Apoptotic Effects on HL60 Human Leukaemia Cells Induced by Lavandin Essential Oil Treatment

Recent scientific investigations have reported a number of essential oils to interfere with intracellular signalling pathways and to induce apoptosis in different cancer cell types. In this paper, Lavandin Essential Oil (LEO), a natural sterile hybrid obtained by cross-breeding L. angustifolia × L. latifolia, was tested on human leukaemia cells (HL60). Based on the MTT results, the reduced cell viability of HL60 cells was further investigated to determine whether cell death was related to the apoptotic process. HL60 cells treated for 24 h with LEO were processed by flow cytometry, and the presence of Annexin V was measured. The activation of caspases-3 was evaluated by western blot and immunofluorescence techniques. Treated cells were also examined by scanning and transmission electron microscopy to establish the possible occurrence of morphological alterations during the apoptotic process. LEO main compounds, such as linalool, linalyl acetate, 1,8-cineole, and terpinen-4-ol, were also investigated by MTT and flow cytometry analysis. The set of obtained results showed that LEO treatments induced apoptosis in a dose-dependent, but not time-dependent, manner on HL60 cells, while among LEO main compounds, both terpinen-4-ol and linalyl acetate were able to induce apoptosis.

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Role of integrins in melanocyte attachment and dendricity

Journal of Cell Science ◽

10.1242/jcs.107.10.2739 ◽

1994 ◽

Vol 107 (10) ◽

pp. 2739-2748 ◽

Cited By ~ 1

Author(s):

M. Hara ◽

M. Yaar ◽

A. Tang ◽

M.S. Eller ◽

W. Reenstra ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Biology ◽

Type Iv Collagen ◽

Cell Attachment ◽

Pigment Cell ◽

Cell Types ◽

Dependent Manner ◽

Flow Cytometry Analysis ◽

Type Iv ◽

Alpha V Beta 3

Integrins are a family of proteins known to mediate attachment of cells to extracellular matrix materials. The substratum specificity and cation dependence of specific integrin heterodimers have been extensively characterized, and to a lesser degree specialized roles in cell attachment versus dendricity have been defined in some cell types. In the past decade, melanocyte attachment rate and morphology have been found to have strong substratum dependence, suggesting a major role for integrins in these processes. In order to investigate this aspect of pigment cell biology, human newborn melanocytes were subjected to flow cytometry analysis and plated on a variety of substrata under conditions known to promote or block the binding of specific integrin pairs. Melanocyte attachment to laminin and type IV collagen was promoted by Mg2+ and Mn2+ but not by Ca2+, in the range of concentrations examined. However, dendrite outgrowth from melanocytes already attached on laminin or type IV collagen was promoted by Ca2+ to a far greater degree than by Mg2+, and Mn2+ had no effect on dendrite outgrowth. Flow cytometry analysis revealed that melanocytes expressed beta 1, alpha 2, alpha 3, alpha 5, alpha 6 and alpha v integrin subunits as well as the alpha v beta 3 heterodimer. The influence of substratum on the profile of integrin expression was minimal, but alpha 6 and beta 1 integrins were observed by confocal microscopy to be expressed over the entire cell surface, while alpha 2, alpha 5 and alpha v beta 3 integrins localized along dendritic processes or at their tips. In accordance with the implications of these distribution patterns, anti-beta 1 and anti-alpha 6 integrin monoclonal antibodies blocked melanocyte attachment to laminin, while anti-alpha 2, anti-alpha 5 and anti-alpha v beta 3 inhibited dendrite outgrowth but did not block substratum attachment on either laminin or type IV collagen. On the basis of these data and the known characteristics of integrin molecules, we conclude that melanocyte attachment to laminin is mediated primarily by alpha 6 beta 1 integrin in a Ca(2+)-independent, Mg(2+)- and/or Mn(2+)-dependent manner, while dendrite outgrowth on laminin and type IV collagen requires extracellular Ca2+ and is mediated by alpha v beta 3 as well as alpha 2 and alpha 5 integrins.

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Chemical Composition, and Cytotoxic, Antioxidant and Antibacterial Activities of the Essential Oil from Ginseng Leaves

Natural Product Communications ◽

10.1177/1934578x1400900637 ◽

2014 ◽

Vol 9 (6) ◽

pp. 1934578X1400900 ◽

Cited By ~ 4

Author(s):

Rui Jiang ◽

Liwei Sun ◽

Yanbing Wang ◽

Jianzeng Liu ◽

Xiaodan Liu ◽

...

Keyword(s):

Essential Oil ◽

Biological Activities ◽

Radical Scavenging ◽

Complex Mixture ◽

Annexin V ◽

Antibacterial Activities ◽

Cytotoxic Activities ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Gram Positive Bacteria

Panax ginseng C.A.Meyer is one of the most valuable traditional Chinese medicines. In this study, the essential oil of ginseng leaves (EOGL), collected using hydrodistillation and analyzed by GC/MS, contained a complex mixture of aliphatic (69.0%), terpenoid (21.5%) and aromatic compounds (2.4%). Among 54 components identified, the major ones were palmitic acid (36.1%), β-farnesene (15.4%), linoleic acid (9.8%) and phytol (5.6%). In the cytotoxicity study, EOGL exhibited obvious cytotoxic activities against different cancer cell lines, including Hela, A549, ZR-75-1, HT-29, SGC7901 and B16 cells. Furthermore, Annexin V-FITC/PI staining assay indicated that EOGL can induce late apoptosis of ZR-75-1 cells, and the percentage of apoptotic cells increased in a concentration-dependent manner (0.9% to 5.6% and 67.4%). In addition to this, we also found that EOGL exhibited weak DPPH radical scavenging (12.0 ± 0.4 mg/mL) and ABTS radical scavenging activities (1.6 ± 0.1 mg/mL), and showed antibacterial activity against the Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis, and the Gram-negative bacterium, Escherichia coli. The data suggest that EOGL, which possesses important biological activities, especially significant anticancer activity, could be a potential medicinal resource.

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Cocaine’s Direct Effect on Either Sickle or Normal Erythrocytes Does Not Increase Phosphatidylserine Exposure, Calcium Pumping, or Scramblase Activity.

Blood ◽

10.1182/blood.v104.11.3692.3692 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3692-3692

Author(s):

Kitty DeJong ◽

Robert Hagar

Keyword(s):

Flow Cytometry ◽

Pulmonary Hypertension ◽

Calcium Influx ◽

Plasma Concentrations ◽

Calcium Ionophore ◽

Annexin V ◽

Cell Types ◽

Red Cell ◽

Sickle Cells ◽

Red Cell Membranes

Hypothesis: Cocaine directly affects the exposure of phosphatidylserine, calcium pumping, and scramblase activity in sickle cells. Background: Cocaine is highly associated with pulmonary hypertension in both normal and sickle cell disease patients. Given the growing interest in pulmonary hypertension as a major morbidity factor, the causes and determinants of pulmonary hypertension need to be elucidated to allow rational study designs. Phosphatidylserine (PS) exposure is known to occur in sickle cells and appears to be relevant for vascular damage. Although the mechanism underlying PS exposure is poorly understood, signal transduction processes appear to play a role. We wondered if cocaine could activate these processes and cause PS exposure, or interfere with the proteins involved in the transbilayer movement of PS. Methods: Sickle and normal erythrocytes were exposed to cocaine HCl (from a 10% topical solution, Roxane Labs) at concentrations between 100 and 1000 ng/ml, which is the range of determined plasma concentrations after cocaine use. PS exposure after cocaine treatment was measured by labeling with fluorescently conjugated annexin V (AV) and analysis by flow cytometry. Alterations in scramblase activity were determined by assessing the percentage of PS-exposing cells following loading of the cells with 0.1 mM calcium using calcium ionophore. Calcium influx and Ca-ATPase-mediated calcium efflux were monitored using the fluorescent probe Fluo4, and flippase activity was assessed using NBD-PS followed by analysis with flow cytometry. Results: Sickle cells differ from normal cells with respect to most of the measured parameters, such as having more PS-exposing cells, lower flippase activity, and alterations in calcium kinetics. However, cocaine did not have any effect on PS exposure, calcium-induced scrambling, calcium influx, calcium pump activity, or flippase activity. The cell scatter patterns did not show any gross changes in red cell shape or density after cocaine treatment. Discussion: The lack of effect implies that cocaine is working through other mechanisms than effects on red cell membranes. Therefore, future studies should focus on other vascular cell types and membrane pathways. Alternatively, cocaine metabolites should be evaluated for their activities on red cells.

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Benzo(a)pyrene, an Active Product of Cigarette Smoke, Role in PLA2 Isoforms Activation in Colon Cancer

Journal of Global Oncology ◽

10.1200/jgo.18.79801 ◽

2018 ◽

Vol 4 (Supplement 2) ◽

pp. 193s-193s

Author(s):

S. Sharma ◽

S. Yadav ◽

S. Rana ◽

P. Avti ◽

K.L. Khanduja

Keyword(s):

Colon Cancer ◽

Cell Viability ◽

Cigarette Smoke ◽

Membrane Integrity ◽

Annexin V ◽

Cell Types ◽

Ros Generation ◽

Dependent Manner ◽

Colon Cells ◽

Ht 29

Background and aim: One of the active combustion product of cigarette smoke, Benzo[a]pyrenes, role in pulmonary cancer is clearly understood. However, its role in gastrointestinal cancer including colon cancer is not clearly understood. Methods: In this study, benzo(a)pyrenes was treated to colon cells to evaluate its role in cell viability, cellular ROS, and gene expression of various PLA2 isoforms was evaluated by FACS and PCR. The identified PLA2 was silenced at the gene level to evaluate its role in cell viability and ROS generation. Results: B(a)P treatment at 1 µg/mL for 48 h to HCT-15 male colon cells significantly reduced the cell viability without affecting HT-29 female colon cells. Higher doses and longer treatment duration with B(a)P showed that female colon cells were highly sensitive than male colon cells. Annexin-V/PI staining for preapoptotic detection showed that B(a)P treatment increased the apoptosis in both the cell types in a concentration and time-dependent manner. The cytosolic ROS (cROS) and superoxide radical (SOR) formation in the female colon cells was significantly higher than male colon cells unlike the mitochondrial ROS (mtROS) production which was significantly higher in male colon cells. Treatment with B(a)P significantly upregulated the IID and IVA PLA2 isoform groups in HCT-15 male colon cells, whereas IB was upregulated in HT-29 female colon cells among the various PLA2 isozyme gene studied (IB, IID, III, IVA, IVB, IVC, VI, X, aiPLA2 and iPLA2). Gene silencing experiments targeting PLA2 IID and IVA in the HCT-15 male colon cells and IB in HT-29 female colon cells showed no effect with B(a)P treatment on the cell proliferation, apoptosis, membrane integrity and free radicals (ROS, mtROS, and SOR) generation. Conclusion: Targeting specific PLA2 isozymes in a cell-specific manner abolished the B(a)P-induced PLA2-mediated oxidative damage–related signaling pathways.

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MDM2 Inhibitor Nutlin-3a Triggers Autophagic Cell Death In Addition to Apoptosis In Leukemia Cell Lines with Wild-Type p53

Blood ◽

10.1182/blood.v116.21.3300.3300 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3300-3300

Author(s):

Seshagiri Duvvuri ◽

Vivian Ruvolo ◽

Duncan H. Mak ◽

Kensuke Kojima ◽

Marina Konopleva ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Western Blot ◽

Cell Lines ◽

Research Funding ◽

Leukemia Cell ◽

Annexin V ◽

Dependent Manner ◽

Autophagic Vacuoles ◽

Leukemia Cell Lines

Abstract Abstract 3300 Background: Nutlin-3a is a small molecule inhibitor of MDM2 and has been shown to induce apoptosis and cell cycle arrest in various cancer models in a p53 dependent manner. Autophagy is a programmed cell death that can occur concurrently with apoptosis or in its absence. There is significant debate whether autophagy is a protective mechanism or a bona fide mechanism of cell death. While autophagy can function as tumor cell defense mechanism against cellular stress induced death, mutation/loss of alleles of certain genes regulating autophagy have been associated with development of cancer (e.g. Beclin-1 in breast cancer [Nature, 1999, 402: 672–676]). Multiple proteins involved in autophagy are transcriptional targets of p53 but Nutlin-3a has not been evaluated for its role in inducing autophagy. Here we present data suggesting that low dose Nutlin-3a induces autophagy in addition to apoptosis in leukemia cell lines in a p53 dependent manner. Methods and results: OCI-AML-3 cells (p53-WT) treated with Nutlin-3a (2.5 and 5.0μM for 48, 72 and 96 hrs) were stained with mono-dansyl-cadaverine (MDC), a dye that accumulates in acidic autophagic vacuoles. OCI-AML-3 cells showed increasing staining with MDC in a dose and time dependent fashion by both flow cytometry (54%, 57% and 51% MDC positive after treatment with Nutlin-3a 5.0μM for 48, 72 and 96 hrs) and by confocal microscopy. Nutlin-3a treated cells also were positive for Annexin-V (flow cytometry 22%, 26% and 36% at 48, 72 and 96 hrs time points), and some of the cells were double-positive for Annexin-V and MDC (9.2%, 5% and 7% at 48, 72 and 96 hrs) suggesting that both apoptosis and autophagy can occur simultaneously. Autophagy induction was confirmed by Transmission Electron Microscopy (TEM). Large, multiple autophagic vacuoles were observed in OCI-AML-3 cells treated with Nutlin-3a. OCI-AML-3 cells with stable p53 knockdown by shRNA or HL-60 cells (p53-null) did not show increased MDC staining by flow cytometry (both cell lines) or autophagic vacuoles by TEM (HL-60) after similar treatment. Western blot analysis showed increases in LC3-II and in conjugation of Atg5/12, early and late autophagy markers respectively, in OCI-AML-3 cells after treatment with Nutlin-3a. Increased expression of the autophagy markers (LC3-II and Atg 5/12 conjugate) were also seen by Western blot analysis in the ALL cell lines REH and NALM-6 (both p53-WT) after treatment with Nutlin-3a. Western blot and/or RT-PCR analysis showed upregulation of other p53 related proteins involved in autophagy e.g. DRAM, AMPK-β, LKB1, pLKB1 in OCI-AML-3 cells treated with Nutlin-3a. As mTOR/Akt pathway inhibits autophagy, analysis of mTOR targets showed downregulation of the total and phospho-ribosomal-S6-protein levels, whereas there was no change in total or phospho-4-EBP-1 levels. Knockdown of Beclin-1 (ATG6), one of the proteins required for initiation of the formation of autophagic vacuoles, caused reduction in autophagic vacuoles (MDC staining by confocal microscopy) in OCI-AML-3 and REH cells without affecting apoptosis induction (Annexin V by flow cytometry). Pharmacologic inhibition of late autophagy by Bafilomycin (10nM for 2 hours) reduced MDC staining in OCI-AML-3 cells treated with Nutlin-3a for 48 hrs (32% without and 9% with Bafilomycin) while having limited inhibition of apoptosis (Annexin V positive 42% without and 33% with Bafilomycin). Conclusion: Nutlin-3a induces autophagy in leukemia cells by a p53 dependent manner. We also demonstrate that autophagy could go hand-in-hand with apoptosis and in a fraction of cells both processes may occur concomitantly. Inhibition of autophagy does not necessarily enhance apoptosis. Disclosures: Andreeff: Roche: Research Funding. Borthakur:ASCO: Research Funding.

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Tanshinone IIA suppresses the progression of lung adenocarcinoma through regulating CCNA2-CDK2 complex and AURKA/PLK1 pathway

Scientific Reports ◽

10.1038/s41598-021-03166-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ziheng Li ◽

Ying Zhang ◽

Yuan Zhou ◽

Fuqian Wang ◽

Chao Yin ◽

...

Keyword(s):

Flow Cytometry ◽

Western Blot ◽

Lung Adenocarcinoma ◽

Annexin V ◽

Tanshinone Iia ◽

Dependent Manner ◽

Information Analysis ◽

Cdk2 Complex ◽

Dual Staining

AbstractLung adenocarcinoma (LUAD) belongs to a subgroup of non-small cell lung cancer (NSCLC) with an increasing incidence all over the world. Tanshinone IIA (TSA), an active compound of Salvia miltiorrhiza Bunge., has been found to have anti-tumor effects on many tumors, but its anti-LUAD effect and its mechanism have not been reported yet. In this study, bio-information analysis was applied to characterize the potential mechanism of TSA on LUA, biological experiments were used to verify the mechanisms involved. TCGA, Pubchem, SwissTargetPrediction, Venny2.1.0, STRING, DAVID, Cytoscape 3.7.2, Omicshare, GEPIA, RSCBPDB, Chem Draw, AutoDockTools, and PyMOL were utilized for analysis in the bio-information analysis and network pharmacology. Our experiments in vitro focused on the anti-LUAD effects and mechanisms of TSA on LUAD cells (A549 and NCI-H1975 cells) via MTT, plate cloning, Annexin V-FITC and PI dual staining, flow cytometry, and western blot assays. A total of 64 differentially expressed genes (DEGs) of TSA for treatment of LUAD were screened out. Gene ontology and pathway analysis revealed characteristic of the DEGs network. After GEPIA-based DEGs confirmation, 46 genes were considered having significant differences. Further, 10 key DEGs (BTK, HSD11B1, ADAM33, TNNC1, THRA, CCNA2, AURKA, MIF, PLK1, and SORD) were identified as the most likely relevant genes from overall survival analysis. Molecular Docking results showed that CCNA2, CDK2 and PLK1 had the lowest docking energy. MTT and plate cloning assays results showed that TSA inhibited the proliferation of LUAD cells in a concentration-dependent manner. Annexin V-FITC and PI dual staining and flow cytometry assays results told that TSA promoted the apoptosis of the two LUAD cells in different degrees, and induced cycle arrest in the G1/S phase. Western blot results showed that TSA significantly down-regulated the expression of CCNA2, CDK2, AURKA, PLK1, and p-ERK. In summary, TSA could suppress the progression of LUAD by inducing cell apoptosis and arresting cell cycle, and these were done by regulating CCNA2-CDK2 complex and AURKA/PLK1 pathway. These findings are the first to demonstrate the molecular mechanism of TSA in treatment of LUAD combination of network bio-information analysis and biological experiments in vitro.

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Abstract 165: Palmitate Induces Dose-Dependent Apoptosis in Chick Embryo Cardiomyocytes

Circulation Research ◽

10.1161/res.111.suppl_1.a165 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Charles C Oh ◽

Jennifer Levy ◽

Wuqiong Ma ◽

Peter Reaven ◽

Raymond Migrino ◽

...

Keyword(s):

Chick Embryo ◽

Cardiac Development ◽

Single Cells ◽

Annexin V ◽

Cell Types ◽

Dependent Manner ◽

Dose Dependent ◽

Pregnant Diabetic ◽

Diabetic Mothers

BACKGROUND: In morbid obesity and type 2 diabetes saturated fatty acid (SFA) levels are markedly increased. Palmitate, a SFA, crosses the placenta easily and may affect embryos of diabetic mothers. Palmitate induces inflammation and apoptosis in many cell types including cardiomyocytes. Transient cardiomyopathy can develop in offspring of diabetic mothers. We are testing the hypothesis that palmitate will increase apoptosis in chick embryo cardiomyocytes, making it vulnerable for later disease. METHODS: Hearts from chick embryos at Hamburger Hamilton stage 31 were harvested and digested into single cells using trypsin then plated. After 48 h of growth, cells were exposed to free palmitate (at ∼2.3:1, FFA:albumin ratio) at high physiological concentrations for 24 h. Apoptosis was measured with Annexin V and cell necrosis with propodium iodide using flow cytometry. RESULTS: Apoptosis was markedly elevated in chick embryo cardiomyocytes exposed to palmitate in a dose dependent manner (see Figure). Percentage of apoptotic cells increased from 11.9% in 0 μM palmitate to 32.0% and 44.1% in 150 μM and 300 μM respectively. The percentage of cells demonstrating non-apoptotic necrosis was low and did not change with increasing palmitate concentration. CONCLUSIONS: In chick embryo cardiomyocytes apoptosis is increased in a dose-dependent manner by brief exposure to palmitate. This may have important implications for intrauterine cardiac development in pregnant diabetic mothers and needs to be further explored.

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Hepatotoxicity During Eltrombopag Administration Might Be Due to Necrosis of Hepatocytes

Blood ◽

10.1182/blood.v118.21.2228.2228 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2228-2228

Author(s):

Kazunori Murai ◽

Shugo Kowata ◽

Akiko Abo ◽

Tatsuo Oyake ◽

Shigeki Ito ◽

...

Keyword(s):

Oxidative Stress ◽

Flow Cytometry ◽

Growth Inhibition ◽

Cell Line ◽

Mtt Assay ◽

Annexin V ◽

Similar Data ◽

Dependent Manner ◽

Inhibitory Effects ◽

Annexin V Assay

Abstract Abstract 2228 Background: Eltrombopag is an oral thrombopoietin receptor agonist for the treatment of immune thrombocytopenic patients who are refractory to the medications including corticosteroid. In RAISE study (The Lancet 2011: 377; 393–402), 79% patients in the eltrombopag group responded to treatment at least once during the study. However, 7% of eltrombopag-treated patients had increase of alanine aminotransferase (ALT) concentration and 4% of total bilirubin. The mechanism of eltrombopag-induced hepatobiliary toxicity remains unknown. In this study, we evaluated the effects of eltrombopag on hepatocytes using HepG2, human hepatocellular carcinoma cell line. Method: Cell proliferation was analyzed by MTT assay. Analysis of apoptosis/necrosis was analyzed by flow cytometry assay using Annexin V and propium iodide (PI) (Annexin-/PI-, viable cells; Annexin+/PI-, early apoptosis cells; Annexin+/PI+, late apoptosis and necrosis cells; Annexin-/PI+, late necrosis cells). Reduced glutathione was measured by DTNB colorimetric method. Murine hepatoma cell line Hepa 1–6 and murine normal liver derived cell line NCTC clone 1469 were also used in this study. Results: HepG2 was incubated with eltrombopag for 72 hrs and then MTT assay was performed. Figure 1a showed the growth inhibition curve of HepG2. HepG2 growth was suppressed by eltrombopag in a dose dependent manner (Figure 1a: without NAC, 12.5 μM 44.3 ± 8.7 %). In the addition of N-acetylcysteine (NAC) canceled the inhibitory effect (Figure 1a: with NAC 10mM: 60.4 ± 22.3 % at 12.5μM eltrombopag, p<0.05). To investigate whether this suppression was due to apoptosis or necrosis, we used PI/ Annexin V assay using flow cytometry. Figure 1b showed that each cell fraction after 72 hrs incubation with eltrombopag. PI positive and PI negative fraction was markedly increased (0μM: 0.7 ± 0.4%, 12.5 μM: 8.0 ± 6.0% p<0.01, 25μM: 39.6 ± 12.2% p<0.001). To confirm that the inhibitory effects of eltrombopag might be due to necrosis, we used IM-54, which is inhibitory molecule against oxidative stress induced necrosis, in MTT assay and PI/ Annexin V assay. As shown in Figure 2 a and 2 b, IM-54 canceled the inhibitory effects of eltrombopag in MTT assay (IM-54 0μM: 18.3 ± 7.5 %, IM-54 10 μM: 33.9 ± 10.6 % at eltrombopag 25μM, p<0.01) and PI/Annexin V assay (IM-54 0μM: 48.3 ± 12.4 %, IM-54: 10 μM 74.2 ± 8.3 % in PI/Annexin V double negative fraction at eltrombopag 25μM, p<0.01). The addition of Caspase-Inhibitor III (Boc-D-FMK) did not cancel the growth inhibition by eltrombopag in MTT assey. We measured GSH concentration of HepG2 cells. After the treatment of eltrombopag for 72 hrs, GSH decreased at 12.5μM of eltrpmbopag, however IM-54 restored the GSH concentration (83 mM at IM-54 0μM and eltrombopag 0 μM, IM-54 0 μM: 42.0 mM and IM-54 10μM: 83.3 mM at eltrombopag 12.5μM). Similar data were obtained in the study using Hepa 1–6 and NCTC clone 1469. Conclusion: Basic structure of eltrombopag is similar to that of 3-bipheyl-carboxylic acid, which is a strong oxidizing agent. Taken together, eltrombopag have an oxidative stress on hepatocytes to result in hepatotoxicity. Disclosures: No relevant conflicts of interest to declare.

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β-Elemene Inhibits Cell Proliferation by Regulating the Expression and Activity of Topoisomerases I and IIαin Human Hepatocarcinoma HepG-2 Cells

BioMed Research International ◽

10.1155/2015/153987 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 5

Author(s):

Min Gong ◽

Ying Liu ◽

Jian Zhang ◽

Ya-jie Gao ◽

Ping-ping Zhai ◽

...

Keyword(s):

Cell Proliferation ◽

Topoisomerase I ◽

Annexin V ◽

Dependent Manner ◽

Dna Breaks ◽

Morphological Alterations ◽

Dna Relaxation ◽

Pbr322 Dna ◽

Topo Ii ◽

Expression And Activity

Objective. To investigate the effects ofβ-Elemene (β-ELE) on the proliferation, apoptosis, and topoisomerase I (TOPO I) and topoisomerase IIα(TOPO IIα) expression and activity of human hepatocarcinoma HepG-2 cells.Methods. After treatment withβ-ELE, morphological alterations of HepG-2 cells were observed under an inverted microscope. Cell proliferation was assessed using an MTT assay, cell cycles were analyzed using flow cytometry, and apoptosis was detected by Annexin V/PI staining. The expression of TOPO I and TOPO IIαwas analyzed by Western blot techniques, and their activity was measured using the TOPO I-mediated, supercoiled pBR322 DNA relaxation and TOPO IIα-mediated Kinetoplast DNA (kDNA) decatenation assays, respectively. Supercoiled pBR322 and kDNA were also used to determine the direct effect ofβ-ELE on DNA breaks.Results.β-ELE significantly inhibited HepG-2 cell proliferation in a dose- and time-dependent manner.β-ELE also induced tumor cell arrest at S phase, induced cell apoptosis, and downregulated the protein expression of TOPO I and TOPO IIαin a dose-dependent manner.β-ELE also inhibited TOPO I- and TOPO IIα-mediated DNA relaxation but did not directly induce DNA breakage at any concentration.Conclusion.β-ELE could inhibit the proliferation of HepG-2 cells and interfere with the expression and activity of TOPO I and TOPO IIα.

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Involvement of Na+/H+ exchanger in desmopressin-induced platelet procoagulant response.

Acta Biochimica Polonica ◽

10.18388/abp.2004_3561 ◽

2004 ◽

Vol 51 (3) ◽

pp. 773-788 ◽

Cited By ~ 6

Author(s):

Maria M Tomasiak ◽

Halina Stelmach ◽

Anna Bodzenta-Łukaszyk ◽

Marian Tomasiak

Keyword(s):

Flow Cytometry ◽

Thrombin Generation ◽

Annexin V ◽

Underlying Mechanism ◽

Dependent Manner ◽

Platelet Volume ◽

Cell Sizing ◽

Dose Dependent ◽

In Vitro Treatment

Desmopressin (DDAVP) action on platelets is associated with the development of procoagulant response but the underlying mechanism of this phenomenon is not known. We investigated whether this effect of DDAVP might be due to activation of plasma membrane Na+/H+ exchanger. The DDAVP-induced platelet procoagulant response, measured as phospholipid-dependent thrombin generation, was dose dependent and significantly weaker than that produced by collagen or monensin (mimics Na+/H+ antiport). Both the DDAVP- and collagen-produced procoagulant responses were less pronounced in the presence of EIPA, an Na+/H+ exchanger inhibitor. Flow cytometry studies revealed that in vitro treatment of platelets with DDAVP or collagen was associated with the appearance of both degranulated (and fragmented) and swollen cells. The DDAVP-evoked rise in size and granularity heterogeneity was similar to that produced by collagen or monensin and was not observed in the presence of EIPA. Using flow cytometry and annexin V-FITC as a probe for phosphatidylserine (PS) we demonstrated increased and uniform binding of this marker to all subsets of DDAVP-treated platelet population. The DDAVP-evoked PS expression was dose dependent, strongly reduced by EIPA and weaker than that caused by monensin or collagen. As judged by optical swelling assay, DDAVP in a dose dependent manner produced a rise in platelet volume. The swelling was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Electronic cell-sizing measurements showed an increase in mean platelet volume and a decrease in platelet count and platelet crit upon treatment with DDAVP. DDAVP elicited a slow (much slower than collagen) alkalinization of platelet cytosol. Altogether the data indicate an involvement of Na+/H+ exchanger in the generation of procoagulant activity in DDAVP-treated platelets.

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