scholarly journals Indole-3-carbinol regulates microglia homeostasis and protects the retina from degeneration

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Amir Saeed Khan ◽  
Thomas Langmann
Keyword(s):  
Retinal Degeneration ◽  
Therapeutic Option ◽  
Retinal Cell ◽  
Subretinal Space ◽  
Western Blots ◽  
Protein Levels ◽  
Qrt Pcr ◽  
Anti Oxidant

Abstract Background Retinal degenerative diseases significantly contribute to visual impairment and blindness. Microglia reactivity is a hallmark of neurodegenerative diseases including retinal cell death and immunomodulation emerges as a therapeutic option. Indole-3-carbinol (I3C) is a natural ligand of aryl hydrocarbon receptor (AhR), with potent immunomodulatory properties. Here, we hypothesized that I3C may inhibit microglia reactivity and exert neuroprotective effects in the light-damaged murine retina mimicking important immunological aspects of retinal degeneration. Methods BV-2 microglia were treated in vitro with I3C followed by lipopolysaccharide (LPS) stimulation to analyze pro-inflammatory and anti-oxidant responses by quantitative real-time PCR (qRT-PCR) and Western blots. Nitric oxide (NO) secretion, caspase 3/7 levels, phagocytosis rates, migration, and morphology were analyzed in control and AhR knockdown cells. I3C or vehicle was systemically applied to light-treated BALB/cJ mice as an experimental model of retinal degeneration. Pro-inflammatory and anti-oxidant responses in the retina were examined by qRT-PCR, ELISA, and Western blots. Immunohistochemical staining of retinal flat mounts and cryosections were performed. The retinal thickness and structure were evaluated by in vivo imaging using spectral domain-optical coherence tomography (SD-OCT). Results The in vitro data showed that I3C potently diminished LPS-induced pro-inflammatory gene expression of I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 and induced anti-oxidants gene levels of NQO1, HMOX1, and CAT1 in BV-2 cells. I3C also reduced LPS-induced NO secretion, phagocytosis, and migration as important functional microglia parameters. siRNA-mediated knockdown of AhR partially prevented the previously observed gene regulatory events. The in vivo experiments revealed that I3C treatment diminished light-damage induced I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 transcripts and also reduced CCL2, I-NOS, IL-1ß, p-NFkBp65 protein levels in mice. Moreover, I3C increased anti-oxidant NQO1 and HMOX1 protein levels in light-exposed retinas. Finally, I3C therapy prevented the accumulation of amoeboid microglia in the subretinal space and protected from retinal degeneration. Conclusions The AhR ligand I3C potently counter-acts microgliosis and light-induced retinal damage, highlighting a potential treatment concept for retinal degeneration.

scholarly journals NLRP7 deubiquitination by USP10 promotes tumor progression and tumor-associated macrophage polarization in colorectal cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Bing Li ◽  
Zhi-Peng Qi ◽  
Dong-Li He ◽  
Zhang-Han Chen ◽  
Jing-Yi Liu ◽  
...  
Keyword(s):  
Tumor Progression ◽  
Acceptor Site ◽  
Protein Levels ◽  
Qrt Pcr ◽  
Chemokine Ligand ◽  
Specific Protease ◽  
Ubiquitin Specific Protease ◽  
Chemokine Ligand 2

Abstract Background NOD-like receptors affect multiple stages of cancer progression in many malignancies. NACHT, LRR, and PYD domain-containing protein 7 (NLRP7) is a member of the NOD-like receptor family, although its role in tumorigenesis remains unclear. By analyzing clinical samples, we found that NLRP7 protein levels were upregulated in colorectal cancer (CRC). We proposed the hypothesis that a high level of NLRP7 in CRC may promote tumor progression. Here, we further investigated the role of NLRP7 in CRC and the underlying mechanism. Methods NLRP7 expression in human CRC and adjacent non-tumorous tissues was examined by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. The effect of NLRP7 in CRC progression was investigated in vitro and in vivo. Proteins interacting with NLRP7 were identified by immunoprecipitation and mass spectrometry analysis while immunofluorescence staining revealed the cellular location of the proteins. Cellular ubiquitination and protein stability assays were applied to demonstrate the ubiquitination effect on NLRP7. Cloning and mutagenesis were used to identify a lysine acceptor site that mediates NLRP7 ubiquitination. Cytokines/chemokines affected by NLRP7 were identified by RNA sequencing, qRT-PCR, and enzyme-linked immunosorbent assay. Macrophage phenotypes were determined using qRT-PCR, flow cytometry, and immunohistochemistry. Results NLRP7 protein levels, but not mRNA levels, were upregulated in CRC, and increased NLRP7 protein expression was associated with poor survival. NLRP7 promoted tumor cell proliferation and metastasis in vivo and in vitro and interacted with ubiquitin-specific protease 10, which catalyzed its deubiquitination in CRC cells. NLRP7 stability and protein levels in CRC cells were modulated by ubiquitination and deubiquitination, and NLRP7 was involved in the ubiquitin-specific protease 10 promotion of tumor progression and metastasis in CRC. K379 was an important lysine acceptor site that mediates NLRP7 ubiquitination in CRC cells. In CRC, NLRP7 promoted the polarization of pro-tumor M2-like macrophages by inducing the secretion of C-C motif chemokine ligand 2. Furthermore, NLRP7 promoted NF-κB nuclear translocation and activation of C-C motif chemokine ligand 2 transcription. Conclusions We showed that NLRP7 promotes CRC progression and revealed an as-yet-unidentified mechanism by which NLRP7 induces the polarization of pro-tumor M2-like macrophages. These results suggest that NLRP7 could serve as a biomarker and novel therapeutic target for the treatment of CRC.

scholarly journals (–)-Epicatechin Suppresses Angiotensin II-induced Cardiac Hypertrophy via the Activation of the SP1/SIRT1 Signaling Pathway

2017 ◽  
Vol 41 (5) ◽  
pp. 2004-2015 ◽  
Author(s):  
Zeng-xiang Dong ◽  
Lin Wan ◽  
Ren-jun Wang ◽  
Yuan-qi Shi ◽  
Guang-zhong Liu ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Signaling Pathway ◽  
Cardiomyocyte Hypertrophy ◽  
Ang Ii ◽  
Beneficial Effects ◽  
Protein Levels ◽  
Qrt Pcr

Background/Aims: Flavonol (–)-epicatechin (EPI) is present in high amounts in cocoa and tea products, and has been shown to exert beneficial effects on the cardiovascular system. However, the precise mechanism of EPI on cardiomyocyte hypertrophy has not yet been determined. In this study, we examined whether EPI could inhibit cardiac hypertrophy. Methods: We utilised cultured neonatal mouse cardiomyocytes and mice for immunofluorescence, immunochemistry, qRT-PCR, and western blot analyses. Results: 1µM EPI significantly inhibited 1µM angiotensin II (Ang II)-induced increase of cardiomyocyte size, as well as the mRNA and protein levels of ANP, BNP and β-MHC in vitro. The effects of EPI were accompanied with an up-regulation of SP1 and SIRT1, and were abolished by SP1 inhibition. Up-regulation of SP1 could block Ang II-induced increase in cardiomyocyte size, as well as the mRNA and protein levels of ANP, BNP and β-MHC, and increase the protein levels of SIRT1 in vitro. Moreover, 1 mg/kg body weight/day EPI significantly inhibited mouse cardiac hypertrophy induced by Ang II, which could be eliminated by SP1 inhibition in vivo. Conclusion: Our data indicated that EPI inhibited AngII-induced cardiac hypertrophy by activating the SP1/SIRT1 signaling pathway.

scholarly journals Inhibition of Androgen Receptor Transcriptional Activity as a Novel Mechanism of Action of Arsenic

2009 ◽  
Vol 23 (3) ◽  
pp. 412-421 ◽  
Author(s):  
Adena E. Rosenblatt ◽  
Kerry L. Burnstein
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Male Infertility ◽  
Transcriptional Activity ◽  
Target Gene ◽  
Nuclear Translocation ◽  
Therapeutic Option ◽  
Protein Levels

Abstract Environmental sodium arsenite is a toxin that is associated with male infertility due to decreased and abnormal sperm production. Arsenic trioxide (ATO), another inorganic trivalent semimetal, is an effective therapy for acute promyelocytic leukemia, and there is investigation of its possible efficacy in prostate cancer. However, the mechanism of arsenic action in male urogenital tract tissues is not clear. Because the androgen receptor (AR) plays an important role in spermatogenesis and prostate cancer, we explored the possibility that trivalent arsenic regulates AR function. We found that arsenic inhibited AR transcriptional activity in prostate cancer and Sertoli cells using reporter gene assays testing several androgen response element-containing regions and by assessing native target gene expression. Arsenic inhibition of AR activity was not due to down-regulation of AR protein levels, decreased hormone binding to AR, disruption of AR nuclear translocation, or interference with AR-DNA binding in vitro. However, chromatin immunoprecipitation studies revealed that arsenic inhibited AR recruitment to an AR target gene enhancer in vivo. Consistent with a deficiency in AR-chromatin binding, arsenic disrupted AR amino and carboxyl termini interaction. Furthermore, ATO caused a significant decrease in prostate cancer cell proliferation that was more pronounced in cells expressing AR compared with cells depleted of AR. In addition, inhibition of AR activity by ATO and by the AR antagonist, bicalutamide, was additive. Thus, arsenic-induced male infertility may be due to inhibition of AR activity. Further, because AR is an important target in prostate cancer therapy, arsenic may serve as an effective therapeutic option.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Exploiting Anti-Inflammation Effects of Flavonoids in Chronic Inflammatory Diseases

2020 ◽  
Vol 26 (22) ◽  
pp. 2610-2619 ◽  
Author(s):  
Tarique Hussain ◽  
Ghulam Murtaza ◽  
Huansheng Yang ◽  
Muhammad S. Kalhoro ◽  
Dildar H. Kalhoro
Keyword(s):  
Oxidative Stress ◽  
Chronic Diseases ◽  
Inflammatory Diseases ◽  
Therapeutic Option ◽  
Cellular Level ◽  
Antiinflammatory Drugs ◽  
Suitable Alternative ◽  
Chronic Inflammatory Diseases

Background: Inflammation is a complex response of the host defense system to different internal and external stimuli. It is believed that persistent inflammation may lead to chronic inflammatory diseases such as, inflammatory bowel disease, neurological and cardiovascular diseases. Oxidative stress is the main factor responsible for the augmentation of inflammation via various molecular pathways. Therefore, alleviating oxidative stress is effective a therapeutic option against chronic inflammatory diseases. Methods: This review article extends the knowledge of the regulatory mechanisms of flavonoids targeting inflammatory pathways in chronic diseases, which would be the best approach for the development of suitable therapeutic agents against chronic diseases. Results: Since the inflammatory response is initiated by numerous signaling molecules like NF-κB, MAPK, and Arachidonic acid pathways, their encountering function can be evaluated with the activation of Nrf2 pathway, a promising approach to inhibit/prevent chronic inflammatory diseases by flavonoids. Over the last few decades, flavonoids drew much attention as a potent alternative therapeutic agent. Recent clinical evidence has shown significant impacts of flavonoids on chronic diseases in different in-vivo and in-vitro models. Conclusion: Flavonoid compounds can interact with chronic inflammatory diseases at the cellular level and modulate the response of protein pathways. A promising approach is needed to overlook suitable alternative compounds providing more therapeutic efficacy and exerting fewer side effects than commercially available antiinflammatory drugs.

scholarly journals All-Trans Retinoic Acid Increases DRP1 Levels and Promotes Mitochondrial Fission

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1202
Author(s):  
Bojjibabu Chidipi ◽  
Syed Islamuddin Shah ◽  
Michelle Reiser ◽  
Manasa Kanithi ◽  
Amanda Garces ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Mitochondrial Fission ◽  
Normal Function ◽  
Mitochondrial Network ◽  
Protein Levels ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Area And Perimeter

In the heart, mitochondrial homeostasis is critical for sustaining normal function and optimal responses to metabolic and environmental stressors. Mitochondrial fusion and fission are thought to be necessary for maintaining a robust population of mitochondria, and disruptions in mitochondrial fission and/or fusion can lead to cellular dysfunction. The dynamin-related protein (DRP1) is an important mediator of mitochondrial fission. In this study, we investigated the direct effects of the micronutrient retinoid all-trans retinoic acid (ATRA) on the mitochondrial structure in vivo and in vitro using Western blot, confocal, and transmission electron microscopy, as well as mitochondrial network quantification using stochastic modeling. Our results showed that ATRA increases DRP1 protein levels, increases the localization of DRP1 to mitochondria in isolated mitochondrial preparations. Our results also suggested that ATRA remodels the mitochondrial ultrastructure where the mitochondrial area and perimeter were decreased and the circularity was increased. Microscopically, mitochondrial network remodeling is driven by an increased rate of fission over fusion events in ATRA, as suggested by our numerical modeling. In conclusion, ATRA results in a pharmacologically mediated increase in the DRP1 protein. It also results in the modulation of cardiac mitochondria by promoting fission events, altering the mitochondrial network, and modifying the ultrastructure of mitochondria in the heart.

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

scholarly journals Long noncoding RNA SNHG14 promotes hepatocellular carcinoma progression by regulating miR-876-5p/SSR2 axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zhibin Liao ◽  
Hongwei Zhang ◽  
Chen Su ◽  
Furong Liu ◽  
Yachong Liu ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Animal Experiments ◽  
Luciferase Reporter ◽  
Western Blot Assay ◽  
Downstream Target ◽  
Protein Levels ◽  
Elevated Expression ◽  
Rna Immunoprecipitation

Abstract Background Aberrant expressions of long noncoding RNAs (lncRNAs) have been demonstrated to be related to the progress of HCC. The mechanisms that SNHG14 has participated in the development of HCC are obscure. Methods Quantitative real-time PCR (qRT-PCR) was used to measure the lncRNA, microRNA and mRNA expression level. Cell migration, invasion and proliferation ability were evaluated by transwell and CCK8 assays. The ceRNA regulatory mechanism of SNHG14 was evaluated by RNA immunoprecipitation (RIP) and dual luciferase reporter assay. Tumorigenesis mouse model was used to explore the roles of miR-876-5p in vivo. The protein levels of SSR2 were measured by western blot assay. Results In this study, we demonstrated that SNHG14 was highly expressed in HCC tissues, meanwhile, the elevated expression of SNHG14 predicted poor prognosis in patients with HCC. SNHG14 promoted proliferation and metastasis of HCC cells. We further revealed that SNHG14 functioned as a competing endogenous RNA (ceRNA) for miR-876-5p and that SSR2 was a downstream target of miR-876-5p in HCC. Transwell, CCK8 and animal experiments exhibited miR-876-5p inhibited HCC progression in vitro and in vivo. By conducting rescue experiments, we found the overexpression of SSR2 or knocking down the level of miR-876-5p could reverse the suppressive roles of SNHG14 depletion in HCC. Conclusion SNHG14 promotes HCC progress by acting as a sponge of miR-876-5p to regulate the expression of SSR2 in HCC.

scholarly journals The Effects of Conjugated Linoleic Acids on Cancer

Processes ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 454 ◽  
Author(s):  
Marko Dachev ◽  
Jana Bryndová ◽  
Milan Jakubek ◽  
Zdeněk Moučka ◽  
Marian Urban
Keyword(s):  
Potential Effect ◽  
Conjugated Linoleic Acids ◽  
Carcinogenic Activity ◽  
Beneficial Effects ◽  
True Value ◽  
Ruminant Animals ◽  
Anti Oxidant ◽  
Cancer Agent

Conjugated linoleic acids (CLA) are distinctive polyunsaturated fatty acids. They are present in food produced by ruminant animals and they are accumulated in seeds of certain plants. These naturally occurring substances have demonstrated to have anti-carcinogenic activity. Their potential effect to inhibit cancer has been shown in vivo and in vitro studies. In this review, we present the multiple effects of CLA isomers on cancer development such as anti-tumor efficiency, anti-mutagenic and anti-oxidant activity. Although the majority of the studies in vivo and in vitro summarized in this review have demonstrated beneficial effects of CLA on the proliferation and apoptosis of tumor cells, further experimental work is needed to estimate the true value of CLA as a real anti-cancer agent.

Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells

2001 ◽  
Vol 360 (3) ◽  
pp. 707-715 ◽  
Author(s):  
Trevor R. PETTITT ◽  
Mark McDERMOTT ◽  
Khalid M. SAQIB ◽  
Neil SHIMWELL ◽  
Michael J. O. WAKELAM
Keyword(s):  
Liquid Chromatography ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Mammalian Cells ◽  
Inducible Promoter ◽  
In Vitro Assays ◽  
Protein Levels ◽  
Intracellular Messenger

Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or −2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography–MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.

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