The Anticancer Effects of the Garlic Organosulfide Diallyl Trisulfide through the Attenuation of B[a]P-Induced Oxidative Stress, AhR Expression, and DNA Damage in Human Premalignant Breast Epithelial (MCF-10AT1) Cells.

Benzo[a]pyrene (B[a]P) is the most characterized polycyclic aromatic hydrocarbon associated with breast cancer. Our lab previously reported that the organosulfur compound (OSC), diallyl trisulfide (DATS), chemoprevention mechanism works through the induction of cell cycle arrest and a reduction in oxidative stress and DNA damage in normal breast epithelial cells. We hypothesize that DATS will inhibit B[a]P-induced cancer initiation in premalignant breast epithelial (MCF-10AT1) cells. In this study, we evaluated the ability of DATS to attenuate B[a]P-induced neoplastic transformation in MCF-10AT1 cells by measuring biological endpoints such as proliferation, clonogenicity, reactive oxygen species (ROS) formation, and 8-hydroxy-2-deoxyguanosine (8-OHdG) DNA damage levels, as well as DNA repair and antioxidant proteins. The results indicate that B[a]P induced proliferation, clonogenic formation, ROS formation, and 8-OHdG levels, as well as increasing AhR, ARNT/HIF-1ß, and CYP1A1 protein expression compared with the control in MCF-10AT1 cells. B[a]P/DATS's co-treatment (CoTx) inhibited cell proliferation, clonogenic formation, ROS formation, AhR protein expression, and 8-OHdG levels compared with B[a]P alone and attenuated all the above-mentioned B[a]P-induced changes in protein expression, causing a chemopreventive effect. This study demonstrates, for the first time, that DATS prevents premalignant breast cells from undergoing B[a]P-induced neoplastic transformation, thus providing more evidence for its chemopreventive effects in breast cancer.

The Garlic Compound, Diallyl Trisulfide, Attenuates Benzo[a]Pyrene-Induced Precancerous Effect through Its Antioxidant Effect, AhR Inhibition, and Increased DNA Repair in Human Breast Epithelial Cells.

Exposure to B[a]P, the most characterized polycyclic aromatic hydrocarbon, significantly increases breast cancer risk. Our lab has previously reported that diallyl trisulfide (DATS), a garlic organosulfur compound (OSC) with chemopreventive and cell cycle arrest properties, reduces lipid peroxides and DNA damage in normal breast epithelial (MCF-10A) cells. In this study, we evaluated the ability of DATS to block the B[a]P-induced initiation of carcinogenesis in MCF-10A cells by examining changes in proliferation, clonogenic formation, reactive oxygen species (ROS) formation, 8-hydroxy-2-deoxyguanosine (8-OHdG) levels, and protein expression of ARNT/HIF-1ß, CYP1A1, and DNA POLß. The study results indicate that B[a]P increased proliferation, clonogenic formation, ROS formation, and 8-OHdG levels, as well as increasing the protein expression of ARNT/HIF-1ß and CYP1A1 compared to the control. Conversely, DATS/B[a]P co-treatment (CoTx) inhibited cell proliferation, clonogenic formation, ROS formation, and 8-OHdG levels compared to B[a]P alone. Treatment with DATS significantly inhibited (p < 0.0001) AhR expression, implicated in the development and progression of breast cancer. The CoTx also attenuated all the above-mentioned B[a]P-induced changes in protein expression. At the same time, it increased DNA POLß protein expression, which indicates increased DNA repair, thus causing a chemopreventive effect. These results provide evidence for the chemopreventive effects of DATS in breast cancer prevention.

Thymoquinone Inhibition of Chemokines in TNF-α-Induced Inflammatory and Metastatic Effects in Triple-Negative Breast Cancer Cells.

The lack of identifiable molecular targets or biomarkers hinders the development of treatment options in triple-negative breast cancer (TNBC). However, natural products offer a promising alternative by targeting inflammatory chemokines in the tumor microenvironment (TME). Chemokines are crucial in promoting breast cancer growth and metastasis and correlate to the altered inflammatory process. In the present study, we evaluated the anti-inflammatory and antimetastatic effects of the natural product thymoquinone (TQ) on TNF-α-stimulated TNBC cells (MDA-MB-231 and MDA-MB-468) to study the cytotoxic, antiproliferative, anticolony, antimigratory, and antichemokine effects using enzyme-linked immunosorbent assays, quantitative real-time reverse transcription-polymerase chain reactions, and Western blots were used in sequence to validate the microarray results further. Four downregulated inflammatory cytokines were identified, CCL2 and CCL20 in MDA-MB-468 cells and CCL3 and CCL4 in MDA-MB-231 cells. Furthermore, when TNF-α-stimulated MDA-MB-231 cells were compared with MDA-MB-468 cells, the two cells were sensitive to TQ's antichemokine and antimetastatic effect in preventing cell migration. It was concluded from this investigation that genetically different cell lines may respond to TQ differently, as TQ targets CCL3 and CCL4 in MDA-MB-231 cells and CCL2 and CCL20 in MDA-MB-468 cells. Therefore, the results indicate that TQ may be recommended as a component of the therapeutic strategy for TNBC treatment. These outcomes stem from the compound's capacity to suppress the chemokine. Even though these findings support the usage of TQ as part of a therapy strategy for TNBC associated with the identified chemokine dysregulations, additional in vivo studies are needed to confirm these in vitro results.

Molecular Mechanisms of the Anti-Inflammatory Effects of Epigallocatechin 3-Gallate (EGCG) in LPS-Activated BV-2 Microglia Cells.

Chronic neuroinflammation is associated with many neurodegenerative diseases, such as Alzheimer's. Microglia are the brain's primary immune cells, and when activated, they release various proinflammatory cytokines. Several natural compounds with anti-inflammatory and antioxidant properties, such as epigallocatechin 3-gallate (EGCG), may provide a promising strategy for inflammation-related neurodegenerative diseases involving activated microglia cells. The objective of the current study was to examine the molecular targets underlying the anti-inflammatory effects of EGCG in activated microglia cells. BV-2 microglia cells were grown, stimulated, and treated with EGCG. Cytotoxicity and nitric oxide (NO) production were evaluated. Immunoassay, PCR array, and WES™ Technology were utilized to evaluate inflammatory, neuroprotective modulators as well as signaling pathways involved in the mechanistic action of neuroinflammation. Our findings showed that EGCG significantly inhibited proinflammatory mediator NO production in LPS-stimulated BV-2 microglia cells. In addition, ELISA analysis revealed that EGCG significantly decreases the release of proinflammatory cytokine IL-6 while it increases the release of TNF-α. PCR array analysis showed that EGCG downregulated MIF, CCL-2, and CSF2. It also upregulated IL-3, IL-11, and TNFS10. Furthermore, the analysis of inflammatory signaling pathways showed that EGCG significantly downregulated mRNA expression of mTOR, NF-κB2, STAT1, Akt3, CCL5, and SMAD3 while significantly upregulating the expression of mRNA of Ins2, Pld2, A20/TNFAIP3, and GAB1. Additionally, EGCG reduced the relative protein expression of NF-κB2, mTOR, and Akt3. These findings suggest that EGCG may be used for its anti-inflammatory effects to prevent neurodegenerative diseases.

The Role of Apoptotic Genes and Protein-Protein Interactions in Triple-negative Breast Cancer.

BACKGROUND/AIM: Compared to other breast cancer types, triple-negative breast cancer (TNBC) has historically had few treatment alternatives. Therefore, exploring and pinpointing potentially implicated genes could be used for treating and managing TNBC. By doing this, we will provide essential data to comprehend how the genes are involved in the apoptotic pathways of the cancer cells to identify potential therapeutic targets. Analysis of a single genetic alteration may not reveal the pathogenicity driving TNBC due to the high genomic complexity and heterogeneity of TNBC. Therefore, searching through a large variety of gene interactions enabled the identification of molecular therapeutic genes. MATERIALS AND METHODS: This study used integrated bioinformatics methods such as UALCAN, TNM plotter, PANTHER, GO-KEEG and PPIs to assess the gene expression, protein-protein interaction (PPI), and transcription factor interaction of apoptosis-regulated genes. RESULTS: Compared to normal breast tissue, gene expressions of BNIP3, TNFRSF10B, MCL1, and CASP4 were downregulated in UALCAN. At the same time, BIK, AKT1, BAD, FADD, DIABLO, and CASP9 was down-regulated in bc-GeneExMiner v4.5 mRNA expression (BCGM) databases. Based on GO term enrichment analysis, the cellular process (GO:0009987), which has about 21 apoptosis-regulated genes, is the top category in the biological processes (BP), followed by biological regulation (GO:0065007). We identified 29 differentially regulated pathways, including the p53 pathway, angiogenesis, apoptosis signaling pathway, and the Alzheimer's disease presenilin pathway. We examined the PPIs between the genes that regulate apoptosis; CASP3 and CASP9 interact with FADD, MCL1, TNF, TNFRSRF10A, and TNFRSF10; additionally, CASP3 significantly forms PPIs with CASP9, DFFA, and TP53, and CASP9 with DIABLO. In the top 10 transcription factors, the androgen receptor (AR) interacts with five apoptosis-regulated genes (p<0.0001; q<0.01), followed by retinoic acid receptor alpha (RARA) (p<0.0001; q<0.01) and ring finger protein (RNF2) (p<0.0001; q<0.01). Overall, the gene expression profile, PPIs, and the apoptosis-TF interaction findings suggest that the 27 apoptosis-regulated genes might be used as promising targets in treating and managing TNBC. Furthermore, from a total of 27 key genes, CASP2, CASP3, DAPK1, TNF, TRAF2, and TRAF3 were significantly correlated with poor overall survival in TNBC (p-value <0.05); they could play important roles in the progression of TNBC and provide attractive therapeutic targets that may offer new candidate molecules for targeted therapy. CONCLUSION: Our findings demonstrate that CASP2, CASP3, DAPK1, TNF, TRAF2, and TRAF3 were substantially associated with the overall survival rate (OS) difference of TNBC patients out of a total of 27 specific genes used in this study, which may play crucial roles in the development of TNBC and offer promising therapeutic interventions.

The Prognostic and Therapeutic Implications of the Chemoresistance Gene BIRC5 in Triple-Negative Breast Cancer.

Chemoresistance affects TNBC patient treatment responses. Therefore, identifying the chemoresistant gene provides a new approach to understanding chemoresistance in TNBC. BIRC5 was examined in the current study as a tool for predicting the prognosis of TNBC patients and assisting in developing alternative therapies using online database tools. According to the examined studies, BIRC5 was highly expressed in 45 to 90% of TNBC patients. BIRC5 is not only abundantly expressed but also contributes to resistance to chemotherapy, anti-HER2 therapy, and radiotherapy. Patients with increased expression of BIRC5 had a median survival of 31.2 months compared to 85.8 months in low-expression counterparts (HR, 1.73; CI, 1.4−2.13; p = 2.5 × 10−7). The overall survival, disease-free survival, relapse-free survival, distant metastasis-free survival, and the complete pathological response of TNBC patients with high expression of BIRC5 who received any chemotherapy (Taxane, Ixabepilone, FAC, CMF, FEC, Anthracycline) and anti-HER2 therapy (Trastuzumab, Lapatinib) did not differ significantly from those patients receiving any other treatment. Data obtained indicate that the BIRC5 promoter region was substantially methylated, and hypermethylation was associated with higher BIRC5 mRNA expression (p < 0.05). The findings of this study outline the role of BIRC5 in chemotherapy-induced resistance of TNBC, further indicating that BIRC5 may serve as a promising prognostic biomarker that contributes to chemoresistance and could be a possible therapeutic target. Meanwhile, several in vitro studies show that flavonoids were highly effective in inhibiting BIRC5 in genetically diverse TNBC cells. Therefore, flavonoids would be a promising strategy for preventing and treating TNBC patients with the BIRC5 molecule.

Anticancer Effects of Thymoquinone through the Antioxidant Activity, Upregulation of Nrf2, and Downregulation of PD-L1 in Triple-Negative Breast Cancer Cells.

The variety of therapies available for treating and preventing triple-negative breast cancer (TNBC) is constrained by the absence of progesterone receptors, estrogen receptors, and human epidermal growth factor receptor 2. Nrf2 (nuclear factor-erythroid 2-related factor), and PD-L1 (program cell death ligand 1), a downstream signaling target, have a strong correlation to oxidative stress and inflammation, major factors in the development and progression of TNBC. In this study, the genetically distinct MDA-MB-231 and MDA-MB-468 TNBC cells were treated with the natural component thymoquinone (TQ). The results show that TQ exhibits considerable antioxidant activity and decreases the generation of H2O2, at the same time increasing catalase (CAT) activity, superoxide dismutase (SOD) enzyme, and glutathione (GSH). Additionally, the results show that TQ treatment increased the levels of the different genes involved in the oxidative stress-antioxidant defense system PRNP, NQO1, and GCLM in both cell lines with significant large-fold change in MDA-MB-468 cells (+157.65 vs. +1.7, +48.87 vs. +2.63 and +4.78 vs. +2.17), respectively. Nrf2 mRNA and protein expression were also significantly increased in TQ-treated TNBC cells despite being higher in MDA-MB-468 cells (6.67 vs. 4.06). Meanwhile, TQ administration increased mRNA levels while decreasing PD-L1 protein expression in both cell lines. In conclusion, TQ modifies the expression of multiple oxidative-stress-antioxidant system genes, ROS, antioxidant enzymes, Nrf2, and PD-L1 protein, pointing to the therapeutic potential and chemopreventive utilization of TQ in TNBC.

Thymoquinone Anticancer Effects Through the Upregulation of NRF2 and the Downregulation of PD-L1 in MDA-MB-231 Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a metastatic nature. TNBC lacks the expression of the progesterone receptor, estrogen receptor, and human epidermal growth factor receptor 2. The absence of these three receptors limits the therapy options. Meanwhile, conventional medication is not very effective in the treatment and prevention of TNBC. Developing innovative therapeutic agents from natural bioactive compounds is a viable option. In the current study, the natural compound thymoquinone (TQ) was used in MDA-MB-231 TNBC cells. A dose response to TQ (0-50 µM) was obtained following a 24-hour exposure. An Annexin V-FITC apoptosis detection was used to study the apoptotic effect of TQ.  With increasing TQ concentrations from 0-20 µM, flow cytometer examination revealed an increase in apoptotic cells. Nearly 80% of the cells studied were in the apoptotic phase at 20 µM. (early and late apoptosis). Meanwhile, at 30 µM, there was a significant drop in apoptotic cells, which may be interpreted as an increase in necrotic cells. Molecular-targeted therapy is a new approach in treating cancer. The effect of TQ on the expression of Nrf2(Nuclear factor erythroid 2- related factor 2) and PD-L1 (Programmed death-ligand 1) was investigated using a specific primary antibody against these proteins. A Western blot analysis confirmed TQ's ability to change the expression of both proteins under investigation. According to normalized data, TQ had the ability to elicit more than 2-fold increase in Nrf2 expression in IFN-γ stimulated MDA-MB-231 cells. In contrast, co-treated cells (IFN-γ + TQ) showed a 65 percent reduction in PD-L1 expression. In conclusion, TQ was suggested as a promising anticancer option for treating  TNBC.

Thymoquinone Alterations of the Apoptotic Gene Expressions and Cell Cycle Arrest in Genetically Distinct Triple-Negative Breast Cancer Cells.

Breast cancer (BC) is the most common cancer in women worldwide, and it is one of the leading causes of cancer death in women. triple-negative breast Cancer (TNBC), a subtype of BC, is typically associated with the highest pathogenic grade and incidence in premenopausal and young African American (AA) women. Chemotherapy, the most common treatment for TNBC today, can lead to acquired resistance and ineffective treatment. Therefore, novel therapeutic approaches are needed to combat medication resistance and ineffectiveness in TNBC patients. Thymoquinone (TQ) is shown to have a cytotoxic effect on human cancer cells in vitro. However, TQ's mode of action and precise mechanism in TNBC disease in vitro have not been adequately investigated. Therefore, TQ's effects on the genetically different MDA-MB-468 and MDA-MB-231 human breast cancer cell lines were assessed. The data obtained show that TQ displayed cytotoxic effects on MDA-MB-468 and MDA-MB-231 cells in a time- and concentration-dependent manner after 24 h, with IC50 values of 25.37 µM and 27.39 µM, respectively. Moreover, MDA-MB-231 and MDA-MB-468 cells in a scratched wound-healing assay displayed poor wound closure, inhibiting invasion and migration via cell cycle blocking after 24 h. TQ arrested the cell cycle phase in MDA-MB-231 and MDA-MB-468 cells. The three cell cycle stages in MDA-MB-468 cells were significantly affected at 15 and 20 µM for G0/G1 and S phases, as well as all TQ concentrations for G2/M phases. In MDA-MB-468 cells, there was a significant decrease in G0/G1 phases with a substantial increase in the S phase and G2/M phases. In contrast, MDA-MB-231 showed a significant effect only during the two cell cycle stages (S and G2/M), at concentrations of 15 and 20 µM for S phases and all TQ values for G2/M phases. The TQ effect on the apoptotic gene profiles indicated that TQ upregulated 15 apoptotic genes in MDA-MB-231 TNBC cells, including caspases, GADD45A, TP53, DFFA, DIABLO, BNIP3, TRAF2/3, and TNFRSF10A. In MDA-MB-468 cells, 16 apoptotic genes were upregulated, including TNFRSF10A, TNF, TNFRSF11B, FADD TNFRSF10B, CASP2, and TRAF2, all of which are important for the apoptotic pathway andsuppress the expression of one anti-apoptotic gene, BIRC5, in MDA-MB-231 cells. Compared to MDA-MB-231 cells, elevated levels of TNF and their receptor proteins may contribute to their increased sensitivity to TQ-induced apoptosis. It was concluded from this study that TQ targets the MDA-MB-231 and MDA-MB-468 cells differently. Additionally, due to the aggressive nature of TNBC and the lack of specific therapies in chemoresistant TNBC, our findings related to the identified apoptotic gene profile may point to TQ as a potential agent for TNBC therapy.

Epigallocatechin-3-Gallate (EGCG): New Therapeutic Perspectives for Neuroprotection, Aging, and Neuroinflammation for the Modern Age.

Alzheimer's and Parkinson's diseases are the two most common forms of neurodegenerative diseases. The exact etiology of these disorders is not well known; however, environmental, molecular, and genetic influences play a major role in the pathogenesis of these diseases. Using Alzheimer's disease (AD) as the archetype, the pathological findings include the aggregation of Amyloid Beta (Aß) peptides, mitochondrial dysfunction, synaptic degradation caused by inflammation, elevated reactive oxygen species (ROS), and cerebrovascular dysregulation. This review highlights the neuroinflammatory and neuroprotective role of epigallocatechin-3-gallate (EGCG): the medicinal component of green tea, a known nutraceutical that has shown promise in modulating AD progression due to its antioxidant, anti-inflammatory, and anti-aging abilities. This report also re-examines the current literature and provides innovative approaches for EGCG to be used as a preventive measure to alleviate AD and other neurodegenerative disorders.

The Anticancer Effects of Flavonoids through miRNAs Modulations in Triple-Negative Breast Cancer.

Triple- negative breast cancer (TNBC) incidence rate has regularly risen over the last decades and is expected to increase in the future. Finding novel treatment options with minimum or no toxicity is of great importance in treating or preventing TNBC. Flavonoids are new attractive molecules that might fulfill this promising therapeutic option. Flavonoids have shown many biological activities, including antioxidant, anti-inflammatory, and anticancer effects. In addition to their anticancer effects by arresting the cell cycle, inducing apoptosis, and suppressing cancer cell proliferation, flavonoids can modulate non-coding microRNAs (miRNAs) function. Several preclinical and epidemiological studies indicate the possible therapeutic potential of these compounds. Flavonoids display a unique ability to change miRNAs' levels via different mechanisms, either by suppressing oncogenic miRNAs or activating oncosuppressor miRNAs or affecting transcriptional, epigenetic miRNA processing in TNBC. Flavonoids are not only involved in the regulation of miRNA-mediated cancer initiation, growth, proliferation, differentiation, invasion, metastasis, and epithelial-to-mesenchymal transition (EMT), but also control miRNAs-mediated biological processes that significantly impact TNBC, such as cell cycle, immune system, mitochondrial dysregulation, modulating signaling pathways, inflammation, and angiogenesis. In this review, we highlighted the role of miRNAs in TNBC cancer progression and the effect of flavonoids on miRNA regulation, emphasizing their anticipated role in the prevention and treatment of TNBC.

Therapeutic Potential of Thymoquinone in Triple-Negative Breast Cancer Prevention and Progression through the Modulation of the Tumor Microenvironment.

To date, the tumor microenvironment (TME) has gained considerable attention in various areas of cancer research due to its role in driving a loss of immune surveillance and enabling rapid advanced tumor development and progression. The TME plays an integral role in driving advanced aggressive breast cancers, including triple-negative breast cancer (TNBC), a pivotal mediator for tumor cells to communicate with the surrounding cells via lymphatic and circulatory systems. Furthermore, the TME plays a significant role in all steps and stages of carcinogenesis by promoting and stimulating uncontrolled cell proliferation and protecting tumor cells from the immune system. Various cellular components of the TME work together to drive cancer processes, some of which include tumor-associated adipocytes, fibroblasts, macrophages, and neutrophils which sustain perpetual amplification and release of pro-inflammatory molecules such as cytokines. Thymoquinone (TQ), a natural chemical component from black cumin seed, is widely used traditionally and now in clinical trials for the treatment/prevention of multiple types of cancer, showing a potential to mitigate components of TME at various stages by various pathways. In this review, we focus on the role of TME in TNBC cancer progression and the effect of TQ on the TME, emphasizing their anticipated role in the prevention and treatment of TNBC. It was concluded from this review that the multiple components of the TME serve as a critical part of TNBC tumor promotion and stimulation of uncontrolled cell proliferation. Meanwhile, TQ could be a crucial compound in the prevention and progression of TNBC therapy through the modulation of the TME.

Proteomic analysis of the effect of the polyphenol pentagalloyl glucose on proteins involved in neurodegenerative diseases in activated BV­2 microglial cells.

Neuroinflammation and microglial activation are two important hallmarks of neurodegenerative diseases. Continuous microglial activation may cause the release of several cytotoxic molecules, including many cytokines that are involved in the inflammatory process. Therefore, attenuating inflammation caused by activated microglia may be an approach for the therapeutic management of neurodegenerative diseases. In addition, many studies have reported that polyphenol pentagalloyl glucose (1,2,3,4,6­penta­O­galloyl­ß­D­glucose; PGG) is a molecule with potent anti­inflammatory effects, such as inhibiting the release of proinflammatory cytokines. Our previous studies revealed that PGG attenuated the expression of two inflammatory cytokines (murine monocyte chemoattractant protein­5 and pro­metalloproteinase­9) in lipopolysaccharide/interferon γ­activated BV­2 microglial cells. Additionally, PGG modulated the NF­κB and MAPK signaling pathways by altering genes and proteins, which may affect the MAPK cascade and NF­κB activation. The aim of the present study was to investigate the ability of PGG to modulate the expression of proteins released in activated BV­2 microglial cells, which may be involved in the pathological process of inflammation and neurodegeneration. Proteomic analysis of activated BV­2 cells identified 17 proteins whose expression levels were significantly downregulated by PGG, including septin­7, ataxin­2, and adenylosuccinate synthetase isozyme 2 (ADSS). These proteins were previously described as being highly expressed in neurodegenerative diseases and/or involved in the signaling pathways associated with the formation and growth of neuronal connections and the control of Alzheimer's disease pathogenesis. The inhibitory effect of PGG on ataxin­2, septin­7 and ADSS was further confirmed at the protein and transcriptional levels. Therefore, the obtained results suggest that PGG, with its potent inhibitory effects on ataxin­2, septin­7 and ADSS, may have potential use in the therapeutic management of neurodegenerative diseases.

The attenuating effects of 1,2,3,4,6 penta-O-galloyl-ß-d-glucose on pro-inflammatory responses of LPS/IFNγ-activated BV-2 microglial cells through NFƙB and MAPK signaling pathways.

BACKGROUND: Overactivated microglial cells exhibit chronic inflammatory response and can lead to the continuous production of pro-inflammatory cytokines, perpetuating inflammation, and ultimately resulting in neuronal injury. 1,2,3,4,6-Penta-O-Galloyl-ß-d-Glucose (PGG), which is a naturally occurring polyphenolic compound, has exhibited anti-inflammatory effect through the inhibition of many cytokines in different experimental models, but its effect on activated microglia cells was never described. In the present study, we investigated PGG effect in proteins involved in the NFƙB and MAPK signaling pathways, which play a central role in inflammation through their ability to induce transcription of pro-inflammatory genes. METHODS: PCR arrays and RT-PCR with individual primers were used to determine the effect of PGG on mRNA expression of genes involved in NFƙB and MAPK signaling pathways. Western blots were performed to confirm PCR results. RESULTS: The data obtained showed that PGG modulated the expression of 5 genes from the NFƙB (BIRC3, CHUK, IRAK1, NFƙB1, NOD1) and 2 genes from MAPK signaling pathway (CDK2 and MYC) when tested in RT-PCR assays. Western blots confirmed the PCR results at the protein level, showing that PGG attenuated the expression of total and phosphorylated proteins (CDK2, CHUK, IRAK1, and NFƙB1) involved in NFƙB and MAPK signaling. CONCLUSION: These findings show that PGG could modulate the expression of genes and proteins involved in the production of pro-inflammatory cytokines in microglia cells.

Thymoquinone increases the expression of neuroprotective proteins while decreasing the expression of pro-inflammatory cytokines and the gene expression NFκB pathway signaling targets in LPS/IFNγ -activated BV-2 microglia cells.

Neuroinflammation and microglial activation are pathological markers of a number of central nervous system (CNS) diseases. Chronic activation of microglia induces the release of excessive amounts of reactive oxygen species (ROS) and pro-inflammatory cytokines. Additionally, chronic microglial activation has been implicated in several neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Thymoquinone (TQ) has been identified as one of the major active components of the natural product Nigella sativa seed oil. TQ has been shown to exhibit anti-inflammatory, anti-oxidative, and neuroprotective effects. In this study, lipopolysaccharide (LPS) and interferon gamma (IFNγ) activated BV-2 microglial cells were treated with TQ (12.5 µM for 24 h). We performed quantitative proteomic analysis using Orbitrap/Q-Exactive Proteomic LC-MS/MS (Liquid chromatography-mass spectrometry) to globally assess changes in protein expression between the treatment groups. Furthermore, we evaluated the ability of TQ to suppress the inflammatory response using ELISArray™ for Inflammatory Cytokines. We also assessed TQ's effect on the gene expression of NFκB signaling targets by profiling 84 key genes via real-time reverse transcription (RT2) PCR array. Our results indicated that TQ treatment of LPS/IFNγ-activated microglial cells significantly increased the expression of 4 antioxidant, neuroprotective proteins: glutaredoxin-3 (21 fold; p < 0.001), biliverdin reductase A (15 fold; p < 0.0001), 3-mercaptopyruvate sulfurtransferase (11 fold; p < 0.01), and mitochondrial lon protease (>8 fold; p < 0.001) compared to the untreated, activated cells. Furthermore, TQ treatment significantly (P < 0.0001) reduced the expression of inflammatory cytokines, IL-2 = 38%, IL-4 = 19%, IL-6 = 83%, IL-10 = 237%, and IL-17a = 29%, in the activated microglia compared to the untreated, activated which expression levels were significantly elevated compared to the control microglia: IL-2 = 127%, IL-4 = 151%, IL-6 = 670%, IL-10 = 133%, IL-17a = 127%. Upon assessing the gene expression of NFκB signaling targets, this study also demonstrated that TQ treatment of activated microglia resulted in >7 fold down-regulation of several NFκB signaling targets genes, including interleukin 6 (IL6), complement factor B (CFB), chemokine (CC motif) ligand 3 (CXCL3), chemokine (CC) motif ligand 5 (CCL5) compared to the untreated, activated microglia. This modulation in gene expression counteracts the >10-fold upregulation of these same genes observed in the activated microglia compared to the controls. Our results show that TQ treatment of LPS/IFNγ-activated BV-2 microglial cells induce a significant increase in expression of neuroprotective proteins, a significant decrease in expression inflammatory cytokines, and a decrease in the expression of signaling target genes of the NFκB pathway. Our findings are the first to show that TQ treatment increased the expression of these neuroprotective proteins (biliverdin reductase-A, 3-mercaptopyruvate sulfurtransferase, glutaredoxin-3, and mitochondrial lon protease) in the activated BV-2 microglial cells. Additionally, our results indicate that TQ treatment decreased the activation of the NFκB signaling pathway, which plays a key role in neuroinflammation. In conclusion, our results demonstrate that TQ treatment reduces the inflammatory response and modulates the expression of specific proteins and genes and hence potentially reduce neuroinflammation and neurodegeneration driven by microglial activation.

Valproate and sodium butyrate attenuate manganese-decreased locomotor activity and astrocytic glutamate transporters expression in mice.

Manganese (Mn) is an essential trace element, but chronic overexposure to this metal, either environmentally or occupationally may cause manganism, a disease analogous to Parkinson's disease. Inhibitors of histone deacetylases, such as valproic acid (VPA) and sodium butyrate (NaB) exert neuroprotective effects in various animal models of neurological disorders. Thus, the present study investigated whether VPA or NaB prevent Mn-induced neurotoxicity by assessing locomotor activities and expression of astrocytic glutamate transporters, glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST), in C57BL/6 mice. C57BL/6 mice were pretreated with VPA (200mg/kg, i.p.) or NaB (1200mg/kg, i.p.) prior to intranasal instillation of Mn (30mg/kg) continually for 21days, followed by open-field and rota-rod behavioral tests and analyses of astrocytic glutamate transporters GLT-1 and GLAST protein/mRNA levels. The results showed that Mn significantly decreased locomotor activity as determined by total distance travelled, stereotypic and ambulatory counts. Mn also significantly decreased rota-rod activity reflecting altered motor coordination. Pretreatment with VPA and NaB with Mn reversed the effects of Mn on the locomotor activity and motor coordination. VPA and NaB also attenuated the Mn-induced decrease in GLT-1 and GLAST mRNA and protein levels in the cerebral cortical and cerebellar regions of mice. These results suggest that VPA and NaB exert protective effects against Mn toxicity seem in vitro are also shown in vivo. VPA and NaB pretreatment in mice enhancing astrocytic glutamate transporter GLT-1 expression as well as locomotor activities. Future research endeavors are warranted to determine if the therapeutic potential of VPA and NaB is via common molecular mechanism, namely, inhibition of histone deacetylases.

The attenuating effects of 1,2,3,4,6 penta-O-galloyl-ß-d-glucose on inflammatory cytokines release from activated BV-2 microglial cells.

Alzheimer's disease (AD) is the most common cause of neurodegeneration and dementia in the elderly. Dysregulated, chronic activation of microglia, the brain's resident macrophages, induces the release of excessive amounts of pro-inflammatory cytokines which has been implicated in the early stages of AD pathology. Therefore, suppressing the expression of these inflammatory mediators may decrease or delay the progression of AD. Many natural compounds derived from plants have shown anti-inflammatory activity. The naturally occurring 1,2,3,4,6 Penta-O-Galloyl-ß-d-Glucose (PGG), is a polyphenolic compound highly enriched in Rhus chinensis Millplant. It is a potent anti-inflammatory agent that act through the inhibition of many cytokines in different experimental models. In the present study, we investigated the role of PGG as an anti-inflammatory agent in LPS/IFNγ activated BV-2 microglia cells. Mouse cytokine antibody arrays were used to assess the effect of PGG on the release of pro-inflammatory cytokines, and ELISA experiments were performed to validate the results from the arrays. The results obtained from the cytokine arrays, and ELISA assays showed that PGG decreased the expression of monocyte chemotactic protein-5 (MCP-5) 8-fold, and pro-matrix metalloproteinase 9 (Pro MMP-9) 10-fold. Both of these cytokines are upregulated during the inflammatory process and have been shown to be involved in brain injury, inflammation, and neurodegeneration. Therefore, these findings suggest that the anti-inflammatory effect of PGG on activated microglia involving the attenuation of MCP-5 and Pro MMP-9 cytokines.

Natural product HTP screening for attenuation of cytokine-induced neutrophil chemo attractants (CINCs) and NO2- in LPS/IFNγ activated glioma cells.

Chronic and acute central nervous system (CNS) inflammation are contributors toward neurological injury associated with head trauma, stroke, infection, Parkinsons or Alzheimers disease. CNS inflammatory illnesses can also contribute toward risk of developing glioblastoma multiforme (GBM). With growing public interest in complementary and alternative medicines (CAMs), we conduct a high throughput (HTP) screening of >1400 natural herbs, plants and over the counter (OTC) products for anti-inflammatory effects on lipopolysaccharide (LPS)/interferon gamma (IFNγ) activated C6 glioma cells. Validation studies were performed showing a pro-inflammatory profile of [LPS 3 µg/ml/ IFNγ 3 ng/ml] consistent with greater release [>8.5 fold] of MCP-1, NO2-, cytokine-induced neutrophil chemo-attractants (CINC) 1, CINC 2a and CINC3. The data show no changes to the following, IL-13, TNF-a, fracktaline, leptin, LIX, GM-CSF, ICAM1, L-Selectin, activin A, agrin, IL-1α, MIP-3a, B72/CD86, NGF, IL-1b, MMP-8, IL-1 R6, PDGF-AA, IL-2, IL-4, prolactin R, RAGE, IL-6, Thymus Chemokine-1, CNTF,IL-10 or TIMP-1. A HTP screening was conducted, where we employ an in vitro efficacy index (iEI) defined as the ratio of toxicity (LC50)/anti-inflammatory potency (IC50). The iEI was precautionary to ensure biological effects were occurring in fully viable cells (ratio > 3.8) independent of toxicity. Using NO2- as a guideline molecule, the data show that 1.77% (25 of 1410 tested) had anti-inflammatory effects with iEI ratios >3.8 and IC50s <250µg/ml. These include reference drugs (hydrocortisone, dexamethasone N6-(1-iminoethyl)-l-lysine and NSAIDS: diclofenac, tolfenamic acid), a histone deacetylase inhibitor (apicidin) and the following natural products; Ashwaganda (Withania somnifera), Elecampagne Root (Inula helenium), Feverfew (Tanacetum parthenium), Green Tea (Camellia sinensis), Turmeric Root (Curcuma longa) Ganthoda (Valeriana wallichii), Tansy (Tanacetum vulgare), Maddar Root (Rubia tinctoria), Red Sandle wood (Pterocarpus santalinus), Bay Leaf (Laurus nobilis, Lauraceae), quercetin, cardamonin, fisetin, EGCG, biochanin A, galangin, apigenin and curcumin. The herb with the largest iEI was Ashwaganda where the IC50/LC50 was 11.1/>1750.0µg/ml, and the compound with the greatest iEI was quercetin where the IC50/LC50 was 10.0/>363.6µg/ml. These substances also downregulate the production of iNOS expression and attenuate CINC-3 release. In summary, this HTP screening provides guideline information about the efficacy of natural products that could prevent inflammatory processes associated with neurodegenerative disease and aggressive glioma tumor growth.

Natural product HTP screening for antibacterial (E.coli 0157:H7) and anti-inflammatory agents in (LPS from E. coli O111:B4) activated macrophages and microglial cells; focus on sepsis.

BACKGROUND: Acute systemic inflammatory response syndrome arising from infection can lead to multiple organ failure and death, with greater susceptibility occurring in immunocompromised individuals. Moreover, sub-acute chronic inflammation is a contributor to the pathology of diverse degenerative diseases (Parkinson's disease, Alzheimer's disease and arthritis). Given the known limitations in Western medicine to treat a broad range of inflammatory related illness as well as the emergence of antibiotic resistance, there is a renewed interest in complementary and alternative medicines (CAMs) to achieve these means. METHODS: A high throughput (HTP) screening of >1400 commonly sold natural products (bulk herbs, cooking spices, teas, leaves, supplement components, nutraceutical food components, fruit and vegetables, rinds, seeds, polyphenolics etc.) was conducted to elucidate anti-inflammatory substances in lipopolysaccharide (LPS) (E. coli serotype O111:B4) monocytes: RAW 264.7 macrophages [peripheral], BV-2 microglia [brain]) relative to hydrocortisone, dexamethasone and L-N6-(1Iminoethyl)lysine (L-NIL). HTP evaluation was also carried out for lethal kill curves against E.coli 0157:H7 1x106 CFU/mL relative to penicillin. Validation studies were performed to assess cytokine profiling using antibody arrays. Findings were corroborated by independent ELISAs and NO2-/iNOS expression quantified using the Griess Reagent and immunocytochemistry, respectively. For robust screening, we developed an in-vitro efficacy paradigm to ensure anti-inflammatory parameters were observed independent of cytotoxicity. This caution was taken given that many plants exert tumoricidal and anti-inflammatory effects at close range through similar signaling pathways, which could lead to false positives. RESULTS: The data show that activated BV-2 microglia cells (+ LPS 1µg/ml) release >10-fold greater IL-6, MIP1/2, RANTES and nitric oxide (NO2-), where RAW 264.7 macrophages (+ LPS 1µg/ml) produced > 10-fold rise in sTNFR2, MCP-1, IL-6, GCSF, RANTES and NO2-. Data validation studies establish hydrocortisone and dexamethasone as suppressing multiple pro-inflammatory processes, where L-NIL suppressed NO2-, but had no effect on iNOS expression or IL-6. The screening results demonstrate relative few valid hits with anti-inflammatory effects at < 250µg/ml for the following: Bay Leaf (Laurus nobilis), Elecampagne Root (Inula helenium), Tansy (Tanacetum vulgare),Yerba (Eriodictyon californicum) and Centipeda (Centipeda minima), Ashwagandha (Withania somnifera), Feverfew (Tanacetum parthenium), Rosemary (Rosmarinus officinalis), Turmeric Root (Curcuma Longa), Osha Root (Ligusticum porteri), Green Tea (Camellia sinensis) and constituents: cardamonin, apigenin, quercetin, biochanin A, eupatorin, (-)-epigallocatechin gallate (EGCG) and butein. Natural products lethal against [E. coli 0157:H7] where the LC50 < 100 µg/ml included bioactive silver hydrosol-Argentyn 23, green tea (its constituents EGCG > Polyphenon 60 > (-)-Gallocatechin > Epicatechin > (+)-Catechin), Grapeseed Extract (Vitis vinifera), Chinese Gallnut (its constituents gallic acid > caffeic acid) and gallic acid containing plants such as Babul Chall Bark (Acacia Arabica), Arjun (Terminalia Arjuna) and Bayberry Root Bark (Morella Cerifera). CONCLUSIONS: These findings emphasize and validate the previous work of others and identify the most effective CAM anti-inflammatory, antibacterial compounds using these models. Future work will be required to evaluate potential combination strategies for long-term use to prevent chronic inflammation and possibly lower the risk of sepsis in immunocompromised at risk populations.

The Antioxidant Effects of Thymoquinone in Activated BV-2 Murine Microglial Cells.

Both neuroinflammation and microglial activation are pathological markers of a number of central nervous system (CNS) diseases. During chronic activation of the microglial cells, the induced release of excessive amounts of reactive oxygen species (ROS) and pro-inflammatory cytokines have been implicated in several neurodegenerative diseases such as Alzheimer's disease. Thymoquinone (TQ), a major bioactive compound of the natural product Nigella sativa seed, has been shown to be effective against numerous oxidative stress-induced and inflammatory disorders as well as possess neuroprotective properties. In this study, we investigated the antioxidant effects of TQ on LPS/IFNγ or H2O2-activated BV-2 microglia by assessing the levels of specific oxidative stress markers, the activities of selected antioxidant enzymes, as well as profiling 84 key genes related to oxidative stress via real-time reverse transcription (RT2) PCR array. Our results showed that in the LPS/IFNγ-activated microglia TQ significantly decreased the cellular production of both superoxide and nitric oxide fourfold (p < 0.0001) and sixfold (p < 0.0001), respectfully. In the H2O2-activated microglia, TQ also significantly decreased the cellular production of superoxide threefold (p < 0.0001) and significantly decreased hydrogen peroxide levels ~20 % (p < 0.05). Moreover, ΤQ treatment significantly decreased the levels oxidative stress in the activated BV-2 as evidenced by the assessed levels of lipid hydroperoxides and glutathione. TQ significantly decreased the levels of lipid hydroperoxides twofold (p < 0.0001) and significantly increased the levels of antioxidant glutathione 2.5-fold (p < 0.0001) in the LPS/IFNγ-activated BV-2 cells. In the H2O2-activated microglia, TQ significantly decreased lipid hydroperoxides eightfold (p < 0.0001) and significantly increased glutathione 15 % (p < 0.05). Activities of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), in the TQ-treated microglial cells also reflected a reduced oxidative stress status in the cellular environment. SOD and CAT activities were sixfold (p < 0.0001) and fivefold (p < 0.0001) lower, respectfully, for the LPS/INFγ-activated microglia treated with TQ in comparison to those that were not. For the H2O2-activated microglia treated with TQ, SOD and CAT activities were fivefold (p < 0.0001) and threefold (p < 0.01) lower, respectfully, compared to the untreated. Furthermore, RT2 PCR array profiling of the selected 84 genes related to oxidative stress confirmed that TQ treatment in the LPS/IFNγ-activated microglia downregulates specific pro-oxidant genes, upregulates specific anti-oxidant genes, and enhances the up- or downregulation of specific genes related to the cells' natural antioxidant defense against LPS/IFNγ activation. These findings suggest that TQ may be utilized as an effective therapeutic agent for delaying the onset and/or slowing/preventing the progression of microglia-derived neurodegeneration propagated by excessive oxidative stress in the CNS.