Time Dependent Appearance of Selected Apoptotic Markers and Usefulness of Their Detection In vitro

Many experiments have demonstrated that some cell lines are resistant to chemically induced apoptosis in vitro, and that apoptosis itself is far from being a homogenous phenomenon. Here we show that 10 μg/ml etoposide elicited only minor changes in Bowes human melanoma cells (temporary decrease in cell viability and proliferation, transient phospatidylserine externalization and caspase-3 activation), which weren’t clearly capable to start apoptotic pathway in the entire treated population. On the other hand, potassium chromate at concentration of 150 μg/ml executed cell death bearing some features of apoptosis (cell blebbing, caspase-3 activation and cytoskeletal changes) but lacking or showing weakly others (DNA fragmentation and phospatidylserine externalization). Our results suggest that in detecting apoptosis several faultproof detection systems are to be used to avoid misleading results and conclusions in each experimental setting.

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Caspase-mediated Cleavage of p130cas in Etoposide-induced Apoptotic Rat-1 Cells

Molecular Biology of the Cell ◽

10.1091/mbc.11.3.929 ◽

2000 ◽

Vol 11 (3) ◽

pp. 929-939 ◽

Cited By ~ 54

Author(s):

Seunghyi Kook ◽

Sang Ryeol Shim ◽

Soo Jeon Choi ◽

Joohong Ahnn ◽

Jae Il Kim ◽

...

Keyword(s):

Extracellular Matrix ◽

Focal Adhesion ◽

Caspase 3 ◽

Morphological Changes ◽

Point Mutations ◽

Membrane Blebbing ◽

Adhesion Sites ◽

Focal Adhesion Proteins ◽

Induced Apoptosis

Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell–cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD416G and DSPD748G) in vitro, and point mutations substituting the Asp of DVPD416G and DSPD748G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD416G generated a 74-kDa fragment, which was in turn cleaved at DSPD748G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas–paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix.

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20-HETE-mediated cytotoxicity and apoptosis in ischemic kidney epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00387.2007 ◽

2008 ◽

Vol 294 (3) ◽

pp. F562-F570 ◽

Cited By ~ 37

Author(s):

Vani Nilakantan ◽

Cheryl Maenpaa ◽

Guangfu Jia ◽

Richard J. Roman ◽

Frank Park

Keyword(s):

Caspase 3 ◽

Fluorescent Protein ◽

Ischemia Reperfusion ◽

Atp Depletion ◽

Cellular Damage ◽

Apoptotic Pathway ◽

Injury Model ◽

Green Fluorescent

20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-PK1 cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-PK1 cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release ( P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the SOD mimetic MnTMPyP (100 μM) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 μM) also inhibited cytotoxicity significantly ( P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase ( P < 0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells ( P < 0.05). This was abolished in the presence of HET-0016 ( P < 0.05) or MnTMPyP ( P < 0.01). These results demonstrate that 20-HETE overexpression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent on enhanced CYP4A generation of free radicals.

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Differential Expression of MicroRNA-19b Promotes Proliferation of Cancer Stem Cells by Regulating the TSC1/mTOR Signaling Pathway in Multiple Myeloma

Cellular Physiology and Biochemistry ◽

10.1159/000494821 ◽

2018 ◽

Vol 50 (5) ◽

pp. 1804-1814 ◽

Cited By ~ 6

Author(s):

Ni Wang ◽

Xiaohua Liang ◽

Weijian Yu ◽

Shihang Zhou ◽

Meiyun Fang

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Cancer Stem Cells ◽

Real Time ◽

Caspase 3 ◽

In Silico Analysis ◽

Luciferase Assay ◽

Downstream Target ◽

Induced Apoptosis

Background/Aims: MiR-19b has been reported to be involved in several malignancies, but its role in multiple myeloma (MM) is still unknown. The objective of this study was to explore the biological mechanism of miR-19b in the progression of MM. Methods: First, we performed real-time polymerase chain reaction (PCR) and Western blot to study the expression of miR-19b, tuberous sclerosis 1 (TSC1), and caspase-3 in different groups. MTT assay was performed to explore the effect of miR-19b on survival and apoptosis of cancer stem cells (CSCs). Computation analysis and luciferase assay were utilized to confirm the interaction between miR-19b and TSC1. Results: A total of 38 participants comprising 20 subjects with MM and 18 healthy subjects as normal controls were enrolled in our study. Real-time PCR showed dramatic upregulation of miR-19b, but TSC1 was evidently suppressed in the MM group. MiR-19b overexpression substantially promoted clonogenicity and cell viability, and further inhibited apoptosis of CSCs in vitro. Furthermore, miR-19b overexpression downregulated the expression of caspase-3, which induced apoptosis. Using in silico analysis, we identified that TSC1 might be a direct downstream target of miR-19b, and this was further confirmed by luciferase assay showing that miR-19b apparently reduced the luciferase activity of wild-type TSC1 3´-UTR, but not that of mutant TSC1 3´-UTR. There was also evident decrease in TSC1 mRNA and protein in CSCs following introduction of miR-19b. Interestingly, reintroduction of TSC1 abolished the miR-19b-induced proliferation promotion and apoptosis inhibition in CSCs. Conclusion: These findings collectively suggest that miR-19b promotes cell survival and suppresses apoptosis of MM CSCs via targeting TSC1 directly, indicating that miR-19b may serve as a potential and novel therapeutic target of MM based on miRNA expression.

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Down-Regulation of Telomerase Activity and Activation of Caspase-3 Are Responsible for Tanshinone I-Induced Apoptosis in Monocyte Leukemia Cells in Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms11062267 ◽

2010 ◽

Vol 11 (6) ◽

pp. 2267-2280 ◽

Cited By ~ 27

Author(s):

Xiao-Dan Liu ◽

Rui-Fang Fan ◽

Yong Zhang ◽

Hong-Zhi Yang ◽

Zhi-Gang Fang ◽

...

Keyword(s):

Caspase 3 ◽

Telomerase Activity ◽

Leukemia Cells ◽

Down Regulation ◽

Tanshinone I ◽

Induced Apoptosis

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Salmonella-Induced Caspase-2 Activation in Macrophages

Journal of Experimental Medicine ◽

10.1084/jem.192.7.1035 ◽

2000 ◽

Vol 192 (7) ◽

pp. 1035-1046 ◽

Cited By ~ 114

Author(s):

Veronika Jesenberger ◽

Katarzyna J. Procyk ◽

Junying Yuan ◽

Siegfried Reipert ◽

Manuela Baccarini

Keyword(s):

Type Iii Secretion ◽

Caspase 3 ◽

Secretion System ◽

Caspase 1 ◽

Chemical Inhibition ◽

Induction Of Apoptosis ◽

Caspase 2 ◽

Induced Apoptosis

The enterobacterial pathogen Salmonella induces phagocyte apoptosis in vitro and in vivo. These bacteria use a specialized type III secretion system to export a virulence factor, SipB, which directly activates the host's apoptotic machinery by targeting caspase-1. Caspase-1 is not involved in most apoptotic processes but plays a major role in cytokine maturation. We show that caspase-1–deficient macrophages undergo apoptosis within 4–6 h of infection with invasive bacteria. This process requires SipB, implying that this protein can initiate the apoptotic machinery by regulating components distinct from caspase-1. Invasive Salmonella typhimurium targets caspase-2 simultaneously with, but independently of, caspase-1. Besides caspase-2, the caspase-1–independent pathway involves the activation of caspase-3, -6, and -8 and the release of cytochrome c from mitochondria, none of which occurs during caspase-1–dependent apoptosis. By using caspase-2 knockout macrophages and chemical inhibition, we establish a role for caspase-2 in both caspase-1–dependent and –independent apoptosis. Particularly, activation of caspase-1 during fast Salmonella-induced apoptosis partially relies on caspase-2. The ability of Salmonella to induce caspase-1–independent macrophage apoptosis may play a role in situations in which activation of this protease is either prevented or uncoupled from the induction of apoptosis.

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Apigenin Suppresses Angiogenesis by Inhibiting Tube Formation and Inducing Apoptosis

Natural Product Communications ◽

10.1177/1934578x1601101005 ◽

2016 ◽

Vol 11 (10) ◽

pp. 1934578X1601101

Author(s):

Hyun Ju Kim ◽

Mok-Ryeon Ahn

Keyword(s):

Umbilical Vein ◽

Chorioallantoic Membrane ◽

Tube Formation ◽

Anticancer Activities ◽

Apoptotic Pathway ◽

Inhibition Of Angiogenesis ◽

Induced Apoptosis ◽

Umbilical Vein Endothelial Cells

Apigenin has been reported to exert angiogenic and anticancer activities in vitro. The mechanism of inhibition of angiogenesis by apigenin, however, has not been well-established. In this study, we investigated whether apigenin not only inhibited tube formation but also induced apoptosis in human umbilical vein endothelial cells (HUVECs). Furthermore, strong antiangiogenic activity of apigenin was observed in the in vivo assay using chick embryo chorioallantoic membrane (CAM). We also analyzed changes in survival signals and the apoptotic pathway through Western blotting. The results indicate that apigenin exerts its antiangiogenic effects through induction of endothelial apoptosis.

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Identification of apoptotic genes mediating TGF-β/Smad3-induced cell death in intestinal epithelial cells using a genomic approach

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00437.2005 ◽

2007 ◽

Vol 292 (1) ◽

pp. G28-G38 ◽

Cited By ~ 18

Author(s):

Yanna Cao ◽

Lu Chen ◽

Weili Zhang ◽

Yan Liu ◽

Harry T. Papaconstantinou ◽

...

Keyword(s):

Caspase 3 ◽

Target Genes ◽

Transforming Growth Factor ◽

Intestinal Epithelial ◽

Cytochrome C Release ◽

Apoptotic Pathway ◽

Genomic Approach ◽

Apoptotic Genes ◽

Induced Apoptosis

Transforming growth factor (TGF)-β-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. Previously, we have shown that TGF-β inhibits the growth of rat intestinal epithelial (RIE)-1 cells. However, RIE-1 cells are relatively resistant to TGF-β-induced apoptosis due to a low endogenous Smad3-to-Akt ratio. Overexpression of Smad3 sensitizes RIE-1 cells (RIE-1/Smad3) to TGF-β-induced apoptosis by altering the Smad3-to-Akt ratio in favor of apoptosis. In this study, we utilized a genomic approach to identify potential downstream target genes that are regulated by TGF-β/Smad3. Total RNA samples were analyzed using Affymetrix oligonucleotide microarrays. We found that TGF-β regulated 518 probe sets corresponding to its target genes. Interestingly, among the known apoptotic genes included in the microarray analyses, only caspase-3 was induced, which was confirmed by real-time RT-PCR. Furthermore, TGF-β activated caspase-3 through protein cleavage. Upstream of caspase-3, TGF-β induced mitochondrial depolarization, cytochrome c release, and cleavage of caspase-9, which suggests that the intrinsic apoptotic pathway mediates TGF-β-induced apoptosis in RIE-1/Smad3 cells.

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Ginsenoside Rb1 Protects Neonatal Rat Cardiomyocytes from Hypoxia/Ischemia Induced Apoptosis and Inhibits Activation of the Mitochondrial Apoptotic Pathway

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2014/149195 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 16

Author(s):

Xu Yan ◽

Jinwen Tian ◽

Hongjin Wu ◽

Yuna Liu ◽

Jianxun Ren ◽

...

Keyword(s):

Caspase 3 ◽

Neonatal Rat ◽

Ginsenoside Rb1 ◽

Caspase 9 ◽

Apoptotic Pathway ◽

Rat Cardiomyocytes ◽

Mitochondrial Apoptotic Pathway ◽

Neonatal Rat Cardiomyocytes ◽

Hypoxia Ischemia

Aim. To investigate the effect of Ginsenoside Rb1 (GS-Rb1) on hypoxia/ischemia (H/I) injury in cardiomyocytesin vitroand the mitochondrial apoptotic pathway mediated mechanism.Methods. Neonatal rat cardiomyocytes (NRCMs) for the H/I groups were kept in DMEM without glucose and serum, and were placed into a hypoxic jar for 24 h. GS-Rb1 at concentrations from 2.5 to 40 µM was given during hypoxic period for 24 h. NRCMs injury was determined by MTT and lactate dehydrogenase (LDH) leakage assay. Cell apoptosis, ROS accumulation, and mitochondrial membrane potential (MMP) were assessed by flow cytometry. Cytosolic translocation of mitochondrial cytochrome c and Bcl-2 family proteins were determined by Western blot. Caspase-3 and caspase-9 activities were determined by the assay kit.Results. GS-Rb1 significantly reduced cell death and LDH leakage induced by H/I. It also reduced H/I induced NRCMs apoptosis induced by H/I, in accordance with a minimal reactive oxygen species (ROS) burst. Moreover, GS-Rb1 markedly decreased the translocation of cytochrome c from the mitochondria to the cytosol, increased the Bcl-2/ Bax ratio, and preserved mitochondrial transmembrane potential (ΔΨm). Its administration also inhibited activities of caspase-9 and caspase-3.Conclusion. Administration of GS-Rb1 during H/Iin vitrois involved in cardioprotection by inhibiting apoptosis, which may be due to inhibition of the mitochondrial apoptotic pathway.

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Volatile Anesthetics Induce Caspase-dependent, Mitochondria-mediated Apoptosis in Human T Lymphocytes In Vitro

Anesthesiology ◽

10.1097/00000542-200506000-00014 ◽

2005 ◽

Vol 102 (6) ◽

pp. 1147-1157 ◽

Cited By ~ 102

Author(s):

Torsten Loop ◽

David Dovi-Akue ◽

Michael Frick ◽

Martin Roesslein ◽

Lotti Egger ◽

...

Keyword(s):

Membrane Potential ◽

T Lymphocytes ◽

Cytochrome C ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Caspase 3 ◽

Volatile Anesthetics ◽

Human T Lymphocytes ◽

Induced Apoptosis

Background Volatile anesthetics modulate lymphocyte function during surgery, and this compromises postoperative immune competence. The current work was undertaken to examine whether volatile anesthetics induce apoptosis in human T lymphocytes and what apoptotic signaling pathway might be used. Methods Effects of sevoflurane, isoflurane, and desflurane were studied in primary human CD3 T lymphocytes and Jurkat T cells in vitro. Apoptosis and mitochondrial membrane potential were assessed using flow cytometry after green fluorescent protein-annexin V and DiOC6-fluorochrome staining. Activity and proteolytic processing of caspase 3 was measured by cleaving of the fluorogenic effector caspase substrate Ac-DEVD-AMC and by anti-caspase-3 Western blotting. Release of mitochondrial cytochrome c was studied after cell fractionation using anti-cytochrome c Western blotting and enzyme-linked immunosorbent assays. Results Sevoflurane and isoflurane induced apoptosis in human T lymphocytes in a dose-dependent manner. By contrast, desflurane did not exert any proapoptotic effects. The apoptotic signaling pathway used by sevoflurane involved disruption of the mitochondrial membrane potential and release of cytochrome c from mitochondria to the cytosol. In addition, the authors observed a proteolytic cleavage of the inactive p32 procaspase 3 to the active p17 fragment, increased caspase-3-like activity, and cleavage of the caspase-3 substrate poly-ADP-ribose-polymerase. Sevoflurane-induced apoptosis was blocked by the general caspase inhibitor Z-VAD.fmk. Death signaling was not mediated via the Fas/CD95 receptor pathway because neither anti-Fas/CD95 receptor antagonism nor FADD deficiency or caspase-8 deficiency were able to attenuate sevoflurane-mediated apoptosis. Conclusion Sevoflurane and isoflurane induce apoptosis in T lymphocytes via increased mitochondrial membrane permeability and caspase-3 activation, but independently of death receptor signaling.

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