Thymoquinone Nanoparticle Induces Apoptosis and Cell Migration Retardation through Modulating of SUMOylation Process Genes in Breast Cancer Cell Line.

Background: Due to the heterogeneity of breast cancer, most advanced-stage patients are resistant to therapy. Disruption of SUMOylation, a post-translational modification, is linked to breast cancer. Objective: This study aimed to assess the impact of thymoquinone nanoparticles (Liposomal-TQ), an anti-cancer drug, combined with doxorubicin (DXR), the most effective chemotherapeutic drug used to treat breast cancer, on the expression of SENP2 and SENP6, two major components involved in the SUMOylation process, in normal and cancerous breast cell lines. Materials and Methods: The MCF7 cell line, a breast cancer cell line, and MCF10, a non-tumor epithelial cell line, were separately treated with Liposomal-TQ and DXR. Cell viability and cell migration were assessed using MTT and scratch tests. Apoptosis analysis was performed using annexin-V/PI staining. Gene expression analysis of SENP2 and SENP6 was conducted using quantitative real-time PCR (RT-qPCR). Additionally, the scratch test evaluated the anti-cell migratory effect of Liposomal-TQ. Results: The findings obtained from RT-qPCR analysis indicated a significant increase in the expression of SENP2 and SENP6 genes in the TQ and DXR treatment groups compared to the control group in MCF7 but not in MCF10 cell lines (p-value < 0.05). Also, after 24 hours of treatment of MCF7 and MCF10 cells with liposomal-TQ, late apoptotic cells were significantly increased compared to the control and liposome groups (p-value < 0.0001) and compared to the control group, both DXR and Liposomal-TQ dramatically reduced the migratory ability of breast cancer cells (p-value = 0.001 and p-value = 0.001, respectively). Conclusion: Our study indicated that Liposomal-TQ promotes apoptosis in breast cancer cells and inhibits cell migration ability. These findings enhance our understanding of the role of Liposomal-TQ in the carcinogenic activities of SENP2 and SENP6 in the SUMOylation pathway of breast cancer.

Neuroprotective effect of Lithospermum officinale callus extract on inflamed primary microglial cells.

BACKGROUND: Plants that have therapeutic features for humans or animals are commonly referred to as "medicinal plants". They produce secondary metabolites with antioxidant, antimicrobial and/or anti-cancer effects. Lithospermum officinale, known as European stone seed, is a famous medicinal herb. However, due to the pyrrolizidine alkaloids (PzAl) in the root extract of L.officinal, there are therapeutic limitations to this herb. Objective This research was devoted to the evaluation of the anti-inflammatory capacity of methanolic extracts of L. officinale callus (LoE) (fresh cells) on rat microglial cells, the immune cells of the Central Nervous System, which play an essential role in the responses to neuroinflammation. METHODS: Primary microglia were obtained from neonatal Wistar rats (1 to 3-days old), and then treated with various concentration of CfA and methanolic extracts of 17 and 31-day-old L. officinale callus before LPS-stimulation. In addition to HPLC analysis of the extracts, viability, nitric oxide production, and evaluation of pro-inflammatory genes and cytokines in the inflamed microglia were investigated by MTT, Griess methos, qrt-PCR, and ELISA. RESULTS: Methanolic extract of the 17-day-old callus of L. officinale exhibited anti-inflammatory effects on LPS-stimulated microglial cells much higher than observed for CfA. The data were further supported by the decreased expression of Nos2, Tnf-α, and Cox-2 mRNA and the suppression of TNF-α and IL-1ß release in the activated microglial cells pretreated with the effective dose of LoE (0.8 mg mL-1). CONCLUSION: It was assumed that the better anti-neuroinflammatory performance of LoE than CfA in LPS-activated primary microglia could be a result of the synergism of the components of the extract and the lipophilic nature of RsA as the main phenolic acid of LoE. Considering that LoE shows a high antioxidant capacity and lacks PzAl, it is anticipated that LoE extract might be considered a reliable substitute to play a key role in the preparation of neuroprotective pharmaceutical formulas, which require in vivo research and further experiments.

Computational study of SENP1 in cancer by novel natural compounds and ZINC database screening.

Introduction: Sentrin-specific protease 1 (SENP1) is a protein whose main function is deSUMOylation. SENP1 inhibits apoptosis, and increases angiogenesis, estrogen and androgen receptor transcription and c-Jun transcription factor, proliferation, growth, cell migration, and invasion of cancer. The in vivo and in vitro studies also demonstrated which natural compounds, especially phytochemicals, minerals, and vitamins, prevent cancer. More than 3,000 plant species have been reported in modern medicine. Natural compounds have many anti-cancerous andanti-turmeric properties such as antioxidative, antiangiogenic, antiproliferative, and pro-apoptotic properties. Methods: In this study, we investigated the interaction of some natural compounds with SENP1 to inhibit its activity. We also screened the ZINC database including natural compounds. Molecular docking was performed, and toxicity of compounds was determined; then, molecular dynamics simulation (MDS) and essential dynamics (ED) were performed on natural compounds with higher free binding energies and minimal side effects. By searching in a large library, virtual screening of the ZINC database was performed using LibDock and CDOCKER, and the final top 20 compounds were allowed for docking against SENP1. According to the docking study, the top three leading molecules were selected and further analyzed by MDS and ED. Results: The results suggest that resveratrol (from the selected compounds) and ZINC33916875 (from the ZINC database) could be more promising SENP1 inhibitory ligands. Discussion: Because these compounds can inhibit SENP1 activity, then they can be novel candidates for cancer treatment. However, wet laboratory experiments are needed to validate their efficacy as SENP1 inhibitors.

Inhibition of NO Production in LPS-Stimulated Primary Rat Glial Cells by Gnidilatimonoein and Extract of Daphne mucronata.

Background: In the CNS, glial cells are involved in neuroinflammation and neuropathic pain. The glial cells are activated by a variety of pathological conditions and release pro-inflammatory mediators, including nitric oxide (NO). Overexpression of iNOS (inducible nitric oxide synthase) and extra NO is detrimental to neurophysiology and neuronal viability. Objectives: This study aimed to examine the effect of Gnidilatimonein isolated from D. mucronata and its leaves extract (as natural phytochemicals) on NO production in the LPS-induced primary glial cells. Materials and Methods: A preparative HPLC method was used to isolate gnidilatimonoein from leaves ethanolic extract. Various doses of Gnidilatimonoein, the ethanolic extract were applied to primary glial cells inflamed by lipopolysaccharide. A Colorimetric test, an MTT assay, and a RT-PCR analysis were then performed to analyze and compare NO production, cell viability, and iNOS expression. Results: Gnidilatimonoein treatment of pretreated primary glial cells significantly inhibited iNOS expression and decreased NO synthesis. Plant extracts also reduced NO production in inflamed microglial and glial at 0.1-3 mg.mL-1. At these concentrations, none of these compounds exerted a cytotoxic effect, suggesting that their anti-inflammatory effects were not due to the death of cells. Conclusion: This study indicates that D. mucronata and its active compound, Gnidilatimonoein, could have restrained effects on the expression of iNOS on the induced glial cells; however, further investigation is warranted.

Anti-Inflammatory Properties of Extract of the Hairy Root of Native Portulaca Oleracea L. and Its Effect on Some Inflammatory Genes Expression in Rat Microglial Cells.

Background: Most herbs play significant roles in the treatment of various diseases. Because dopamine functions in the anti-inflammatory process and the presence of this substance in Portulaca Oleracea L. native plant, investigating this plant's anti-inflammatory properties in treating neurological diseases is interesting. Objectives: The objective of this study was to estimate the NO production and the expression level of inflammatory genes in lipopolysaccharide (LPS)-treated microglial cells affected by P. oleracea L. extraction. Materials and Methods: P. oleracea L. hairy root extract was isolated, and the primary microglial cell of the rat was isolated from glial cells and confirmed by immunocytochemistry analysis. Microglial cells were pretreated with different concentrations of P. oleracea L. extract and then treated with 1 µg.mL-1 LPS. The control group did not receive any treatment. The NO level in culture supernatants was measured by the Griess method. The mRNA expression levels of iNOS (inducible Nitric oxide synthase) and TNF-α (tumor necrosis factor-alpha) in LPS-treated microglial cells were evaluated using Real-Time PCR. Results: The present study determined that 0.1 mg. mL-1 of the P. oleracea L. extract decreased the NO production in rat microglial cells. Different concentrations of the P. oleracea L. extract had no prominent effects on LPS-treated cell viability. The results of real-time PCR indicated that P. oleracea L extracts suppressed the mRNA expression levels of iNOS and TNF-α in LPS-treated cells. MTT assay determined that P. oleracea L. extract was not cytotoxic, and the anti-inflammatory P. oleracea L. extract effects observed were not because of cell death. Conclusion: P. oleracea L. extract might be helpful as an anti-inflammatory agent in treating inflammatory diseases.

Electrospun Nanofibrous Scaffolds of Polycaprolactone/Gelatin Reinforced with Layered Double Hydroxide Nanoclay for Nerve Tissue Engineering Applications.

Nerve tissue engineering (NTE) is an effective approach for repairing damaged nerve tissue. In this regard, nanoparticle-incorporated electrospun scaffolds have aroused a great deal of interest in NTE applications. In this study, layered double hydroxide (LDH)-incorporated polycaprolactone (PCL)/gelatin (Gel) nanofibrous scaffolds were fabricated by an electrospinning technique. The physicochemical, mechanical, and biological properties of the scaffolds were examined. Also, the phase identification, morphology, and elemental composition were studied using X-ray diffraction, scanning electron microscopy, and energy-dispersive X-ray spectroscopy, respectively. The results revealed that the inclusion of LDH nanoparticles into the PCL/Gel scaffold has improved its mechanical strength and elongation at the break, while the degradation rate was enhanced in comparison with the pure PCL/Gel mat. The LDH-enriched electrospun PCL/Gel scaffolds exhibited a considerable impact on cell attachment and proliferation. The gene expression results showed that the neuron-specific (γγ) enolase (NSE) gene expression was significantly decreased in the scaffolds containing 1 and 10 wt % LDH compared to the scaffold without LDH, whereas in the scaffold with 0.1 wt % LDH, a slight increase in expression was observed. It can be deduced that electrospun PCL/Gel scaffolds containing LDH with optimum concentration can be a promising candidate for nerve tissue engineering applications.

Synergy between sublethal doses of shikonin and metformin fully inhibits breast cancer cell migration and reverses epithelial-mesenchymal transition.

BACKGROUND: Shikonin is a natural multipotent anti-tumorigenic compound. We investigated potential synergy between shikonin and anti-diabetic metformin against tumorigenic properties of breast cancer cell line MCF-7. METHODS AND RESULTS: The IC50 of shikonin and metformin was determined after a single treatment of two cell lines MCF-7 and MDA-MB-231. We then measured optimal doses of each drug, used in combination, in MCF-7 cells. These sub-IC50 doses were co-applied for all subsequent combined treatments to evaluate their synergistic effects on MCF-7 tumorigenic properties. Next, we examined expression levels of the genes crucial for apoptosis, cell growth, and EMT using RT-PCR or real-time PCR and monitored CD44/CD24 ratios using flow cytometry. Binding energies between shikonin and growth molecules were measured by in silico simulation. Shikonin caused significantly reduced cell survival that was accelerated by the synergizing presence of metformin. Drug combination induced apoptosis and ROS levels while fully blocking cell migration and reverting EMT. RT-PCR showed strong suppression of BCL-2 but induction of BAX and PTEN. Prolonged shikonin treatment caused a total loss of the nuclear membrane, whereas metformin prevented this damage while promoting apoptotic morphologies. Our real-time PCR detected reduced levels of EMT genes but increases in the anti-EMT gene CDH1. Combined treatment also reduced CD44/CD24 ratios in favor of chemosensitivity. Binding energies strongly favored shikonin interactions with growth-signaling molecules. CONCLUSIONS: Shikonin and metformin synergize in inhibiting the tumorigenic activities of MCF-7 cells including their proliferation, invasiveness, and EMT with a potential to inhibit multidrug resistance.

Identification of Natural Products as SENP2 Inhibitors for Targeted Therapy in Heart Failure.

Aims: Sentrin-specific protease -2 (SENP2) is involved in deSUMOylation. Increased deSUMOylation in murine hearts by SENP2 upregulation resulted in cardiac dysfunction and congenital heart defects. Natural compounds via regulating cell proliferation and survival, induce cell cycle cessation, cell death, apoptosis, and producing reactive oxygen species and various enzyme systems cause disease prevention. Then, natural compounds can be suitable inhibitors and since SENP2 is a protein involved in heart disease, so our aim was inhibition of SENP2 by natural products for heart disease treatment. Material and methods: Molecular docking and molecular dynamics simulation of natural products i.e. Gallic acid (GA), Caffeic acid (CA), Thymoquinone (TQ), Betanin, Betanidin, Fisetin, and Ebselen were done to evaluate the SENP2 inhibitory effect of these natural products. The toxicity of compounds was also predicted. Results: The results showed that Betanin constituted a stable complex with SENP2 active site as it revealed low RMSD, high binding energy, and hydrogen bonds. Further, as compared to Ebselen, Betanin demonstrated low toxicity, formed a stable complex with SENP2 via four to seven hydrogen bonds, and constituted more stable MD plots. Therefore, depending upon the outcomes presented herein, Betanin significantly inhibited SENP2 and hence may be considered as a suitable natural compound for the treatment of heart failure. Further clinical trials must be conducted to validate its use as a potential SENP2 inhibitor.

Identification of novel anti-cancer agents, applying in silico method for SENP1 protease inhibition.

The SENP1 (Sentrin-Specific Protease1) is essential for desumoylation. SENP1 plays an essential role in many diseases such as cardiovascular disease, diabetes and cancer via targeting GATA2, NEMO, Pin1, SMAD4 and HIF-1α for deSUMOylation. Considering that, over expression of SENP1 was reported in cancer, thus an optional inhibitor of SENP1 can restitute the balance to the skewed system of SUMO and act as an effective therapeutic agent. The purpose of this study was to select and to sort inhibitors with a stronger binding affinity with SENP1. Molecular docking of SENP1 with natural compounds including Gallic acid, Caffeic acid, Thymoquinone, Thymol, Betaine, Alkannin, Ellagic acid, Betanin, Shikonin, Betanidin and Momordin IC was performed using AutoDock 4, then docking complexes for molecular dynamics (MD) simulation with GROMACS 4.6.5 were applied. Results with RMSD, RMSF, SASA, DSSP, gyrate, H-bond, ADMET and TOPKAT measurements, binding energy and structural features were surveyed. Among those, Gallic acid has shown the most significant results including RMSD and RMSF plots with high stability, high hydrogen bonds, high binding energy and the highest intermolecular bonds with SENP1. Gallic acid demonstrated strong connections and results of toxicity better than Momordin as control. Gallic acid is a phenolic compound which affects several pharmacological and biochemical pathways and has strong antioxidant, anti-inflammatory, antimutagenic and anticancer properties. Further research can improve the appropriate use of plant products drastically. Basic, pre-clinical and clinical research on Gallic acid may provide a roadmap for its ultimate application in the field of cancer prevention.Communicated by Ramaswamy H. Sarma.

Computational drug repurposing of bethanidine for SENP1 inhibition in cardiovascular diseases treatment.

AIMS: Bethanidine (BW467C60) is a newly presented strong adrenergic neuron blocking factor which has a hypotensive operation in man. SENPs are essential for maintaining a balance between SUMOylation and deSUMOylation which can be disturbed by changing the expression of (sentrin-specific proteases) SENPs. SENP1 is the most studied isoform of SENPs. Hypertrophic stimuli can increase SENP1 expression using calcium/calcineurin-NFAT3 signaling in heart. Moreover, SENP1 expression may positively relate to the expression of mitochondrial genes of the heart, and can cause the heart and mitochondrial dysfunction. MATERIALS AND METHODS: In order to inhibit SENP1 using Bethanidine, molecular docking and molecular dynamics (MD) simulation of SENP1 with Bethanidine were performed. Molecular docking showed that Bethanidine can inhibit SENP1. KEY FINDINGS: MD Simulation showed that Bethanidine constitutes a stable complex with SENP1 as was evident from RMSD, RMSF, H-bond and DSSP plots. Free binding energy and the interaction patterns were obtained from molecular docking, and MD trajectory exhibited Bethanidine can be a potential drug candidate for SENP1 inhibition. SIGNIFICANCE: This study supplies enough evidences that Bethanidine is a potential inhibitor of SENP1 and can be applied for the treatment of cardiovascular diseases.

Evidence of Omics, Immune Infiltration, and Pharmacogenomic for SENP1 in the Pan-Cancer Cohort.

Sentrin specific-protease 1 (SENP1) is a protein involved in deSUMOylation that is almost overexpressed in cancer. SENP1 has a determinative role in the activation of transcription programs in the innate immune responses and the development B of and C lymphocytes. We found, SENP1 possibly plays a critical role in immune infiltration and acts as an expression marker in PAAD, ESCA, and THYM. CD4+ T cells, CD8+ T cells, and macrophages were more key-related immune cells, indicating that SENP1 might be introduced as a potential target for cancer immunotherapy. We further showed that dysregulation of SENP1 is powerfully associated with decreased patient survival and clinical stage. Total SENP1 protein also increases in cancer. SENP1 is also controlled by transcription factors (TFs) CREB1, KDM5A, REST, and YY1 that regulates apoptosis, cell cycle, cell proliferation, invasion, tumorigenesis, and metastasis. These TFs were in a positive correlation with SENP1. MiR-138-5p, miR-129-1-3p, and miR-129-2-3p also inhibit tumorigenesis through targeting of SENP1. The SENP1 expression level positively correlated with the expression levels of UBN1, SP3, SAP130, NUP98, NUP153 in 32 tumor types. SENP1 and correlated and binding genes: SAP130, NUP98, and NUP153 activated cell cycle. Consistent with this finding, drug analysis was indicated SENP1 is sensitive to cell cycle, apoptosis, and RTK signaling regulators. In the end, SENP1 and its expression-correlated and functional binding genes were enriched in cell cycle, apoptosis, cellular response to DNA damage stimulus. We found that the cell cycle is the main way for tumorigenesis by SENP1. SENP1 attenuates the effect of inhibitory drugs on the cell cycle. We also introduced effective FDA-Approved drugs that can inhibit SENP1. Therefore in the treatments in which these drugs are used, SENP1 inhibition is a suitable approach. This study supplies a wide analysis of the SENP1 across The Cancer Genome Atlas (CGA) cancer types. These results suggest the potential roles of SENP1 as a biomarker for cancer. Since these drugs and the drugs that cause to resistance are applied to cancer treatment, then these two class drugs can use to inhibition of SENP1.

Withdrawal Notice: Neuroprotective Effect of Lithospermum officinale callus Extract on Inflamed Primary Microglial Cells

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Betanin purification from red beetroots and evaluation of its anti-oxidant and anti-inflammatory activity on LPS-activated microglial cells.

Microglial activation can release free radicals and various pro-inflammatory cytokines, which implicates the progress of a neurodegenerative disease. Therefore suppression of microglial activation can be an appropriate strategy for combating neurodegenerative diseases. Betanin is a red food dye that acts as free radical scavenger and can be a promising candidate for this purpose. In this study, purification of betanin from red beetroots was carried out by normal phase colum chromatography, yielding 500 mg of betanin from 100 g of red beetroot. The purified betanin was evaluated by TLC, UV-visible, HPLC, ESI-MASS, FT-IR spectroscopy. Investigation on the inhibitory effect of betanin on activated microglia was performed using primary microglial culture. The results showed that betanin significantly inhibited lipopolysaccharide induced microglial function including the production of nitric oxide free radicals, reactive oxygen species, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1ß). Moreover, betanin modulated mitochondrial membrane potential, lysosomal membrane permeabilization and adenosine triphosphate. We further investigated the interaction of betanin with TNF-α, IL-6 and Nitric oxide synthase (iNOS or NOS2) using in silico molecular docking analysis. The docking results demonstrated that betanin have significant negative binding energy against active sites of TNF-α, IL-6 and iNOS.

Anti-oxidant and Selective Anti-proliferative Effects of the Total Cornicabra Olive Polyphenols on Human Gastric MKN45 Cells.

BACKGROUND: According to the epidemiological studies, consuming olive products can decrease the incidence of the different types of cancers mostly due to the high anti-oxidant properties of their polyphenolic compounds. OBJECTIVES: To evaluate the anti-oxidant and anti-proliferative potentials of the olive fruits total polyphenols on the gastric adenocarcinoma MKN45 cells in comparison to the normal Hu02 cells. MATERIALS AND METHODS: The total phenolic content of the olive fruits and radical scavenging activity were determined by Folin and 2,2-diphenyl-1-picrylhydrazyl (DPPH) tests respectively. MTT assay was performed for the evaluation of the cell viability. Intracellular reactive oxygen species (ROS) level was measured using DCFH-DA. Statistical analysis was performed using SPSS 16 statistical software. RESULTS: Treatment of the MKN45 cells with the phenolic compounds extracted from olive fruits decreased growth and viability of the cells in a dose- and time-dependent manner. In addition, treatment of the MKN45 cells with a combination of the phenolic compounds extracts and cytarabine further decreased cell compared to monotherapy of the cells with each compound alone. Mechanistically, we showed that the anti-cancer effects of the olive polyphenols in the MKN45 cells are mediated through depletion of ROS. Similarly, polyphenolic extracts were found to decrease ROS level in the normal cells at the concentrations of 500 and 1000 µg.mL-1 and short treatment times (6 h), but the viability of these cells did not significantly change. At high concentrations (2000 µg.mL-1) of the phenolic extracts or at longer times of incubation (12 h), however, both ROS levels and the viability of the cells were significantly decreased in the normal cells. CONCLUSIONS: The olive fruits polyphenolic extract modulates ROS levels and selectively targets cancerous cells at low concentrations. Also, the effects of cytarabine could be potentiated by the olive fruits polyphenols. Thus, for a combined protocol of cancer cell therapy, olive fruit polyphenolic compound could be proposed as a proper candidate.

Preliminary In Vitro Effects of CD8+ T Lymphocyte Specific for the CD20 Alternative Splicing D393-CD20 Peptide Expressed on Burkitt Lymphoma Cells.

The effective discovery of clinically relevant tumor antigens holds a fundamental role for the development of new diagnostic tools and anticancer immunotherapies. D393-CD20 mRNA is absent from normal resting B cells but present in various malignant or transformed B cells. CD8+T lymphocytes play a central role in immunity to cancer. In this study, we want use from T CD8+ against D393-CD20 for effect in RAMOS cell line. After isolation and expanding of specific TCD8 + Lymphocyte against D393-CD20 antigen, for examining the effect of specialized T lymphocyte clone of D393-CD20 antigen on RAMOS cell line, we co-cultured them together, and the rate of apoptosis were examined by flow cytometry and cytotoxicity techniques by using MTT technique. We observed that specialized TCD8+ lymphocyte of D393-CD20 antigen can induce apoptosis in malignant B-lymphocytes, and this antigen can be a proper target for immunotherapy.

Thymoquinone Effects on Cell Viability, Apoptosis and VEGF-A Gene Expression Level in AGS(CRL-1739) Cell Line.

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.

Anti­inflammatory effect of Migri­Heal® in an in vitro inflammatory model of primary mixed glial cells.

Migri­Heal®, is a novel herbal remedy that was introduced for the treatment of migraine headaches. Previous studies revealed that this drug may reduce nitric oxide (NO) in an in vitro inflammatory model. The aim of the present study was to investigate the anti­inflammatory effect of Migri­Heal® on primary mix glial cells stimulated with LPS. In the current study, neonatal rat primary mix glial cells were isolated from the mixed glial cultures via shaking, and cultured in Dulbecco's' modified Eagle's medium supplemented with 10% fetal bovine serum. Following pretreatment with Migri­Heal® (25, 75, 100, 150, 200 and 300 µg/ml) and cells were treated with LPS (10 µg/ml) for 1 h, and incubated for 48 h. The present study determined that 150 µg/ml Migri­Heal® significantly reduced the production of NO in rat mix glial cells stimulated with 10 µg/ml LPS. Migri­Heal® also suppressed mRNA expression level of LPS­induced inducible nitric oxide synthase and tumor necrosis factor α, which was accompanied by inhibition of the transcription factor nuclear factor­κB. Additionally, MTT assay determined that Migri­Heal® was not cytotoxic, suggesting that the anti­inflammatory effects of Migri­Heal® observed were not due to cell death. In conclusion, the findings of the present study demonstrated that Migri­Heal® may be useful as a potential anti­inflammatory agent in inflammatory diseases. However, additional studies are required to confirm these findings.

Novel Epigenetic Controlling of Hypoxia Pathway Related to Overexpression and Promoter Hypomethylation of TET1 and TET2 in RPE Cells.

CpG methylation of DNA takes part in a specific epigenetic memory that plays crucial roles in the differentiation and abnormality of the cells. The methylation pattern aberration of genomes is affected in three ways, namely DNA methyltransferase (DNMT), ten-eleven translocation (TET), and methyl-binding domain (MBD) proteins. Of these, TET enzymes have recently been demonstrated to be master modifier enzymes in the DNA methylation process. Additionally, recent studies emphasize that not only epigenetic phenomena play a role in controlling hypoxia pathway, but the hypoxia condition also triggers hypomethylation of genomes that may help with the expression of hypoxia pathway genes. In this study, we suggested that TET1 and TET2 could play a role in the demethylation of genomes under chemical hypoxia conditions. Herein, the evaluating methylation status and mRNA expression of mentioned genes were utilized through real-time PCR and methylation-specific PCR (MSP), respectively. Our results showed that TET1 and TET2 genes were overexpressed (P < 0.05) under chemical hypoxia conditions in Retinal Pigment Epithelial (RPE) cells, whereas the promoter methylation status of them were hypomethylated in the same condition. Therefore, chemical hypoxia not only causes overexpression of TET1 and TET2 but also could gradually do promoter demethylation of same genes. This is the first study to show the relationship between epigenetics and the expression of mentioned genes related to hypoxia pathways. Furthermore, it seems that these associations in RPE cells are subjected to chemical hypoxia as a mechanism that could play a crucial role in methylation pattern changes of hypoxia-related diseases such as cancer and ischemia. J. Cell. Biochem. 118: 3193-3204, 2017. © 2017 Wiley Periodicals, Inc.

Toxicity of serum albumin on microglia upon seeding effect of amyloid peptide.

We demonstrate in vitro cross-seeding of bovine serum albumin (BSA) in the presence of Aß25-35 and their cytotoxic effects on microglial cells. To investigate the cross-seeding of BSA in the presence of Aß25-35 fibrils, we examined how Aß25-35 fibrils can function as seeds to trigger and accelerate BSA fibrillogenesis using ThT, intrinsic fluorescence, ANS fluorescence and transmission electron microscopy (TEM). Moreover, the effects of these fibrils on microglial viability were measured using MTT and Annexin V/propidium iodide (PI) staining. Although Aß25-35 is toxic against microglia, it acted as seed and affected the aggregation pathway and accelerated the fibrillogenesis of BSA in vitro, resulted in an enhanced cytotoxic effect in comparison with Aß25-35 or BSA alone. These observations thought to be helpful to understand the molecular mechanism of enhanced toxicity due to the coexistence of the aggregation prone proteins/peptides,. then cross-seeding effect on microglial cells that may involve in neurodegenerative diseases such as Alzheimer's disease (AD).

Probable Chemical Hypoxia Effects on Progress of CNV Through Induction of Promoter CpG Demethylation and Overexpression of IL17RC in Human RPE Cells.

PURPOSE: To survey the changes of promoter CpG methylation status and mRNA expression of IL17RC (interleukin 17 receptor C) gene in retinal pigment epithelium (RPE) cells under chemical hypoxia condition for choroidal neovascularization (CNV) modeling in vitro. MATERIALS AND METHODS: RPE cells were cultured in both untreated as a control group and treated by cobalt chloride media as a hypoxia group for various concentrations (100-150µM) and times (24-36 hrs.) To confirm chemical hypoxia condition, mRNA expression of HIF (Hypoxia Inducible Factor) -1α, -2α, and Vascular Endothelial Growth Factor (VEGF) was compared between two groups by Real-time PCR. Also, in normoxia and hypoxia conditions, IL17RC expression changes and promoter CpG methylation status were evaluated by Real-time PCR and methylation-specific PCR (MSP) techniques, respectively. RESULTS: Overexpression of HIF-1α, HIF-2α, and VEGF was significant in hypoxia versus normoxia conditions. Our data showed overexpression of IL17RC (2.1- to 6.3-fold) and decreasing of its promoter methylation in comparison with hypoxia and normoxia conditions. It was found that there are significant association between promoter methylation status and expression of IL17RC in chemical hypoxia condition. CONCLUSION: Therefore, methylation of IL17RC could play as a marker in CNV and degeneration of RPE cells in vitro. Additionally, HIF-α and methylation phenomena may be considered as critical targets for blocking in angiogenesis of age-related degeneration in future studies.