scholarly journals Fluorescein-labeled Tyramide Strongly Enhances the Detection of Low Bromodeoxyuridine Incorporation Levels

1997 ◽  
Vol 45 (2) ◽  
pp. 315-319 ◽  
Author(s):  
Jimmy Van heusden ◽  
Paul de Jong ◽  
Frans Ramaekers ◽  
Hélène Bruwiere ◽  
Marcel Borgers ◽  
...  
Keyword(s):  
Signal Amplification ◽  
Enzymatic Reaction ◽  
Cancer Prognosis ◽  
Brdu Incorporation ◽  
Computer Assisted ◽  
Tyramide Signal Amplification ◽  
Amplification Method ◽  
Brdu Labeling

Immunocytochemical detection of bromodeoxyuridine (BrdU) labeling can be hampered by low BrdU incorporation levels. We describe here an amplification method for weak BrdU immunosignals. The tyramide signal amplification method based on catalyzed reporter deposition (CARD) uses fluorescein-labeled tyramide as a substrate for horseradish peroxidase. The enzyme catalyzes the formation of highly reactive tyramide radicals with a very short half-life, resulting in the binding of fluorescein-conjugated tyramide only at the site of the enzymatic reaction. MCF-7 cells were grown in vitro in medium containing charcoal-stripped fetal bovine serum supplemented by growth factors. Under these culture conditions, the BrdU immunosignal was hard to detect but could be enhanced specifically by the tyramide signal amplification system, resulting in clear-cut differences between BrdU-negative and BrdU-positive cells. This enabled rapid and objective quantification of the BrdU labeling index without the risk of underestimating the number of cells in S-phase. Therefore, this amplification of BrdU immunosignals might also prove valuable for in vivo cancer prognosis, cell kinetics studies, and computer-assisted image analyses.

scholarly journals Identification of DNA-Synthesizing Bacterial Cells in Coastal North Sea Plankton

2002 ◽  
Vol 68 (11) ◽  
pp. 5728-5736 ◽  
Author(s):  
Annelie Pernthaler ◽  
Jakob Pernthaler ◽  
Martha Schattenhofer ◽  
Rudolf Amann
Keyword(s):  
North Sea ◽  
Signal Amplification ◽  
Sea Water ◽  
Fold Increase ◽  
Brdu Incorporation ◽  
Bacterial Cells ◽  
Tyramide Signal Amplification ◽  
Fab Fragment ◽  
Phytoplankton Primary Production ◽  
Brdu Labeling

ABSTRACT We describe a method for microscopic identification of DNA-synthesizing cells in bacterioplankton samples. After incubation with the halogenated thymidine analogue bromodeoxyuridine (BrdU), environmental bacteria were identified by fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-linked oligonucleotide probes. Tyramide signal amplification was used to preserve the FISH staining during the subsequent immunocytochemical detection of BrdU incorporation. DNA-synthesizing cells were visualized by means of an HRP-labeled antibody Fab fragment and a second tyramide signal amplification step. We applied our protocol to samples of prefiltered (pore size, 1.2 μm) North Sea surface water collected during early autumn. After 4 h of incubation, BrdU incorporation was detected in 3% of all bacterial cells. Within 20 h the detectable DNA-synthesizing fraction increased to >14%. During this period, the cell numbers of members of the Roseobacter lineage remained constant, but the fraction of BrdU-incorporating Roseobacter sp. cells doubled, from 24 to 42%. In Alteromonas sp. high BrdU labeling rates after 4 to 8 h were followed by a 10-fold increase in abundance. Rapid BrdU incorporation was also observed in members of the SAR86 lineage. After 4 h of incubation, cells affiliated with this clade constituted 8% of the total bacteria but almost 50% of the visibly DNA-synthesizing bacterial fraction. Thus, this clade might be an important contributor to total bacterioplankton activity in coastal North Sea water during periods of low phytoplankton primary production. The small size and low ribosome content of SAR86 cells are probably not indications of inactivity or dormancy.

Cardiovascular Effects of Micromeria graeca (L.) Benth. ex Rchb in Normotensive and Hypertensive Rats

2020 ◽  
Vol 20 (8) ◽  
pp. 1253-1261
Author(s):  
Mourad Akdad ◽  
Mohamed Eddouks
Keyword(s):  
Blood Pressure ◽  
Cardiovascular System ◽  
Arterial Blood Pressure ◽  
Antihypertensive Activity ◽  
Computer Assisted ◽  
Hypertensive Rats ◽  
Vasorelaxant Effect ◽  
Arterial Blood

Aims: The present study was performed in order to analyze the antihypertensive activity of Micromeria graeca (L.) Benth. ex Rchb. Background: Micromeria graeca (L.) Benth. ex Rchb is an aromatic and medicinal plant belonging to the Lamiaceae family. This herb is used to treat various pathologies such as cardiovascular disorders. Meanwhile, its pharmacological effects on the cardiovascular system have not been studied. Objective: The present study aimed to evaluate the effect of aqueous extract of aerial parts of Micromeria graeca (AEMG) on the cardiovascular system in normotensive and hypertensive rats. Methods: In this study, the cardiovascular effect of AEMG was evaluated using in vivo and in vitro investigations. In order to assess the acute effect of AEMG on the cardiovascular system, anesthetized L-NAME-hypertensive and normotensive rats received AEMG (100 mg/kg) orally and arterial blood pressure parameters were monitored during six hours. In the sub-chronic study, rats were orally treated for one week, followed by blood pressure assessment during one week of treatment. Blood pressure was measured using a tail-cuff and a computer-assisted monitoring device. In the second experiment, isolated rat aortic ring pre-contracted with Epinephrine (EP) or KCl was used to assess the vasorelaxant effect of AEMG. Results: Oral administration of AEMG (100 mg/kg) provoked a decrease of arterial blood pressure parameters in hypertensive rats. In addition, AEMG induced a vasorelaxant effect in thoracic aortic rings pre-contracted with EP (10 μM) or KCl (80 mM). This effect was attenuated in the presence of propranolol and methylene blue. While in the presence of glibenclamide, L-NAME, nifedipine or Indomethacin, the vasorelaxant effect was not affected. Conclusion: This study showed that Micromeria graeca possesses a potent antihypertensive effect and relaxes the vascular smooth muscle through β-adrenergic and cGMP pathways.

scholarly journals Cell kinetics of human tumors by in vitro bromodeoxyuridine labeling.

1989 ◽  
Vol 37 (9) ◽  
pp. 1449-1454 ◽  
Author(s):  
J S Meyer ◽  
J Nauert ◽  
S Koehm ◽  
J Hughes
Keyword(s):  
Tritiated Thymidine ◽  
S Phase ◽  
Dna Flow Cytometry ◽  
Breast Carcinomas ◽  
Brdu Labeling ◽  
The Central Nervous System ◽  
Labeling Method ◽  
Kinetics Of

We labeled active S-phase cells in primary breast carcinomas with a modified 5-bromo-2'-deoxyuridine (BrdU) procedure using a silver-enhanced colloidal gold visualization step. Separate samples of 29 tumors were labeled with BrdU or tritiated thymidine ([3H]-dThd), and the labeling indices (LI) from the two methods were equivalent (Spearman's correlation coefficient = 0.96). Three breast carcinomas were incubated in various mixes of both BrdU and [3H]-dThd and developed sequentially for each. Paired photomicrographs showed that the same nuclei were labeled by either precursor. The in vitro method yielded LIs similar to those reported after in vivo pulse BrdU labeling for tumors of the central nervous system. The BrdU LI correlated significantly (r = 0.76, p less than 0.001) with % S-phase by DNA flow cytometry in 33 breast carcinomas. The BrdU labeling method is simpler and more rapid than the [3H]-dThd procedure (1-2 days for completion for the former, 7-10 days for the latter), and it provides an equivalent measurement of proliferative index.

scholarly journals ATP:Mg2+ shapes condensate properties of rRNA-NPM1 in vitro nucleolus model and its partitioning of ribosomes

2021 ◽  
Author(s):  
N. Amy Yewdall ◽  
Alain A. M. André ◽  
Merlijn H. I. van Haren ◽  
Frank H. T. Nelissen ◽  
Aafke Jonker ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Enzymatic Reaction ◽  
Atp Depletion ◽  
Gel Point ◽  
Dependent Manner ◽  
Biophysical Properties ◽  
Temperature Dependent ◽  
Quantitative Fluorescence

Nucleoli have viscoelastic gel-like condensate dynamics that are not well represented in vitro. Nucleoli models, such as those formed by nucleophosmin 1 (NPM1) and ribosomal RNA (rRNA), exhibit condensate dynamics orders of magnitude faster than in vivo nucleoli. Here we show that an interplay between magnesium ions (Mg2+) and ATP governs rRNA dynamics, and this ultimately shapes the physical state of these condensates. Using quantitative fluorescence microscopy, we demonstrate that increased RNA compaction occurs in the condensates at high Mg2+ concentrations, contributing to the slowed RNA dynamics. At Mg2+ concentrations above 7 mM, rRNA is fully arrested and the condensates are gels. Below the critical gel point, NPM1-rRNA droplets age in a temperature-dependent manner, suggesting that condensates are viscoelastic materials, undergoing maturation driven by weak multivalent interactions. ATP addition reverses the dynamic arrest of rRNA, resulting in liquefaction of these gel-like structures. Surprisingly, ATP and Mg2+ both act to increase partitioning of NPM1-proteins as well as rRNA, which influences the partitioning of small client molecules. By contrast, larger ribosomes form a halo around NPM1-rRNA coacervates when Mg2+ concentrations are higher than ATP concentrations. Within cells, ATP levels fluctuate due to biomolecular reactions, and we demonstrate that a dissipative enzymatic reaction can control the biophysical properties of in vitro condensates through depletion of ATP. This enzymatic ATP depletion also reverses the formation of the ribosome halos. Our results illustrate how cells, by changing local ATP concentrations, may regulate the state and client partitioning of RNA-containing condensates such as the nucleolus.

scholarly journals Association of CD44−/CD24− Breast Cancer Cells with Late Stage Tumor Recurrence

2020 ◽  
Author(s):  
Xinbo Qiao ◽  
Yixiao Zhang ◽  
Lisha Sun ◽  
Qingtian Ma ◽  
Jie Yang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cells ◽  
Tumor Metastasis ◽  
Cancer Prognosis ◽  
Molecular Events ◽  
Cell Conversion ◽  
Tissue Specimens ◽  
Distant Tumor

AbstractTumor metastasis remains the main cause of breast cancer-related deaths, especially the later breast cancer distant metastasis. This study assessed CD44−/CD24− tumor cells in 576 tissue specimens for associations with clinicopathological features and metastasis and then investigated the underlying molecular events. The data showed that level of CD44−/CD24− cells was associated with later postoperative distant tumor metastasis. Furthermore, CD44−/CD24− triple negative cells could spontaneously convert into CD44+/CD24− cancer stem cells (CSCs) with properties similar to CD44+/CD24− CSCs from parental MDA-MB-231 cells in terms of gene expression, tumor cell xenograft formation, and lung metastasis in vitro and in vivo. Single-cell RNA sequencing identified RHBDL2 as a regulator that enhanced spontaneous CD44+/CD24− CSC conversion, whereas knockdown of RHBDL2 expression inhibited YAP/NF-κB signaling and blocked spontaneous CD44−/CD24− cell conversion to CSCs. These data suggested that the level of CD44−/CD24− tumor cells could predict breast cancer prognosis, metastasis, and response to adjuvant therapy.

Proliferation and differentiation potential of rat forebrain oligodendroglial progenitors both in vitro and in vivo

Development ◽  
1991 ◽  
Vol 111 (4) ◽  
pp. 1061-1080 ◽  
Author(s):  
R. Hardy ◽  
R. Reynolds
Keyword(s):  
Progenitor Cell ◽  
Neonatal Rat ◽  
Myelin Proteins ◽  
Brdu Incorporation ◽  
Cell Type ◽  
Differentiation Potential ◽  
Serum Free ◽  
Rat Forebrain

We have followed the development of the O-2A progenitor cell from the neonatal rat forebrain, both in dissociated cell culture and in cryostat sections, using immunocytochemical techniques employing a panel of antibodies that recognise the cells at different stages of their development. This included the monoclonal antibody LB1, which binds to the surface ganglioside GD3 expressed on O-2A progenitor cells. In secondary cultures enriched for O-2A progenitors maintained in a serum-free chemically defined medium, a large proportion of the cells are primed to differentiate into oligodendroglia and go on to express the oligodendroglial specific surface glycolipid galactocerebroside (GC) and then the myelin proteins CNP and MBP. However, a significant proportion of immature bipolar GD3+ cells remained after 6 days in secondary culture. It appears that not all the O-2A progenitors in our cultures differentiate immediately and some cells remain in an undifferentiated state and divide to replenish progenitor numbers. We have also identified in our cultures a small apolar GD3- cell, which when isolated differentiated into a GD3+ bipolar O-2A progenitor cell. We have termed this cell type a preprogenitor. The differentiation of this cell type into O-2A progenitors may be the source of the immature GD3+ cells present at the later stages of our secondary cultures. The proliferative profile of the cultures was studied using 5′bromo-2-deoxyuridine (BrdU) incorporation as an index of mitosis. Only the immature, bipolar O-2A progenitors were seen to divide at any time in serum-free culture. Neither the more mature multipolar O-2A cells nor the oligodendroglia were seen to divide. The developmental profile of the O-2A cells in the rat forebrain in vivo showed a largely similar progression to that in culture, with a time lag of at least 6 days between GD3 expression and the onset of myelination. BrdU incorporation studies in vivo also showed that the GD3+ progenitor cell is mitotic whereas the GC(+)-expressing oligodendroglia is not. We have shown that there are several significant alterations in the timing of antigen expression in both O-2A progenitors and oligodendroglia in vitro compared to that seen in vivo.

scholarly journals Computational Chemogenomics Drug Repositioning Strategy Enables the Discovery of Epirubicin as a New Repurposed Hit for Plasmodium falciparum and P. vivax

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Letícia Tiburcio Ferreira ◽  
Juliana Rodrigues ◽  
Gustavo Capatti Cassiano ◽  
Tatyana Almeida Tavella ◽  
Kaira Cristina Peralis Tomaz ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
In Silico ◽  
Antimalarial Activity ◽  
Drug Repositioning ◽  
Dna Gyrase ◽  
Plasmodium Yoelii ◽  
Computer Assisted ◽  
Content Type

ABSTRACT Widespread resistance against antimalarial drugs thwarts current efforts for controlling the disease and urges the discovery of new effective treatments. Drug repositioning is increasingly becoming an attractive strategy since it can reduce costs, risks, and time-to-market. Herein, we have used this strategy to identify novel antimalarial hits. We used a comparative in silico chemogenomics approach to select Plasmodium falciparum and Plasmodium vivax proteins as potential drug targets and analyzed them using a computer-assisted drug repositioning pipeline to identify approved drugs with potential antimalarial activity. Among the seven drugs identified as promising antimalarial candidates, the anthracycline epirubicin was selected for further experimental validation. Epirubicin was shown to be potent in vitro against sensitive and multidrug-resistant P. falciparum strains and P. vivax field isolates in the nanomolar range, as well as being effective against an in vivo murine model of Plasmodium yoelii. Transmission-blocking activity was observed for epirubicin in vitro and in vivo. Finally, using yeast-based haploinsufficiency chemical genomic profiling, we aimed to get insights into the mechanism of action of epirubicin. Beyond the target predicted in silico (a DNA gyrase in the apicoplast), functional assays suggested a GlcNac-1-P-transferase (GPT) enzyme as a potential target. Docking calculations predicted the binding mode of epirubicin with DNA gyrase and GPT proteins. Epirubicin is originally an antitumoral agent and presents associated toxicity. However, its antiplasmodial activity against not only P. falciparum but also P. vivax in different stages of the parasite life cycle supports the use of this drug as a scaffold for hit-to-lead optimization in malaria drug discovery.

The Rho Family GTPase Cdc42 Regulates Multiple Hematopoietic Stem/Progenitor Cell Functions and Erythropoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 32-32
Author(s):  
Lei Wang ◽  
Linda Yang ◽  
Marie–Dominique Filippi ◽  
David A. Williams ◽  
Yi Zheng
Keyword(s):  
Bone Marrow ◽  
Fetal Liver ◽  
Brdu Incorporation ◽  
Low Density ◽  
Actin Assembly ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Rho Family

Abstract The Rho family GTPase Cdc42 has emerged as a key signal transducer in cell regulation. To investigate its physiologic function in hematopoiesis, we have generated mice carrying a gene targeted null allele of cdc42gap, a major negative regulatory gene of Cdc42 and mice with conditional targeted cdc42 allele (cdc42flox/flox). Deletion of the respective gene products in mice was confirmed by PCR genotyping and Western blotting. Low-density fetal liver or bone marrow cells from Cdc42GAP−/− mice displayed ~3 fold elevated Cdc42 activity and normal RhoA, Rac1 or Rac2 activity, indicating that cdc42gap deletion has a specific effect on Cdc42 activity. The Cdc42GAP-deficient hematopoietic stem/progenitor cells (HSC/Ps, Lin−c-Kit+) generated from Cdc42GAP−/− E14.5 fetal liver and the Cdc42−/− HSC/Ps derived by in vitro expression of Cre via a retrovirus vector from Cdc42flox/flox low density bone marrow showed a growth defect in liquid culture that was associated with increased apoptosis but normal cell cycle progression. Cdc42GAP-deficient HSC/Ps displayed impaired cortical F-actin assembly with extended actin protrusions upon exposure to SDF–1 in vitro and a punctuated actin structure after SCF stimulation while Cdc42−/− but not wild type HSC/Ps responded to SDF-1 in inducing membrane protrusions. Both Cdc42−/− and Cdc42GAP−/− HSC/Ps were markedly decreased in adhesion to fibronectin. Moreover, both Cdc42−/− and Cdc42GAP−/− HSC/Ps showed impaired migration in response to SDF-1. These results demonstrate that Cdc42 regulation is essential for multiple HSC/P functions. To understand the in vivo hematopoietic function of Cdc42, we have characterized the Cdc42GAP−/− mice further. The embryos and newborns of homozygous showed a ~30% reduction in hematopoietic organ (i.e. liver, bone marrow, thymus and spleen) cellularity, consistent with the reduced sizes of the animals. This was attributed to the increased spontaneous apoptosis associated with elevated Cdc42/JNK/Bid activities but not to a proliferative defect as revealed by in vivo TUNEL and BrdU incorporation assays. ~80% of Cdc42GAP−/− mice died one week after birth, and the surviving pups attained adulthood but were anemic. Whereas Cdc42GAP−/− mice contained small reduction in the frequency of HSC markers and normal CFU-G, CFU-M, and CFU-GM activities, the frequency of BFU-E and CFU-E were significantly reduced. These results suggest an important role of Cdc42 in erythropoiesis in vivo. Taken together, we propose that Cdc42 is essential for multiple HSC/P functions including survival, actin cytoskeleton regulation, adhesion and migration, and that deregulation of its activity can have a significant impact on erythropoiesis. Cdc42 regulates HSC/P functions and erythropoiesis Genotype/phenotype Apoptosis increase Adhesion decrease Migration decrease F-actin assembly HSC frequency decrease BFU-E, CFU-E decrease The numbers were indicated as fold difference compared with wild type. ND:not determined yet. Cdc42GAP−/− 2.43, p<0.005 0.97, p<0.01 1.01, p<0.01 protrusion (SDF-1); punctruated (SCF) 0.34, p<0.05 0.92, p<0.01; 0.38, p<0 Cdc42−/− 3.68, p<0.005 0.98, p<0.001 3.85, p<0.005 protrusion (SDF-1) ND ND

The Role of Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) in Hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1362-1362
Author(s):  
Yi Shen ◽  
Valerie Barbier ◽  
Ingrid G Winkler ◽  
Jean Hendy ◽  
Jean-Pierre Levesque
Keyword(s):  
Endothelial Cells ◽  
B Cells ◽  
Biologically Active ◽  
Brdu Incorporation ◽  
Tissue Inhibitor ◽  
Over Expression ◽  
Qrt Pcr ◽  
Cell Counts

Abstract Matrix metalloproteinase (MMP) activity is regulated by tissue inhibitor of metalloproteinases (TIMPs). We found that while TIMP-1 and -2 expressions were unaffected, and TIMP-4 was not expressed, TIMP-3 mRNA expression decreased 10-fold within the bone marrow (BM) during G-CSF induced mobilization. In addition, through reverse zymography, the level of biologically active TIMP-3 protein was also shown to decrease during mobilization. Down-regulation of TIMP-3 may contribute to the accumulation of active MMPs within the BM, allowing for the release of hematopoieticstem/progenitor cells (HSPC) from the BM matrix. By qRT-PCR we have shown 10-fold greater TIMP-3 expression in endosteal mRNA compared to central BM mRNA in mouse femur (p=0.008). To assess which bone associated cell populations expressed the majority of TIMP-3, pooled bones were crushed, collagenase treated and FACS sorted. Mesenchymal progenitors (CD45-Lin-Sca1+) expressed the highest level of TIMP-3 followed by endothelial cells (CD45-Lin-CD31+) and mature osteoblasts (CD45-Lin-Sca1-CD51+). Erythroid progenitors (CD45+Ter119+Kit+), megakaryocyte progenitors (CD45+CD41+Kit+) and megakaryocytes (CD45+CD41+Kit−) from BM were also found to express TIMP-3, but at a level at least 10-fold lower than those of non-hematopoietic stromal cells. All other BM hematopoietic cell types tested were negative for TIMP-3 expression. Immunohistofluorescence on bone sections validated TIMP-3 expression in megakaryocytes, endothelial cells and osteoblasts. Expression of TIMP-3 in mouse platelets was confirmed by reverse zymography. To investigate TIMP-3 function we over-expressed huTIMP-3 in mice via retroviral transduction with MND-X-IRES-eGFP (MXIE) retroviral vector. BM cells retrovirally transduced with MXIE-huTIMP-3 or empty MXIE control was transplanted into lethally irradiated congenic mice. Engraftment and transduction levels were determined by GFP expression. At 3-months post-transplant there were no significant differences in body weight, total blood, spleen or BM cell counts between the two groups. qRT-PCR data showed that over-expressing huTIMP-3 did not alter the expression level of endogenous mTIMP-3. Flow cytometry analysis showed that in mice transduced with MXIE-huTIMP-3, the frequency of GFP+ B cells (CD11b-B220+) was reduced by 50% in the blood from 23.88±12.00% to 11.94±7.85% (p=0.0315) and by 64% in the BM from 25.06±13.78% to 9.02±7.67% (p=0.0188) when compared to MXIE controls. Conversely, the frequency of GFP+ huTIMP-3 expressing myeloid cells (CD11b+) was significantly increased in the blood from 55.69±17.13% to77.91±6.31% (p=0.0005), BM from 58.67±16.32% to 77.32±12.02% (p=0.0244) and spleen from 14.07±3.75% to 28.82±6.85% (p=0.0002). Unexpectedly, the frequency of untransduced GFP- myeloid and B cells were similar between the two groups. Although huTIMP-3 over-expression did not significantly alter the number of GFP+ HSPC (Linage-Sca1+Kit+, LSK) per femur (MXIE 0.03±0.03%, MXIE-huTIMP-3 0.01±0.01%, p=0.1139), LSK turnover in huTIMP-3 over-expressing cells was increased in vivo from 4.36±2.83% to 13.31±5.61% (p=0.0159) as determined by BrdU incorporation following 3 days of BrdU administration. Similarly, a trend was also observed in vitro after 12days of culture, LSK sorted from MXIE-huTIMP-3 mice proliferate faster than MXIE controls from 2.55^6cells/ml±1.05 to 9.6^6cells/ml±0.54 (p=0.1). In summary, huTIMP-3 over-expression in mice increased HSPC proliferation in vivo and in vitro. And whilst the huTIMP-3 over-expression in mice was not at a sufficient level to observe a global effect on total BM haematopoiesis, our data suggests that forced huTIMP-3 over-expression in vivo skews differentiation towards myelopoiesis at the detriment of lymphopoiesis.

Using epigenetic reprogramming to target triple-negative breast cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14565-e14565
Author(s):  
D. Sharma ◽  
B. B. Knight ◽  
R. Yacoub ◽  
T. Liu ◽  
L. Taliaferro-Smith ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Hormone Receptors ◽  
Triple Negative ◽  
Combined Treatment ◽  
Brdu Incorporation ◽  
Breast Cancers

e14565 Background: The outcome for patients with breast cancer has been significantly improved by the use of targeted agents. The prognosis of triple negative (TN) breast cancers, which do not express hormone receptors (ER, PR) or Her2, is poor, because of an aggressive clinical course and lack of targeted therapeutic agents. Epigenetic silencing of specific genes has been observed in breast cancer and some of these genes are more important due to available targeted therapies such as ER. Since all endocrine therapies are designed to block ER function in some way, the identification of new therapies or strategies that could sensitize TN breast cancers to existing endocrine therapy could provide a revolutionary means of treating this aggressive subtype of cancer Methods: We examined the efficacy of combined treatment of HDAC inhibitor LBH589 and DNMT inhibitor decitabine to regenerate ER and PR in TN breast cancer cells using RT-PCR and immunoblotting. Changes in growth and proliferation of TN breast cancer cells in response to LBH589 and decitabine treatment were determined by XTT, BrdU incorporation and colony formation assay. Changes in apoptotic proteins were determined by western blotting. Athymic nude mice were used to establish pre-clinical models for TN breast cancer cells and effectiveness of combined treatment of LBH589 and decitabine was determined. Tumors biopsies were analyzed for ER and PR re-expression by western blot analysis and immunohistochemistry at the end of the treatment. Results: Combined treatment of LBH589 and decitabine resulted in re-expression of ER and PR in TN breast cancers in vitro and in vivo. Although re-expression of ER and PR were noted following LBH589 treatment alone, re-expression was more robust with the combination. TN breast cancer cells showing re-expressed ER can be targeted with tamoxifen. Tamoxifen inhibits growth of TN breast cancer cells re- expressing ER by triggering apoptosis. Conclusions: The importance of epigenetic events such as DNA methylation and HDAC inhibition in tumor progression is becoming increasingly evident. A trial evaluating the ability of LBH589 and decitabine to re- express ER, which can then be targeted by tamoxifen, is planned in patients with metastatic TN breast cancer. No significant financial relationships to disclose.

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