Insulin and IGF-1 Mediated Inhibition of Apoptosis in CHO Cells Grown in Suspension in a Protein-free Medium

2007 ◽  
Vol 35 (3) ◽  
pp. 349-352 ◽  
Author(s):  
Lars Adamson ◽  
Erik Walum
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cell Viability ◽  
Cho Cells ◽  
Chinese Hamster ◽  
B Cell Lymphoma ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
P53 Expression ◽  
Product Level

When Chinese hamster ovary (CHO) cells were grown in suspension and deprived of serum, 40% of them became apoptotic after 72 hours, as determined by flow cytometry analysis of TUNEL-labelled cells. Cell viability, assessed by erythrocin B staining, decreased correspondingly. An increase in the total fraction of cells expressing interleukin converting enzyme (ICE; caspase 1), B-cell lymphoma 2 protein (Bcl-2,) and Bcl-2 associated x protein (Bax) was shown by antibody probing and subsequent flow cytometry. The p53 tumour suppressor gene product level remained low within the cell population. Insulin-like growth factor-1 (IGF-1) inhibited cell death in a concentration-dependent manner, and at 20ng/ml, cell viability was maintained close to 100% and no apoptotic cells were detected. Also, insulin was shown to inhibit cell death — at 1.0μg/ml, cell viability was 95%, whereas 10% of the cells stained for apoptosis. At the highest concentrations of IGF-1 and insulin, the expression of ICE, Bcl-2 and Bax was fully suppressed, whereas the p53 product level increased, despite still being detectable in a minority of cells. Under these conditions, IGF-1 may increase p53 expression to restrain abnormal cell proliferation. It is concluded that special attention should be paid to exposure and culture conditions that induce acquired susceptibility to a toxic insult, during the development and validation of cell-based assays.

Combination Of Inhibitors Against DNA Methyltransferases and Histone Deacetylases Synergistically Suppresses Cell Viability In Acute Myeloid Cells In a p53-Dependent Manner

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4894-4894 ◽  
Author(s):  
Anilkumar Gopalakrishnapillai ◽  
Sonali P Barwe ◽  
E. Anders Kolb
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Dna Methyltransferase ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
Hdac Inhibitors ◽  
Dna Methyltransferases ◽  
Dependent Manner ◽  
P53 Expression ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) accounts for 18% of childhood leukemia diagnoses. Although, the 5-year survival rate for children with AML is estimated at 64%, novel therapeutic options are required to improve the outcome for certain subsets of AML that are refractory. The contribution of epigenetic modifiers to the pathogenesis of AML in children is becoming evident following the discovery of mutations in patients, which affect DNA and/or histone methylation. Although DNA methyltransferase (DNMT) inhibitors have been approved in AML, their efficacy as single agents has been limited. This could be due to the crosstalk between DNA methylation and histone modifications that together regulate gene expression. This raises the possibility that optimal re-expression of silenced tumor suppressor genes in AML requires treatment with both DNMT and histone deacetylase (HDAC) inhibitors. We evaluated the effect of a combination of DNA methyltransferase inhibitor azacytidine with HDAC inhibitors panobinostat or romidepsin on the viability of three pediatric AML cell lines- MV4;11 (expressing wild-type p53), THP-1 (with no detectable p53 mRNA) and AML-193 (with mutated p53). We observed that azacytidine as a single-agent only moderately reduced cell viability of MV4;11 cells (IC50 – 7 uM), and it failed to induce cell death in THP-1 and AML-193 cells. However, azacytidine in combination with panobinostat and romidepsin was more effective at reducing cell viability in MV4;11 and THP-1 cells (with a combination index of 0.5 to 0.6). AML-193 cells were almost insensitive to cell death induced by the combination treatment. This suggests that p53 is required for induction of cell death by azacytidine and panobinostat/romidepsin combination. Although THP-1 cells have no detectable p53 expression, other studies have reported the upregulation of p53 following stimulation with nitric oxide or 7-oxysterols. The effect on cell viability in THP-1 cells is likely due to the reexpression of silenced p53 in these cells. Taken together, our data suggests that combining azacytidine with panobinostat/romidepsin induces synergistic cell death in a p53-dependent manner. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Quaternary structures of opsin in live cells revealed by FRET spectrometry

2016 ◽  
Vol 473 (21) ◽  
pp. 3819-3836 ◽  
Author(s):  
Ashish K. Mishra ◽  
Megan Gragg ◽  
Michael R. Stoneman ◽  
Gabriel Biener ◽  
Julie A. Oliver ◽  
...  
Keyword(s):  
Cho Cells ◽  
Quaternary Structure ◽  
Chinese Hamster ◽  
Resonance Energy ◽  
Photoreceptor Cells ◽  
Live Cells ◽  
Dependent Manner ◽  
Rod Photoreceptor ◽  
Concentration Dependent Manner ◽  
Oligomeric Forms

Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that initiates phototransduction in the retina. The receptor consists of the apoprotein opsin covalently linked to the inverse agonist 11-cis retinal. Rhodopsin and opsin have been shown to form oligomers within the outer segment disc membranes of rod photoreceptor cells. However, the physiological relevance of the observed oligomers has been questioned since observations were made on samples prepared from the retina at low temperatures. To investigate the oligomeric status of opsin in live cells at body temperatures, we utilized a novel approach called Förster resonance energy transfer spectrometry, which previously has allowed the determination of the stoichiometry and geometry (i.e. quaternary structure) of various GPCRs. In the current study, we have extended the method to additionally determine whether or not a mixture of oligomeric forms of opsin exists and in what proportion. The application of this improved method revealed that opsin expressed in live Chinese hamster ovary (CHO) cells at 37°C exists as oligomers of various sizes. At lower concentrations, opsin existed in an equilibrium of dimers and tetramers. The tetramers were in the shape of a near-rhombus. At higher concentrations of the receptor, higher-order oligomers began to form. Thus, a mixture of different oligomeric forms of opsin is present in the membrane of live CHO cells and oligomerization occurs in a concentration-dependent manner. The general principles underlying the concentration-dependent oligomerization of opsin may be universal and apply to other GPCRs as well.

A microplate assay for measuring cell death in C2C12 cells

2018 ◽  
Vol 96 (5) ◽  
pp. 702-706 ◽  
Author(s):  
Tanes I. Lima ◽  
Leonardo R. Silveira
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Fluorescence Analysis ◽  
High Sensitivity ◽  
Accurate Method ◽  
C2c12 Cells ◽  
Dependent Manner ◽  
Microplate Assay ◽  
Concentration Dependent Manner

The main goal of this study was to develop a straightforward and rapid microplate assay for measuring propidium iodide (PI) in C2C12 cells. The PI method has proven to be an efficient quantitative assay for analyzing cell viability through PI fluorescence analysis. Importantly, the protocol takes less than 30 min and the results are reproducible. C2C12 cells were exposed to an increasing concentration of palmitate for a period of 24 h to induce cell death, and the PI fluorescence increased in a concentration-dependent manner. Evaluation of mitochondrial function and the production of reactive oxygen species confirmed the deleterious effects of palmitate. Also, the microplate PI assay demonstrated high sensitivity, as indicated by the detection of modest fluctuations in cell viability in response to catalase overexpression in palmitate-treated cells. The microplate PI assay, therefore, offers an accurate method for use in in-vitro studies.

scholarly journals Overexpression of Myo1e promotes albumin endocytosis by mouse glomerular podocytes mediated by Dynamin

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8599
Author(s):  
Huijun Shen ◽  
Yu Bao ◽  
Chunyue Feng ◽  
Haidong Fu ◽  
Jianhua Mao
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Fluorescence Intensity ◽  
Annexin V ◽  
Underlying Mechanism ◽  
Immunofluorescence Assay ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Foot Process ◽  
Albumin Endocytosis

Background As a fundamental process internalizing molecules from the plasma membrane, endocytosis plays a crucial role in podocyte biology. Our previous study has identified that overexpression of Myole may enhance podocyte endocytosis. However, its potential mechanism has been not well understand. Thus, we aimed to analyze whether albumin endocytosis by mouse glomerular podocytes is dependent on Myo1e expression. Also, we aimed to elucidate whether the underlying mechanism is mediated by Dynamin. Methods Firstly, mouse podocyte cells (MPC5) were treated with different concentrations of FITC-bovine serum albumin (BSA). The fluorescence intensity and cell viability were detected by flow cytometry and MTT assays, respectively. Afterwards, the optimal concentration of FITC-BSA was determined. Secondly, MPC5 cells were treated with Myole overexpression or knockdown. Cell morphology was observed under microscope. Immunofluorescence assay was used to determine the expression of F-actin. The protein expression of nephrin and podocin was detected by western blot. Flow cytometry was used to detect MPC5 cell apoptosis with annexin V. Finally, MPC5 cells were treated with Myole overexpression and/or Dynasore (a GTPase inhibitor of Dynamin). The fluorescence intensity was detected using flow cytometry assay. Results MPC5 endocytosis BSA was elevated with a concentration-dependent manner. MTT results showed that MPC5 cell viability was inhibited with a concentration-dependent manner. Myo1e overexpression promoted podocyte endocytic FITC-BSA, which was contrary to its knockdown. Under microscope, after inhibition of Myo1e, podocyte foot process fusion was observed. Myo1e overexpression promoted the expression of cytoskeleton F-actin and podocyte-specific molecules (nephrin and podocin) in podocyte endocytic FITC-BSA. Furthermore, we found that Myo1e promoted the apoptosis of podocytes. Dynasore attenuated the increase in endocytosis of FITC-BSA induced by Myo1e overexpression, suggesting that podocytes might mediate albumin endocytosis via Myo1e-Dynamin-Albumin. Conclusion Our findings revealed that overexpression of Myo1e promotes albumin endocytosis in mouse glomerular podocyte endocytic albumin mediated by Dynamin.

Z-Guggulsterone Downregulates Survivin and Induces Cell Death in Large B Cell Lymphoma Cells In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4752-4752
Author(s):  
Maria K. Angelopoulou ◽  
Konstantinos Lilakos ◽  
Vassilios Salpeas ◽  
Sotirios Sachanas ◽  
Penelope Korkolopoulou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cell Viability ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Survivin Expression ◽  
B Cell Lymphoma ◽  
Lymphoma Cells

Abstract Introduction: Survivin is a member of the Inhibitor of Apoptosis Proteins and has recently gained attention as a possible therapeutic target in malignancies, due to its dual role both as an antiapoptotic protein and as a cell cycle regulator. It is overexpressed in malignant cells and confers resistance to chemotherapy and other stimuli triggering apoptosis. Z-Guggulsterone (Z-GGS) is a plant sterol, which has been used in inflammatory conditions and has been recognized as a potent NF-kB suppressor. Since Survivin, as well as other antiapoptotic proteins, are under NFkB regulation, we studied the effect of Z-GGS on two B-cell lymphoma cell lines. Methods: DB and HT cell lines were treated with increasing concentrations (10μM, 20μM and 30μM) of Z-GGS, for 24, 48 and 72 hours. Survivin expression was tested with Flow Cytometry and Survivin transcripts were measured with quantitative real time PCR using the Universal Probe Library hydrolysis probes and expressed as Survivin/abl ratio. Cell viability was assessed with the MTT assay. Results: Both cell lines were positive for Survivin at baseline by flow cytometry (66% of total cells for DB and 95% for HT). Treatment of DB cells with 10, 20 and 30μM Z-GGS resulted in a 44%, 49% and 68% reduction of Survivin expression at 24 hours, respectively, whereas the effect on HT was less prominent with a 10% reduction at 24 hours with 30μM Z-GGS. Survivin transcripts decreased as well, with the maximum effect observed at 72 hours with 30μM Z-GGS for both cell lines: Survivin/abl was 0.009 for untreated cells vs 0.0008 with 30μM Z-GGS for DB cells and 0.0135 vs 0.0005 for HT cells. Linearity was observed for increasing concentrations of Z-GGS at 72 hours. Cell viability was practically unaffected at any time point with 10 and 20μM Z-GGS for both cell lines, whereas 30 μM Z-GGS resulted in a 63% and 78% cell death at 48 and 72 hours respectively for DB cells and 67% and 83% for HT cells. Conclusions: The steroid Z-GGS downregulates Survivin expression in B-lymphoma cells in vitro and induces cell death at 30μM concentration. Further experiments will clarify its possible role in the treatment of B-cell malignancies.

scholarly journals Human Neuronal Cell Lines as An In Vitro Toxicological Tool for the Evaluation of Novel Psychoactive Substances

2021 ◽  
Vol 22 (13) ◽  
pp. 6785
Author(s):  
Valeria Sogos ◽  
Paola Caria ◽  
Clara Porcedda ◽  
Rafaela Mostallino ◽  
Franca Piras ◽  
...  
Keyword(s):  
Cell Death ◽  
Neuronal Cell ◽  
In Vitro Study ◽  
Dependent Manner ◽  
Novel Psychoactive Substances ◽  
Psychoactive Substances ◽  
Concentration Dependent Manner ◽  
Mechanisms Of Toxicity ◽  
Neuronal Cell Lines

Novel psychoactive substances (NPS) are synthetic substances belonging to diverse groups, designed to mimic the effects of scheduled drugs, resulting in altered toxicity and potency. Up to now, information available on the pharmacology and toxicology of these new substances is very limited, posing a considerable challenge for prevention and treatment. The present in vitro study investigated the possible mechanisms of toxicity of two emerging NPS (i) 4′-methyl-alpha-pyrrolidinoexanophenone (3,4-MDPHP), a synthetic cathinone, and (ii) 2-chloro-4,5-methylenedioxymethamphetamine (2-Cl-4,5-MDMA), a phenethylamine. In addition, to apply our model to the class of synthetic opioids, we evaluated the toxicity of fentanyl, as a reference compound for this group of frequently abused substances. To this aim, the in vitro toxic effects of these three compounds were evaluated in dopaminergic-differentiated SH-SY5Y cells. Following 24 h of exposure, all compounds induced a loss of viability, and oxidative stress in a concentration-dependent manner. 2-Cl-4,5-MDMA activates apoptotic processes, while 3,4-MDPHP elicits cell death by necrosis. Fentanyl triggers cell death through both mechanisms. Increased expression levels of pro-apoptotic Bax and caspase 3 activity were observed following 2-Cl-4,5-MDMA and fentanyl, but not 3,4-MDPHP exposure, confirming the different modes of cell death.

scholarly journals Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

2020 ◽  
Vol 22 (1) ◽  
pp. 202
Author(s):  
Josephin Glück ◽  
Julia Waizenegger ◽  
Albert Braeuning ◽  
Stefanie Hessel-Pras
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Pyrrolizidine Alkaloids ◽  
Defense Mechanism ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Dependent Manner ◽  
Human Hepatoma Cell

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure–activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.

scholarly journals Cytotoxic Effects of Flubendazole in Human Lung Cancer Cell Line A549

2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Elham Hoveizi ◽  
Fatemeh Fakharzadeh Jahromi
Keyword(s):  
Lung Cancer ◽  
Cell Viability ◽  
Human Lung ◽  
A549 Cells ◽  
Beclin 1 ◽  
Human Lung Cancer ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Acridine Orange Staining

Background: The development of effective anticancer drugs is a significant health issue. Previous studies showed that members of the benzimidazole family have anticancer effects on several cancers Objectives: The present study investigated the cytotoxic effect of flubendazole on A549 human lung cancer cells. Methods: The A549 cells were treated with flubendazole at 1, 2, 5, and 10 µM concentrations for three days. Cell viability was measured by the MTT assay and Acridine orange staining. Also, the expressions of P62 and Beclin -1 were analyzed by qRT-PCR analysis. Results: Cell viability of A549 cells, in a concentration-dependent manner, showed significant differences between the treatment and control groups, and the IC50 value was determined to be 2 µM. Also, flubendazole reduced the expression of P62 and increased the expression of Beclin 1 in treated cells. Conclusions: Flubendazole induces cell death in A549 cells in a dose and time-dependent manner and can offer new factors in lung cancer therapeutic strategies.

scholarly journals Anticancer effects of mifepristone on human uveal melanoma cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Prisca Bustamante Alvarez ◽  
Alexander Laskaris ◽  
Alicia A. Goyeneche ◽  
Yunxi Chen ◽  
Carlos M. Telleria ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Progesterone Receptor ◽  
Dna Fragmentation ◽  
Cell Lines ◽  
Caspase 3 ◽  
Annexin V ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Free Dna

Abstract Background Uveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds. Methods The effects of incremental concentrations of MF (0, 5, 10, 20, or 40 μM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR. Results MF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor. Conclusion This study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

Assessment of the immunomodulatory effect of Aloe vera polysaccharides extracts on macrophages functions

2020 ◽  
pp. 408
Author(s):  
Berenice Aranda-Cuevas ◽  
Jorge Tamayo- Cortez ◽  
Lourdes Vargas y Vargas ◽  
Ignacio Islas- Flores ◽  
Víctor Arana- Argáez ◽  
...  
Keyword(s):  
Cell Viability ◽  
Phagocytic Activity ◽  
Rainy Season ◽  
Aloe Vera ◽  
Peritoneal Macrophages ◽  
Dry Season ◽  
Immunomodulatory Effect ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Proliferative Effect

The present study evaluates the immunomodulatory effect of high molecular weight fractions of Aloe vera polysaccharides harvested during the dry season (March-April) and the rainy season (August-September). Peritoneal macrophages (MΦs) secluded from Balb/c mice underwent treatment with A. vera leaves extract and acemannan standard (the major component found in A. vera) and stimulated with lipopolysaccharides (LPS). Macrophage cell viability was assessed by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide method. Phagocytic activity was also evaluated in peritoneal macrophages, such as the production of nitric oxide and interleukin 6 (IL-6). In the results, found that the A. vera polysaccharides harvested during the rainy season stimulated the phagocytic activity with greater intensity than dry season and improvement NO and IL-6 production. No cytotoxic effect was found on cell viability and they cause a significant proliferative effect on macrophages in a concentration-dependent manner. It can be concluded that the A. vera polysaccharides harvested during the rainy season possessed a stronger immunostimulatory effect compared to the extracts from leaves obtained during dry seasons in a concentration-dependent manner without aff at the cell viability of macrophages.

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