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.

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.

Antioxidant and Anti-Inflammatory Mechanisms of Cardamonin through Nrf2 Activation and NF-kB Suppression in LPS-Activated BV-2 Microglial Cells.

Chronic oxidative stress (OS) and inflammation are implicated in developing and progressing neurodegenerative diseases (NDs). The chronic activation of microglia cells leads to the overproduction of several substances, including nitric oxide and reactive oxygen species, which can induce neurodegeneration. Natural compounds have recently been investigated for their potential to protect cells from OS and to improve many disease-related conditions. Cardamonin (CD) is a bioactive compound in many plants, such as Alpinia katsumadai and Alpinia conchigera. The present study examined the effects of CD on LPS-activated BV-2 microglial cells. The cell viability results showed that the increasing concentrations of CD, ranging from 0.78 to 200 µM, induced BV-2 cell cytotoxicity in a dose-response manner. In the nitric oxide assay, CD concentrations of 6.25 to 25 µM reduced the release of nitric oxide in LPS-activated BV-2 cells by 90% compared to those treated with LPS only (p ≤ 0.0001). CD (6.25 µM) significantly decreased the cellular production of SOD (3-fold (p ≤ 0.05)) and increased the levels of expression of CAT (2.5-fold (p ≤ 0.05)) and GSH (2-fold (p ≤ 0.05)) in the LPS-activated BV-2 cells. Furthermore, on RT-PCR arrays, CD (6.25 µM) downregulated mRNA expression of CCL5/RANTES (5-fold), NOS2 (2-fold), SLC38A1 (3-fold), TXNIP (2-fold), SOD1 (2-fold), SOD2 (1.5-fold) and upregulated GSS (1.9-fold), GCLC (1.7-fold) and catalase (2.9-fold) expression, indicating CD efficacy in modulating genes involved in OS and inflammation. Furthermore, CD (6.25 µM) increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and lowered the levels of Kelch-like ECH-associated protein 1 (Keap1), indicating that this may be the signaling responsible for the elevation of antioxidant factors. Lastly, the results showed that CD (6.25 µM) modulated genes and proteins associated with the NF-kB signaling, downregulating genes related to excessive neuroinflammation. These results imply that CD may be a potential compound for developing therapeutic and preventive agents in treating neurodegeneration induced by excessive OS and inflammation.

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.

Anticancer Effects of Fucoxanthin through Cell Cycle Arrest, Apoptosis Induction, and Angiogenesis Inhibition in Triple-Negative Breast Cancer Cells.

The absence of progesterone receptors, estrogen receptors, and human epidermal growth factor receptor-2 restricts the therapy choices for treating triple-negative breast cancer (TNBC). Moreover, conventional medication is not highly effective in treating TNBC, and developing effective therapeutic agents from natural bioactive compounds is a viable option. In this study, the anticancer effects of the natural compound fucoxanthin were investigated in two genetically different models of TNBC cells: MDA-MB-231 and MDA-MB-468 cells. Fucoxanthin had a significant anticancer effect in both cell lines at a concentration range of 1.56-300 µM. The compound decreased cell viability in both cell lines with higher potency in MDA-MB-468 cells. Meanwhile, proliferation assays showed similar antiproliferative effects in both cell lines after 48 h and 72 h treatment periods. Flow cytometry and Annexin V-FITC apoptosis assay revealed the ability of fucoxanthin to induce apoptosis in MDA-MB-231 only. Cell cycle arrest analysis showed that the compound also induced cell cycle arrest at the G1 phase in both cell lines, accompanied by more cell cycle arrest in MDA-MB-231 cells at S-phase and a higher cell cycle arrest in the MDA-MB-468 cells at G2-phase. Wound healing and migration assay showed that in both cell lines, fucoxanthin prevented migration, but was more effective in MDA-MB-231 cells in a shorter time. In both angiogenic cytokine array and RT-PCR studies, fucoxanthin (6.25 µM) downregulated VEGF-A and -C expression in TNF-α-stimulated (50 ng/mL) MDA-MB-231, but not in MDA-MB-468 cells on the transcription and protein levels. In conclusion, this study shows that fucoxanthin was more effective in MDA-MB-231 TNBC cells, where it can target VEGF-A and VEGF-C, inhibit cell proliferation and cell migration, and induce cell cycle arrest and apoptosis-the most crucial cellular processes involved in breast cancer development and progression.

The Anticancer Effects of Marine Carotenoid Fucoxanthin through Phosphatidylinositol 3-Kinase (PI3K)-AKT Signaling on Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks specific targets such as estrogen, progesterone, and HER2 receptors. TNBC affects one in eight women in the United States, making up 15-20% of breast cancer cases. Patients with TNBC can develop resistance to chemotherapy over time, leading to treatment failure. Therefore, finding other options like natural products is necessary for treatment. The advantages of using natural products sourced from plants as anticancer agents are that they are less toxic, more affordable, and have fewer side effects. These products can modulate several cellular processes of the tumor microenvironment, such as proliferation, migration, angiogenesis, cell cycle arrest, and apoptosis. The phosphatidyl inositol 3-kinase (PI3K)-AKT signaling pathway is an important pathway that contributes to the survival and growth of the tumor microenvironment and is associated with these cellular processes. This current study examined the anticancer effects of fucoxanthin, a marine carotenoid isolated from brown seaweed, in the MDA-MB-231 and MDA-MB-468 TNBC cell lines. The methods used in this study include a cytotoxic assay, PI3K-AKT signaling pathway PCR arrays, and Wes analysis. Fucoxanthin (6.25 µM) + TNF-α (50 ng/mL) and TNF-α (50 ng/mL) showed no significant effect on cell viability compared to the control in both MDA-MB-231 and MDA-MB-468 cells after a 24 h treatment period. PI3K-AKT signaling pathway PCR array studies showed that in TNF-α-stimulated (50 ng/mL) MDA-MB-231 and MDA-MB-468 cells, fucoxanthin (6.25 µM) modulated the mRNA expression of 12 genes, including FOXO1, RASA1, HRAS, MAPK3, PDK2, IRS1, EIF4EBP1, EIF4B, PTK2, TIRAP, RHOA, and ELK1. Additionally, fucoxanthin significantly downregulated the protein expression of IRS1, EIF4B, and ELK1 in MDA-MB-231 cells, and no change in the protein expression of EIF4B and ELK1 was shown in MDA-MB-468 cells. Fucoxanthin upregulated the protein expression of RHOA in both cell lines. The modulation of the expression of genes and proteins of the PI3K-AKT signaling pathway may elucidate fucoxanthin's effects in cell cycle progression, apoptotic processes, migration, and proliferation, which shows that PI3K-AKT may be the possible molecular mechanism for fucoxanthin's effects. In conclusion, the results obtained in this study elucidate fucoxanthin's molecular mechanisms and indicate that fucoxanthin may be considered a promising candidate for breast cancer-targeted therapy.

Astrocytic transcription factor REST upregulates glutamate transporter EAAT2, protecting dopaminergic neurons from manganese-induced excitotoxicity.

Chronic exposure to high levels of manganese (Mn) leads to manganism, a neurological disorder with similar symptoms to those inherent to Parkinson's disease. However, the underlying mechanisms of this pathological condition have yet to be established. Since the human excitatory amino acid transporter 2 (EAAT2) (glutamate transporter 1 in rodents) is predominantly expressed in astrocytes and its dysregulation is involved in Mn-induced excitotoxic neuronal injury, characterization of the mechanisms that mediate the Mn-induced impairment in EAAT2 function is crucial for the development of novel therapeutics against Mn neurotoxicity. Repressor element 1-silencing transcription factor (REST) exerts protective effects in many neurodegenerative diseases. But the effects of REST on EAAT2 expression and ensuing neuroprotection are unknown. Given that the EAAT2 promoter contains REST binding sites, the present study investigated the role of REST in EAAT2 expression at the transcriptional level in astrocytes and Mn-induced neurotoxicity in an astrocyte-neuron coculture system. The results reveal that astrocytic REST positively regulates EAAT2 expression with the recruitment of an epigenetic modifier, cAMP response element-binding protein-binding protein/p300, to its consensus binding sites in the EAAT2 promoter. Moreover, astrocytic overexpression of REST attenuates Mn-induced reduction in EAAT2 expression, leading to attenuation of glutamate-induced neurotoxicity in the astrocyte-neuron coculture system. Our findings demonstrate that astrocytic REST plays a critical role in protection against Mn-induced neurotoxicity by attenuating Mn-induced EAAT2 repression and the ensuing excitotoxic dopaminergic neuronal injury. This indicates that astrocytic REST could be a potential molecular target for the treatment of Mn toxicity and other neurological disorders associated with EAAT2 dysregulation.

Sanguinarine Inhibition of TNF-α-Induced CCL2, IKBKE/NF-κB/ERK1/2 Signaling Pathway, and Cell Migration in Human Triple-Negative Breast Cancer Cells.

Angiogenesis is a process that drives breast cancer (BC) progression and metastasis, which is linked to the altered inflammatory process, particularly in triple-negative breast cancer (TNBC). In targeting inflammatory angiogenesis, natural compounds are a promising option for managing BC. Thus, this study was designed to determine the natural alkaloid sanguinarine (SANG) potential for its antiangiogenic and antimetastatic properties in triple-negative breast cancer (TNBC) cells. The cytotoxic effect of SANG was examined in MDA-MB-231 and MDA-MB-468 cell models at a low molecular level. In this study, SANG remarkably inhibited the inflammatory mediator chemokine CCL2 in MDA-MB-231 and MDA-MB-468 cells. Furthermore, qRT-PCR confirmed with Western analysis studies showed that mRNA CCL2 repression was concurrent with reducing its main regulator IKBKE and NF-κB signaling pathway proteins in both TNBC cell lines. The total ERK1/2 protein was inhibited in the more responsive MDA-MB-231 cells. SANG exhibited a higher potential to inhibit cell migration in MDA-MB-231 cells compared to MDA-MB-468 cells. Data obtained in this study suggest a unique antiangiogenic and antimetastatic effect of SANG in the MDA-MB-231 cell model. These effects are related to the compound's ability to inhibit the angiogenic CCL2 and impact the ERK1/2 pathway. Therefore, SANG use may be recommended as a component of the therapeutic strategy for TNBC.

Anticancer Effects of Fucoxanthin through Cell Cycle Arrest, Apoptosis Induction, Angiogenesis Inhibition, and Autophagy Modulation.

Cancer accounts for one in seven deaths worldwide and is the second leading cause of death in the United States, after heart disease. One of the standard cancer treatments is chemotherapy which sometimes can lead to chemoresistance and treatment failure. Therefore, there is a great need for novel therapeutic approaches to treat these patients. Novel natural products have exhibited anticancer effects that may be beneficial in treating many kinds of cancer, having fewer side effects, low toxicity, and affordability. Numerous marine natural compounds have been found to inhibit molecular events and signaling pathways associated with various stages of cancer development. Fucoxanthin is a well-known marine carotenoid of the xanthophyll family with bioactive compounds. It is profusely found in brown seaweeds, providing more than 10% of the total creation of natural carotenoids. Fucoxanthin is found in edible brown seaweed macroalgae such as Undaria pinnatifida, Laminaria japonica, and Eisenia bicyclis. Many of fucoxanthin's pharmacological properties include antioxidant, anti-tumor, anti-inflammatory, antiobesity, anticancer, and antihypertensive effects. Fucoxanthin inhibits many cancer cell lines' proliferation, angiogenesis, migration, invasion, and metastasis. In addition, it modulates miRNA and induces cell cycle growth arrest, apoptosis, and autophagy. Moreover, the literature shows fucoxanthin's ability to inhibit cytokines and growth factors such as TNF-α and VEGF, which stimulates the activation of downstream signaling pathways such as PI3K/Akt autophagy, and pathways of apoptosis. This review highlights the different critical mechanisms by which fucoxanthin inhibits diverse cancer types, such as breast, prostate, gastric, lung, and bladder development and progression. Moreover, this article reviews the existing literature and provides critical supportive evidence for fucoxanthin's possible therapeutic use in cancer.

Transcriptome Profile Analysis of Triple-Negative Breast Cancer Cells in Response to a Novel Cytostatic Tetrahydroisoquinoline Compared to Paclitaxel.

The absence of chemotherapeutic target hormone receptors in breast cancer is descriptive of the commonly known triple-negative breast cancer (TNBC) subtype. TNBC remains one of the most aggressive invasive breast cancers, with the highest mortality rates in African American women. Therefore, new drug therapies are continually being explored. Microtubule-targeting agents such as paclitaxel (Taxol) interfere with microtubules dynamics, induce mitotic arrest, and remain a first-in-class adjunct drug to treat TNBC. Recently, we synthesized a series of small molecules of substituted tetrahydroisoquinolines (THIQs). The lead compound of this series, with the most potent cytostatic effect, was identified as 4-Ethyl-N-(7-hydroxy-3,4-dihydroisoquinolin-2(1H)-yl) benzamide (GM-4-53). In our previous work, GM-4-53 was similar to paclitaxel in its capacity to completely abrogate cell cycle in MDA-MB-231 TNBC cells, with the former not impairing tubulin depolymerization. Given that GM-4-53 is a cytostatic agent, and little is known about its mechanism of action, here, we elucidate differences and similarities to paclitaxel by evaluating whole-transcriptome microarray data in MDA-MB-231 cells. The data obtained show that both drugs were cytostatic at non-toxic concentrations and caused deformed morphological cytoskeletal enlargement in 2D cultures. In 3D cultures, the data show greater core penetration, observed by GM-4-53, than paclitaxel. In concentrations where the drugs entirely blocked the cell cycle, the transcriptome profile of the 48,226 genes analyzed (selection criteria: (p-value, FDR p-value < 0.05, fold change -2< and >2)), paclitaxel evoked 153 differentially expressed genes (DEGs), GM-4-53 evoked 243 DEGs, and, of these changes, 52/153 paclitaxel DEGs were also observed by GM-4-53, constituting a 34% overlap. The 52 DEGS analysis by String database indicates that these changes involve transcripts that influence microtubule spindle formation, chromosome segregation, mitosis/cell cycle, and transforming growth factor-ß (TGF-ß) signaling. Of interest, both drugs effectively downregulated "inhibitor of DNA binding, dominant negative helix-loop-helix" (ID) transcripts; ID1, ID3 and ID4, and amphiregulin (AREG) and epiregulin (EREG) transcripts, which play a formidable role in cell division. Given the efficient solubility of GM-4-53, its low molecular weight (MW; 296), and capacity to penetrate a small solid tumor mass and effectively block the cell cycle, this drug may have future therapeutic value in treating TNBC or other cancers. Future studies will be required to evaluate this drug in preclinical models.

Manganese Accumulation in the Brain via Various Transporters and Its Neurotoxicity Mechanisms.

Manganese (Mn) is an essential trace element, serving as a cofactor for several key enzymes, such as glutamine synthetase, arginase, pyruvate decarboxylase, and mitochondrial superoxide dismutase. However, its chronic overexposure can result in a neurological disorder referred to as manganism, presenting symptoms similar to those inherent to Parkinson's disease. The pathological symptoms of Mn-induced toxicity are well-known, but the underlying mechanisms of Mn transport to the brain and cellular toxicity leading to Mn's neurotoxicity are not completely understood. Mn's levels in the brain are regulated by multiple transporters responsible for its uptake and efflux, and thus, dysregulation of these transporters may result in Mn accumulation in the brain, causing neurotoxicity. Its distribution and subcellular localization in the brain and associated subcellular toxicity mechanisms have also been extensively studied. This review highlights the presently known Mn transporters and their roles in Mn-induced neurotoxicity, as well as subsequent molecular and cellular dysregulation upon its intracellular uptakes, such as oxidative stress, neuroinflammation, disruption of neurotransmission, α-synuclein aggregation, and amyloidogenesis.

The Antiproliferative Effects of Flavonoid MAO Inhibitors on Prostate Cancer Cells.

Prostate cancer (PCa) patients commonly experience clinical depression. Recent reports indicated that monoamine oxidase-A (MAO-A) levels elevate in PCa, and antidepressant MAO-Is show anti-PCa properties. In this work, we aimed to find potential drugs for PCa patients suffering from depression by establishing novel anti-PCa reversible monoamine oxidase-A inhibitors (MAO-AIs/RIMA); with an endeavor to understand their mechanism of action. In this investigation, twenty synthesized flavonoid derivatives, defined as KKR compounds were screened for their inhibitory potentials against human MAO-A and MAO-B isozymes. Meanwhile, the cytotoxic and antiproliferative effects were determined in three human PCa cell lines. MAO-A-kinetics, molecular docking, SAR, cell morphology, and cell migration were investigated for the most potent compounds. The screened KKRs inhibited MAO-A more potently than MAO-B, and non-toxically inhibited LNCaP cell proliferation more than the DU145 and PC3 cell lines, respectively. The results showed that the three top MAO-AI KKRs compounds (KKR11, KKR20, and KKR7 (IC50s 0.02-16 µM) overlapped with the top six antiproliferative KKRs against LNCaP (IC50s ~9.4 µM). While KKR21 (MAO-AI) and KKR2A (MAO-I) were ineffective against the PCa cells. Furthermore, KKR21 and KKR11 inhibited MAO-A competitively (Kis ≤ 7.4 nM). Molecular docking of the two compounds predicted shared hydrophobic and distinctive hydrophilic interactions-between the KKR molecule and MAO-A amino acid residues-to be responsible for their reversibility. The combined results and SAR observations indicated that the presence of specific active groups-such as chlorine and hydroxyl groups-are essential in certain MAO-AIs with anti-PCa effects. Additionally, MAO-A inhibition was found to be associated more with anti-PCa property than MAO-B. Distinctively, KKR11 [(E)-3-(3,4-dichlorophenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one] exhibited anti-metastatic effects on the DU145 cell line. The chlorine substitution groups might play vital roles in the KKR11 multiple actions. The obtained results indicated that the flavonoid derivative KKR11 could present a novel candidate for PCa patients with depression, through safe non-selective potent inhibition of MAOs.

The Benzopyrone Biochanin-A as a reversible, competitive, and selective monoamine oxidase B inhibitor.

BACKGROUND: Monoamine oxidase-B (MAO-B) inhibitors are widely used in the treatment of Parkinson's disease. They increase vital monoamine neurotransmitters in the brain. However, there is a need for safer natural reversible MAO inhibitors with MAO-B selectivity. Our previous studies showed that Psoralea corylifolia seeds (PCS) extract contains compounds that inhibit monoamine oxidase-B. METHODS: In this study, six of PCS constituents sharing a benzopyrone structure were investigated. The compounds Biochanin-A (BIO-A), isopsoralen, 6-prenylnaringenin, neobavaisoflavone, psoralen, and psoralidin, were tested for their ability to inhibit recombinant human MAO-A and B (hMAO-A and hMAO-B) isozymes. The ability of these compounds to inhibit MAO-A and MAO-B were compared to that of PCS ethanolic extract (PCSEE) using spectrophotometric assays and confirmed by luminescence assays. The highly potent and selective MAO-B inhibitor, BIO-A, was further investigated for both isozymes reversibility and enzyme kinetics. Molecular docking studies were used to predict the bioactive conformation and molecular interactions of BIO-A with both isozymes. RESULTS: The data obtained indicate that benzopyrones inhibited hMAO-A and hMAO-B with different degrees as confirmed with the luminescence assay. BIO-A inhibited hMAO-B with high potency and selectivity in the present study (IC50 = 0.003 µg/mL) and showing 38-fold more selectivity than PCSEE (hMAO-B IC50 = 3.03 µg/mL, 17-fold selectivity) without affecting hydrogen peroxide. Furthermore, BIO-A reversibly and competitively inhibited both hMAOs with significantly lower inhibitory constant (Ki) in hMAO-B (3.8 nM) than hMAO-A (99.3 nM). Our docking studies indicated that the H-bonds and hydrophobic interactions at the human MAO-A and MAO-B active sites contributed to the reversibility and selectivity of BIO-A. CONCLUSIONS: The data obtained indicate that BIO-A is a potent, reversible and selective MAO-B inhibitor and may be recommended for further investigation in its possible use in the therapeutic management of Parkinson's and Alzheimer's diseases.

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.

Design and evaluation of novel oxadiazole derivatives as potential prostate cancer agents.

Various 1,3,4-oxadiazole derivatives have been synthesized and their antiproliferative properties have been studied. The in vitro screening was performed against androgen dependent (LNCaP) and androgen independent (PC-3) prostate cancer cell lines. Most of the compounds showed promising activity. Among them, compounds 2d (IC50=0.22 and 1.3µM) and 2a (IC50=8.34 and 2,5µM) have shown significant activities on PC-3 and LNCaP cell lines respectively. To investigate the mechanism of cell death we performed cell apoptosis staining and cell cycle arrest assay on more sensitive PC-3 cell lines on 2d. The results demonstrated that 2d induced apoptosis and shifted the cells to the sub G0/G1 and S phase. Our study evidently identified the potency of compound 2d as potential anti-prostate cancer agent.

Synthesis of "neoprofen", a rigidified analogue of ibuprofen, exemplifying synthetic methodology for altering the 3-D topology of pharmaceutical substances.

3,3-Dimethylcyclopentanes (neopentylenes) are ubiquitous in Nature but largely absent from synthetic pharmaceutical libraries. Neopentylenes define a hydrophobic and rigid 3-D topology with distinct molecular pharmacology, as exemplified here with two neopentylene-fused analogues of the synthetic anti-inflammatory drug, ibuprofen.

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.

Neurotrophic Effects of Mu Bie Zi (Momordica cochinchinensis) Seed Elucidated by High-Throughput Screening of Natural Products for NGF Mimetic Effects in PC-12 Cells.

Post-mitotic central nervous system (CNS) neurons have limited capacity for regeneration, creating a challenge in the development of effective therapeutics for spinal cord injury or neurodegenerative diseases. Furthermore, therapeutic use of human neurotrophic agents such as nerve growth factor (NGF) are limited due to hampered transport across the blood brain barrier (BBB) and a large number of peripheral side effects (e.g. neuro-inflammatory pain/tissue degeneration etc.). Therefore, there is a continued need for discovery of small molecule NGF mimetics that can penetrate the BBB and initiate CNS neuronal outgrowth/regeneration. In the current study, we conduct an exploratory high-through-put (HTP) screening of 1144 predominantly natural/herb products (947 natural herbs/plants/spices, 29 polyphenolics and 168 synthetic drugs) for ability to induce neurite outgrowth in PC12 dopaminergic cells grown on rat tail collagen, over 7 days. The data indicate a remarkably rare event-low hit ratio with only 1/1144 tested substances (<111.25 µg/mL) being capable of inducing neurite outgrowth in a dose dependent manner, identified as; Mu Bie Zi, Momordica cochinchinensis seed extract (MCS). To quantify the neurotrophic effects of MCS, 36 images (n = 6) (average of 340 cells per image), were numerically assessed for neurite length, neurite count/cell and min/max neurite length in microns (µm) using Image J software. The data show neurite elongation from 0.07 ± 0.02 µm (controls) to 5.5 ± 0.62 µm (NGF 0.5 µg/mL) and 3.39 ± 0.45 µm (138 µg/mL) in MCS, where the average maximum length per group extended from 3.58 ± 0.42 µm (controls) to 41.93 ± 3.14 µm (NGF) and 40.20 ± 2.72 µm (MCS). Imaging analysis using immunocytochemistry (ICC) confirmed that NGF and MCS had similar influence on 3-D orientation/expression of 160/200 kD neurofilament, tubulin and F-actin. These latent changes were associated with early rise in phosphorylated extracellular signal-regulated kinase (ERK) p-Erk1 (T202/Y204)/p-Erk2 (T185/Y187) at 60 min with mild changes in pAKT peaking at 5 min, and no indication of pMEK involvement. These findings demonstrate a remarkable infrequency of natural products or polyphenolic constituents to exert neurotrophic effects at low concentrations, and elucidate a unique property of MCS extract to do so. Future research will be required to delineate in depth mechanism of action of MCS, constituents responsible and potential for therapeutic application in CNS degenerative disease or injury.

Biological evaluation of synthetic chalcone and flavone derivatives as anti-inflammatory agents.

Flavonoids and chalcones are natural plant derived compounds with inherent therapeutic value for a range of human pathologies. In this study, a series of 24 substituted chalcones and flavones were synthesized and subsequently screened for anti-inflammatory effects on lipopolysaccharide (1 µg/ml)-activated BV-2 microglial cells by assessing initial production/release of nitric oxide (NO). The data obtained eliminate the majority of compounds as weak or non-effective, whereas 2'-hydroxy-3,4,5,3',4'-pentamethoxychalcone (1) and 2'-hydroxy-3,4,5-trimethoxychalcone (2) were potent, having an IC50 of 1.10 and 2.26 µM, respectively; with greater potency than L-N6-(1-iminoethyl)lysine selective iNOS inhibitor (IC50 = 3.1 µM) but less than steroidal dexamethasone (IC50 < 200 nM). The most potent compound (chalcone 1) attenuated NO parallel to reducing iNOS protein expression, events also corresponding to reduction of IL-1α, IL-10 and IL-6 pro-inflammatory cytokines. These findings suggest that the presence of electron donating groups OH and OCH3 on both A and B rings of synthetic compounds correlate to stronger anti-inflammatory potency.

Synthesis and biological evaluation of substituted N-[3-(1H-pyrrol-1-yl)methyl]-1,2,5,6-tetrahydropyridin-1-yl]benzamide/benzene sulfonamides as anti-inflammatory agents.

The pharmacological activities of tetrahydropyridine (THP) derivatives are dependent on the substituent ring moiety. In this study, we investigate the anti-inflammatory activities of 12 newly synthesized substituted N-[3-(1H-pyrrol-1-yl)methyl]-1,2,5,6-tetrahydrobenzamide/benzene sulfonamides (9a-l) in murine BV-2 microglial cells. All compounds were initially screened for attenuation of nitric oxide (NO) production in lipopolysaccharide (LPS) (1 µg/mL)-activated microglial cells. The data show that only SO2 -substituted THPs were effective at sub-lethal concentrations (IC50 values of 12.92 µM (9i), 14.64 µM (9j), 19.63 µM (9k)) relative to L-N6-(1-iminoethyl)lysine positive control (IC50 = 3.1 µM). The most potent SO2 -substituted compound (9i) also blocked the LPS-inducible nitric oxide synthase (iNOS) and attenuated the release of several cytokines including IL-1α, IL-10, and IL-6. These findings establish the moderate immuno-modulating effects of SO2 -substituted THP derivatives.