scholarly journals In Vitro Effects of Arthrocen, an Avocado/Soy Unsaponifiables Agent, on Inflammation and Global Gene Expression in Human Monocytes

2017 ◽  
Vol 9 (4) ◽  
pp. 31 ◽  
Author(s):  
Jared F Taylor ◽  
Ramin Goudarzi ◽  
Puya G Yazdi ◽  
Brian Allen Pedersen
Keyword(s):  
Synergistic Effect ◽  
Mrna Level ◽  
Global Gene Expression ◽  
Transcript Level ◽  
Symptomatic Treatment ◽  
Transcriptional Level ◽  
Vitro Assay ◽  
Protein Levels ◽  
Model Cell

Osteoarthritis (OA) is the most common form of arthritis. Symptomatically characterized by stiffness and pain, OA is a chronic degenerative disease of joints. Of note, there is growing interest in the potential use of plant-based compounds for symptomatic treatment of OA. Arthrocen is a plant-derived agent consisting of a one to two ratio of avocado and soy unsaponifiable extracts. In order to decipher the potential mechanisms of Arthrocen's action at the molecular level, we employed an in vitro assay using cultured human THP-1 cells (a model cell line for monocytes) to study its effects. By pairing protein arrays enriched for inflammatory markers, transcriptomic pathway analysis using RNA-Sequencing, and eicosanoid specific lipidomics, we have begun to unravel its potential mechanism of action. Specifically, we found that Arthrocen can attenuate the inflammatory response at the transcript level while inducing significant changes in numerous cytokines. Furthermore, we discovered that while Arthrocen alone did not increase IL-8 or MCP-1 levels, its presence had a synergistic effect on the observed increase in response to LPS stimulation. Additionally, this synergistic effect of Arthrocen on LPS stimulation of IL-8 and MCP-1 protein levels was also observed at the mRNA level and suggests a regulatory mechanism at the transcriptional level. Interestingly, Arthrocen induced no changes in any of the eicosanoids studied. This multi-omics approach implies that Arthrocen functions at the level of gene transcription to dampen inflammation mediated by monocytes in OA.

scholarly journals Exposure of Toluene Diisocyanate Induces DUSP6 and p53 through Activation of TRPA1 Receptor

2022 ◽  
Vol 23 (1) ◽  
pp. 517
Author(s):  
Soee Kim ◽  
Min Kim ◽  
Jung-Suk Sung
Keyword(s):  
Manufacturing Industry ◽  
Mrna Level ◽  
Global Gene Expression ◽  
Receptor Activation ◽  
Toluene Diisocyanate ◽  
Protein Levels ◽  
Dual Specificity ◽  
Specific Antagonist ◽  
The Impact ◽  
Cancerous Cells

Toluene diisocyanate (TDI), a major intermediate agent used in the manufacturing industry, causes respiratory symptoms when exposed to the human body. In this study, we aimed to determine the molecular mechanism of TDI toxicity. To investigate the impact of TDI exposure on global gene expression, we performed transcriptomic analysis of human bronchial epithelial cells (BEAS-2B) after TDI treatment. Differentially expressed genes (DEGs) were sorted and used for clustering and network analysis. Among DEGs, dual-specificity phosphatase 6 (DUSP6) was one of the genes significantly changed by TDI exposure. To verify the expression level of DUSP6 and its effect on lung cells, the mRNA and protein levels of DUSP6 were analyzed. Our results showed that DUSP6 was dose-dependently upregulated by TDI treatment. Thereby, the phosphorylation of ERK1/2, one of the direct inhibitory targets of DUSP6, was decreased. TDI exposure also increased the mRNA level of p53 along with its protein and activity which trans-activates DUSP6. Since TRPA1 is known as a signal integrator activated by TDI, we analyzed the relevance of TRPA1 receptor in DUSP6 regulation. Our data revealed that up-regulation of DUSP6 mediated by TDI was blocked by a specific antagonist against TRPA1. TDI exposure attenuated the apoptotic response, which suggests that it promotes the survival of cancerous cells. In conclusion, our results suggest that TDI induces DUSP6 and p53, but attenuates ERK1/2 activity through TRPA1 receptor activation, leading to cytotoxicity.

scholarly journals Evaluation of biomarkers for in vitro prediction of drug-induced nephrotoxicity in RPTEC/TERT1 cells

2020 ◽  
Vol 9 (2) ◽  
pp. 91-100 ◽  
Author(s):  
Xuan Qiu ◽  
Yufa Miao ◽  
Xingchao Geng ◽  
Xiaobing Zhou ◽  
Bo Li
Keyword(s):  
Transcriptional Activation ◽  
Messenger Rna ◽  
Aristolochic Acid ◽  
Kidney Injury ◽  
Drug Induced ◽  
Vitro Assay ◽  
Protein Levels ◽  
Aristolochic Acid I

Abstract There have been intensive efforts to identify in vivo biomarkers that can be used to monitor drug-induced kidney damage before significant impairment occurs. Kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, clusterin, β2-microglobulin and cystatin C (CysC) have been validated as clinical or preclinical biomarkers in urinary and plasma predictive of acute and chronic kidney injuries and diseases. A high-throughput in vitro assay predictive of nephrotoxicity could potentially be implemented in early drug discovery stage to reduce attrition at later stages of drug development. To assess the potential of these known in vivo biomarkers for in vitro evaluation of drug-induced nephrotoxicity, we selected four nephrotoxic agents (cisplatin, cyclosporin, aristolochic acid I and gentamicin) and detected their effects on the protein levels of nephrotoxic biomarkers in RPTEC/TERT1 cells. The protein levels of clusterin, CysC, GSTπ and TIMP-1 significantly increased in the conditioned media of RPTEC/TERT1 cells treated with cisplatin, cyclosporin, aristolochic acid I and gentamicin. The messenger RNA levels of clusterin, CysC, GSTπ and TIMP-1 also increased in RPTEC/TERT1 cells treated with cisplatin, cyclosporin, aristolochic acid I and gentamicin, indicating that drug-induced upregulation involves transcriptional activation. Taken together, the results clearly demonstrate that among the known in vivo nephrotoxic biomarkers, clusterin, CysC, GSTπ and TIMP-1 can be effectively used as in vitro biomarkers for drug-induced nephrotoxicity in RPTEC/TERT1 cells.

scholarly journals TIMP-2 regulates proliferation, invasion and STAT3-mediated cancer stem cell-dependent chemoresistance in ovarian cancer cells

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Ruth M. Escalona ◽  
Maree Bilandzic ◽  
Patrick Western ◽  
Elif Kadife ◽  
George Kannourakis ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Lines ◽  
Cancer Progression ◽  
Mrna Level ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Ovarian Cancer Cells ◽  
Knock Down ◽  
Protein Levels ◽  
Response To Chemotherapy

Abstract Background The metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression. This study aimed to determine the role of TIMP-2 in ovarian cancer progression and chemoresistance by reducing TIMP-2 expression in vitro in Fallopian tube secretory epithelial (FT282) and ovarian cancer (JHOS2 and OVCAR4) cell lines. Methods FT282, JHOS2 and OVCAR4 cells were transiently transfected with either single or pooled TIMP-2 siRNAs. The expression of different genes after TIMP-2 knock down (T2-KD) or in response to chemotherapy was determined at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence. Sensitivity of the cell lines in response to chemotherapy after TIMP-2 knock down was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Ethynyl-2′-deoxyuridine (EdU) assays. Cell invasion in response to TIMP-2 knockdown was determined by xCELLigence. Results Sixty to 90 % knock down of TIMP-2 expression was confirmed in FT282, OVCAR4 and JHOS2 cell lines at the mRNA and protein levels. TIMP-2 knock down did not change the mRNA expression of TIMP-1 or TIMP-3. However, a significant downregulation of MMP-2 in T2-KD cells occurred at both the protein and activation levels, compared to Control (Cont; scrambled siRNA) and Parental cells (P, transfection reagent only). In contrast, membrane bound MT1-MMP protein levels were significantly upregulated in T2-KD compared to Cont and P cells. T2-KD cells exhibited enhanced proliferation and increased sensitivity to cisplatin and paclitaxel treatments. Enhanced invasion was observed in the T2-KD-JOSH2 and OVCAR4 cells but not in T2-KD-FT282 cells. Treatment with cisplatin or paclitaxel significantly elevated the expression of TIMP-2 in Cont cells but not in T2-KD cells, consistent with significantly elevated expression of chemoresistance and CSC markers and activation of STAT3. Furthermore, a potent inhibitor of STAT3 activation, Momelotinib, suppressed chemotherapy-induced activation of P-STAT3 in OVCAR4 cells with concomitant reductions in the expression of chemoresistance genes and CSC markers. Conclusions The above results suggest that TIMP-2 may have a novel role in ovarian cancer proliferation, invasion and chemoresistance.

Rapamycin inhibits the growth of cholangiocarcinoma cells in vitro

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 15153-15153 ◽  
Author(s):  
T. Sawada ◽  
T. Okada ◽  
K. Kubota
Keyword(s):  
Synergistic Effect ◽  
Cell Lines ◽  
Mtt Assay ◽  
Mrna Level ◽  
Dependent Manner ◽  
In Vitro Methods ◽  
Proliferative Effect ◽  
Cholangiocarcinoma Cell ◽  
Dose Dependent

15153 Background: In the present study, anti-neoplastic effect of rapamycin against cholangiocarcinoma was studied in vitro. Methods: Expression of mTOR in 4 cholangiocarcinoma cell lines, TFK1, HuCCT1, NOZW, and OZ was evaluated by real-time PCR. Then, the four cholangiocarcinoma cell lines were cultured with rapamycin (0, 25, 50, 100, 200 nM), gemcitabine (0, 0.5, 1, 2 μM), or both, and anti-proliferative effect was evaluated by MTT assay. Results: All the four cholangiocarcinoma cell lines expressed endogenous mTOR- mRNA. Level of expression was the highest in HuCCT1 (65.8), and the lowest in TFK1 (17.6). Then, rapamycin significantly inhibited the growth of all the four cholangiocarcinoma cell lines, in dose-dependent manner. Gemcitabine inhibited the growth of NOZW (48.4%) and HuCCT1 (48.9%), but less efficiently in TFK1 (5.9%) and OZ (27.4%). Furthermore, synergistic anti-proliferative effect of rapamycin and gemcitabine was observed in TFK1 (39.1%), NOZW (38.9%), and OZ (47.1%), not in HuCCT1 (18.9%). Conclusion: Rapamycin effectively inhibited the growth of the cholangiocarcinoma cell lines, and synergistic effect with gemcitabine was observed in three of the four cell lines. No significant financial relationships to disclose.

scholarly journals Enhanced Cerebral Expression of MCT1 and MCT2 in a Rat Ischemia Model Occurs in Activated Microglial Cells

2009 ◽  
Vol 29 (7) ◽  
pp. 1273-1283 ◽  
Author(s):  
Tiago JTP Moreira ◽  
Karin Pierre ◽  
Fumihiko Maekawa ◽  
Cendrine Repond ◽  
Aleta Cebere ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Level ◽  
Protein Levels ◽  
Microglial Cell Line ◽  
Tnf Α ◽  
Ischemic Episode ◽  
Isolectin B4 ◽  
Factor Α ◽  
Necrosis Factor

Monocarboxylate transporters (MCTs) are essential for the use of lactate, an energy substrate known to be overproduced in brain during an ischemic episode. The expression of MCT1 and MCT2 was investigated at 48 h of reperfusion from focal ischemia induced by unilateral extradural compression in Wistar rats. Increased MCT1 mRNA expression was detected in the injured cortex and hippocampus of compressed animals compared to sham controls. In the contralateral, uncompressed hemisphere, increases in MCT1 mRNA level in the cortex and MCT2 mRNA level in the hippocampus were noted. Interestingly, strong MCT1 and MCT2 protein expression was found in peri-lesional macrophages/microglia and in an isolectin B4+/S100β+ cell population in the corpus callosum. In vitro, MCT1 and MCT2 protein expression was observed in the N11 microglial cell line, whereas an enhancement of MCT1 expression by tumor necrosis factor-α (TNF-α) was shown in these cells. Modulation of MCT expression in microglia suggests that these transporters may help sustain microglial functions during recovery from focal brain ischemia. Overall, our study indicates that changes in MCT expression around and also away from the ischemic area, both at the mRNA and protein levels, are a part of the metabolic adaptations taking place in the brain after ischemia.

The Regulation of Natural Food Dyes Curcumin on the Amyloidogenic Pathway of APP

2013 ◽  
Vol 781-784 ◽  
pp. 1160-1163
Author(s):  
Xiong Zhang ◽  
Jie Yun Sun ◽  
Hong Mei Zhang ◽  
Lu Si ◽  
Yu Li
Keyword(s):  
Time Course ◽  
Mrna Level ◽  
Time Dependent ◽  
Hek293 Cells ◽  
Natural Food ◽  
Inhibition Effect ◽  
Dose Time ◽  
Protein Levels ◽  
Food Dyes

As a promising prevention and therapeutic intervention in Alzheimer’s disease, natural food dyes curcumin could obviously inhibit the generation of Aβ, but the mechanism is not fully defined. This study aims to investigate the effects of curcumin on the amyloidogenic pathway of APP in vitro. Plasmids APPswe and BACE1-mychis were transiently co-transfected into SH-SY5Y and HEK293 cells. Then, they were treated with curcumin at 0, 1.25, 5, 20 μmol/L for 24 h, or with curcumin at 5μmol/L for 0, 12, 24 and 48 h for the time course assay. Our findings showed that curcumin could inhibit the expression of the APP at mRNA level; the expression of the BACE1 and C99 at mRNA and protein levels, furthermore it could inhibit the generation of Aβ40/42, and those changes were dose-time dependent (p<0.05). Our study indicates that Aβ40/42 generation inhibition effect of curcumin might due to its influence on amyloidogenic pathway. This may provide important experimental basis for AD treatment with curcumin.

High Glucose/Thrombin-Induced Endothelial Inflammation Via Microrna-146a and Nox4 Regulation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5952-5952
Author(s):  
Ching-Tien Peng ◽  
Wan Yu Lo ◽  
Huang Joe Wang
Keyword(s):  
High Glucose ◽  
Conflicts Of Interest ◽  
Mrna Level ◽  
In Silico Analysis ◽  
Fold Increase ◽  
Ros Generation ◽  
Aortic Endothelial Cells ◽  
Protein Levels ◽  
Endothelial Inflammation

Abstract Diabetes is associated with hyperglycemia and increased thrombin generation. It is unknown whether high glucose (HG)/thrombin can modulate the expression of NAPDH oxidase (Nox) subtypes in human aortic endothelial cells (HAECs). Besides, we investigate whether miR-146a is involved in endothelial cell inflammation. We observed that HG (25 mmol/l) exerted a synergistic effect with thrombin (2 U/ml) for induction of Nox4 mRNA level in HAECs. The increased Nox4 mRNA was associated with increased Nox4 protein and ROS production. We also demonstrated that HG/thrombin treatment increased interleukin-8 and interleukin-6 protein levels. Besides, HG/thrombin treatment caused an 11.43-fold increase of THP-1 adhesion to HAECs. In Silico analysis identified homology between miR-146a and the 3’-UTR of the human Nox4 mRNA, suggesting a potential regulation of Nox4 by miR-146a. Furthermore, HG/thrombin treatment decreased miR146a expression to 58% of the control, indicating an impaired feedback restrain of HG/thrombin-induced endothelial inflammation. MiR-146a mimic transfection prevented HG/thrombin-induced upregulation of Nox4 mRNA, Nox4 protein, and ROS generation. In addition, inflammatory phenotypes were attenuated in miR-146a mimic-transfected HAECs. In conclusion, miR-146a is involved in the regulation of endothelial inflammation via modulation of Nox4 in an in-vitro milieu mimicking diabetic atherothrombosis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Overexpression of DDR1 Promotes Migration, Invasion, Though EMT-Related Molecule Expression and COL4A1/DDR1/MMP-2 Signaling Axis

2020 ◽  
Vol 19 ◽  
pp. 153303382097327
Author(s):  
Xin Xie ◽  
Hongchao He ◽  
Ning Zhang ◽  
Xiaojing Wang ◽  
Wenbin Rui ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Molecular Mechanisms ◽  
Mrna Level ◽  
Migration And Invasion ◽  
Rt Pcr ◽  
Protein Levels ◽  
Signaling Axis ◽  
Slug Expression ◽  
Mrna And Protein Expression

Purpose: Discoidin domain receptor 1 (DDR1) belongs to a novel class of receptor tyrosine kinases. Previous evidence indicates that DDR1 overexpression promotes the aggressive growth of bladder cancer (BC) cells. This study aimed to investigate the molecular mechanisms by which DDR1 influences BC. Methods: DDR1 was transfected into human BC RT4 cells. DDR1, COL4A1, and MMP-2 expression in 30 BC tissues and paired adjacent tissues were examined by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. Transwell assays were conducted to determine cell migration and invasion. RT-PCR and western blot (WB) were also used to measure the DDR1, COL4A1, MMP-2, and EMT-related gene (ZEB1 and SLUG) expression in RT4 cells after DDR1 overexpression. Results: COL4A1 and MMP-2 interacted with DDR1 in the PPI network. RT-PCR and immunohistochemistry results showed that both mRNA and protein levels of DDR1 and COL4A1 were significantly increased in BC tissue, while the expression of MMP-2 was increased only at the mRNA level ( P < 0.05). Overexpression of DDR1 in RT4 cells significantly promoted their migratory and invasive capabilities in vitro ( P < 0.05). Moreover, overexpression of DDR1 in RT4 cells increased the mRNA and protein expression of ZEB1, SLUG, COL4A1, and MMP-2 ( P < 0.01). DDR1-mediated migration and invasion of RT4 cells were reversed after COL4A1-siRNA treatment. Conclusion: DDR1 may be a potential therapeutic target in BC patients.

Suppression of DPYD expression in RKO Cells via DNA methylation in the regulatory region of the DPYD promoter: a potentially important epigenetic mechanism regulating DPYD expression

2007 ◽  
Vol 85 (3) ◽  
pp. 337-346 ◽  
Author(s):  
Xue Zhang ◽  
Richie Soong ◽  
Kangsheng Wang ◽  
Lin Li ◽  
James R. Davie ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Trichostatin A ◽  
Dihydropyrimidine Dehydrogenase ◽  
Mrna Level ◽  
Regulatory Region ◽  
Cancer Cell Line ◽  
Epigenetic Mechanism ◽  
Colorectal Cancer Cell Line ◽  
Transcriptional Level

Dihydropyrimidine dehydrogenase (DPD) is one of the factors that determine the efficacy and toxicity of 5-fluorouracil. Variations in DPD activity may result from alterations at the transcriptional level of the DPYD gene. Heterogeneity in DPYD expression has been reported, but the molecular mechanisms responsible for this remain unclear. We investigated methylation of the DPYD promoter as a mechanism for transcriptional regulation of DPYD in the RKO colorectal cancer cell line. We demonstrate that the active transcription machinery for DPYD is present in RKO cells, but promoter binding of Sp1, a transactivator of DPYD, was inhibited, which on subsequent examination was shown to be associated with dense promoter methylation. Treatment with 5-aza-2′-deoxycytidine alone or the combination of 5-aza-2′-deoxycytidine and trichostatin A induced demethylation of the promoter and markedly increased the DPYD mRNA level in RKO cells but not in unmethylated WiDr cells. Furthermore, in vitro methylation of the DPYD promoter decreased promoter activity. These data suggest an important role for methylation in DPYD suppression. The transcriptional suppression of DPYD by methylation may be responsible for the increased 5-fluorouracil sensitivity observed in some patients. This may also provide insight into the mechanism underlying the downregulation of DPYD in some colorectal cancers.

P19INK4D links endomitotic arrest and megakaryocyte maturation and is regulated by AML-1

Blood ◽  
2008 ◽  
Vol 111 (8) ◽  
pp. 4081-4091 ◽  
Author(s):  
Laure Gilles ◽  
Romain Guièze ◽  
Dominique Bluteau ◽  
Véronique Cordette-Lagarde ◽  
Catherine Lacout ◽  
...  
Keyword(s):  
Ploidy Level ◽  
Molecular Mechanisms ◽  
Transcriptional Level ◽  
Moderate Increase ◽  
Protein Levels ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Ploidy Class

Abstract The molecular mechanisms that regulate megakaryocyte (MK) ploidization are poorly understood. Using MK differentiation from primary human CD34+ cells, we observed that p19INK4D expression was increased both at the mRNA and protein levels during ploidization. p19INK4D knockdown led to a moderate increase (31.7% ± 5%) in the mean ploidy of MKs suggesting a role of p19INK4D in the endomitotic arrest. This increase in ploidy was associated with a decrease in the more mature MK population (CD41highCD42high) at day 9 of culture, which was related to a delay in differentiation. Inversely, p19INK4D overexpression in CD34+ cells resulted in a decrease in mean ploidy level associated with an increase in CD41 and CD42 expression in each ploidy class. Confirming these in vitro results, bone marrow MKs from p19INK4D KO mice exhibited an increase in mean ploidy level from 18.7N (± 0.58N) to 52.7N (± 12.3N). Chromatin immunoprecipitation assays performed in human MKs revealed that AML-1 binds in vivo the p19INK4D promoter. Moreover, AML-1 inhibition led to the p19INK4D down-regulation in human MKs. These results may explain the molecular link at the transcriptional level between the arrest of endomitosis and the acceleration of MK differentiation.

Sign in / Sign up

Export Citation Format

Share Document