TQ and MK-801 concentrations. We analyzed cell viability, apoptosis, and alteration of mitochondria. Cell viability significantly decreased depending on doses of TQ and MK-801. Apoptosis and mitochondrial dysfunctions induced by low and high doses of TQ and MK-801. Our study emphasizes the need for further safety evaluation of MK-801 due to the potential toxicity risk of TQ and MK-801. Optimal and toxic doses of TQ and MK-801 were determined for the treatment of colon cancer. It should be considered as a possibility that colon cancer can be treated with TQ and MK-801., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Kidney disease is a common health problem worldwide. Acute or chronic injuries may interfere with kidney functions, eventually resulting in irreversible kidney damage. A number of recent studies have shown that the plant-derived natural products have an extensive potential for renal protection. Thymoquinone (TQ) is an essential compound derived from Nigella Sativa (NS), which is widely applied in the Middle East as a folk medicine. Previous experiments have demonstrated that TQ has a variety of potential pharmacological effects, including anti-oxidant, antibacterial, antitumor, immunomodulatory, and neuroprotective activities. In particular, the prominent renal protective efficacy of TQ has been demonstrated in both in vivo and in vitro experiments. TQ can prevent acute kidney injuries from various xenobiotics through anti-oxidation, anti-inflammatory, and anti-apoptosis effects. In addition, TQ exhibited significant pharmacological effects on renal cell carcinoma, renal fibrosis, and urinary calculi. The essential mechanisms involve scavenging ROS and increasing anti-oxidant activity, decreasing inflammatory mediators, inducing apoptosis, and inhibiting migration and invasion. The purpose of this review is to conclude the pharmacological effects and the potential mechanisms of TQ in renal protection, shedding new light on the exploration of medicinal phyto-protective agents targeting kidneys.

The treatments available for non-small cell lung cancer exert various side effects in patients, and the burden of treatment cost is high. Therefore, exploring the alternative system of medicines, including therapies based on natural compounds, has become inevitable in developing anticancer therapeutics. This study used an integrated approach involving in-silico and in-vitro methods to explore natural compounds targeting Bax and Bcl2 for their apoptotic potential. Molecular docking followed by molecular dynamics (MD) simulation of thymoquinone (Tq) and quercetin (Qu) with Bax and Bcl2 were carried out to explore their interactions and stability under explicit solvent conditions. Tq and Qu showed appreciable binding affinities toward Bax (-6.2 and -7.1 kcal/mol, respectively) and Bcl2 (-5.6 and -6.4 kcal/mol, respectively) with well-organized conformational fitting compatibility. The MD simulation results revealed the development of stable complexes maintained by various noncovalent interactions that were preserved throughout the 100 ns trajectories. Further studies with these compounds were carried out using various in-vitro experimental approaches like MTT assay, apoptotic assay, and Western blot. IC 50 values of Tq and Qu alone in A549 cells were found to be 45.78 and 35.69 µM, while in combination, it comes down to 22.49 µM, which is quite impressive. Similarly, in apoptosis assay, a combination of Tq and Qu shows 50.9% early apoptosis compared to Tq (40.6%) and Qu (33.3%) when taken alone. These assays signify their apoptotic induction potential, whereas both compounds significantly reduce the expression of antiapoptotic protein Bcl2 and induce proapoptotic Bax, suggestive of sensitizing NSCLS cells toward apoptosis., (© 2021 Wiley Periodicals LLC.)

Melanoma is a highly aggressive and treatment-resistant cancer, and the incidence and mortality rates are increasing worldwide. Thymoquinone (TQ) is the active component of Nigella sativa seed extracts and exerts anticancer effects in various cancer cells. However, the anticancer effects of TQ on melanoma and the underlying molecular mechanisms remain elusive. In this study, TQ treatment induced apoptosis in SK-MEL-28 cells. Interestingly, constitutive phosphorylation of Janus kinase 2 (Jak2) and signal transducer and activator of transcription 3 (STAT3) was markedly decreased following TQ treatment. Furthermore, TQ treatment downregulated STAT3-dependent genes including cyclin D1, D2, and D3 and survivin. Moreover, inhibition of Jak2/STAT3 using AG490, an inhibitor of Jak2 or genetic ablation of STAT3, abrogated the expression of target genes. TQ increased the levels of reactive oxygen species (ROS), whereas pretreatment with N-acetyl cysteine (NAC), a ROS scavenger, prevented the suppressive effect of TQ on Jak2/STAT3 activation and protected SK-MEL-28 cells from TQ-induced apoptosis. TQ administration further attenuated the growth of SK-MEL-28 tumor xenografts. Taken together, TQ induced apoptosis of SK-MEL-28 by hindering the Jak2/STAT3 signaling pathway through ROS generation. Our results support further development of TQ as a potential anticancer therapeutic agent for treating melanoma., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: Cancer treatment using a targeted inducer of apoptosis like tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) faced the obstacle of resistance, thus providing a plus drug like Thymoquinone (TQ) could be of great interest to tackle breast cancer cells. The aim of the present work is to examine the genetic modulation impacts of the TRAIL receptors and apoptotic markers upon the combinatorial remedy of TRAIL plus TQ on human breast cancer cell lines., Methods: To achieve this rationale, the protein content-based cytotoxicity using SRB assay, as well as the genetic expressions of the TRAIL receptors (DR4 and DR5) and apoptotic markers (Bcl-2, Cas-8, and FADD) using real time qRT-PCR technique were preceded against breast cancer MCF-7 and MDA-MB-231 cancerous cell lines., Results: The current study showed that the combination therapy of TQ+TRAIL significantly inhibited the protein content-based proliferation of MDA-MB-231 cells more than MCF-7 cells. The synergistic effect of them significantly up-regulated the genetic expressions of DR4, DR5, Cas-8, and FADD genes and inhibited the genetic expression of the Bcl-2 gene in the proposed cell lines treated for 24 h. The induction of the apoptotic genes using the combined therapy was stimulated by the elevation of the reactive oxygen species (ROS); nitric oxide (NO) and malondialdehyde (MDA) levels., Conclusions: The synergistic influence between TQ which induced the DR5 and TRAIL, facilitating the connection between TRAIL and its receptors on the cancerous cell membrane. Hence, the proposed combination therapy induced the ROS-mediated apoptotic stimulus.

Objective: Cancer is one of the leading causes of mortality in both developed and developing nations. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is characterized by its ability to selectively trigger apoptosis in cancer cells. TRAIL-based interventions have led to the development of recombinant human (rhTRAIL) as a promising therapy for different types of human cancer. Thymoquinone (TQ) has been shown to exert anticancer effect. The aim of the current study is to investigate the anticancer effect of the combinatorial therapy of TRAIL+TQ against human breast cancer cells., Methods: To achieve this hypothesis, cytotoxicity using MTT assay, as well as apoptosis and cell cycle using flow cytometric technique were preceded against breast cancer MCF-7 and MDA-MB-231 cancerous cell lines., Results: The current study showed that TRAIL induced cell cycle arrest and apoptosis. Moreover, it inhibited proliferation of MDA-MB-231 cells more than MCF-7 cells. Adding TQ to TRAIL increased the chemo-sensitivity of MDA-MB-231, while overcame the MCF-7 resistance to TRAIL., Conclusion: In conclusion, there is a synergistic effect between TRAIL and TQ playing a therapeutic role in killing resistant breast cancer cells.

Objective: The natural compound, thymoquinone (TQ) has demonstrated potential anticancer properties in inhibiting cell proliferation and promoting apoptosis in myeloid leukemia cells, breast cancer cells, and others. However, the effect mechanism of TQ on AML cells still not fully understood. In this study, the authors examined the effects of TQ on the expression of JAK/STAT-negative regulator genes SOCS-1, SOCS-3, and SHP-1, and their consequences on cell proliferation and apoptosis in HL60 leukemia cells., Methods: MTT and trypan blue exclusion tests were conducted to determine the 50% inhibitory concentration (IC50) and cell proliferation. FITC Annexin and Guava® reagent were used to study the cell apoptosis and examine the cell cycle phases, respectively. The expression of JAK/STAT-negative regulator genes, SOCS-1, SOCS-3, and SHP-1, was investigated using reverse transcriptase- quantitative PCR (RT-qPCR)., Results: TQ demonstrated a potential inhibition of HL60 cell proliferation and a significant increase in apoptotic cells in dose and time-dependent manner. TQ significantly induced cycle arrest at G0-G1 phase (P < 0.001) and enhanced the re-expression of JAK/STAT-negative regulator genes., Conclusion: TQ potentially inhibited HL60 cell proliferation and significantly increased apoptosis with re-expression of JAK/STAT-negative regulator genes suggesting that TQ could be a new therapeutic candidate for leukemia therapy., .

Doxorubicin (Dox) induces senescence in numerous cancer cell types, but these senescent cancer cells relapse again if they are not eliminated. On this principle, we investigated the apoptotic effect of thymoquinone (TQ), the active ingredient of Nigella sativa seeds and costunolide (COS), the active ingredient of Costus speciosus , on the senescent colon (Sen-HCT116) and senescent breast (Sen-MCF7) cancer cell lines in reference to their corresponding proliferative cells to rapidly eliminate the senescent cancer cells. The senescence markers of Sen-HCT116 and Sen-MCF7 were determined by a significant decrease in bromodeoxyuridine (BrdU) incorporation and significant increases in SA-β-gal, p53, and p21 levels. Then proliferative, Sen-HCT116, and Sen-MCF7 cells were subjected to either TQ (50 µM) or COS (30 µM), the Bcl2-associated X protein (Bax), B-cell lymphoma 2 (Bcl2), caspase 3 mRNA expression and its activity were established. Results revealed that TQ significantly increased the Bax/Bcl2 ratio in HCT116 + Dox5 + TQ, MCF7 + TQ, and MCF7 + Dox5 + TQ compared with their corresponding controls. COS significantly increased the Bax/Bcl2 ratio in HCT116 + Dox5 + TQ and MCF7 + Dox5 + TQ compared with their related controls. Also, TQ and COS were significantly increased caspase 3 activity and cell proliferation of Sen-HCT116 and Sen-MCF7. The data revealed a higher sensitivity of senescent cells to TQ or COS than their corresponding proliferative cells.

Background: Breast cancer intimidates the contemporary medical advances, attempting to revolutionize cancer therapeutics. While patients suffering an advanced breast cancer are dependent on mono drugs, yet the build out of resistance leading to treatment fails has become inevitable., Methods: Cell viability Assay with MTT revealed the "IC50" concentrations of the drugs in both cancer as well as PBMC. Cell cycle arrest, flow cytometric ROS analysis & apoptosis evaluation pointed out the efficacy of the dual drug. Wound Healing, Transwell Migration & Immunocytochemistry indicated anti-migratory potential of TQ-Emo while expression patterns of Cl-Cas3, p53, Bax, Bcl2 & the stemness markers further vouched the potential of the combinatorial drug. Furthermore, validation of tumor inhibitory effect was earned by an ex-ovo xenograft model., Results: Dual dosage enhanced apoptosis through ROS generation, anti- migratory effect by targeting FAK &Integrins, displaying effective stemness control by assessing regulatory proteins- Oct4, Sox2, Nanog, ALDH1/2. Ex-ovo xenograft model validated tumor regression. Our study thereby deals with devastating effects of cancer drug resistance while trying to abate enhanced migratory potential & stemness, utilizing the synergism of the combinable therapy., Conclusion: TQ/Emo inhibited breast cancer proliferation synergistically while enhancing cytotoxicity, inducing apoptosis on MCF-7 cells while curbing migration & stemness., General Significance: Employment of the combinatorial phytochemicals, Thymoquinone & Emodin attempted to achieve deliverables like reduced cellular toxicity, drug resistance, anti-migratory potency & stemness. Besides, decreased p-FAK expression or regression in Mammosphere & tumor size in ex-ovo xenograft model is indicative of the better anti-tumorigenic potential of the dual formulation., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Currently, there are limited effective treatment options for renal cell carcinoma (RCC), due to its poor responses to conventional therapies. Instead of using extrinsic anti-cancer drugs, cancer cell-intrinsic reactive oxygen species (ROS) can be a weapon of RCC treatment. In the present study, we found that the phytochemical thymoquinone (TQ), a bioactive natural product obtained from the black cumin seeds of Nigella sativa, generates intracellular ROS in human renal cancer Caki-1 cells. Treatment of Caki-1 cells with high concentration of TQ up-regulated pro-apoptotic p53 and Bax expression, while downregulated anti-apoptotic Bcl-2 and Bcl-xl expression. Simultaneously, TQ suppressed the pro-oncogenic JAK2/STAT3 pathway, resulting in decreased expression of Bcl-2, Bcl-xl, cyclin D1, cyclin D2, and survivin. Thus, TQ can integrate between apoptosis and the pro-survival JAK2/STAT3 pathway through the Bcl family members, collectively magnifying Caki-1 cell apoptosis. However, treatment with the ROS scavenger N-acetyl cysteine significantly blocked TQ-induced apoptosis as well as incorporated signaling pathways, supporting that its pro-oxidant property is crucial for Caki-1 cell apoptosis. Moreover, TQ reduced the tumor xenograft growth of Caki-1 cells in nude mice. Taken together, these data suggest that TQ is a prominent anti-cancer drug to treat human RCC by enhancing apoptosis through its pro-oxidant nature., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The present study evaluated biochemical endpoints characterizing acrylamide (ACR) neurotoxicity in the cortex of rats, following the possible neuroprotective activity of thymoquinone (TQ), an active constituent of Nigella sativa. ACR (50 mg/kg, intraperitoneal [i.p.]) concurrently with TQ (2.5, 5 and 10 mg/kg, i.p.) for 11 days were administered to rats. As positive control, vitamin E was used. After 11 days of injections, narrow beam test (NBT) was performed. The levels of reduced glutathione (GSH) and malondialdehyde (MDA) were measured and Western blotting was done for mitogen-activated protein kinases (MAPKinases) and apoptosis pathways proteins in the rats' cortex. Additionally, Evans blue assay was done to evaluate the integrity of blood brain barrier (BBB). Administration of ACR significantly induced gait abnormalities. A significant decrease and increase in the levels of GSH and MDA was observed in the cortex of ACR-treated rats, respectively. The elevation in the levels of caspases 3 and 9, glial fibrillary acidic protein (GFAP) content, and Bax/Bcl-2, P-P38/P38 and P-JNK/JNK ratios accompanied by reduction in myelin basic protein (MBP) content and P-ERK/ERK ratio were noticed in the ACR group. TQ (5 mg/kg) improved gait abnormalities, and restored these changes. ACR affected the integrity of BBB while TQ was able to maintain the integrity of this barrier. TQ reversed the alterations in the protein contents of MAP kinase and apoptosis signaling pathways as well as MBP and GFAP contents, induced by ACR. It protected against ACR-mediated neurotoxicity, partly through its antioxidant and antiapoptotic properties.

Fasciolosis is a neglected tropical disease caused by the liver fluke Fasciola gigantica. The absence of successful vaccine and emerging resistance in flukes against the drug of choice, triclabendazole, has necessitated the search for alternatives including phyto-therapeutic approaches. Curcumin and thymoquinone, the active ingredients of Curcuma longa and Nigella sativa plants respectively, were first screened for their binding affinity with Glutathione-S-transferase (GST) molecule through in silico molecular docking followed by in vitro treatment of worms with varying concentrations of the test compounds. The in silico molecular docking of curcumin and thymoquinone with sigma GST revealed strong hydrogen bonding as well as hydrophobic interactions with high fitness scores but showing inter-specific differences. The in vitro treatment of F. gigantica worms with both curcumin and thymoquinone resulted in a significant increase in the generation of reactive oxygen species (ROS) whereas the level of reduced glutathione, a primary redox regulator, was found to be significantly decreased (p < 0.05). The two compounds not only inhibited the GST activity, which is an important detoxification enzyme and also a key drug/vaccine target for the control of fasciolosis but also significantly inhibited the activity of antioxidant enzymes glutathione peroxidase and glutathione reductase that are vital in maintenance of redox homeostasis. The immunohistochemistry performed using anti sigma GST polyclonal antibodies revealed that both the compounds used in the present study significantly reduced immunofluorescence in the vitellaria, developing eggs present in the ovary and the intestinal caecae indicating inhibition of GST enzyme in these regions of the worms. Further, following treatment with curcumin and thymoquinone, chromatin condensation and DNA fragmentation was also observed in F. gigantica worms. In conclusion, both curcumin and thymoquinone generated oxidative stress in the worms by production of ROS and significantly inhibiting their antioxidant and detoxification ability. The oxidative stress along with induction of apoptotic like events would compromise the survival ability of worms within the host. However, further studies are required to establish their anthelmintic potential alone and in combination with the commonly used anthelmintic drugs under in vivo conditions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Thymoquinone (TQ), a natural anticancer agent exerts cytotoxic effects on several tumors by targeting multiple pathways, including apoptosis. Difluoromethylornithine (DFMO), an irreversible inhibitor of the ornithine decarboxylase (ODC) enzyme, has shown promising inhibitory activities in many cancers including leukemia by decreasing the biosynthesis of the intracellular polyamines. The present study aimed to investigate the combinatorial cytotoxic effects of TQ and DFMO on human acute T lymphoblastic leukemia Jurkat cells and to determine the underlying mechanisms. Here, we show that the combination of DFMO and TQ significantly reduced cell viability and resulted in significant synergistic effects on apoptosis when compared to either DFMO or TQ alone. RNA-sequencing showed that many key epigenetic players including Ubiquitin-like containing PHD and Ring finger 1 (UHRF1) and its 2 partners DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) were down-regulated in DFMO-treated Jurkat cells. The combination of DFMO and TQ dramatically decreased the expression of UHRF1, DNMT1 and HDAC1 genes compared to either DFMO or TQ alone. UHRF1 knockdown led to a decrease in Jurkat cell viability. In conclusion, these results suggest that the combination of DFMO and TQ could be a promising new strategy for the treatment of human acute T lymphoblastic leukemia by targeting the epigenetic code.

Thymoquinone (TQ) is a highly perspective chemotherapeutic agent against gliomas and glioblastomas because of its ability to cross the blood-brain barrier and its selective cytotoxicity for glioblastoma cells compared to primary astrocytes. Here, we tested the hypothesis that TQ-induced mild oxidative stress provokes C6 glioma cell apoptosis through redox-dependent alteration of MAPK proteins. We showed that low concentrations of TQ (20-50 μM) promoted cell-cycle arrest and induced hydrogen peroxide generation as a result of NADH-quinone oxidoreductase 1-catalyzed two-electron reduction of this quinone. Similarly, low concentrations of TQ efficiently conjugated intracellular GSH disturbing redox state of glioma cells and provoking mitochondrial dysfunction. We demonstrated that high concentrations of TQ (70-100 μM) induced reactive oxygen species generation due to its one-electron reduction. TQ provoked apoptosis in C6 glioma cells through mitochondrial potential dissipation and permeability transition pore opening. The identified TQ modes of action on C6 glioma cells open up the possibility of considering it as a promising agent to enhance the sensitivity of cancer cells to standard chemotherapeutic drugs.

Thymoquinone (TQ) loaded monodispersed mesoporous silica nanoparticles (TQ-MSNPs) with size of 188 ± 3 nm were prepared and characterized using DLS, TEM and FTIR. These TQ-MSNPs overcome the limitations of free TQ like hydrophobicity, low aqueous and photo stability and thus enhance its anticancer activity. In vitro release kinetics showed biphasic drug release where up to 50% was released in first 8 h and subsequently 98% released after 48 h. Enhanced cytotoxicity of TQ-MSNPs was observed against MCF-7 and HeLa cell lines as compared to free TQ. DAPI and Annexin V-FITC/PI staining confirmed the induction of apoptosis in cancer cells following treatment with TQ-MSNPs. Also, TQ-MSNPs exhibited enhanced anti-invasion properties against both cell lines as very low concentration of loaded TQ imparts similar benefits as free TQ. Both TQ and TQ-MSNPs exerted their cytotoxicity via reactive oxygen species (ROS) generation, as addition of an antioxidant NAC attenuated their killing activity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Thymoquinone is a phytochemical compound isolated from Nigella sativa and has various biological effects, including anti-inflammation, antioxidation, and anticancer. Here, we further investigated the anticancer effects and associated molecular mechanism of 2-methyl-5-isopropyl-1,4-benzoquinone (thymoquinone) on human renal carcinoma cell lines 786-O and 786-O-SI3 and transitional carcinoma cell line BFTC-909. Results showed that thymoquinone significantly reduced cell viability, inhibited the colony formation of renal cancer cells, and induced cell apoptosis and mitochondrial membrane potential change in both cancer cells. In addition, thymoquinone also triggered the production of reactive oxygen species (ROS) and superoxide and the activation of apoptotic and autophagic cascade. ROS inhibition suppressed the caspase-3 activation and restored the decreased cell viability of 786-O-SI3 in response to thymoquinone. Autophagy inhibition did not restore the cell viability of 786-O-SI3 suppressed by thymoquinone. Moreover, thymoquinone suppressed the cell sphere formation and the expression of aldehyde dehydrogenase, Nanog, Nestin, CD44, and Oct-4 in 786-O-SI3 cells. The tumor-bearing model showed that thymoquinone in vivo inhibited the growth of implanted 786-O-SI3 cell. All these findings indicate that thymoquinone inhibits the proliferation of 786-O-SI3 and BFTC-909 cell possibly due to the induction of ROS/superoxide and the consequent apoptosis, suggesting that thymoquinone may be a potential anticancer supplement for genitourinary cancer., (© 2019 Wiley Periodicals, Inc.)

Black seed (Nigella sativa) oil has been used in various dermatological applications, and its major constituent, thymoquinone (TQ) has been shown to exhibit antiproliferative activity against various cancer cells. In this study, we tried to provide a mechanistic basis of apoptosis induced by TQ. Skin squamous carcinoma A431 cells were treated with TQ to monitor the apoptosis induced by TQ. Western blot analysis was performed to detect expression of apoptotic or anti-apoptotic proteins. Cell viability and apoptosis were measured by using the MTT test and FACS analysis, respectively. The induction of intracellular reactive oxygen species (ROS) by TQ was evaluated by 2',7'-dichlorofluorescein diacetate staining. In vivo xenograft study was followed to confirm the antiproliferative effect of TQ. Treatment of A431 cells with TQ-induced apoptosis, which was associated with the induction of p53 and Bax, inhibition of Mdm2, Bcl-2, and Bcl-xl expression, and activation of caspase-9, -7, and -3. TQ inhibited the constitutive phosphorylation and DNA binding activity of signal transducer and activator of transcription-3 (STAT3) in A431 cells by blocking the phosphorylation of the upstream kinase, Src. Moreover, the expression of STAT3 target gene products, cyclin D1 and survivin, was attenuated by TQ treatment. The generation of ROS was increased during TQ-induced apoptosis, and the pretreatment of N-acetyl cysteine, a ROS scavenger, reversed the apoptotic effect of TQ. In vivo study with NOD scid gamma (NSG) mice confirmed the inhibitory effect of TQ on the growth of A431 cells. Our results provide the first demonstration that TQ induces the apoptosis of A431 cells through generation of ROS and inhibition of STAT3 signaling., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Oral squamous cell carcinoma (OSCC) is the sixth most prevalent cancer type in the world, with a 5-year survival rate of only 40% to 50%. Finding effective anti-tumor drug candidates is quite necessary for the progression of OSCC therapy. In this study, the role of Thymoquinone (TQ) in OSCC cells was studied. It was confirmed that TQ can inhibit the proliferation, migration and invasion of KB cells, and can also achieve apoptosis by inhibiting the activation of PI3K/Akt pathway. Therefore, TQ is an effective candidate for the treatment of OSCC.

Background: Cancer is one of the most fatal diseases across the world and it was reported that 90% of cancer fatality depends on its angiogenesis potential. Black seed or Nigella sativa L. is a medicinal plant native to southwest Asia. N. sativa has been used for medicinal purposes for centuries and predominantly has bioactive components like Thymoquinone, which is used as a candidate for anti-cancer and anti-angiogenesis drugs., Methods: Callus was induced from leaf tissue, after that alcoholic extracts were prepared from three-month-old calluses. Thymoquinone content was measured by HPLC methods. AGS cell line was cultured and treated with standard Thymoquinone and extracts from callus. Then, cell proliferation, expression of angiogenic factor (VEGF-A gene), and apoptosis test were done by MTT assay, real-time PCR and Annexin-v kit, respectively., Results: HPLC found the maximum amount of Thymoquinone in the extract of leaf calluses, which were grown in the dark. MTT assay revealed that particular doses of extracts reduced cell proliferation. Real-time and Fluorescence- Activated Cell Sorting (FACS) results demonstrated that standard Thymoquinone and callus extracts down-regulated the VEGF-A gene expression, and all three induced apoptosis in the AGS cell line., Conclusion: It has been shown that TQ has pro-apoptotic and anti-metastatic effects on stomach cancer cell line, and these properties can introduce it as an anti-cancer drug in the near future., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Thymoquinone (TQ), the major active compound isolated from black seed oil (Nigella sativa), has been reported to exhibit anti-inflammatory and anticancer abilities. However, the exact molecular mechanism underlying the anticancer effect of TQ is still poorly understood, especially in regard to TQ's effect on endoplasmic reticulum stress-mediated apoptosis., Methods: The cytotoxicity of TQ on T24 and 253J bladder cancer cells was analyzed by MTT assay, colony formation assay and Annexin V-FITC/PI staining, while SV-HUC-1, the SV-40 immortalized human uroepithelial cell line, was used as a normal control. The change in mitochondrial membrane potential was assessed by JC-1 staining. mRNA expression and protein expression were detected by qPCR and western blotting. Caspase-3 activity was detected by colorimetric assay., Key Finding: TQ has a significant cytotoxicity on bladder cancer cells and can inhibit their proliferation and induce apoptosis. The protein changes of Bcl-2, Bax, cytochrome c and endoplasmic reticulum stress-related proteins (GRP78, CHOP, and caspase-12) revealed that the anticancer effect of TQ was associated with mitochondrial dysfunction and the endoplasmic reticulum stress pathway. Pretreatment with a pan-caspase inhibitor, Z-VAD-fmk, or an ER stress inhibitor, 4-PBA, or knockdown of CHOP by shRNA can partly reverse the pro-apoptotic effect of TQ by enhancing the expression of the anti-apoptotic protein Bcl-2, blocking the release of cytochrome c and the translocation of Bax from the cytoplasm to mitochondria., Significance: Our findings provide the first demonstration of the anticancer effect of TQ on bladder cancer, and the relationship between ER stress and mitochondrial dysfunction was clearly understood when the apoptosis progressed is revealed., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Opioids bind to specific receptors that are located in the central nervous system (CNS) and many other organs such as cardiovascular tissue. Morphine binds to opioid receptors and can induce oxidative stress under some certain conditions. Thymoquinone (TQ) has shown many therapeutic effects such as anti-inflammatory, antioxidant and immunomodulatory ones. Considering the oxidative effects of morphine, antioxidant effects of TQ and effects of oxidative damage in various types of biomolecules, the present study was conducted to determine the effect of morphine plus TQ on the expression of apoptotic genes in the heart of male mice. Hence we used real-time PCR to identify alterations in mRNA expression of genes involved in apoptotic pathway, including p53, Bax and Bcl-2 between the morphine-treated and TQ plus morphine-treated mice. Serum nitric oxide (NO) (Griess assay) and total antioxidant capacity (TAC) were analyzed and compared. In the morphine group, compared to control group, a significant increase in P53 and Bax mRNA expression and a significant decrease in Bcl-2 mRNA expression were observed (p &lt; 0.01). In TQ plus morphine groups, NO was decreased (P &lt;0 .001) and TAC levels were increased significantly (P &lt; .001). Interestingly, TQ (9 and 18 mg/kg) plus morphine caused a significant decrease in p53 and Bax and a significant increase in Bcl2 mRNA expression, compared to morphine-treated group (p &lt; 0.01). Collectively, the results of this study indicated that TQ, as an antioxidant, can improve the apoptotic effects induced by morphine in the heart tissue of mice.

Background: Prognosis of patients with melanoma brain metastasis is poor despite various chemotherapeutic agents. Researchers focus on finding effective treatment with a low risk of toxicity. Thymoquinone (TQ) has been found to be effective on different types of cancer. However, no data exist regarding the effect of TQ in intracerebral melanoma. The purpose of this study was to assess the effect of TQ in B16-F10 melanoma cell in vitro and intracerebral melanoma in vivo., Methods: The mechanisms of efficacy were investigated using adenosine triphosphate assay for cytotoxicity, flow cytometry, and acridine orange staining for apoptosis, comet assay for genotoxicity, CM-H 2 DCF-DA (2,7-dichlorodihydrofluorescein) for intracellular reactive oxygen species (ROS) generation and ELISA methods for inflammatory cytokines. Western blotting was performed to assess the expressions of p-JAK2, p-STAT3, caspase-3, Bax, Bcl-2, and survivin. In addition, the effect of TQ was investigated in a model system of intracerebral melanoma in syngeneic mice., Results: The median survival was improved by TQ in mice with intracerebral melanoma compared with the control group (16 days vs 9 days; P = 0.008). Cytotoxicity was enhanced by TQ in B16-F10 cells in a dose-dependent manner. TQ also induced apoptosis, DNA damage, and increased intracellular ROS. TQ inhibited p-STAT3, resulting in apoptosis through regulation of proapoptotic and antiapoptotic proteins., Conclusions: Our findings suggest that TQ would be an effective treatment in intracerebral metastatic lesions. This warrants further investigation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: Adult T-cell leukemia (ATL) is an aggressive form of malignancy caused by human T- cell lymphotropic virus 1 (HTLV-1). Currently, there is no effective treatment for ATL. Thymoquinone has been reported to have anti-cancer properties., Objective: The aim of this study is to investigatthe effects of TQ on proliferation, apoptosis induction and the underlying mechanism of action in both HTLV-1 positive (C91-PL and HuT-102) and HTLV-1 negative (CEM and Jurkat) malignant T-lymphocytes., Materials and Methods: Cells were incubated with different thymoquinone concentrations for 24h. Cell cytotoxicity was assayed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit. Cell proliferation was determined using CellTiter 96® Non-Radioactive Cell Proliferation. Cell cycle analysis was performed by staining with propidium iodide. Apoptosis was assessed using cell death ELISA kit. The effect of TQ on p53, p21, Bcl-2 protein expression was determined using Western blot analysis while TGF mRNA expression was determined by RT-PCR., Results: At non-cytotoxic concentrations of TQ, it resulted in the inhibition of proliferation in a dose dependent manner. Flow cytometric analysis revealed a shift in the cell cycle distribution to the PreG1 phase which is a marker of apoptosis. Also TQ increase DNA fragmentation. TQ mediated its anti-proliferative effect and apoptosis induction by an up-regulation of TGFβ1, p53 and p21 and a down-regulation of TGF-α and Bcl-2α., Conclusion: Thymoquinone presents antiproliferative and proapoptotic effects in ATL cells. For this reason, further research is required to investigate its possible application in the treatment of ATL., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Context: Thyroid cancers (TCs) are the most common endocrine malignancies. There were two problems with the current cancer chemotherapy: the ineffectiveness of treatment due to resistance to cancer cell, and the toxic effect on normal cells., Aims: This study was aimed to determine the effects of thymoquinone (TQ) and genistein (Gen) phytotherapeutics on telomerase activity, angiogenesis, and apoptosis in follicular and anaplastic thyroid cancer cells (TCCs)., Materials and Methods: Cell viability, caspase-3 (CASP-3) activity, and messenger RNA (mRNA) expression levels of human telomerase reverse transcriptase (hTERT), phosphatase and tensin homolog (PTEN), nuclear factor-kappa B (NF-kB), cyclin-dependent kinase inhibitor 1 (p21), and vascular endothelial growth factor-A (VEGF-A) genes were analyzed., Results: It was found that TQ and Gen treatment on TCCs caused a statistically significant decrease of cell viability, and mRNA expression levels of hTERT, VEGF-A, and NF-kB genes, but a statistically significant increase of PTEN and p21 mRNA expression levels. In addition, TQ and Gen treatment also caused a statistically significant increase active CASP-3 protein level in TCCs. Moreover, our results demonstrated that, when compared with follicular TCCs, anaplastic TCCs were more sensitive to the treatment of TQ and Gen., Conclusions: Based on these results, two agents can be good options as potential phytochemotherapeutics against TCCs., Competing Interests: There are no conflicts of interest.

Background: Thymoquinone (TQ) is the major constituent of Nigella sativa seed and has shown biological activity in various human carcinomas. However, few studies have reported its effect on esophageal carcinoma (EC)., Aims: To explore the chemosensitive effect and mechanism of TQ in augmentation of cisplatin (DDP)-induced apoptosis of EC, both in vitro and in vivo., Methods: The viability and apoptosis of esophageal carcinoma cells were detected by the Cell Counting Kit-8 assay, flow cytometry, and Hoechst 33258 staining. The expression levels of JAK2, p-JAK2, STAT3, p-STAT3, Bax, Bcl-2, Cyclin D1, Survivin, and caspase-3, 7, 9 were evaluated by western blot analysis. The histological changes were examined by TUNEL technique and immunohistochemical analysis., Results: TQ enhanced the proapoptotic effect of DDP in human esophageal carcinoma cell line Eca-109, while blocking the activation of JAK2/STAT3 signaling pathway. The apoptosis of esophageal carcinoma cells was induced via blocking the activation of JAK2/STAT3 by using a molecular inhibitor (WP1066). Consistent with the in vivo and in vitro results, TQ increased cellular apoptosis and enriched the chemosensitivity of DDP., Conclusions: TQ along with DDP may regulate the progression of EC and has potential to be a chemotherapeutic agent in EC.

This study was undertaken to evaluate the protective effect of thymoquinone (TQ), the bioactive compound of Nigella sativa seeds, against warm ischemia-reperfusion (I/R) injury in liver. Rats were given an oral administration of a vehicle solution (sham group) or TQ at the appropriate dose (10, 20, 30 and 40mg/kg) for ten days consecutively. Following, they were subjected to 60min of partial hepatic ischemia followed by 24h of reperfusion. .Transaminase activities, histopathological changes, TNFα and antioxidant parameters were evaluated. Also, endoplasmic reticulum (ER) stress, mitochondrial damage and apoptosis were studied. In addition, ERK and P38 phosphorylation was determined by Western blot technique. We found that TQ at 30mg/kg is the effective dose to protect rat liver against I/R injury. Moreover, 30mg/kg of TQ prevented histological damages, inflammation and oxidative stress. Interestingly, it decreased the expression of ER stress parameters including GRP78, CHOP and caspase-12. In parallel, it improved mitochondrial function and attenuated the expression of apoptotic parameters. Furthermore, TQ significantly enhanced ERK and P38 phosphorylation. In conclusion, we demonstrated the potential of TQ to protect the rat liver against I/R injury through the prevention of ER stress and mitochondrial dysfunction. These effects implicate the prevention of oxidative stress., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Aims: Sodium nitrite is a widely used color fixative and preservative. However, it has been reported to exert deleterious toxic effects on various body organs. Moreover, thymoquinone (TQ), the active constituent of Nigella sativa oil is known to possess beneficial antioxidant and anti-inflammatory effects. The present study was conducted to evaluate the potential protective effects of TQ against sodium nitrite-induced renal toxicity., Main Methods: Male Sprague-Dawley rats were treated with sodium nitrite (80mg/kg, po, daily) in presence or absence of TQ (25 and 50mg/kg, po, daily). Morphological changes in renal sections were assessed by staining with Hematoxylin/Eosin and Periodic acid-Schiff. Renal homogenate was used for measurement of oxidative stress markers (MDA and GSH), inflammatory markers (CRP, TNF-α, IL-6, IL-1β), anti-inflammatory cytokines (IL-10 and IL-4) and apoptotic markers (caspase-3/caspase-8/caspase-9)., Key Findings: Treatment with sodium nitrite significantly increased markers of renal dysfunction, oxidative stress, inflammation and apoptosis. These effects were markedly attenuated by TQ in dose dependent manner., Significance: TQ has a potential protective effect against sodium nitrite-induced renal toxicity. This can be attributed to its ability to dampen oxidative stress, restore the normal balance between pro- and anti-inflammatory cytokines and protect renal tissue form extrinsic and intrinsic apoptosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

OBJECTIVE Ischemia-reperfusion (I/R) injury of the spinal cord following thoracoabdominal aortic surgery remains the most devastating complication, with a life-changing impact on the patient. Thymoquinone (TQ), the main constituent of the volatile oil from Nigella sativa seeds, is reported to possess strong antioxidant, antiinflammatory, and antiapoptotic properties. This study investigated the effects of TQ administration following I/R injury to the spinal cord. METHODS Thirty-two rats were randomly allocated into 4 groups. Group 1 underwent only laparotomy. For Group 2, aortic clip occlusion was introduced to produce I/R injury. Group 3 was given 30 mg/kg of methylprednisolone intraperitoneally immediately after the I/R injury. Group 4 was given 10 mg/kg of TQ intraperitoneally for 7 days before induction of spinal cord I/R injury, and administration was continued until the animal was euthanized. Locomotor function (Basso, Beattie, and Bresnahan scale and inclined plane test) was assessed at 24 hours postischemia. Spinal cord tissue samples were harvested to analyze tissue concentrations of malondialdehyde, nitric oxide, tumor necrosis factor-α, interleukin-1, superoxide dismutase, glutathione-peroxidase, catalase, and caspase-3. In addition, histological and ultrastructural evaluations were performed. RESULTS Thymoquinone treatment improved neurological outcome, which was supported by decreased levels of oxidative products (malondialdehyde and nitric oxide) and proinflammatory cytokines (tumor necrosis factor-α and interleukin-1), increased activities of antioxidant enzymes (superoxide dismutase, glutathione-peroxidase, and catalase), as well as reduction of motor neuron apoptosis. Light microscopy and electron microscopy results also showed preservation of tissue structure in the treatment group. CONCLUSIONS As shown by functional, biochemical, histological, and ultrastructural analysis, TQ exhibits an important protective effect against I/R injury of the spinal cord.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. The transcription factor NF-κB is overexpressed in human MB and is a critical factor for MB tumor growth. NF-κB is known to regulate the expression of interleukin-8 (IL-8), the chemokine that enhances cancer cell growth and resistance to chemotherapy. We have recently shown that thymoquinone (TQ) suppresses growth of hepatocellular carcinoma cells in part by inhibiting NF-κB signaling. Here we sought to extend these studies in MB cells and show that TQ suppresses growth of MB cells in a dose- and time-dependent manner, causes G2M cell cycle arrest, and induces apoptosis. TQ significantly increased generation of reactive oxygen species (ROS), while pretreatment of MB cells with the ROS scavenger N-acetylcysteine (NAC) abrogated TQ-induced cell death and apoptosis, suggesting that TQ-induced cell death and apoptosis are oxidative stress-mediated. TQ inhibitory effects were associated with inhibition of NF-κB and altered expression of its downstream effectors IL-8 and its receptors, the anti-apoptotic Bcl-2, Bcl-xL, X-IAP, and FLIP, as well as the pro-apoptotic TRAIL-R1, caspase-8, caspase-9, Bcl-xS, and cytochrome c. TQ-triggered apoptosis was substantiated by up-regulation of the executioner caspase-3 and caspase-7, as well as cleavage of the death substrate poly(ADP-ribose)polymerase. Interestingly, pretreatment of MB cells with NAC or the pan-caspase inhibitor zVAD-fmk abrogated TQ-induced apoptosis, loss of cyclin B1 and NF-κB activity, suggesting that these TQ-mediated effects are oxidative stress- and caspase-dependent. These findings reveal that TQ induces both extrinsic and intrinsic pathways of apoptosis in MB cells, and suggest its potential usefulness in the treatment of MB.

Aim: To elucidate the mechanism of thymoquinone (TQ)-induced apoptosis in human gastric cancer cells in vitro and in vivo., Methods: HGC27, BGC823, and SGC7901 cells were cultured in vitro and treated with TQ (0, 10, 25, 50, 75, 100, 125 μmol/L) for 12 h, 24 h, and 36 h, and then the proliferation inhibitory rates were detected by methylthiazole tetrazolium assay. Apoptosis was observed after Hoechst staining. The protein expressions of signal transducer and activator of transcription (STAT)3, p-STAT3, STAT5, p-STAT5, phospho-janus-activated kinase 2 (JAK2), JAK2, p-Src, Src, glyceraldehyde-3-phosphate dehydrogenase, lamin-A, survivin, Cyclin D, Bcl-2, Bax, peroxisome proliferator activated receptor, and caspase-3,7,9 were detected by western blot. Cell cycle and apoptosis were determined with flow cytometry. TQ induced dose-dependent apoptotic cell death in HGC27 cells was measured by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) analysis and Hoechst 33258., Results: TQ inhibited the phosphorylation of STAT3 but not STAT5. TQ-induced downregulation of STAT3 activation was associated with a reduction in JAK2 and c-Src activity. TQ also downregulated the expression of STAT3-regulated genes, such as Bcl-2, cyclin D, survivin, and vascular endothelial growth factor, and activated caspase-3,7,9. Consistent with the in vitro results, TQ was significantly effective as an antitumor agent in a xenograft tumor mouse model., Conclusion: This study provides strong evidence that downregulation of the STAT3 signaling pathway mediates TQ-induced apoptosis in gastric cancer.

Nonalcoholic steatohepatitis (NAFLD) is a progressive form of liver disease that leads to advanced fibrosis. The present study was designed to assess the hepatoprotective effect of thymoquinone (TQ) on liver functions, insulin resistance, and PPAR-γ expression in NAFLD. Rats were divided into two main groups: one fed with normal rat chow diet and the other with high-fat high-cholesterol diet group for 6 weeks. Every group was subdivided into three subgroups (n = 8): treated with saline, low dose TQ (10 mg/kg), high dose TQ (20 mg/kg). High fat high cholesterol diet caused marked liver damage as noted in histopathology and significant increase in liver index, liver enzymes. There was significant increase in the insulin resistance, serum cholesterol, triglyceride, PPAR-γ gene overexpression with significant decrease in HDL. Additionally, oxidative stress increased by measuring MDA associated with significant decrease in serum total antioxidant capacity. As markers of inflammation, hepatic TNF-α was significantly increased with decrease in IL10. Further, there was increase in BAX protein with decrease in Bcl as compared to control group. This model of 6 weeks high-fat high-cholesterol diet showed minimal fibrosis as noticed by increase MMP2 and Masson trichrome satin. Co-treatment with TQ improved all previous parameters. High dose was more effective, although mostly non-statistically significant. TQ may have a promising agent to improve hepatic steatosis, oxidative stress; inflammatory, apoptotic status, fibrosis and so prevent liver damage in patients with NAFLD. Although PPAR-γ was significantly under-expressed by TQ, insulin resistance was improved significantly suggesting a role of liver damage.

Thymoquinone (TQ) is the active ingredient of Nigella sativa which has a therapeutic potential in cancer therapy and prevention. In this study, TQ has been shown to induce specific cytotoxicity and apoptosis and to inhibit wound healing in triple-negative breast cancer cell line. TQ also inhibited cancer growth in a mouse tumor model. Moreover, TQ and paclitaxel (Pac) combination inhibited cancer growth in cell culture and in mice. Genes involved in TQ and TQ-Pac-mediated cytotoxicity were studied using focused real-time PCR arrays. After bioinformatic analysis, genes in apoptosis, cytokine, and p53 signaling categories were found to be modulated with a high significance in TQ-treated cells (p < 10(-28), p < 10(-8), and p < 10(-6), respectively). Important to note, TQ has been found to regulate the genes involved in the induction of apoptosis through death receptors (p = 5.5 × 10(-5)). Additionally, tumor suppressor genes such as p21, Brca1, and Hic1 were highly upregulated by TQ and TQ-Pac combination. Interestingly, when cells were treated with high dose TQ, several growth factors such as Vegf and Egf were upregulated and several pro-apoptotic factors such as caspases were downregulated possibly pointing out key pathways manipulated by cancer cells to resist against TQ. In cells treated with the combination of TQ and Pac, genes in apoptosis cascade (p < 10(-12)), p53 signaling (p = 10(-5)), and JAK-STAT signaling (p < 10(-3)) were differentially expressed. TQ has also been shown to induce protein levels of cleaved Caspase-3, Caspase-7, and Caspase-12 and PARP and to reduce phosphorylated p65 and Akt1. The in vivo therapeutic potential of TQ-Pac combination and the genetic network involved in this synergy have been shown for the first time to the best of our knowledge.

Cadmium (Cd), an environmental and industrial pollutant, generates free radicals responsible for oxidative stress. Cd can also lead to various renal toxic damage such as the proximal tubules and glomerulus dysfunction. Thymoquinone (TQ) is the main constituent of the essential oil obtained from black seeds (Nigella sativa) and has various pharmacological effects. The aim of the present study was to examine the nephroprotective, anti-oxidant, and anti-apoptotic effect of the TQ against Cd-induced nephrotoxicity. A total of 24 male Wistar albino rats were divided into three groups: control, Cd-treated, and Cd-treated with TQ; each group contain eight animals. The Cd-treated group was injected subcutaneously with CdCl2 dissolved in saline in the amount of 2 ml/kg/day for 30 days, resulting in a dosage of 1 mg/kg Cd. The rats in TQ-treated groups were given TQ (50 mg/kg body weight) once a day orally together with first Cd injection during the study period. The histopathological studies in the kidney of rats also showed that TQ markedly reduced the toxicity of Cd and preserved the normal histological architecture of the renal tissue. Immunohistochemical analysis revealed that TQ significantly decreased the Cd-induced over expression of nuclear factor-κB in renal tissue. Furthermore, TQ treatment resulted in decreased the number of apoptotic cells. TQ significantly suppressed lipid peroxidation, compensated deficits in the anti-oxidant defenses (reduced superoxide dismutase, glutathione peroxidase and catalase activities) in renal tissue resulted from Cd administration. These findings suggest that the nephroprotective potential of TQ in Cd toxicity might be due to its anti-oxidant and anti-apoptotic properties, which could be useful for achieving optimum effects in Cd-induced nephrotoxicity.

The objective of this study was to examine the effects of thymoquinone (TQ), which has antioxidant properties in the experimental testicular I/R model in rats in terms of its anti-apoptotic, proliferative and biochemical attributes. In our study, 24 male rats were divided into three groups: control group, I/R group and I/R+TQ group. Testicular torsion was created by rotating the left testis 720° in a clockwise direction. The ischaemia period was 4 h, and an orchiectomy was performed after 4 h of detorsion. Spermatogenesis and the mean seminiferous tubule diameter were significantly decreased in the I/R groups compared to the control group. Furthermore, TQ-treated animals displayed an improved histological appearance in the I/R group. It was also observed that treatment with TQ increased the activity of PCNA, which decreased as a result of I/R, and this treatment also reduced the number of TUNEL-positive cells. The I/R+TQ group showed a decrease in malondialdehyde levels and an increase in the activities of superoxide dismutase, catalase and glutathione peroxidase in comparison with the I/R group. It could be concluded that cytoprotective effects of TQ on the I/R testicles are via reduction of apoptosis, oxidative stress and lipid peroxidation., (© 2015 Blackwell Verlag GmbH.)

We aimed to investigate the protective role of thymoquinone (TQ) by targeting its antiapoptotic and antioxidant properties against kidney damage induced by arsenic in rats. We have used the 24 male Sprague-Dawley rats. Rats were divided into three groups. Physiological serum in 10 mL/kg dose as intragastric was given to the control group. Sodium arsenite (10 mg/kg, intragastric by gavage for fifteen days) was given to the arsenic group. Sodium arsenite (10 mg/kg, intragastric by gavage for fifteen days) and TQ (10 mg/kg, intragastric by gavage for 15 days) was given to the arsenic + TQ group. After 15 days, the animals' kidneys were taken theirs, then we have performed histological and apoptotic assessment. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) enzyme activities and malondialdehyde (MDA) levels have examined as the oxidative stress parameters. We have determined the levels of arsenic. Increased renal injury and apoptotic cells have been detected in the arsenic group. Degenerative changes in the arsenic + TQ group were diminished. Although the MDA levels were augmented in the arsenic group, SOD, CAT and GSH-Px enzyme activities were lessened than the other groups. Our findings suggest that TQ may impede the oxidative stress, the cells have been damaged and also the generation of apoptotic cells arisen from arsenic. TQ plays a protective role against arsenic-induced toxicity in kidney and may potentially be used as a remedial agent.

Chemotherapy causes unwanted side effects and chemoresistance, limiting its effectiveness. Therefore, phytochemicals are now used as alternative treatments. Thymoquinone (TQ) is used to treat different cancers, including colon cancer. The irinotecan-resistant (CPT-11-R) LoVo colon cancer cell line was previously constructed by stepwise CPT-11 challenges to untreated parental LoVo cells. TQ dose-dependently increased the total cell death index and activated apoptosis at 2 μM, which then diminished at increasing doses. The possibility of autophagic cell death was then investigated. TQ caused mitochondrial outer membrane permeability (MOMP) and activated autophagic cell death. JNK and p38 inhibitors (SP600125 and SB203580, respectively) reversed TQ autophagic cell death. TQ was also found to activate apoptosis before autophagy, and the direction of cell death was switched toward autophagic cell death at initiation of autophagosome formation. Therefore, TQ resulted in caspase-independent, autophagic cell death via MOMP and activation of JNK and p38 in CPT-11-R LoVo colon cancer cells.

Aim: Thymoquinone (TQ), the predominant bioactive constituent of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against multifarious tumors. However, the meticulous mechanism of TQ on Akt mediated survival pathway is still unrevealed in breast cancer. Here, we investigated TQ's mechanism of action against PI3K/Akt signaling and its downstream targets by modulating proteins translational machinery, leading to apoptosis in cancer cells., Main Methods: MDA-MB-468 and T-47D cells were treated with TQ and evaluated for its anticancer activity through phase distribution and western blot. Modulatory effects of TQ on Akt were affirmed through kinase and drug potential studies., Key Findings: Studies revealed G1 phase arrest till 24h incubation with TQ while extended exposure showed phase shift to subG1 indicating apoptosis, supported by suppression of cyclin D1, cyclin E and cyclin dependent kinase inhibitor p27 expression. Immunoblot and membrane potential studies revealed mitochondrial impairment behind apoptotic process with upregulation of Bax, cytoplasmic cytochrome c and procaspase-3, PARP cleavage along with Bcl-2, Bcl-xL and survivin downregulation. Moreover, we construed the rationale behind mitochondrial dysfunction by examining the phosphorylation status of PDK1, PTEN, Akt, c-raf, GSK-3β and Bad in TQ treated cells, thus ratifying the involvement of Akt in apoptosis. Further, the consequential effect of Akt inhibition by TQ is proven by translational repression through deregulated phosphorylation of 4E-BP1, eIF4E, S6R and p70S6K., Significance: Our observations for the first time may provide a new insight for the development of novel therapies for Akt overexpressed breast cancer by TQ., (© 2013.)

Silymarin and thymoquinone exert neuroprotective effects, although their combined effects in focal cerebral ischemia/reperfusion (I/R) models are unknown. We compared the effect of silymarin and thymoquinone in an I/R rat model. Wistar rats were divided into five groups: SHAM, REP (I/R), SIR (200 mg/kg silymarin+I/R), TIR (3 mg/kg thymoquinone+I/R), and STIR (200 mg/kg silymarin+3-mg thymoquinone+I/R). The rats underwent bilateral carotid artery occlusion for 30 min and neurological assessments 24 h thereafter. Apoptosis was evaluated using anti-caspase-3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assays. Astrocyte activation was determined using an anti-GFAP antibody. Total antioxidant status (TAS), total oxidant status (TOS), and trimethylamine N-oxide (TMAO) levels were measured. SHAM and REP rats had the lowest and highest neurological scores, respectively (p = 0.001). REP rats showed greater deterioration than SIR, TIR, and STIR rats. SIR, TIR, and STIR rats had fewer TUNEL and caspase-3-positive cells than REP rats (p<0.05). GFAP expression was higher in REP rats (p<0.05) than in SIR, TIR, and STIR rats (p<0.05). SIR and TIR rats showed higher TAS than REP rats (p<0.05). SIR, TIR, and STIR rats had lower TMAO values than REP and SHAM rats (p<0.05). Silymarin/thymoquinone reduces impairment, apoptosis, and astrocyte activation. Combination therapy reduces TMAO levels. [ABSTRACT FROM AUTHOR]

Chemotherapeutic drugs have demonstrated effectiveness in treating various neoplastic conditions; however, they can also have detrimental effects on male gonadal function and fertility. Consequently, interest has grown in identifying novel approaches that can mitigate chemotherapy-induced testicular damage. Thymoquinone (TQ), the chief active component of the volatile oil of Nigella sativa (NS), has a wide range of therapeutic properties, including antioxidant, anti-inflammatory and anti-apoptotic effects. The aim of this systematic review was to identify experimental animal studies that have evaluated the protective effects of TQ against testicular complications associated with chemotherapy. In accordance with the preferred reporting items for systematic review and meta-analyses (PRISMA) guidelines, a thorough search was performed across several databases (PubMed, EBSCOhost, Sage and Scopus) to identify experimental studies published from 2010 to May 2022 that focused on rodent models and compared the effects of TQ versus other chemotherapeutic drugs. Eight studies met the inclusion criteria, comparing TQ with methotrexate (MTX), 6-mercaptopurine (6-MP), cyclophosphamide (CPA), bleomycin (BL), doxorubicin (DOX) or busulfan (BUS). The results of these studies consistently demonstrated that TQ significantly improved sperm parameters, the levels of oxidative stress (OS) markers, apoptosis markers, and hormones and testicular histopathology, indicating that TQ has protective effects against chemotherapy-induced damage. TQ mitigated chemotherapy-induced testicular toxicity by decreasing lipid peroxidation and enhancing the activity of antioxidant enzymes within chemotherapy-treated testes. These findings highlight the potential of TQ as a therapeutic agent that can ameliorate testicular complications associated with chemotherapy, thereby providing a basis for further research and potential therapeutic applications. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma is the most common primary malignant tumor of the liver, and its incidence is increasing worldwide. There is a need to develop new therapeutic strategies to treat the disease. In this study, we synthesised the oxime derivative of thymoquinone and investigated cytotoxicity, genotoxicity, and apoptosis in hepatocellular cancer cells. The synthesised thymoquinone-oxime structure was confirmed by NMR. After incubating the hepatocellular cancer cell line for 24 h, the cytotoxicity ATP by luminometric, intracellular reactive oxygen species, and intracellular calcium by fluorometric. The mitochondrial membrane potential was determined by flow cytometry. DNA damage by alkaline single-cell gel electrophoresis, and apoptosis damage by acridine orange/ethidium bromide double dye method. Concentrations of thymoquinone-oxime statistically increased cytotoxicity, intracellular reactive oxygen species, intracellular calcium, apoptosis, and DNA damage in a concentration-dependent manner. Mitochondrial membrane potential and glutathione levels are also decreased. These findings show that thymoquinone-oxime has an anti-tumor effect on hepatocellular carcinoma cells. Among many herbs, Black seed (Nigella sativa) belonging to the Ranunculaceae family has been recognised worldwide as one of the most valuable nutrient-rich herbs There is no relevant data on the effect of the newly synthesised oxime derivative of TQ (TQ-ox) in cancer cells HEP-G2 A new TQ derivative has a higher anti-tumor effect on hepatocellular carcinoma [ABSTRACT FROM AUTHOR]

Background: In the present experiment, we evaluated the impact of thymoquinone (TQ) and paclitaxel (PTX) treatment on MDA-MB-231 cell line growth inhibition via controlling apoptosis/autophagy. Materials and results: MDA-MB-231cells were exposed to PTX (0, 25, 50, 75, and 100 nM), TQ (0, 25, 50, 75, and 100 µM), and combinations for 48 h. After the MTT assessment, dose-response curves and IC50 values were calculated, and the combination synergism was evaluated using the Compusyn software. Following the treatment with PTX, TQ, and combinations at IC50 doses, the expression of apoptosis and autophagy genes was assessed in cells. The GraphPad Prism program was used to analyze the data, and Tukey's test at p < 0.05 was then run. PTX, TQ, and their combinations inhibited MDA-MB-231cell proliferation and viability dose-dependently. TQ reduced the effective concentration (IC50) of PTX in co-treatment groups. PTX and TQ showed antagonistic effects when cell proliferation declined above 70%. Antagonistic effects shifted into additive and synergistic effects upon increasing PTX concentration, indicated by diminished cell proliferation below 70%. PTX-TQ co-treatment significantly enhanced P53 and BAX expression while reducing Bcl-2 expression. Also, their combination increased Beclin-1, ATG-5, and ATG-7 expression in treated cells. Conclusion: Effective concentrations of TQ and PTX had synergic effects and inhibited breast cancer cells via prompting apoptosis and autophagy in vitro. [ABSTRACT FROM AUTHOR]

Abstract Colorectal neoplasms are one of the deadliest diseases among all cancers worldwide. Thymoquinone (TQ) is a natural compound of Nigella sativa that has been used in traditional medicine against a variety of acute/chronic diseases such as asthma, bronchitis, rheumatism, headache, back pain, anorexia, amenorrhea, paralysis, inflammation, mental disability, eczema, obesity, infections, depression, dysentery, hypertension, gastrointestinal, cardiovascular, hepatic, and renal disorders. This review aims to present a detailed report on the studies conducted on the anti-cancer properties of TQ against colorectal cancer, both in vitro and in vivo. TQ stands as a promising natural therapeutic agent that can enhance the efficacy of existing cancer treatments while minimizing the associated adverse effects. The combination of TQ with other anti-neoplastic agents promoted the efficacy of existing cancer treatments. Further research is needed to acquire a more comprehensive understanding of its exact molecular targets and pathways and maximize its clinical usefulness. These investigations may potentially aid in the development of novel techniques to combat drug resistance and surmount the obstacles presented by chemotherapy and radiotherapy. Graphical Abstract

In the present study, a self-nano-emulsifying drug delivery system (SNEDDS) was developed to evaluate the efficiency of thymoquinone (TQ) in hepatic ischemia/reperfusion. SNEDDS was pharmaceutically characterized to evaluate droplet size, morphology, zeta potential, thermodynamic stability, and dissolution/diffusion capacity. Animals were orally pre-treated during 10 days with TQ-loaded SNEDDS. Biochemical analyses, hematoxylin–eosin staining, indirect immunofluorescence, and reverse transcription polymerase chain reaction (RT-PCR) were carried out to assess cell injury, oxidative stress, inflammation, and apoptosis. The TQ formulation showed good in vitro characteristics, including stable nanoparticle structure and size with high drug release rate. In vivo determinations revealed that TQ-loaded SNEDDS pre-treatment of rats maintained cellular integrity by decreasing transaminase (ALT and AST) release and preserving the histological characteristics of their liver. The antioxidant ability of the formulation was proven by increased SOD activity, reduced MDA concentration, and iNOS protein expression. In addition, this formulation exerted an anti-inflammatory effect evidenced by reduced plasma CRP concentration, MPO activity, and gene expressions of TLR-4, TNF-α, NF-κB, and IL-6. Finally, the TQ-loaded SNEDDS formulation promoted cell survival by enhancing the Bcl-2/Bax ratio. In conclusion, our results indicate that TQ encapsulated in SNEDDS significantly protects rat liver from I/R injury. [ABSTRACT FROM AUTHOR]