Binding and detoxification ability of lactobacillus acidophilus towards di-n-butyl phthalate: Change of MAPK pathway in Caco-2 cell model

AbstractBackgroundThe mechanism of pancreatic cancer(PA) is not fully understanded. In our last report, TRPM2 plays a promising role in pancreatic cancer. However, the mechanism of TRPM2 is still unknown in this dismal disease. This study was designed to investigate the role and mechanism of TRPM2 in pancreatic cancer.MethodsTRPM2 overexpressed and siRNA plasmid were created and transfected with pancreatic cancer cell line(BxPC-3) to construct the cell model. We employed CCK-8, Transwell, scratch wound, and nude mice tumor bearing model to investigate the role of TRPM2 in pancreatic cancer. Besides, we collected the clinical data, tumor tissue sample(TT) and para-tumor sample(TP) from the pancreatic cancer patients treated in our hospital. We analyzed the mechanism of TRPM2 in pancreatic cancer by transcriptome analysis, Westernblot, and PCR.ResultsOverexpressed TRPM2 could promote pancreatic cancer in proliferation, migration, and invasion ability in no matter the cell model or nude mice tumor bearing model. TRPM2 level is highly negative correlated to the overall survival and progression-free survival time in PA patients, however, it is significantly increased in PA tissue as the tumor stage increases. The transcriptome analysis, GSEA analysis, Westernblot, and PCR results indicates TRPM2 is highly correlated with PKC/MAPK pathways.ConclusionTRPM2 could directly activate PKCα by calcium or indirectly activate PKCε and PKCδ by increased DAG in PC, which promote PC by downstream MAPK/MEK pathway activation.

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Abstract 19583: Autonomous and Non-Autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes

Circulation ◽

10.1161/circ.130.suppl_2.19583 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Rebecca Josowitz ◽

Sonia Mulero-Navarro ◽

Christine Falce ◽

Ninette Cohen ◽

Erik M Ullian ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Cell Model ◽

Mapk Pathway ◽

Therapeutic Option ◽

Induced Pluripotent Stem Cell ◽

Mapk Signaling ◽

Calcium Handling ◽

Cardiomyocyte Hypertrophy ◽

Cellular Interactions ◽

Braf Mutant

Hypertrophic cardiomyopathy (HCM) is a pathological disorder predominantly due to mutations in sarcomeric components. Germline mutations in BRAF cause a developmental syndrome called cardio-facio-cutaneous syndrome (CFCS), in which 40% of patients also develop HCM. Since the role of the RAS/MAPK pathway in HCM is still unclear, we generated a human induced pluripotent stem cell model for CFCS from three patients with activating BRAF T599R or Q257R mutations. In order to examine hiPSC-derived cell-type specific phenotypes and cellular interactions underpinning HCM, we generated a method to purify cardiomyocytes and non-cardiomyocytes simultaneously by cell sorting based on SIRPα and CD90 expression. Purified BRAF-mutant SIRPα+/CD90- cells were >95% cardiomyocytes, displayed cellular hypertrophy with pro-hypertrophic gene expression, and dysregulation of the RAS/MAPK pathway. BRAF-mutant cardiomyocytes also displayed intrinsic calcium handling defects, including increased calcium transient irregularity and increased stored calcium within the sarcoplasmic reticulum. In addition, purified BRAF-mutant SIRPα-/CD90+ cells, which were fibroblast-like, displayed activation of the RAS/MAPK pathway and exhibited a pro-fibrotic phenotype. Cross-culture studies revealed that BRAF-mutant fibroblast-like cells critically modulate cardiomyocyte hypertrophy through TGFβ paracrine signaling, as TGFβ inhibition prevented cardiomyocyte hypertrophy induced by BRAF-mutant fibroblast-like cells. Additionally, inhibition of RAS/MAPK signaling was capable of rescuing BRAF-mutant cardiomyocyte hypertrophy and cardiomyocyte-intrinsic calcium handling abnormalities. Thus, we show for the first time that cell autonomous and non-autonomous defects underlie RASopathy-associated HCM. As fibroblast activation has been documented previously in sarcomeric HCM, our findings suggest that cardiac fibroblasts may contribute to pathologic hypertrophy in addition to causing fibrosis in primary forms of HCM. TGFβ inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies.

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Human Erythrocytes Exposed to Phthalates and Their Metabolites Alter Antioxidant Enzyme Activity and Hemoglobin Oxidation

International Journal of Molecular Sciences ◽

10.3390/ijms21124480 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4480 ◽

Cited By ~ 3

Author(s):

Paulina Sicińska ◽

Kinga Kik ◽

Bożena Bukowska

Keyword(s):

Hydroxyl Radical ◽

Cell Model ◽

Human Life ◽

Redox Balance ◽

Human Erythrocytes ◽

In Vitro Toxicity ◽

Mechanisms Of Toxicity ◽

Butyl Phthalate ◽

And Function

Phthalates used as plasticizers have become a part of human life because of their important role in various industries. Human exposure to these compounds is unavoidable, and therefore their mechanisms of toxicity should be investigated. Due to their structure and function, human erythrocytes are increasingly used as a cell model for testing the in vitro toxicity of various xenobiotics. Therefore, the purpose of our study was to assess the effect of selected phthalates on methemoglobin (metHb), reactive oxygen species (ROS) including hydroxyl radical levels, as well as the activity of antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), in human erythrocytes. Erythrocytes were incubated with di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP), and their metabolites, i.e., mono-n-butyl phthalate (MBP) and monobenzyl phthalate (MBzP), at concentrations ranging from 0.5 to 100 µg/mL for 6 or 24 h. This study shows that the analyzed phthalates disturbed the redox balance in human erythrocytes. DBP and BBP, at much lower concentrations than their metabolites, caused a statistically significant increase of metHb and ROS, including hydroxyl radical levels, and changed the activity of antioxidant enzymes. The studied phthalates disturbed the redox balance in human erythrocytes, which may contribute to the accelerated removal of these cells from the circulation.

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Expression of Melatonin and Dopamine D3 Receptor Heteromers in Eye Ciliary Body Epithelial Cells and Negative Correlation with Ocular Hypertension

Cells ◽

10.3390/cells9010152 ◽

2020 ◽

Vol 9 (1) ◽

pp. 152 ◽

Cited By ~ 3

Author(s):

Irene Reyes-Resina ◽

Hanan Awad Alkozi ◽

Anna del Ser-Badia ◽

Juan Sánchez-Naves ◽

Jaume Lillo ◽

...

Keyword(s):

Epithelial Cells ◽

Ciliary Body ◽

Cell Model ◽

Mapk Pathway ◽

Melatonin Receptors ◽

Specific Cell ◽

Label Free ◽

Clear Trend ◽

Receptor Heteromers ◽

Dopamine D3

Background: Experiments in the late nineties showed an inverse relationship in the eye levels of melatonin and dopamine, thereby constituting an example of eye parameters that are prone to circadian variations. The underlying mechanisms are not known but these relevant molecules act via specific cell surface dopamine and melatonin receptors. This study investigated whether these receptors formed heteromers whose function impact on eye physiology. We performed biophysical assays to identify interactions in heterologous systems. Particular heteromer functionality was detected using Gi coupling, MAPK activation, and label-free assays. The expression of the heteroreceptor complexes was assessed using proximity ligation assays in cells producing the aqueous humor and human eye samples. Dopamine D3 receptors (D3Rs) were identified in eye ciliary body epithelial cells. We discovered heteromers formed by D3R and either MT1 (MT1R) or MT2 (MT2R) melatonin receptors. Heteromerization led to the blockade of D3R-Gi coupling and regulation of signaling to the MAPK pathway. Heteromer expression was negatively correlated with intraocular hypertension. Conclusions: Heteromers likely mediate melatonin and dopamine actions in structures regulating intraocular pressure. Significant expression of D3R–MT1R and D3R–MT1R was associated with normotensive conditions, whereas expression diminished in a cell model of hypertension. A clear trend of expression reduction was observed in samples from glaucoma cases. The trend was marked but no statistical analysis was possible as the number of available eyes was 2.

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Xanthohumol Impairs the PMA-Driven Invasive Behaviour of Lung Cancer Cell Line A549 and Exerts Anti-EMT Action

Cells ◽

10.3390/cells10061484 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1484

Author(s):

Adrianna Sławińska-Brych ◽

Magdalena Mizerska-Kowalska ◽

Sylwia Katarzyna Król ◽

Andrzej Stepulak ◽

Barbara Zdzisińska

Keyword(s):

Lung Cancer ◽

Cell Line ◽

Cell Model ◽

Mapk Pathway ◽

A549 Cells ◽

In Vivo Studies ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

E Cadherin

Xanthohumol (XN), the main prenylated flavonoid from hop cones, has been recently reported to exert significant proapoptotic, anti-proliferative, and growth inhibitory effects against lung cancer in both in vitro and in vivo studies. However, its anti-metastatic potential towards this malignancy is still unrevealed. Previously, we indicated that the human lung adenocarcinoma A549 cell line was sensitive to XN treatment. Therefore, using the same tumour cell model, we have studied the influence of XN on the phorbol-12-myristate-13-acetate (PMA)-induced cell migration and invasion. The effects of XN on the expression/activity of pro-invasive MMP-9 and MMP-2 and the expression of MMP inhibitors, i.e., TIMP-1 and TIMP-2 (anti-angiogenic factors), were evaluated. Additionally, the influence of XN on the production of the key pro-angiogenic cytokine, i.e., VEGF, and the release of TGF-β, which is both a pro-angiogenic cytokine and an epithelial-mesenchymal transition (EMT) stimulator, was studied. Furthermore, the influence of XN on the expression of EMT-associated proteins such as E-cadherin and α-E-catenin (epithelial markers), vimentin and N-cadherin (mesenchymal markers), and Snail-1 (transcriptional repressor of E-cadherin) was studied. To elucidate the molecular mechanism underpinning the XN-mediated inhibition of metastatic progression in PMA-activated cells, the phosphorylation levels of AKT, FAK, and ERK1/2 kinases, which are signalling molecules involved in EMT program activation, were assayed. The results showed that XN in non-cytotoxic concentrations impaired the PMA-driven migratory and invasive capacity of A549 cells by decreasing the level of expression of MMP-9 and concomitantly increasing the expression of the TIMP-1 protein, i.e., a specific blocker of pro-MMP-9 activation. Moreover, XN decreased the PMA-induced production of VEGF and TGF-β. Furthermore, the XN-treatment counteracted the PMA-induced EMT of the A549 cells by the upregulation of E-cadherin and α-E-catenin and the downregulation of N-cadherin, vimentin, and Snail-1 expression. The proposed mechanism underlying the anti-invasive XN activity involved the inhibition of the ERK/MAPK pathway and suppression of FAK and PI3/AKT signalling. Our results suggesting migrastatic properties of XN against lung cancer cells require further verification in in vivo assays.

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Murine immunomodulation of IL-10 and IL-12 induced by new isolates from avian type 2 Lactobacillus acidophilus

Canadian Journal of Microbiology ◽

10.1139/w07-056 ◽

2007 ◽

Vol 53 (8) ◽

pp. 944-956 ◽

Cited By ~ 8

Author(s):

Christian Bleau ◽

Roland Savard ◽

Lucie Lamontagne

Keyword(s):

Lactobacillus Acidophilus ◽

Mapk Pathway ◽

Lactobacillus Rhamnosus ◽

Peritoneal Macrophages ◽

Lymphoid Cells ◽

Mitogen Activated Protein Kinase ◽

Bacterial Isolates ◽

Necrosis Factor Alpha ◽

Mitogen Activated Protein

Several bacterial and immunogenic factors are involved in the host response to probiotic strains of Lactobacillus . Here, we report the isolation of new intestinal lactobacilli from chicken, with different immunomodulating properties on lymphoid cells from SJL and C57BL/6 mice. Analysis of biochemical markers in the Lactobacillus acidophilus CBA4P, CBA3P, and TPA3P isolates reveal that these bacterial isolates belong to the type 2 prototype, although they differ from each other. The effect of conditioned media (CM) from SJL- and C57BL/6-derived peritoneal macrophages incubated with the 3 sonicated bacterial isolates from chicken, as well as with Lactobacillus rhamnosus 9595, Escherichia coli lipopolysaccharide, or Staphylococcus aureus peptidoglycan were compared. Our results show that the CM of macrophages from C57BL/6 and SJL mice treated with the CBA4P isolate stimulated syngeneic splenic lymphocytes at a level similar to the one induced with CM from peptidoglycan-stimulated macrophages. In contrast, the CM from TPA3P- and CBA3P-treated macrophages promoted low or no stimulation of lymphoid cells. Incubation of splenic cells with CM from macrophages treated with L. rhamnosus or TPA3P led to a relative decrease in the percentages of splenic CD4+ T cells, whereas the relative percentages of B cells increased. The CBA4P and CBA3P isolates induced higher levels of gamma interferon when compared with the TPA3P isolate. The effects of the lactobacilli isolates differed according to the mouse strain used but correlated with the production of macrophagic tumor necrosis factor alpha and interleukins 6, 10, and 12 and with the modulation of the p38 mitogen-activated protein kinase (MAPK). Taken together, these results indicate that the immunomodulating properties of the new L. acidophilus isolates depend on their capacity to induce production of interleukins 10 and 12 by macrophages, which is under genetic control and depends on the p38 MAPK pathway.

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TRPM2 promotes pancreatic cancer by PKC/MAPK pathway

Cell Death and Disease ◽

10.1038/s41419-021-03856-9 ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Rui Lin ◽

Xunxia Bao ◽

Hui Wang ◽

Sibo Zhu ◽

Zhongyan Liu ◽

...

Keyword(s):

Pancreatic Cancer ◽

Wound Healing ◽

Western Blot ◽

Nude Mice ◽

Transcriptome Analysis ◽

Cell Model ◽

Mapk Pathway ◽

Migration And Invasion ◽

Tumor Bearing ◽

Scratch Wound

AbstractThe mechanism of pancreatic cancer (PA) is not fully understanded. In our last report, TRPM2 plays a promising role in pancreatic cancer. However, the mechanism of TRPM2 is still unknown in this dismal disease. This study was designed to investigate the role and mechanism of TRPM2 in pancreatic cancer. TRPM2 overexpressed and siRNA plasmid were created and transfected with pancreatic cancer cell line (BxPC-3) to construct the cell model. We employed CCK-8, Transwell, scratch wound, and nude mice tumor-bearing model to investigate the role of TRPM2 in pancreatic cancer. Besides, we collected the clinical data, tumor tissue sample (TT) and para-tumor sample (TP) from the pancreatic cancer patients treated in our hospital. We analyzed the mechanism of TRPM2 in pancreatic cancer by transcriptome analysis, western blot, and PCR. We blocked the downstream PKC/MEK pathway of TRPM2 to investigate the mechanism of TRPM2 in pancreatic cancer by CCK8, scratch wound healing, and transwell assays. Overexpressed TRPM2 could promote pancreatic cancer in proliferation, migration, and invasion ability in no matter the cell model or nude mice tumor-bearing model. TRPM2 level is highly negative correlated to the overall survival and progression-free survival time in PA patients, however, it is significantly increased in PA tissue as the tumor stage increases. The transcriptome analysis, GSEA analysis, western-blot, and PCR results indicate TRPM2 is highly correlated with PKC/MAPK pathways. The experiments of PKC/MEK inhibitors added to TRPM2 overexpressed BxPC-3 cell showed that significant inhibition of PA cells happened in CCK8, transwell, and wound-healing assay. TRPM2 may directly activate PKCα by calcium or indirectly activate PKCε and PKCδ by increased DAG in PA, which promote PA by downstream MAPK/MEK pathway activation.

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MMP-Inhibitory Effects of Flavonoid Glycosides from Edible Medicinal Halophyte Limonium tetragonum

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/6750274 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Min Joo Bae ◽

Fatih Karadeniz ◽

Jung Hwan Oh ◽

Ga Hyun Yu ◽

Mi-Soon Jang ◽

...

Keyword(s):

Cell Model ◽

Mapk Pathway ◽

Flavonoid Glycosides ◽

Inhibitory Effects ◽

Bioactive Substances ◽

Effect Of Solvent ◽

Potential Source ◽

Mouse Melanoma Cell ◽

Pathway Activation ◽

Stimulation Of

Limonium tetragonum has been well-known for its antioxidative properties as a halophyte. This study investigated the antimetastasis effect of solvent-partitioned L. tetragonum extracts (LTEs) and isolated compounds on HT1080 mouse melanoma cell model with a focus on matrix metalloproteinase (MMP) activity and TIMP and MAPK pathways. Upregulation and stimulation of MMPs result in elevated degradation of extracellular matrix which is part of several complications such as metastasis, cirrhosis, and arthritis. The anti-MMP capacity of LTEs was confirmed by their MMP-inhibitory effects, regulation of MMP and TIMP expression, and suppression of MAPK pathway. Among all tested LTEs, 85% aq. MeOH and n-BuOH were found to be most active fractions which later yielded two known flavonoid glycosides, myricetin 3-galactoside and quercetin 3-o-beta-galactopyranoside. Anti-MMP potential of the compounds was confirmed by their ability to regulate MMP expression through inhibited MAPK pathway activation. These results suggested that L. tetragonum might serve as a potential source of bioactive substances with effective anti-MMP properties.

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Enhanced survival effect of pyruvate correlates MAPK and NF-κB activation in hydrogen peroxide-treated human endothelial cells

Journal of Applied Physiology ◽

10.1152/japplphysiol.00797.2003 ◽

2004 ◽

Vol 96 (2) ◽

pp. 793-801 ◽

Cited By ~ 52

Author(s):

Yong-Jin Lee ◽

Il-Jun Kang ◽

Rolf Bünger ◽

Young-Hee Kang

Keyword(s):

Reactive Oxygen Species ◽

Hydrogen Peroxide ◽

Therapeutic Potential ◽

Cell Model ◽

Mapk Pathway ◽

Umbilical Vein ◽

Reactive Oxygen ◽

Antioxidant Potential ◽

Mitogen Activated Protein Kinase ◽

Oxygen Species

We recently reported that pyruvate inhibited translocation and activation of p53 caused by DNA damage due to oxidant injury (Lee YJ, Kang IJ, Bünger R, and Kang YH. Microvasc Res 66: 91-101, 2003); this was associated with increased expression of apoptosis-related bcl-2 and decreased expression of bax gene. This study attempted to delineate possible regulatory sites and mechanisms of antiapoptotic pyruvate, focusing on reactive oxygen species-mediated signaling in a human umbilical vein endothelial cell model. We compared the effects of the cytosolic reductant l-lactate and malate-aspartate shuttle blocker aminooxyacetate, both of which increase cytosolic NADH, on the downstream signaling pathway. Hydrogen peroxide (0.5 mM H2O2) depleted intracellular total glutathione that was prevented by pyruvate but not by l-lactate or aminooxyacetate. Activation of caspase-3 and the cleavage of procaspase-6 and procaspase-7 were strongly inhibited by pyruvate but markedly enhanced by l-lactate and aminooxyacetate, implicating redox-related antiapoptotic mechanisms of pyruvate. Western blot analysis and immunochemical data revealed that H2O2-induced transactivation of nuclear factor-κB (NF-κB) was also inhibited by pyruvate but not by l-lactate or aminooxyacetate. In addition, H2O2 downregulated extracellular signal-regulated kinase (ERK1/2) and phosphorylated p38 mitogen-activated protein kinase (MAPK), effects that were fully reversed by pyruvate within 2 h. Collectively, these findings indicate that pyruvate can protect cellular glutathione, thus enhancing cellular antioxidant potential, and that enhanced antioxidant potential can desensitize NF-κB transactivation due to reactive oxygen species, suggesting possible metabolic redox relations to NF-κB. Furthermore, pyruvate blocked the p38 MAPK pathway and activated the ERK pathway in an apparently redox-sensitive manner, which may regulate expression of genes believed to prevent apoptosis and promote cell survival. Thus pyruvate may have therapeutic potential for reducing endothelial dysfunction and improving survival during oxidative stress.

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