scholarly journals Novel Subtilase Cytotoxin Produced by Shiga-Toxigenic Escherichia coli Induces Apoptosis in Vero Cells via Mitochondrial Membrane Damage

2009 ◽  
Vol 77 (7) ◽  
pp. 2919-2924 ◽  
Author(s):  
Gen Matsuura ◽  
Naoko Morinaga ◽  
Kinnosuke Yahiro ◽  
Reiko Komine ◽  
Joel Moss ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Membrane Damage ◽  
Annexin V ◽  
Vero Cells ◽  
Caspase 8 ◽  
Caspase Inhibitors ◽  
Induced Apoptosis ◽  
Subtilase Cytotoxin

ABSTRACT Subtilase cytotoxin (SubAB) is an AB5 cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G1 phase. Here we show that SubAB, but not the catalytically inactive mutant SubAB(S272A), induced apoptosis in Vero cells, as detected by DNA fragmentation and annexin V binding. SubAB induced activation of caspase-3, -7, and -8. Caspase-3 appeared earlier than caspase-8, and by use of specific caspase inhibitors, it was determined that caspase-3 may be upstream of caspase-8. A general caspase inhibitor blocked SubAB-induced apoptosis, detected by annexin V binding. SubAB also stimulated cytochrome c release from mitochondria, which was not suppressed by caspase inhibitors. In HeLa cells, Apaf-1 small interfering RNA inhibited caspase-3 activation, suggesting that cytochrome c might form an apoptosome, leading to activation of caspase-3. These data suggested that SubAB induced caspase-dependent apoptosis in Vero cells through mitochondrial membrane damage.

scholarly journals Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

2015 ◽  
Vol 83 (6) ◽  
pp. 2338-2349 ◽  
Author(s):  
J. Funk ◽  
N. Biber ◽  
M. Schneider ◽  
E. Hauser ◽  
S. Enzenmüller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Vero Cells ◽  
Toxic Activity ◽  
Content Type ◽  
B Subunits ◽  
Induced Apoptosis ◽  
The Individual ◽  
Apoptotic Effects ◽  
Subtilase Cytotoxin

In this study, the cytotoxicity of the recently described subtilase variant SubAB2-2of Shiga toxin-producingEscherichia coliwas determined and compared to the plasmid-encoded SubAB1and the chromosome-encoded SubAB2-1variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD50) differ for the individual variants. Highest cytotoxicity was shown for SubAB1. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA1exhibited cytotoxic effects in the absence of the respective B1subunit. A more detailed investigation in the human HeLa cell line revealed that SubA1alone induced apoptosis, while the B1subunit alone did not induce cell death.

scholarly journals Autoregulatory Feedback Mechanism of P38MAPK/Caspase-8 in Photodynamic Therapy-Hydrophilic/Lipophilic Tetra-α-(4-carboxyphenoxy) Phthalocyanine Zinc-Induced Apoptosis of Human Hepatocellular Carcinoma Bel-7402 Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Yu Wang ◽  
Chunhui Xia ◽  
Wei Chen ◽  
Yuhang Chen ◽  
Yiyi Wang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Photodynamic Therapy ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Human Hepatocellular Carcinoma ◽  
Caspase 8 ◽  
Induced Apoptosis

Photodynamic therapy (PDT) is a novel and promising antitumor treatment. Our previous study showed that hydrophilic/lipophilic tetra-α-(4-carboxyphenoxy) phthalocyanine zinc- (TαPcZn-) mediated PDT (TαPcZn-PDT) inhibits the proliferation of human hepatocellular carcinoma Bel-7402 cells by triggering apoptosis and arresting cell cycle. However, mechanisms of TαPcZn-PDT-induced apoptosis of Bel-7402 cells have not been fully clarified. In the present study, therefore, effect of TαPcZn-PDT on apoptosis, P38MAPK, p-P38MAPK, Caspase-8, Caspase-3, Bcl-2, Bid, Cytochrome c, and mitochondria membrane potential in Bel-7402 cells without or with P38MAPK inhibitor SB203580 or Caspase-8 inhibitor Ac-IEFD-CHO was investigated by haematoxylin and eosin (HE) staining assay, flow cytometry analysis of annexin V-FITC/propidium iodide (PI) double staining cells and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and immunoblot assay. We found that TαPcZn-PDT resulted in apoptosis induction, activation of P38MAPK, Caspase-8, Caspase-3, and Bid, downregulation of Bcl-2, release of Cytochrome c from mitochondria, and disruption of mitochondrial membrane potential in TαPcZn-PDT-treated Bel-7402 cells. In contrast, SB203580 or Ac-IEFD-CHO attenuated induction of apoptosis, activation of P38MAPK, Caspase-8, Caspase-3, and Bid, downregulation of Bcl-2, release of Cytochrome c from mitochondria, and disruption of mitochondrial membrane potential in TαPcZn-PDT-treated Bel-7402 cells. Taken together, we conclude that Caspase-3, Bcl-2, Bid, and mitochondria are involved in autoregulatory feedback of P38MAPK/Caspase-8 during TαPcZn-PDT-induced apoptosis of Bel-7402 cells.

scholarly journals Esculetin induces apoptosis of SMMC-7721 cells through IGF-1/PI3K/Akt-mediated mitochondrial pathways

2017 ◽  
Vol 95 (7) ◽  
pp. 787-794 ◽  
Author(s):  
Juan Li ◽  
Shuang Li ◽  
Xiuli Wang ◽  
Hongxin Wang
Keyword(s):  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Annexin V ◽  
Coumarin Derivative ◽  
Caspase 9 ◽  
Cytochrome C Release ◽  
Dapi Staining ◽  
Cancer Cell Growth ◽  
Induced Apoptosis ◽  
Apoptotic Effects

Esculetin (6,7-dihydroxycoumarin) is a coumarin derivative extracted from natural plants and has been reported to have anticancer activity. However, the mechanism by which esculetin prevents human hepatic cancer cell growth is still largely unknown. In this study, we investigated the effect of esculetin on human hepatocellular carcinoma (HCC) SMMC-7721 cells and explored the cell signal mechanism. Our data indicated that esculetin induced apoptosis in SMMC-7721 cells, which were supported by DAPI staining and Annexin V/PI staining. Meanwhile, esculetin increased the activities of caspase-3 and caspase-9, promoted bax expression, decreased bcl-2 expression, and triggered collapse of mitochondrial membrane potential, and increased cytochrome c release from mitochondria. In addition, the inactivation of IGF-1, PI3K, and Akt was observed after esculetin administration. Furthermore, pretreatment with IGF-1 before esculetin administration abrogated the pro-apoptotic effects of esculetin, while PI3K inhibitor increased the pro-apoptotic effects of esculetin. These results indicated that esculetin induced the apoptosis of SMMC-7721 cells through IGF-1/PI3K/Akt-regulated mitochondrial dysfunction.

Vitamin C inhibits FAS-induced apoptosis in monocytes and U937 cells

Blood ◽  
2003 ◽  
Vol 102 (1) ◽  
pp. 336-343 ◽  
Author(s):  
Isabel Perez-Cruz ◽  
Juan M. Carcamo ◽  
David W. Golde
Keyword(s):  
Immune System ◽  
Vitamin C ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Fas Ligand ◽  
Membrane Integrity ◽  
Caspase 8 ◽  
Human Monocytes ◽  
Fas Receptor ◽  
Induced Apoptosis

Abstract The FAS receptor—FAS ligand system is a key apoptotic pathway for cells of the immune system. Ligation of the FAS-receptor (CD95) induces apoptosis by activation of pro—caspase-8 followed by downstream events, including an increase in reactive oxygen species (ROS) and the release of proapoptotic factors from the mitochondria, leading to caspase-3 activation. We investigated the role of vitamin C in FAS-mediated apoptosis and found that intracellular accumulation of pharmacologic concentrations of vitamin C inhibited FAS-induced apoptosis in the monocytic U937 cell line and in fresh human monocytes. Cells were loaded with vitamin C by exposure to dehydroascorbic acid (DHA), thereby circumventing in vitro artifacts associated with the poor transport and pro-oxidant effects of ascorbic acid (AA). Vitamin C inhibition of FAS-mediated apoptosis was associated with reduced activity of caspase-3, -8, and -10, as well as diminished levels of ROS and preservation of mitochondrial membrane integrity. Mechanistic studies indicated that the major effect of vitamin C was inhibition of the activation of caspase-8 with no effect on it enzymatic activity. An independent action of high intracellular concentrations of vitamin C on mitochondrial membrane stabilization was also detected. These studies illuminate the nature of redox-dependent signaling in FAS-induced apoptosis of human monocytes and suggest that vitamin C can modulate the immune system by inhibiting FAS-induced monocyte death. (Blood. 2003;102:336-343)

scholarly journals Protective Effect of Thrombopoietin on CoCl2-Induced Apoptosis of HUVEC Cells through PI3-k/Akt Pathways

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4969-4969 ◽  
Author(s):  
Jieyu Ye ◽  
Jun-yan Wang ◽  
En-yu Liang ◽  
Mo Yang
Keyword(s):  
Protective Effect ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Annexin V ◽  
Akt Pathway ◽  
Human Endothelial Cells ◽  
Apoptotic Effect ◽  
Huvec Cells ◽  
Induced Apoptosis ◽  
Active Caspase

Abstract Endothelial damage is a serious syndrome following by stem cell transplantation. However, a useful treatment for attenuating the periods of this disorder was still under explored. Our previous studies have demonstrated that in patients with acute inflammation states, serum thrombopoietin (TPO) levels were significant higher compared with healthy subjects (181.11 ± 35.38 vs 96.13 ± 9.7 pg/ml, p<0.001), which may act as an acute response protein to protect the body. TPO is now recognized as a potent proliferative factor for outer hematopoietic system, such as cardiomyocytes and neurocytes. This study is aim to investigate the protective effect of TPO on human endothelial cells, and to explore its potential mechanism. Four experimental groups were set up using human umbilical vein endothelial (HUVEC) cells, which are: Normal control, CoCl2 alone (800 μmol/L), TPO(100 μg/L) + CoCl2 and TPO alone groups. CoCl2 alone decreased the viability of HUVEC cells in a dose dependent manner (r=-0.997), while addition of TPO significantly rescued this effect (n=5, p<0.01). We further tested the anti-apoptotic effect of TPO on HUVEC cells. CoCl2 alone was able to induced apoptosis as demonstrated by Annexin V (n=4, p<0.001), active-caspase-3 (n=4, p<0.01) and Mitochondrial Membrane potential (JC-1) assays (n=4, p<0.01). Consistently, TPO plus CoCl2 showed a significant anti-apoptotic effect compared with CoCl2 alone group in Annexin V (n=4, p<0.01), active-caspase-3 (n=4, p=0.039) and Mitochondrial Membrane potential (JC-1) assays (n=4, p=0.038). In order to find out the underlying mechanism, we further tested the expression of phospho-Akt (p-Akt). Our results demonstrated that CoCl2 alone suppressed the PI-3k/Akt pathway. TPO plus CoCl2 was shown to activate the expression of p-Akt by western blot. In summary, our findings showed that TPO had a protective effect on human endothelial cells. This effect was likely mediated by activation of PI-3k/Akt pathway, which leads to anti-apoptosis on these cells. This may provide us a new clue to identify novel strategy in treating endothelial syndromes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Shikonin Inhibits Cholangiocarcinoma Cell Line QBC939 by Regulating Apoptosis, Proliferation, and Invasion

2021 ◽  
Vol 30 ◽  
pp. 096368972097916
Author(s):  
Chang Liu ◽  
Li-Qian Xuan ◽  
Kai Li ◽  
Zhuo Feng ◽  
Chan Lv ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Growth Factor Receptor ◽  
Annexin V ◽  
Caspase 8 ◽  
Dependent Manner ◽  
Invasion Assay ◽  
Egfr Expression ◽  
Transwell Invasion Assay ◽  
Induced Apoptosis ◽  
Proliferation And Invasion

This study was designed to clarify whether Shikonin causes proliferation, apoptosis, and invasion in cholangiocarcinoma cells and to investigate the mechanism of action. QBC939 cells were cultured with different doses of Shikonin, and then 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium assay was used to detect cell viability. Apoptosis of cells was detected using flow cytometry with Annexin V/propidium iodide (PI) assay after being stained with Hoechst 33242. The role of Shikonin on the invasive and metastasis ability was detected using Transwell invasion assay. Real-time polymerase chain reaction and Western blotting were used to detect the expression of caspase-3, caspase-8, epidermal growth factor receptor (EGFR), and matrix metalloproteinase (MMP)-9. Shikonin inhibited proliferation and invasive ability of QBC939 cells in a dose-dependent manner; at the same time, apoptosis of cells was also observed in a concentration-dependent fashion. Moreover, Annexin V/PI assay and Transwell invasion assay results indicated that Shikonin induced apoptosis and invasion inhibitory probably due to upregulation of caspase-3 and caspase-8 expression and downregulation of MMP-9 and EGFR expression in a concentration-dependent fashion. Shikonin could enhance apoptosis and inhibit proliferation and invasion of QBC939 cells; such biological behaviors mainly occurred via upregulating the expression of caspase-3 and caspase-8 and downregulating the expression of MMP-9 and EGFR.

scholarly journals Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis

2016 ◽  
Vol 291 (22) ◽  
pp. 11843-11851 ◽  
Author(s):  
Kai Huang ◽  
Jingjing Zhang ◽  
Katelyn L. O'Neill ◽  
Channabasavaiah B. Gurumurthy ◽  
Rolen M. Quadros ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Caspase 8 ◽  
Membrane Association ◽  
Induced Apoptosis

scholarly journals Inclusion Complex of Zerumbone with Hydroxypropyl-β-Cyclodextrin Induces Apoptosis in Liver Hepatocellular HepG2 Cells via Caspase 8/BID Cleavage Switch and Modulating Bcl2/Bax Ratio

2013 ◽  
Vol 2013 ◽  
pp. 1-16 ◽  
Author(s):  
Nabilah Muhammad Nadzri ◽  
Ahmad Bustamam Abdul ◽  
Mohd Aspollah Sukari ◽  
Siddig Ibrahim Abdelwahab ◽  
Eltayeb E. M. Eid ◽  
...  
Keyword(s):  
Inclusion Complex ◽  
Mitochondrial Membrane ◽  
Morphological Study ◽  
Caspase 3 ◽  
Anticancer Agent ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Solubility In Water ◽  
Anticancer Effects ◽  
First Time

Zerumbone (ZER) isolated fromZingiber zerumbetwas previously encapsulated with hydroxypropyl-β-cyclodextrin (HPβCD) to enhance ZER’s solubility in water, thus making it highly tolerable in the human body. The anticancer effects of this new ZER-HPβCD inclusion complex via apoptosis cell death were assessed in this study for the first time in liver hepatocellular cells, HepG2. Apoptosis was ascertained by morphological study, nuclear stain, and sub-G1 cell population accumulation with G2/M arrest. Further investigations showed the release of cytochrome c and loss of mitochondrial membrane potential, proving mitochondrial dysfunction upon the ZER-HPβCD treatment as well as modulating proapoptotic and anti-apototic Bcl-2 family members. A significant increase in caspase 3/7, caspase 9, and caspase 8 was detected with the depletion of BID cleaved by caspase 8. Collectively, these results prove that a highly soluble inclusion complex of ZER-HPβCD could be a promising anticancer agent for the treatment of hepatocellular carcinoma in humans.

scholarly journals Azithromycin Promotes the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells after Stimulation with TNF-α

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Tingting Meng ◽  
Ying Zhou ◽  
Jingkun Li ◽  
Meilin Hu ◽  
Xiaomeng Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Alkaline Phosphatase Activity ◽  
Periodontal Ligament ◽  
Caspase 3 ◽  
Caspase 8 ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Induced Apoptosis

Background and Objective. This study investigated the effects and underlying mechanisms of azithromycin (AZM) treatment on the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) after their stimulation with TNF-α in vitro. Methods. PDLSCs were isolated from periodontal ligaments from extracted teeth, and MTS assay was used to evaluate whether AZM and TNF-α had toxic effects on PDLSCs viability and proliferation. After stimulating PDLSCs with TNF-α and AZM, we analyzed alkaline phosphatase staining, alkaline phosphatase activity, and alizarin red staining to detect osteogenic differentiation. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed to detect the mRNA expression of osteogenic-related genes, including RUNX2, OCN, and BSP. Western blotting was used to measure the NF-κB signaling pathway proteins p65, phosphorylated p65, IκB-α, phosphorylated IκB-α, and β-catenin as well as the apoptosis-related proteins caspase-8 and caspase-3. Annexin V assay was used to detect PDLSCs apoptosis. Results. TNF-α stimulation of PDLSCs decreased alkaline phosphatase and alizarin red staining, alkaline phosphatase activity, and mRNA expression of RUNX2, OCN, and BSP in osteogenic-conditioned medium. AZM enhanced the osteogenic differentiation of PDLSCs that were stimulated with TNF-α. Western blot analysis showed that β-catenin, phosphorated p65, and phosphorylated IκB-α protein expression decreased in PDLSCs treated with AZM. In addition, pretreatment of PDLSCs with AZM (10 μg/ml, 20 μg/ml) prevented TNF-α-induced apoptosis by decreasing caspase-8 and caspase-3 expression. Conclusions. Our results showed that AZM promotes PDLSCs osteogenic differentiation in an inflammatory microenvironment by inhibiting the WNT and NF-κB signaling pathways and by suppressing TNF-α-induced apoptosis. This suggests that AZM has potential as a clinical therapeutic for periodontitis.

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

2005 ◽  
Vol 102 (6) ◽  
pp. 1147-1157 ◽  
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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