Investigation of the Role of Neurokinin-1 Receptor Inhibition Using Aprepitant in the Apoptotic Cell Death through PI3K/Akt/NF-κB Signal Transduction Pathways in Colon Cancer Cells

Background. Colorectal cancer (CRC) is recognized as one of the most common malignancies with a high mortality rate worldwide, supporting the necessity for an effective novel antitumor drug to improve current therapy’s effectiveness. Substance P (SP) is the essential member of the tachykinins (TKs) family, which binds to the specific receptors, known as neurokinin-1 receptor (NK1R), exerting its multiple influences such as tumor cell proliferation, angiogenesis, and metastasis. Aprepitant, as a specific NK1R antagonist, is suggested as a novel antitumor agent, promoting apoptotic processes in tumor cells; however, the exact antitumor mechanism of aprepitant on molecular signaling in CRC is not entirely known. Method. The resazurin assay was conducted to assess the cytotoxic effects of aprepitant on the viability of the CRC cell line (SW480). The level of reactive oxygen species (ROS) was measured after 24-hour treatment with SP and aprepitant. PI/annexin V-FITC staining was conducted to assess apoptosis. Also, the expression of NF-κB antiapoptotic target genes and proapoptotic p53 target genes was measured by real-time- (RT-) PCR assay. Western blotting assay was performed to determine the expression of PI3k/AKT/NF-κB proteins. Results. We found that aprepitant stimulates apoptotic cell death and attenuates the PI3K/Akt pathway and its downstream proapoptotic target gene, including NF-κB in SW480 cells. Also, the obtained results from the quantitative RT-PCR assay showed that aprepitant could decrease the level of mRNA of NF-κB antiapoptotic target genes. Conclusion. Towards this end, this study suggests that SP/NK1R system plays a vital role in the development of CRC, and pharmaceutical targeting of NK1R using aprepitant might be a promising treatment against CRC.

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Aprepitant Promotes Caspase-Dependent Apoptotic Cell Death and G2/M Arrest through PI3K/Akt/NF-κB Axis in Cancer Stem-Like Esophageal Squamous Cell Carcinoma Spheres

BioMed Research International ◽

10.1155/2021/8808214 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Hossein Javid ◽

Amir R. Afshari ◽

Farnaz Zahedi Avval ◽

Jahanbakhsh Asadi ◽

Seyed Isaac Hashemy

Keyword(s):

Cell Death ◽

Apoptotic Cell ◽

Single Agent ◽

Annexin V ◽

Apoptotic Cell Death ◽

Anticancer Activities ◽

M Cell ◽

Apoptotic Pathway ◽

Neurokinin 1

The antagonists of the neurokinin-1 receptor (NK1R) are known for their anti-inflammatory, anxiolytic, antiemetic, and anticancer activities. Aprepitant, a nonpeptide NK1R antagonist, is used in nausea and vomiting, the most common side effects of cancer chemotherapy in patients. It has been established that NK1R activation by substance P (SP), which links cancer promotion and progression to a neurokinin-mediated environment, became one mechanism that corresponds to the mitogenesis of tumor cells. Therefore, this study is aimed at explaining and evaluating the anticancer impacts of aprepitant on esophageal squamous cancer cell (ESCC) spheres by using in vitro experiments, such as resazurin, ROS, annexin-V binding, RT-PCR, and Western blot analysis. As a result, we showed that aprepitant had strong antiproliferative and cytotoxic effects on ESCC cell spheres. Also, aprepitant caused significant G2-M cell cycle arrest depending on concentration increase. Further, exposure of cells to this agent resulted in caspase -8/-9-dependent apoptotic pathway activation by modifying the expression of genes involved in apoptosis. Besides, treatment of the cells by aprepitant abrogates of the PI3K/Akt pathway, as shown by reducing the level of Akt, induces apoptotic cell death. In summary, pharmacological inhibition of NK1R with aprepitant seems to have a significant chance of treating ESCC as a single agent or in conjunction with other chemotherapeutic drugs.

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Biochemical Characterisation of an In Vitro Model of Hepatocellular Apoptotic Cell Death

Alternatives to Laboratory Animals ◽

10.1177/026119290903700210 ◽

2009 ◽

Vol 37 (2) ◽

pp. 209-218 ◽

Cited By ~ 10

Author(s):

Mathieu Vinken ◽

Elke Decrock ◽

Elke De Vuyst ◽

Luc Leybaert ◽

Tamara Vanhaecke ◽

...

Keyword(s):

Cell Death ◽

In Vitro Model ◽

Caspase 3 ◽

Fas Ligand ◽

Apoptotic Cell ◽

Annexin V ◽

Apoptotic Cell Death ◽

Biochemical Characterisation ◽

Vitro Model

This study was set up to critically evaluate a commonly-used in vitro model of hepatocellular apoptotic cell death, in which freshly isolated hepatocytes, cultured in a monolayer configuration, are exposed to a combination of Fas ligand and cycloheximide for six hours. A set of well-acknowledged cell death markers was addressed: a) cell morphology was studied by light microscopy; b) apoptotic and necrotic cell populations were quantified by in situ staining with Annexin-V, Hoechst 33342 and propidium iodide (PI); c) apoptotic and necrotic activities were monitored by probing caspase 3-like activity and measuring the extracellular leakage of lactate dehydrogenase (LDH), respectively; and d) the expression of apoptosis regulators was investigated by immunoblotting. The initiation of apoptosis was evidenced by the activation of caspase 8 and caspase 9, and increased Annexin-V reactivity. Progression through the apoptotic process was confirmed by the activation of caspase 3 and Bid, the enhanced expression of Bax, and the occurrence of nuclear fragmentation. Late transition to a necrotic appearance was demonstrated by an increased number of PI-positive cells and augmented extracellular release of LDH. Thus, the in vitro model allows the study of the entire course of Fas-mediated hepatocellular apoptotic cell death, which is not possible in vivo. This experimental system can serve a broad range of in vitro pharmaco-toxicological purposes, thereby directly assisting in the reduction of animal experimentation.

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Flow Cytometry and FTIR Spectroscopy for Detection of Early Apoptosis in Human T Cells

Proceedings ◽

10.3390/proceedings2251558 ◽

2018 ◽

Vol 2 (25) ◽

pp. 1558 ◽

Cited By ~ 1

Author(s):

Günnur Güler ◽

Ayten Nalbant

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Cell Death ◽

Ftir Spectroscopy ◽

Apoptotic Cell ◽

Annexin V ◽

Apoptotic Cell Death ◽

Biochemical Changes ◽

Human T Cells ◽

Early Apoptosis

Apoptosis, a programmed cell death, has a vital role in various cellular processes. Apoptotic cells exhibit morphological and biochemical changes, detected by a variety of assays (caspases, mitochondrial dyes, DNA laddering). Flow cytometry is a powerful tool for detection of apoptotic cell death and allows information about the cell size and molecules associated with cell-bound antibodies. Recently, Fourier transform infrared (FTIR) spectroscopy as rapid and low-cost tool has been extensively used for cellular studies, providing information on cellular structures. The aim of this study was to detect early apoptosis and obtain further insights into the capability of FTIR spectroscopy, comparing the results with flow cytometry. In this study, apoptotic cell death was induced in human Jurkat T cells with Camptothecin (CPT), a DNA topoisomerase I inhibitor. Cells were cultured with 4µM CPT in RPMI (with 5% FCS) for 24 h. Immunoflourescence labeling for multicolor flow cytometry was accomplished with Annexin V concomitantly with 7-AAD. The same cells were also analyzed with ATR-FTIR spectroscopy. Flow cytometry data represents that the cells are Annexin V positive but 7AAD negative. This indicates that cells are in the early apoptotic stage, only externalization of phosphatidylserine exists on the plasma membrane. FTIR data reveals that membrane phospholipids and proteins undergo changes; fatty acid acyl chains are disordered and increased in mobility after treatment, which result from the early apoptosis process after CPT-treatment, confirmed by the flow cytometry. A combined study of flow cytometry and FTIR spectroscopy for analysis of apoptosis in human T cells exhibited compatible and complementary results. Existence of biophysical and biochemical changes in T cells after treatment were also demonstrated.

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Divergent Roles of Histone Deacetylases in Human Adult Eythropoiesis.

Blood ◽

10.1182/blood.v106.11.130.130 ◽

2005 ◽

Vol 106 (11) ◽

pp. 130-130

Author(s):

Kohshi Ohishi ◽

Naoyuki Katayama ◽

Kentaro Yamamura ◽

Yuji Heike ◽

Yoichi Takaue ◽

...

Keyword(s):

Cell Death ◽

Histone Deacetylases ◽

Colony Formation ◽

Apoptotic Cell ◽

Annexin V ◽

Viable Cell ◽

Apoptotic Cell Death ◽

Erythroid Progenitors ◽

Human Adult ◽

Cd34 Cells

Abstract Histone deacetylases (HDACs) plays a regulatory role in the expression and activity of transcription factors. Several studies suggested that HDACs influence the proliferation and differentiation of hematopoietic progenitors. Here, we studied the role of HDACs in human adult erythropoiesis, using a HDAC inhibitor FK228 [depsipeptide]. We first examined the effect of FK228 on the growth of erythroid progenitors by incubating G-CSF-mobilized peripheral blood CD34+ cells (500 cells/well) in semisolid serum-free cultures containing IL-3, SCF, or IL-3+SCF in the presence or absence of FK228. Addition of FK228 (0.5 ng/ml) to the cultures with IL-3 and SCF+IL-3 resulted in 32-fold and 12-fold increases, respectively, in the number of erythroid colonies that consist of CD36+ glycophorin A (GPA)low/negative cells, compared with the cultures without FK228. FK228 also increased the number of cells comprising erythroid colonies in the cultures with IL-3 and SCF+IL-3. No colony growth was detected with SCF and SCF+FK228. We next examined the effect of FK228 on BFU-E colony formation by incubating CD34+ cells with EPO or SCF+EPO in the presence or absence of FK228. While BFU-E colony formation was observed with SCF+EPO but not with EPO alone, addition of FK228 to the cultures containing SCF+EPO almost completely inhibited BFU-E colony formation. To further elucidate the effect of FK228 on BFU-E progenitors that grow in an EPO-dependent fashion, CD34+ cells were cultured for 7 days with SCF, Flt3L, TPO, and IL-3, and CD36+ cells were purified. Although BFU-E colony formation was induced by incubation of the CD36+ cells with EPO, it was strongly repressed by the presence of FK228. Suspension cultures of CD36+ cells revealed that viable cell numbers in the cultures with EPO+FK228 rapidly declined from 2 days of cultures, in contrast to an increase in the number of erythroid cells in the cultures with EPO. Annexin V/propidium iodide assay showed that, upon exposure to FK228, a remarkably higher fraction of the CD36+ erythroid progenitors expressed an annexin V+PI− phenotype, indicative of an apoptotic cell death, compared with the control cultures (71 ± 8% vs 4 ± 1%). On the other hand, even though FK228 blocked the BFU-E colony formation from CD34+ cells in the presence of SCF+EPO, FK228 did not significantly increase the population of cells that undergo an apoptotic cell death in CD34+ cells. These findings suggest that HDACs, while playing an essential role in SCF/EPO- and EPO-dependent erythropoiesis, negatively regulate the action of IL-3 and IL-3+SCF on the growth of early erythroid progenitors. Our results indicate that HDACs have diverse functions in human adult erythropoiesis.

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Chronic Lymphocytic Leukemia Apoptotic Cell Death Induced by Glucocorticoids Is Mediated by BIM and GILZ and Can Be Predicted by FKBP5 Basal Expression Levels.

Blood ◽

10.1182/blood.v114.22.1236.1236 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1236-1236

Author(s):

Maria Joao Baptista ◽

Ana Muntañola ◽

Carles Codony ◽

Eva Calpe ◽

Eva Fernandez ◽

...

Keyword(s):

Cell Death ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

Lymphocytic Leukemia ◽

Mrna Levels ◽

Rt Pcr ◽

Genome Wide ◽

Apoptotic Effect ◽

Apoptotic Activity ◽

Induced Apoptosis

Abstract Abstract 1236 Poster Board I-258 Glucocorticoids (GC) apoptotic effect in chronic lymphocytic leukemia (CLL) cells is known for many years. However, in CLL their use is often confined to their immunosuppressive activity in order to control autoimmune phenomena or as palliation. The fact that GC, particularly dexamethasone (DEX), can overcome p53 mediated resistance to therapy has renewed the interest in the use of GC as therapeutic agents in CLL. GC apoptotic induced cell death mechanism seems to depend on cell type and few studies were performed in CLL. GC increasing importance as apoptotic agent in CLL prompted us to analyze DEX apoptotic activity in CLL cells in order to clarify GC apoptotic mechanisms. For this, peripheral blood samples from 45 patients with CLL were selected for in vitro studies. Patients were analyzed for IGHV mutational status and ZAP-70 expression. Tumour cells were cultured over 24 hours with DEX at 13.25uM and viability was then determined by surface annexin V binding and propidium iodide (PI) staining flow cytometry analysis. To determine early apoptotic signal onset, BIM mRNA GC induced expression was quantified at different time points by quantitative RT-PCR. Genome-wide expression profile of CLL cells was done to discriminate genes involved in DEX apoptotic action. After 24 hours of exposure to therapeutic concentrations of DEX, cell viability was higher in mutated cases (M-CLL) than in unmutated IGHV cases (UM-CLL) (85.6% vs 69.5% in mean, respectively; p=0.000). mRNA BIM expression after 24 hours of treatment with DEX correlated with induced apoptotic cell death (R=0.496; p=0.000). As a consequence, UM-CLL had higher levels of induced mRNA levels of BIM than M-CLL cases (p=0.036). Time course experiments have shown that at 6h after DEX treatment BIM mRNA levels were induced 3 times without influence on cell viability. Genome-wide expression analysis of 12 CLL cases (7 UM-CLL, 5 M-CLL) was done after 6 hours of DEX treatment and was compared to the baseline gene expression. In both groups many genes were up and down regulated by DEX (UM-CLL 3359 genes and M-CLL 1008 genes, p adjusted < 0.01). Analysis of genes involved in the GC pathway revealed that basal mRNA levels of FKBP5, a protein essential to maintain the GC receptor (GR) complex suitable for GC binding, was more expressed in UM-CLL than in M-CLL cases (1.85 times, p= 0.027). Analysis by quantitative RT-PCR performed in 45 CLL patients to validate micro-array data confirmed that at baseline, FKPB5 expression was higher in UM-CLL than M-CLL (0.97 vs. 0.74 arbitrary units, respectively; p=0.042). The same was observed at protein level, WB analysis of FKBP5 basal levels showed that UM-CLL cases expressed more this protein than M-CLL (Fig. 1). In addition to that, genome-wide analysis revealed that GILZ, a glucocorticoid-induced leucine zipper, was differently induced by DEX in the studied groups. GILZ mRNA was less induced in M-CLL cases than in UM-CLL cases (difference fold change=0.52, p=0.0005). These results were also confirmed in 45 CLL cases by quantitative RT-PCR: in M-CLL, GILZ was induced 3.67 times whilst it was induced 5.62 times in UM-CLL cases (p=0.0001). In conclusion, treatment with GC induces more apoptotic cell death in UM-CLL than in M-CLL. As a downstream effect, BIM expression after GC exposure correlates with GC-induced apoptosis. Moreover, GC apoptotic effect in CLL is the result of several cell pathways imbalance as revealed by gene expression analysis. GILZ induction was proved to be necessary for DEX induced apoptosis in other cell types like multiple myeloma cell lines. In CLL, GILZ differential induction was observed at different degrees in GC-responders and non-responders. Finally, FKBP5 expression, upstream effecter of the GC pathway, correlated with cellular effect of GC and can be used to predict GC apoptotic activity in CLL cases. Figure 1 WB analysis of FKBP5 expression in CLL cells. Figure 1. WB analysis of FKBP5 expression in CLL cells. Disclosures No relevant conflicts of interest to declare.

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Use of Flow Cytometry for Detection of Apoptotic Cell Death in Th17 Cells

Proceedings ◽

10.3390/proceedings2251560 ◽

2018 ◽

Vol 2 (25) ◽

pp. 1560

Author(s):

Ayten Nalbant

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Plasma Membrane ◽

Dna Fragmentation ◽

Peripheral Blood ◽

Th17 Cells ◽

Apoptotic Cell ◽

Annexin V ◽

Apoptotic Cell Death ◽

Membrane Changes

Flow cytometry (FC) is a powerful and reliable system for cell death studies. It allows to study the molecular changes in both the surface and cytoplasmic part of the cells. Depending on the laser range of flow cytometry, it is possible to collect a large number of information about a single cell by combining multiple labeling strategies. With these assets, flow cytometry allows scientists to accelerate their research. Flow cytometry is also widely used in clinical studies. Therefore, we used this powerful tool to study human T helper 17 (Th17) cell apoptosis. Newly discovered Th17 cells are important players of immune response regulation. They are also involved in different types of pathologies including autoimmune diseases and cancer. There are intensive data in the literature about molecules that are involved in Th17 differentiation signaling networks, but the apoptotic and survival molecular mechanisms of this cells are not fully understood yet. Therefore, apoptosis of Th17 cells were measured by Flow cytometry. Healthy human subjects have been invited to study with the informed consent which is approved by the ethics committee. Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from peripheral blood of healthy volunteers. Phenotypically characterized and sorted naïve CD4+ T cells from PBMC were cultured under Th17 polarizing conditions. IL-17, IL-22 or CCR6 molecules were used to monitor Th17 cells. Apoptotic cell death of Th17 cells were measured by plasma membrane changes and DNA fragmentation. During differentiation stages, plasma membrane changes were monitored by Annexin V concomitantly with 7AAD. At day 7, there were Annexin V positive cells in Th17 cells. Apoptotic cell death occurs through sequential events including caspase activation and DNA fragmentation. DNA fragmentation data showed that Th17 cells were not apoptotic compared to negative control cultures. This finding suggested that survival molecules of Th17 cells interferes with this apoptotic process.

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