Abnormal Expression of TRAF Adapter Proteins in Waldenstrom’s Macroglobulinemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4640-4640
Author(s):  
Xavier Leleu ◽  
Lian Xu ◽  
Zachary R. Hunter ◽  
Anne-Sophie Moreau ◽  
Xiaoying Jia ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Low Grade ◽  
Adapter Proteins ◽  
Rt Pcr ◽  
Growth And Survival ◽  
Healthy Donors ◽  
Sirna Knockdown

Abstract Background: Waldenström’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma with as yet unknown genetic basis for its pathogenesis. Several TNF family members (CD40L, APRIL and BAFF/BLYS) are known to regulate WM growth and survival. TRAFs are a novel family of adapter proteins that facilitate pro-apoptotic (TACI) or pro-survival/differentiation (CD40, BAFFR, BCMA) receptor signaling mediated by TNF family ligands. Therefore, understanding the TRAF system in WM may yield important clues about WM growth and survival. Methods: WM cell lines (BCWM.1 and WSU-WM), IgM secreting low-grade lymphoma cell lines (MEK1, RL, Namalwa), and primary bone marrow CD19+ selected lymphoplasmacytic cells (LPC) from 20 WM patients and 6 healthy donors were evaluated for TRAF (TRAF 2, 3, 5, 6) expression using semi quantitative RT-PCR and/or western blot analysis. Results: The TNF familiy receptors CD40, BAFFR, BCMA, and TACI were expressed in all cell lines tested as well as in CD19+ selected LPC from WM patients and healthy donors. Moreover, TRAF 2, 3, 5, 6 were expressed in all cell lines by both RT-PCR and western blot analysis. In contrast, we observed loss or abnormally low expression of both TRAF 2 and 5 in 6/20 (30%) patients, whilst TRAF 3 was absent or abnormally low in 3/30 (15%) patients. TRAF 6 was expressed in all patients. Among healthy donors, we observed expression of all TRAF adapter proteins. Conclusion: Up to one third of WM patients demonstrate loss of TRAF 2 and 5 adapter proteins which facilitate signaling through the pro-apoptotic receptor TACI. Ongoing studies including gene sequencing and siRNA knockdown models are delineating a role for TRAF loss in the pathogenesis of WM.

The Spermatozoa Protein, SLLP1, Is a Novel Cancer-Testis Antigen in Hematologic Malignancies.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4281-4281
Author(s):  
Zhiqing Wang ◽  
Yana Zhang ◽  
Arabinda Mandal ◽  
Jian Zhang ◽  
Francis J. Giles ◽  
...  
Keyword(s):  
Immune Responses ◽  
Western Blot ◽  
Tumor Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Hematologic Malignancies ◽  
Myeloma Cell ◽  
Rt Pcr ◽  
Healthy Donors ◽  
Cancer Testis

Abstract SLLP1 is a unique non-bacteriolytic c-lysozyme-like protein isolated from human spermatozoa. Antisera to SLLP1 blocks binding in the hamster egg penetration assay, suggesting that SLLP1 may be involved in sperm/egg adhesion. A recent study by dot blot analysis on RNA showed that SLLP1 was expressed only in the testis and in Burkitt lymphoma Raji cell line, suggesting that further studies are warranted to determine and characterize SLLP1 expression in tumor cells, in particular, fresh tumor specimens. Using a pair of sequence-specific primers in RT-PCR, we found that SLLP1 transcripts could be detected in 5/8 myeloma cell lines, suggesting that SLLP1 may be expressed in tumor cells from some hematologic malignancies. When we applied the investigations to 52 primary hematologic malignant specimens, SLLP1 transcripts were detected in 6/17 myeloma, 4/14 CML, 3/11 CLL, 2/9 AML and 0/1 hairy cell leukemia. In contrast, SLLP1 transcripts were not detected in the peripheral blood (n=12) or bone marrow (n=3) from any healthy donors. The specificity of the PCR products was confirmed by either sequence analysis or restriction digest with Pvu II. SLLP1 transcripts were translated into its corresponding protein in these tumor cells. Using tumor cell lysate in Western blot analysis, we detected SLLP1 protein in the myeloma cell lines and also in fresh malignant specimens, although positivities were only observed in specimens with high RT-PCR signals. All PCR-negative specimens were also negative in Western blot analysis. The specificity of the Western blot signals were confirmed in all cases by blocking assays with a high concentration of recombinant SLLP1 protein. We next investigated the expression of SLLP1 in a large panel of normal tissues using RT-PCR and real time quantitative PCR. Both approaches showed that SLLP1 is a novel Cancer-Testis antigen in hematologic malignancies. SLLP1 was detected, at a level of 8206 copies/0.25 mcg total RNA, only in normal testis. We also found that the SLLP1 mRNA copy numbers in fresh hematologic tumor specimens were up to 2316 copies/0.25 mcg total RNA, i.e. more than 25% of the level found in normal testis. We cloned and generated SLLP1 recombinant protein from E coli and used the purified recombinant SLLP1 in an ELISA system to detect anti-SLLP1 antibodies. Using sera from 24 healthy donors and the mean + 2SD as the cut-off signal intensities, we found that high titer IgG antibodies directed at SLLP1 could be detected in the sera from 2/9 AML, 5/23 CLL, 6/27 CML and 14/51 myeloma patients. The specificity of the antibodies was confirmed in Western blot analysis. Probably due to the decreased sensitivity of the detection system in Western blot analysis, only 1/2 AML, 3/5 CLL, 4/6 CML and 7/14 myeloma SLLP1 antibody+ sera produced a signal in the Western blot analysis. Interesting, IgG2 was by far the commonest SLLP1 antibodies in these patients. There was a good correlation between SLLP1 gene expression and immune responses. In summary, SLLP1 is a novel CT antigen in hematologic malignancies and is capable of eliciting B-cell immune responses in vivo in cancer-bearing patients. Our results support SLLP1 as a protein target appropriate for further in vitro study to define its suitability for immunotherapy.

Inhibition Of H3K27-Methylome As a Novel Therapeutic Strategy In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3162-3162
Author(s):  
Maria Gkotzamanidou ◽  
Masood A Shammas ◽  
Jun Qi ◽  
Mehmet Kemal Samur ◽  
Teru Hideshima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inhibitory Effect ◽  
Epigenetic Changes ◽  
Healthy Donors ◽  
Equity Ownership ◽  
Induced Apoptosis

Abstract Multiple myeloma (MM) remains incurable, despite recent therapeutic advances; newer insights into the pathogenic mechanisms that cause this disease and additional therapies are urgently needed. Recent studies of the epigenome and in particular the methylome, have shown that myeloma is characterized by widespread epigenetic changes. Epigenetic changes may precede genetic mutations and genomic instability; Ubiquitously Transcribed Tetratricopeptide Repeat Protein X (UTX), a histone H3K27 demethylase may represent such an example. Previous studies have shown inactivating somatic mutations in UTX (KDM6A) in 10% of MM cases. Aberrant methylation of core histone tails and deregulation of the corresponding enzymes such as UTX and JMJD3 have been implicated in leukemia as well as other types of cancers, but their role in MM remains unknown. We evaluated the activity of the selective Jumonji H3K27 demethylase (UTX/JMJD3) inhibitor, GSK-J4, in MM. In a panel of 15 human MM cell lines (HMCLs) including cell lines resistant to bortezomib and dexamethasone, GSK-J4 induced significant survival and proliferation inhibition, as measured by luminescence-based viability assay (CTG), MTT (inhibition>68% in 48 hours) and 3H thymidine uptake, with the exception of OPM2 that was resistant up to 5 uM concentration. GSK-J4 induced apoptosis as measured by flow cytometry upon staining with Annexin-V/Propidium Iodide. The compound did not induce cytotoxicity in PBMCs from healthy donors and normal human skin fibroblasts. The inhibitory effect of GSK-J4 was observed also in CD138+ primary plasma cells from newly diagnosed MM patients (n=5) compared to PBMCs from healthy donors (n=9) (p<0.001). The compound also resulted in significant inhibition of MM cell colony formation in soft agar after 2 weeks in compared to controls. Moreover, interaction between MM cells and bone marrow stromal cells from MM patients or HS-5 stromal cell line did not overcome the inhibitory effect of GSK-J4 in all the HMCLs (KMS-12BM, LP-1, MM1S, OPM2, RPMI-8226, H929 and INA6) tested. To further investigate the mechanism of GSK-J4-induced apoptosis, we evaluate the activation of caspases 3/7, 8 and 9, and observed significant activation of caspase 3/7 and 9, indicating the involvement of intrinsic apoptotic pathway into the GSK-J4 function, as also confirmed by western blot analysis of Bcl-xl, p53, and Bax. We evaluated the enzymatic activity of UTX/JMJD3 in HMCLs by using a fluometric-based assay. The sensitivity of the HMCLs to demethylase inhibitor was directly related with UTX/JMJD3 activity (p<0.05). As the methylation of H3K27 mark plays a major role in the maintenance of active and silent states of gene expression in important cellular processes, we next mapped H3K27me3 and H3K27me2 chromatin modifications by genome-wide chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) analysis in RPMI8226 and KMS-12BM cells before and after GSK-J4 treatment and in UTX knockdown cells. Transcription factors of OSKM complex: Oct4, Sox2, and Nanog were found to be targets of aberrant demethylation after treatment with GSK-J4, indicating possible involvement of H3K27 demethylases in pathogenesis of MM through the regulation of “stemness” genes. Western blot analysis showed that the inhibitor reduced the expression of these genes. In conclusion, we demonstrate a significant beneficial impact of inhibition of H3K27 demethylation in MM. These results provide new insights into the mechanism of altering methylome as a potent epigenetic intervention in treatment of MM. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

Prima-1Met Combined with Bortezomib Has Synergistic Anti-Myeloma Activity By Modulation of Apoptosis and Cell Cycle Regulating Genes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4213-4213
Author(s):  
Priya Khoral ◽  
Robert J Guo ◽  
Jahangir Abdi ◽  
Hong Chang
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Western Blot ◽  
Real Time ◽  
Cell Lines ◽  
Blot Analysis ◽  
Drug Combination ◽  
Western Blot Analysis ◽  
Rt Pcr ◽  
Novel Therapeutic

Abstract INTRODUCTION Multiple Myeloma (MM) is a plasma-cell malignancy characterized by dismal prognosis and a high level of relapse, thus novel therapeutic approaches are needed. PRIMA-1Met is a novel small molecule showing anti-tumour activity and currently in clinical phase I-II trials. We recently demonstrated that PRIMA-1Met has potent anti-MM activity in vitro and in vivo. Bortezomib (BTZ) is a proteasome inhibitor that has been successfully used for treating some cases of relapsed MM. The aim of the current study is to determine whether PRIMA-1Met could be used in combination with BTZ to enhance the cytotoxic effects in myeloma cells. METHODS Using three different MM cell lines (LP1, U266 and 8226), we established dose response curves for both PRIMA-1Met and BTZ, and tested drug cytotoxicity using MTT assays. We then tested drug cytotoxicity of a range of concentrations of the drugs in combination. The Chou Talay method was used to determine whether or not the drug combinations were synergistic. A gene expression array was used to investigate the mechanism of the drug combination's effects. Total RNA was isolated from MM cell pellets, then synthesized cDNAs were applied to real time RT-PCR gene expression arrays containing 84 genes of interest. The genes selected were involved in apoptotic as well as cell growth and proliferation pathways. After normalization to 4 different housekeeping genes, fold changes in gene expression were analyzed in both drug treated and control samples using the 2-ΔΔCt algorithm. Western blot analysis was used to further investigate proteins of interest. RESULTS Cell viability of 8226, LP1 and U266 cells treated with individual concentrations of PRIMA-1Met (10uM) and BTZ (10nM) was on average 65%, 45% and 72.5%, respectively. However, combination of above doses reduced viability to 20% in 8226 and LP1, and to 40% in U266. The Chou Talay method identified this drug combination as synergistic in 2 out of the three tested cell lines, with Combination Index (CI) values of 0.72 in 8226 and 0.582 in U266. The gene expression analysis in real time RT-PCR indicated that the drug combination resulted in downregulation of genes involved in cell cycle and proliferation (CCND1, CDK4, CDK6, CDK2, IGFIR), genes from the Bcl-2 family of apoptosis regulation (Bcl-2, Bcl-XL, Mcl-1), as well as MDM2 from the p53 signalling pathway, and MYC, which is involved in both apoptosis and cell cycle progression. Western blot analysis revealed up-regulation of cleaved caspase-3 and -9, implying involvement of the intrinsic apoptotic pathway in the drug combination's activity. CONCLUSION Our results reveal that PRIMA-1Met synergistically enhances the anti-MM effect of BTZ, leading to a significantly higher level of MM cell death. Real time RT-PCR gene array analysis offers some insight into the mechanism of this combination's effect, implicating apoptotic, cell cycle and growth regulating genes. Our study provides framework for further evaluation of this drug combination as a novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Therapeutic Potential of Axl Blockade in BCR-ABL Negative Myeloproliferative Neoplasms (MPN)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3063-3063
Author(s):  
Antonia Beitzen-Heineke ◽  
Isabel Ben Batalla ◽  
Nikolaus Berenbrok ◽  
Sarina Paesler ◽  
Victoria Gensch ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Therapeutic Potential ◽  
Brdu Incorporation ◽  
Additive Effects ◽  
Healthy Donors

Abstract Axl, a member of the TAM family of receptor tyrosine kinases, mediates survival and therapy resistance of different cancer cells. The Axl ligand growth-arrest specific gene 6 (Gas6) was discovered to promote proliferation of leukemia cells in acute and chronic myeloid leukemia and Axl was identified as a potential therapeutic target in these diseases. Based on these data we investigated the role of Axl in BCR-ABL negative myeloproliferative neoplasms (MPN) and the therapeutic potential of Axl blockade in this group of diseases. We studied the effects of Axl blockade using the small molecule Axl inhibitor BGB324 and performing a lentivirus shRNA mediated knockdown of Axl in human SET-2 and murine BaF3-Jak2V617F MPN cell lines. Pharmacologic Axl blockade resulted in a significant dose dependent decrease in viability of MPN cell lines as measured by WST-1 cell viability assay. Annexin+ staining revealed an increased rate of apoptotic cells upon BGB324 treatment for SET-2 (increase by 15% at 1µM, p<0.001) and BaF3-Jak2V617F cells (increase by 54% at 2µM, p<0.05). Moreover, Western Blot analysis showed higher levels of cleaved caspase 3 in BGB324 treated SET-2 cells and decreased levels of anti-apoptotic bcl-2 in BGB324 treated BaF3-Jak2V617F cells. Additionally, BrdU incorporation assays showed a dose dependent decrease in proliferating cells upon treatment with BGB324 in MPN cell lines (p<0.05). Genetic knockdown of Axl in SET-2 cells decreased cell viability by 75% (p<0.01), increased apoptosis levels as measured by Annexin+ staining by 61% (p<0.05) and decreased proliferation as measured by BrdU incorporation by 35% (p<0.001) compared to control-transduced cells. Furthermore, Western Blot analysis revealed that genetic knockdown of Axl resulted in decreased phosphorylation of Stat3 and Stat5 compared to control-transduced cells. Combined Axl and Jak2 blockade, using BGB324 and the Jak2-inhibitor ruxolitinib, showed additive effects on reducing cell viability in SET-2 and BaF3-Jak2V617F cells (p<0.01 and p<0.001, respectively). Western Blot analysis identified inhibition of Stat5 by BGB324 single treatment in SET-2 cells whereas additive effects of combined Axl and Jak2 blockade resulted from additional inhibition of Stat3. In BaF3-Jak2V617F cells, BGB324 single treatment resulted in downstream inhibition of Akt signaling whereas additive effects of combined Axl and Jak2 blockade were exerted via additional inhibition of Stat5, Stat3 and Erk. The finding that BGB324 inhibits growth of MPN cells was further corroborated in vivo. A xenograft tumor model with SET-2 cells was set up in vivo. SET-2 tumor bearing mice treated with BGB324 50mg/kg showed a slower tumor growth (n=8, p<0.01), with a 60% reduction of tumor weight compared to vehicle treated mice (n=8/8, p<0.01). As a second in vivo model, a systemic model of Jak2V617F driven disease was used. After intravenous injection of BaF3-Jak2V617F cells, mice were treated with 50mg/kg BGB324 or vehicle starting the day after inoculation. BGB324 treated mice had a longer overall survival compared to vehicle treated mice (n=10/11, p*<0.05). Furthermore, to evaluate the potential of BGB324 in primary MPN cells, peripheral blood mononuclear cells (PBMC) were isolated from MPN patients and healthy donors. Western Blot analysis showed higher levels of Axl expression by PBMC from MPN patients compared to PBMC from healthy donors. Moreover, colony-forming assays with PBMC were performed in the presence of different concentrations of BGB324. Here, a higher reduction in the number of colony forming units (BFU-E and CFU-GEMM) was observed in samples from MPN patients compared to healthy donors upon treatment with 1µM (77% vs. 5%, respectively; p<0.001) or 2µM (100% vs. 60%, respectively; p<0.01) of BGB324 (n=5/5). In conclusion, these data indicate therapeutic potential of Axl blockade in BCR-ABL negative MPN as monotherapy and in combination with Jak2-inhibition, supporting the need for clinical investigation. Disclosures von Amsberg: Novartis: Honoraria, Speakers Bureau; Ipson: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau. Loges:BerGenBio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

scholarly journals NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells

2000 ◽  
Vol 278 (2) ◽  
pp. G197-G206 ◽  
Author(s):  
J. Praetorius ◽  
D. Andreasen ◽  
B. L. Jensen ◽  
M. A. Ainsworth ◽  
U. G. Friis ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Intracellular Ph ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mrna Level ◽  
Rt Pcr ◽  
Acid Extrusion ◽  
Molecular Expression ◽  
Nhe Isoforms

Na+/H+-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na+-dependent processes to acid extrusion, 2) sensitivity to Na+/H+ exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pHi) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na+ concentration ([Na+]o) during pHirecovery decreased H+ efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na+. The Na+/H+exchange inhibitors ethylisopropylamiloride and amiloride inhibited H+ efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na+]o and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na+/H+ exchange by isoforms NHE1, NHE2, and NHE3.

scholarly journals Integrin β3 Mediates the Endothelial-to-Mesenchymal Transition via the Notch Pathway

2018 ◽  
Vol 49 (3) ◽  
pp. 985-997 ◽  
Author(s):  
Weisen Wang ◽  
Zhi Wang ◽  
Dingyuan Tian ◽  
Xi Zeng ◽  
Yangdong Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Notch Signaling ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Notch Signaling Pathway ◽  
Rt Pcr ◽  
Mesenchymal Transition ◽  
Integrin Β3 ◽  
Endothelial To Mesenchymal Transition ◽  
Tgf Β1

Background/Aims: Neointimal hyperplasia is responsible for stenosis, which requires corrective vascular surgery, and is also a major morphological feature of many cardiovascular diseases. This hyperplasia involves the endothelial-to-mesenchymal transition (EndMT). We investigated whether integrin β3 can modulate the EndMT, as well as its underlying mechanism. Methods: Integrin β3 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). The expression of endothelial markers and mesenchymal markers was determined by real-time reverse transcription PCR (RT-PCR), immunofluorescence staining, and western blot analysis. Notch signaling pathway components were detected by real-time RT-PCR and western blot analysis. Cell mobility was evaluated by wound-healing, Transwell, and spreading assays. Fibroblast-specific protein 1 (FSP-1) promoter activity was determined by luciferase assay. Results: Transforming growth factor (TGF)-β1 treatment or integrin β3 overexpression significantly promoted the EndMT by downregulating VE-cadherin and CD31 and upregulating smooth muscle actin α and FSP-1 in HUVECs, and by enhancing cell migration. Knockdown of integrin β3 reversed these effects. Notch signaling was activated after TGF-β1 treatment of HUVECs. Knockdown of integrin β3 suppressed TGF-β1-induced Notch activation and expression of the Notch downstream target FSP-1. Conclusion: Integrin β3 may promote the EndMT in HUVECs through activation of the Notch signaling pathway.

scholarly journals Investigation into imbalance of Th1/Th2 cells in cirrhotic, hypersplenic rats

2019 ◽  
Vol 48 (3) ◽  
pp. 030006051988944 ◽  
Author(s):  
Yunfu Lv ◽  
Yejuan Li ◽  
Ning Liu ◽  
Yonghong Dong ◽  
Jie Deng
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Chemokine Receptors ◽  
Immunohistochemical Staining ◽  
Th2 Cells ◽  
Intragastric Administration ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Chemokine Receptor Ccr3

Objectives To evaluate the Th1/Th2 cell profile in spleens of cirrhotic and hypersplenic rats by investigating the expression of Th1-associated chemokine receptors CXCR3, CCR5 and Th2-associated chemokine receptor CCR3. Methods Experimental liver cirrhosis and hypersplenism were induced in rats by the intragastric administration of carbon tetrachloride (CCl4; 40% solution [0.3 ml/100g, twice/week for 8 weeks]) and confirmed by pathology and hemogram. Presence of the three chemokine receptors was investigated by real-time polymerase chain reaction (RT-PCR), immunohistochemical staining, and western blot analysis. Results By comparison with control animals (n=10), RT-PCR demonstrated that CXCR3 and CCR5-mRNA levels were significantly elevated in the hypersplenic rats (n=26) and CCR3-mRNA levels were lower. Immunohistochemical staining showed that by comparison with controls, the mean density of the Th1-associated CXCR3 and CCR5 receptors was significantly increased but there was no difference between groups in Th2-associated CCR3 receptors. Western blot analysis showed that by comparison with controls, hypersplenic rats had higher levels of CXCR3 and CCR5 protein but lower levels of CCR3 protein. Conclusions The abnormal expression of Th1-associated chemokine receptors in spleens of rats with cirrhosis and hypersplenism induced by CCL4 suggests that a functional imbalance between Th1/Th2 cells may play a role in the pathogenesis of hypersplenism.

scholarly journals Melatonin Reduces Inflammatory Injury through Inhibiting NF-κB Activation in Rats with Colitis

2005 ◽  
Vol 2005 (4) ◽  
pp. 185-193 ◽  
Author(s):  
Jun-Hua Li ◽  
Jie-Ping Yu ◽  
Hong-Gang Yu ◽  
Xi-Ming Xu ◽  
Liang-Liang Yu ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Rt Pcr ◽  
Colon Tissue ◽  
Inflammatory Injury ◽  
Proinflammatory Mediators ◽  
Proinflammatory Molecule ◽  
Colitis Model

Proinflammatory mediators are important in the pathogenesis of IBD, which are regulated by activation of NF-κB. The aim of this study was to investigate whether melatonin reduces inflammatory injury and inhibits proinflammatory molecule and NF-κB in rats with colitis. Rat colitis model was established by TNBS enema. NF-κB p65, TNF-α, ICAM-1, and IκBα in colon tissue were examined by immunohistochemistry, EMSA, RT-PCR, and Western blot analysis. Expression of proinflammatory molecule and activation of NF-κB were upregulated and IκB level decreased in rats with colitis. Melatonin reduces colonic inflammatory injury through downregulating proinflammatory molecule mediated by NF-κB inhibition and blockade of IκBα degradation.

Sign in / Sign up

Export Citation Format

Share Document