In Vitro Assays for Caspase-3 Activation and DNA Fragmentation

2000 ◽  
pp. 177-182 ◽  
Author(s):  
Xuesong Liu ◽  
Xiaodong Wang
Keyword(s):  
Dna Fragmentation ◽  
Caspase 3 ◽  
In Vitro Assays

scholarly journals Granzyme B Short-Circuits the Need for Caspase 8 Activity during Granule-Mediated Cytotoxic T-Lymphocyte Killing by Directly Cleaving Bid

2000 ◽  
Vol 20 (11) ◽  
pp. 3781-3794 ◽  
Author(s):  
Michele Barry ◽  
Jeffrey A. Heibein ◽  
Michael J. Pinkoski ◽  
Siow-Fong Lee ◽  
Richard W. Moyer ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Caspase 3 ◽  
Cytotoxic T Lymphocyte ◽  
Granzyme B ◽  
Serine Proteinase ◽  
Target Cells ◽  
Caspase 8 ◽  
Intact Cells ◽  
Whole Cells

ABSTRACT Cytotoxic T lymphocytes (CTL) can trigger an apoptotic signal through the Fas receptor or by the exocytosis of granzyme B and perforin. Caspase activation is an important component of both pathways. Granzyme B, a serine proteinase contained in granules, has been shown to proteolytically process and activate members of the caspase family in vitro. In order to gain an understanding of the contributions of caspases 8 and 3 during granule-induced apoptosis in intact cells, we have used target cells that either stably express the rabbitpox virus-encoded caspase inhibitor SPI-2 or are devoid of caspase 3. The overexpression of SPI-2 in target cells significantly inhibited DNA fragmentation, phosphatidylserine externalization, and mitochondrial disruption during Fas-mediated cell death. In contrast, SPI-2 expression in target cells provided no protection against granzyme-mediated apoptosis, mitochondrial collapse, or cytolysis, leading us to conclude that SPI-2-inhibited caspases are not an essential requirement for the granzyme pathway. Caspase 3-deficient MCF-7 cells were found to be resistant to CTL-mediated DNA fragmentation but not to CTL-mediated cytolysis and loss of the mitochondrial inner membrane potential. Furthermore, we demonstrate that granzyme B directly cleaves the proapoptotic molecule Bid, bypassing the need for caspase 8 activation of Bid. These results provide evidence for a two-pronged strategy for mediating target cell destruction and provide evidence of a direct link between granzyme B activity, Bid cleavage, and caspase 3 activation in whole cells.

scholarly journals Porphyra-334, a mycosporine-like amino acid, attenuates UV-induced apoptosis in HaCaT cells

2017 ◽  
Vol 67 (2) ◽  
pp. 257-264 ◽  
Author(s):  
Sung-Suk Suh ◽  
Se Kyung Oh ◽  
Sung Gu Lee ◽  
Il-Chan Kim ◽  
Sanghee Kim
Keyword(s):  
Caspase 3 ◽  
Biological Activities ◽  
Inhibitory Effect ◽  
In Vitro Assays ◽  
Control Group ◽  
Hacat Cells ◽  
Dramatic Decrease ◽  
Induced Apoptosis ◽  
Active Caspase

Abstract The main aim of the current research was to study the effect of porphyra-334, one of mycosporine-like amino acids (MAAs), well known as UV-absorbing compounds, on UVinduced apoptosis in human immortalized keratinocyte (HaCaT) cells. Due to their UV-screening capacity and ability to prevent UV-induced DNA damage, MAAs have recently attracted considerable attention in both industry and research in pharmacology. Herein, human HaCaT cells were used to determine the biological activities of porphyra- 334 by various in vitro assays, including proliferation, apoptosis and Western blot assays. The proliferation rate of UV-irradiated HaCaT cells was significantly decreased compared to the control group. Pretreatment with porphyra- 334 markedly attenuated the inhibitory effect of UV and induced a dramatic decrease in the apoptotic rate. Expression of active caspase-3 protein was increased in response to UV irradiation, while caspase-3 levels were similar between cells treated with porphyra-334 and the non-irradiated control group. Taken together, our data suggest that porphyra-334 inhibits UV-induced apoptosis in HaCaT cells through attenuation of the caspase pathway.

Increase in DNA fragmentation and apoptosis-related gene expression in frozen-thawed bovine blastocysts

Zygote ◽  
2006 ◽  
Vol 14 (2) ◽  
pp. 125-131 ◽  
Author(s):  
Sae Young Park ◽  
Eun Young Kim ◽  
Xiang Shun Cui ◽  
Jin Cheol Tae ◽  
Won Don Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Fragmentation ◽  
Caspase 3 ◽  
Expression Patterns ◽  
Survival Rates ◽  
Related Gene ◽  
Hsp 70 ◽  
Frozen Embryos ◽  
Apoptosis Related Gene

SummaryEvaluation of apoptosis and expression level of apoptosis-related genes is useful for examining the variation in embryo quality according to environmental change. The objective of this study was to investigate DNA fragmentation and apoptosis-related gene expression patterns in frozen-thawed bovine blastocysts.In vitroproduced day 7 blastocysts were frozen by two different vitrification methods (conventional 0.25 ml straw or MVC straw). After thawing, DNA fragmentation of surviving embryos was examined by TUNEL assay, and the expression patterns of their apoptotic genes (survivin, Fas, Hsp 70 and caspase-3) were evaluated using real-time quantitative reverse transcriptase polymerase chain reaction.In vitrosurvival rates of frozen-thawed embryos were higher following the MVC vitrification method (88.2% re-expanded at 24 h, 77.1% hatching at 48 h) than the conventional (C) vitrification method (77.0% re-expanded at 24 h, 66.7% hatching at 48 h). However, both vitrified methods resulted in a significantly higher apoptotic index (C vitrification method 11.9%, MVC vitrification method 11.0%) than in non-frozen embryos (3.0%). Expression levels of survivin, Fas, caspase-3, and Hsp 70 were also increased in the frozen-thawed embryos compared with non-frozen embryos. These results indicate that the cryopreservation procedure might cause damage that results in an increase in DNA fragmentation and apoptosis-related gene transcription, reducing developmental capacity of frozen-thawed embryos.

Anti-cancer Effect of Cyanidin-3-glucoside from Mulberry via Caspase-3 Cleavage and DNA Fragmentation in vitro and in vivo

2017 ◽  
Vol 17 (11) ◽  
Author(s):  
Eugene Cho ◽  
Eun Y. Chung ◽  
Hye-Yeon Jang ◽  
On-Yu Hong ◽  
Hee S. Chae ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Caspase 3 ◽  
Anti Cancer

Abstract TP265: AATF Protects Neuron Against Ischemia via Regulating PAR/AIF Pathway

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Wei Jin ◽  
Wei Xu ◽  
Jing Chen ◽  
Xiaoxiao Zhang ◽  
Chuancheng Ren
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Western Blot ◽  
Dna Fragmentation ◽  
Caspase 3 ◽  
Immunofluorescent Staining ◽  
Dependent Manner ◽  
Cellular Death ◽  
Cell Death Pathway

Abstract: Apoptosis antagonizing transcription factor (AATF) exerts an effect against oxidative stress, DNA damage and cellular apoptosis. However, its role in neuronal ischemia or hypoxia damage has not been elucidated yet. Present study investigated the neuroprotective roles and mechanisms of AATF under ischemia and hypoxia in vivo and in vitro. Focal cerebral ischemia of rat was generated by distal middle cerebral artery occlusion (dMCAO) model, SH-SY5Y cells were used to generate oxygen glucose deprivation (OGD) model in vitro. Western blot and immunofluorescent staining were used to investigate the expression changes of AATF. CCK-8 and LDH were performed to evaluate cellular survival and cytotoxicity. Overexpression and interference lentivirus vectors were performed to regulate the expression of AATF in SH-SY5Y cells. DHE staining that measured by flow cytometry was performed to investigate cellular superoxide anion levels. 8-OHdG expression and AP sites measurement were used to evaluate DNA damage. DNA Ladder and TUNEL staining were employed to evaluate DNA fragmentation. MNNG and DPQ were respectively used to agitate or antagonist caspase-3 independent PCD (programmed cell death) pathway, STS and Z-VAD-fmk were respectively used to agitate or antagonist caspase-3 dependent PCD pathway. Western blot was performed to investigate the expression of Poly(ADP-ribose) polymers (PAR) and apoptosis inducing factor (AIF) in different cellular components, Co-IP (co-immunoprecipitation) was used to test the interaction of AIF, H2AX and CypA (Cyclophilin A). We found that AATF was increased in cortical neurons after brain ischemia (P<0.001). Besides, AATF was upregulated in OGD-treated SH-SY5Y cells in a time-dependent manner (P=0.007). Additionally, overexpressing AATF ameliorated OGD-induced cellular death (P < 0.001) and cytotoxicity (P = 0.001), and AATF interference exacerbated OGD-induced cellular death (P=0.033) and cytotoxicity (P=0.006). We also found that AATF overexpression suppressed cellular DNA fragmentation (P=0.003) but did not ameliorate oxidative stress and DNA damage. Moreover, we discovered that overexpressing AATF suppressed PAR/AIF signaling pathway via binding with AIF.

Induction of Caspase-Activated Deoxyribonuclease Activity after Focal Cerebral Ischemia and Reperfusion

2002 ◽  
Vol 22 (1) ◽  
pp. 15-20 ◽  
Author(s):  
Yumin Luo ◽  
Guodong Cao ◽  
Wei Pei ◽  
Cristine O'Horo ◽  
Steven H. Graham ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Caspase 3 ◽  
Focal Cerebral Ischemia ◽  
Focal Ischemia ◽  
Brain Cell ◽  
Ischemic Brain ◽  
Cell Extracts ◽  
Apoptosis Assay ◽  
Deoxyribonuclease Activity

Deoxyribonucleic acid fragmentation at nucleosomal junctions is a hallmark of neuronal apoptosis in ischemic brain injury, for which the mechanism is not fully understood. Using the in vitro cell-free apoptosis assay, the authors found that caspase-3–dependent deoxyribonuclease activity caused internucleosomal DNA fragmentation in brain-cell extracts in a rat model of transient focal ischemia. This in vitro deoxyribonuclease activity was completely inhibited by purified inhibitor of caspase-activated deoxyribonuclease protein, the specific endogenous inhibitor of caspase-activated deoxyribonuclease, or by caspase-activated deoxyribonuclease immunodepletion. The induction of the deoxyribonuclease activity was correlated with caspase-3 activation and caspase-3–mediated degradation of inhibitor of caspase-activated deoxyribonuclease. Furthermore, inhibiting caspase-3–like protease activity prevented the endogenous induction of internucleosomal DNA fragmentation in the ischemic brain. These results suggest that caspase-3–dependent caspase-activated deoxyribonuclease activity plays an important role in mediating DNA fragmentation after focal ischemia.

Comparison of In Vitro Assays of Cellular Toxicity in the Human Hepatic Cell Line HepG2

2005 ◽  
Vol 11 (2) ◽  
pp. 184-193 ◽  
Author(s):  
Silvia Miret ◽  
Els M. De Groene ◽  
Werner Klaffke
Keyword(s):  
Cell Death ◽  
Hepg2 Cells ◽  
Caspase 3 ◽  
Fluorometric Assay ◽  
In Vitro Assays ◽  
Mitochondrial Activity ◽  
Cytotoxicity Test ◽  
Wide Range

Cytotoxicity testing allows determining whether a compound or extract contains significant quantities of biologically harmful chemicals. Cytotoxicity test methods are useful for screening because they serve to separate toxic from nontoxic materials, providing predictive evidence of compound safety. However, a wide range of assays measuring different aspects of cell death is available in the market, but it is difficult to determine which one(s) to use when evaluating a selection of compounds. The objective of this study was to compare different commercially available in vitro assays for cytotoxicity in HepG2 cells according to its sensitivity, reproducibility, simplicity, cost, and speed. The assays evaluated included Alamar Blue for the measurement of mitochondrial activity, ATPlite and ViaLight for the determination of cellular adenosine triphosphate (ATP), ToxiLight as an indicator of cellular necrosis, and Caspase-3 Fluorometric Assay, Apo-ONE Caspase-3/7 Homogeneous Assay, and Caspase-Glo for the determination of caspase-3/7 activity. All assays were performed using 4 compounds of previously reported cytotoxic activity: DMSO, butyric acid, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and camptothecine. Overall, it was concluded that the best way to evaluate the potential cytotoxicity of a compound is to employ a battery of assays that focus on different aspects of cell death. In this case, the focus has been on ATP levels, cell necrosis, and capsase-3/7 activation. Many other kits are commercially available in the market for these and other aspects of necrosis and/or apoptosis. However, the use of ViaLight Plus, ToxiLight, and Caspase-3 Fluorometric Assay resulted in the most useful combination when working with HepG2 cells.

scholarly journals In Vitro Anticancer Effect of Gedunin on Human Teratocarcinomal (NTERA-2) Cancer Stem-Like Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Luxmiga Tharmarajah ◽  
Sameera Ranganath Samarakoon ◽  
Meran Keshawa Ediriweera ◽  
Poorna Piyathilaka ◽  
Kamani Hemamamla Tennekoon ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Fragmentation ◽  
Real Time Pcr ◽  
Caspase 3 ◽  
Mononuclear Cells ◽  
Cell Model ◽  
Morphological Changes ◽  
Peripheral Blood Mononuclear ◽  
Client Proteins

Gedunin is one of the major compounds found in the neem tree(Azadirachta indica). In the present study, antiproliferative potential of gedunin was evaluated in human embryonal carcinoma cells (NTERA-2, a cancer stem cell model) and peripheral blood mononuclear cells (PBMCs), using Sulforhodamine (SRB) and WST-1 assays, respectively. The effects of gedunin on expression of heat shock protein 90 (HSP90), its cochaperone Cdc37, and HSP client proteins (AKT, ErbB2, and HSF1) were evaluated by real-time PCR. Effects of gedunin on apoptosis were evaluated by (a) apoptosis associated morphological changes, (b) caspase 3/7 expression, (c) DNA fragmentation, (d) TUNEL assay, and (e) real-time PCR of apoptosis related genes (Bax,p53,andsurvivin). Gedunin showed a promising antiproliferative effect in NTERA-2 cells with IC50values of 14.59, 8.49, and 6.55 μg/mL at 24, 48, and 72 h after incubations, respectively, while exerting a minimal effect on PBMCs. Expression of HSP90, its client proteins, andsurvivinwas inhibited andBaxandp53were upregulated by gedunin. Apoptosis related morphological changes, DNA fragmentation, and increased caspase 3/7 activities confirmed the proapoptotic effects of gedunin. Collectively, results indicate that gedunin may be a good drug lead for treatment of chemo and radiotherapy resistant cancer stem cells.

scholarly journals Inhibitory Effect of Cuphea aequipetala Extracts on Murine B16F10 Melanoma In Vitro and In Vivo

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Ashanti Concepcion Uscanga-Palomeque ◽  
Pablo Zapata-Benavides ◽  
Santiago Saavedra-Alonso ◽  
Diana Elisa Zamora-Ávila ◽  
Moisés Armides Franco-Molina ◽  
...  
Keyword(s):  
Oral Administration ◽  
Dna Fragmentation ◽  
Aqueous Extract ◽  
Murine Model ◽  
Caspase 3 ◽  
Methanolic Extract ◽  
B16f10 Cell ◽  
B16f10 Cells

Cuphea aequipetala (C. aequipetala) has been used in Mexican traditional medicine since prehispanic times to treat tumors. In this paper, we evaluated the antiproliferative and apoptotic effect of the methanolic and aqueous extracts of C. aequipetala on several cancer cell lines including the B16F10 cell line of murine melanoma and carried a murine model assay. In vitro assay analyzed the effect in the cellular cycle and several indicators of apoptosis, such as the caspase-3 activity, DNA fragmentation, phosphatidylserine exposure (Annexin-V), and induction of cell membrane permeabilization (propidium iodide) in the B16F10 cells. In vivo, groups of C57BL/6 female mice were subcutaneously injected with 5x105 B16F10 cells and treated with 25 mg/mL of C. aequipetala extracts via oral. Aqueous and methanolic extracts showed a cytotoxic effect in MCF-7, HepG2, and B16F10 cell lines. The methanolic extract showed more antiproliferative effect with less concentration, and for this reason, the in vitro experiments were only continued with it. This extract was able to induce accumulation of cells on G1 phase of the cell cycle; moreover, it was able to induce DNA fragmentation and increase the activity of caspase-3 in B16F10 cells. On the other hand, in the murine model of melanoma, the aqueous extract showed a greater reduction of tumor size in comparison with the methanolic extract, showing an 80% reduction versus one of around 31%, both compared with the untreated control, indicating a better antitumor effect of C. aequipetala aqueous extract via oral administration. In conclusion, the in vitro data showed that both C. aequipetala extracts were able to induce cytotoxicity through the apoptosis pathway in B16F10 cells, and in vivo, the oral administration of aqueous extract reduces the melanoma tumoral mass, suggesting an important antitumoral effect and the perspective to search for effector molecules involved in it.

scholarly journals Prolactin Suppresses Glucocorticoid-Induced Thymocyte Apoptosis in Vivo

Endocrinology ◽  
2003 ◽  
Vol 144 (5) ◽  
pp. 2102-2110 ◽  
Author(s):  
Nithya Krishnan ◽  
Olivier Thellin ◽  
Donna J. Buckley ◽  
Nelson D. Horseman ◽  
Arthur R. Buckley
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Dna Fragmentation ◽  
Caspase 3 ◽  
Elevated Serum ◽  
Annexin V ◽  
Lymphoid Cells ◽  
Immune System Development

The hypothesis that prolactin (PRL) functions as an immunomodulator was based on studies showing lymphocyte PRL receptors, and its effects on growth, differentiation, and apoptosis in lymphoid cells. However, studies of PRL (PRL−/−) and PRL receptor knockout mice indicated that PRL was not required for immune system development or function under basal conditions. Because PRL maintains survival in glucocorticoid (GC)-treated Nb2-T lymphocytes in vitro, and PRL and GCs are elevated during stress, we investigated whether PRL protected T cells in vivo from GC-induced apoptosis. Adrenalectomized mice [PRL −/−, undetectable PRL; pituitary grafted PRL−/− (PRL−/−Graft), elevated PRL; and PRL+/−, normal PRL] were treated with dexamethasone (DEX) or PBS. Thymocytes and splenocytes were isolated and annexin V labeling of phosphatidylserine, DNA fragmentation, and caspase-3 activation were assessed as indices of apoptosis. Total thymocytes and CD4+ and CD8+ T cells obtained from DEX-treated PRL−/− mice exhibited significantly increased annexin V binding. In contrast, binding was not altered by DEX in PRL−/−Graft thymocytes. In addition, DEX induced classic DNA fragmentation in PRL−/− thymocytes. Elevated serum PRL reduced this effect. Thymocytes from DEX-treated PRL−/− mice exhibited increased caspase-3 activation, which was inhibited in cells from PRL−/−Graft mice. Finally, elevated expression of X-linked inhibitor of apoptosis, XIAP, was observed in thymi from DEX-treated PRL −/−Graft mice. This is the first demonstration that elevated PRL antagonizes apoptosis in thymocytes exposed to GCs in vivo. These observations suggest that, under conditions of increased GCs, such as during stress, elevated PRL functions physiologically to maintain survival and function of T-lymphocytes.

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