scholarly journals Luteolin Induces Apoptosis and Autophagy in Mouse Macrophage ANA-1 Cells via the Bcl-2 Pathway

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Yuexia Liao ◽  
Yang Xu ◽  
Mengyao Cao ◽  
Yuanyuan Huan ◽  
Lei Zhu ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Inflammatory Diseases ◽  
Flow Cytometric Analysis ◽  
Beclin 1 ◽  
Caspase 8 ◽  
Mouse Macrophage ◽  
Expression Levels ◽  
Traditional Medicines ◽  
Flow Cytometric ◽  
Induced Apoptosis

Plants rich in luteolin have been used as Chinese traditional medicines for inflammatory diseases, hypertension, and cancer. However, little is known about the effect of luteolin on the apoptosis or autophagy of the macrophages. In this study, mouse macrophage ANA-1 cells were incubated with different concentrations of luteolin. The viability of the cells was determined by an MTT assay, apoptosis was determined by flow cytometric analysis, the level of cell autophagy was observed by confocal microscopy, and the expression levels of apoptotic or autophagic and antiapoptotic or antiautophagic proteins were detected by Western blot analysis. The results showed that luteolin decreased the viability of ANA-1 cells and induced apoptosis and autophagy. Luteolin induced apoptosis accompanied by downregulation of the expression of Bcl-2 and upregulation of the expression of caspase 3 and caspase 8. And luteolin increased FITC-LC3 punctate fluorescence accompanied by the increased expression levels of LC3-I, ATG7, and ATG12, while it suppressed the expression level of Beclin-1. Luteolin treatment resulted in obvious activation of the p38, JNK, and Akt signaling pathways, which is important in modulating apoptosis and autophagy. Thus, we concluded that luteolin induced the apoptosis and autophagy of ANA-1 cells most likely by regulating the p38, JNK, and Akt pathways, inhibiting the activity of Bcl-2 and Beclin-1 and upregulating caspase 3 and caspase 8 expression. These results provide novel insights into a therapeutic strategy to prevent and possibly treat macrophage-related diseases through luteolin-induced apoptosis and autophagy.

scholarly journals Flow Cytometry Reveals the Nature of Oncotic Cells

2019 ◽  
Vol 20 (18) ◽  
pp. 4379
Author(s):  
Anna Vossenkamper ◽  
Gary Warnes
Keyword(s):  
Cell Death ◽  
Sodium Azide ◽  
Caspase 3 ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
Regulated Cell Death ◽  
Damage Response ◽  
Induced Apoptosis ◽  
Apoptosis And Necrosis ◽  
Specific Pathway

The term necrosis is commonly applied to cells that have died via a non-specific pathway or mechanism but strictly is the description of the degradation processes involved once the plasma membrane of the cell has lost integrity. The signalling pathways potentially involved in accidental cell death (ACD) or oncosis are under-studied. In this study, the flow cytometric analysis of the intracellular antigens involved in regulated cell death (RCD) revealed the phenotypic nature of cells undergoing oncosis or necrosis. Sodium azide induced oncosis but also classic apoptosis, which was blocked by zVAD (z-Vla-Ala-Asp(OMe)-fluoromethylketone). Oncotic cells were found to be viability+ve/caspase-3–ve/RIP3+ve/–ve (Receptor-interacting serine/threonine protein kinase 3). These two cell populations also displayed a DNA damage response (DDR) phenotype pH2AX+ve/PARP–ve, cleaved PARP induced caspase independent apoptosis H2AX–ve/PARP+ve and hyper-activation or parthanatos H2AX+ve/PARP+ve. Oncotic cells with phenotype cell viability+ve/RIP3–ve/caspase-3–ve showed increased DDR and parthanatos. Necrostatin-1 down-regulated DDR in oncotic cells and increased sodium azide induced apoptosis. This flow cytometric approach to cell death research highlights the link between ACD and the RCD processes of programmed apoptosis and necrosis.

scholarly journals Different expression levels of TNF receptors on the rheumatoid synovial macrophages derived from surgery and a synovectomy as detected by a new flow cytometric analysis

2009 ◽  
Vol 60 (1-3) ◽  
pp. 161-164 ◽  
Author(s):  
Hiroaki Ida ◽  
Toshiyuki Aramaki ◽  
Hideki Nakamura ◽  
Keita Fujikawa ◽  
Kazuhiko Arima ◽  
...  
Keyword(s):  
Flow Cytometric Analysis ◽  
Tnf Receptors ◽  
Expression Levels ◽  
Flow Cytometric

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.

Brucea javanica Oil Induces Apoptosis in T24 Bladder Cancer Cells via Upregulation of Caspase-3, Caspase-9, and Inhibition of NF-κB and COX-2 Expressions

2010 ◽  
Vol 38 (03) ◽  
pp. 613-624 ◽  
Author(s):  
Guo-Guang Lou ◽  
Hang-Ping Yao ◽  
Li-Ping Xie
Keyword(s):  
Caspase 3 ◽  
Flow Cytometric Analysis ◽  
Morphological Characteristics ◽  
Control Group ◽  
Caspase 9 ◽  
Brucea Javanica ◽  
Flow Cytometric ◽  
T24 Cells ◽  
Bladder Cancer Cells ◽  
Cox 2

The potential molecular mechanism of Brucea javanica oil in the induction of apoptosis of T24 bladder cancer cells was investigated in vitro. T24 cells were divided into two groups: one, treated with B. javanica oil and the other, untreated. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and 4 mM glutamine. The morphological characteristics of T24 cells were examined microscopically at the 2nd and 5th day of the culture. The drug toxicity spectrum ( IC 50) was estimated by the MTT assay, and viability of T24 cells was assessed on the basis of the percentage of T24 apoptotic cells, as determined by Annexin/PI staining and flow cytometric analysis. The expression of caspase-3, capase-9, NF-κB p65, and COX-2 was analyzed by Western blotting. Morphological characteristics of the cells on the 2nd day showed apoptosis of the treated T24 cells; it was more apparent in the cells on the 5th day. B. javanica oil decreased the cell viability at the testing concentrations spectrum (5–0.156 mg/ml), and this viability was significantly higher as compared to the control group. In this concentration spectrum, B. javanica oil also induced apoptosis of T24 cells, which was analyzed by annexin/PI staining and flow cytometric analysis. These results were also statistically significant as compared to those of the control group. The expressions of caspase-3 and caspase-9 were low in the control T24 cells, while the expressions of NF-κB and COX-2 were high in normal T24 cells. Treatment with B. javanica oil significantly induced the expressions of caspase-3 and caspase-9 proteins in T24 cells, whereas the expressions of NF-κB and COX-2 proteins were inhibited. B. javanica oil significantly reduced the viability of T24 cells and induced T24 cell apoptosis. The molecular mechanism underlying these effects may be the activation of caspase apoptotic pathway by upregulation of the expression of caspase-3 and caspase-9 proteins and inhibition of the expression of NF-κB and COX-2 proteins.

scholarly journals Protective Effects of Shenfuyixin Granule on H2O2-Induced Apoptosis in Neonatal Rat Cardiomyocytes

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Xinlu Wang ◽  
Xuanxuan Hao ◽  
Youping Wang ◽  
Bin Li ◽  
Lin Cui ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Caspase 3 ◽  
Neonatal Rat ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Rat Cardiomyocytes ◽  
Expression Levels ◽  
Apoptosis Rate ◽  
Neonatal Rat Cardiomyocytes ◽  
Mitochondrion Membrane Potential

Shenfuyixin granule (SFYXG, i.e., Xinshuaikang granule) is a prescription, commonly used in the clinical experience, which plays a significant role in the treatment of heart failure. The purpose of this present research was to investigate the protective effect of SFYXG, and the mechanism about anti-H2O2-induced oxidative stress and apoptosis in the neonatal rat cardiomyocytes. Myocardial cells, as is well known, were divided into 4 groups: normal, model, SFYXG, and coenzyme Q10 group, respectively. Cells viability was determined by MTT assay. Flow cytometry and AO/EB staining were implemented to test the apoptosis rate and intracellular reactive oxygen species (ROS) level. Mitochondrion membrane potential (MMP) was evaluated by JC-1 fluorescence probe method. The myocardial ultrastructure of mitochondrion was measured by electron microscope. The related mRNA expression levels of Bax, Bcl-2, Caspase-3, caspase-8, and caspase-9 were detected by real-time polymerase chain reaction (PCR). Also, the expression levels of Bax and Bcl-2 protein were detected by Western blot, and the expression levels of caspase-3, caspase-8, and caspase-9 protein were tested by caspase-Glo®3 Assay, caspase-Glo®8 Assay, and caspase-Glo®9 Assay, respectively. GAPDH was used as the internal reference gene/protein. The results revealed that SFYXG (0.5 mg/ml) raised the viability of myocardial cell, weakened the apoptosis rate and ROS level, corrected the mitochondrion membrane potential stability, and improved cell morphology and ultrastructure of myocardial mitochondrion. Furthermore, SFYXG upregulated the antiapoptosis gene of Bcl-2, but downregulated the proapoptosis genes of Bax, caspase-3, and caspase-9. In conclusion, SFYXG could appear to attenuate myocardial injury by its antioxidative and antiapoptosis effect.

scholarly journals Expression levels of caspase-3 and gasdermin E and their involvement in the occurrence and prognosis of lung cancer

2021 ◽  
Author(s):  
yuanli huang ◽  
GuangHui Zhang ◽  
Qing Zhu ◽  
Xia Wu ◽  
LIGao Wu
Keyword(s):  
Lung Cancer ◽  
Caspase 3 ◽  
Clinical Stage ◽  
The Body ◽  
Caspase 8 ◽  
Survival Prognosis ◽  
Expression Levels ◽  
Protein Levels ◽  
Normal Tissues ◽  
Related Proteins

Abstract Background Pyroptosis plays a dual role in the development of cancer and malignancy, and as such, may potentially be a new target for cancer treatment. However, the inflammatory response to pyroptosis may have adverse effects on the body. The roles of gasdermin E (GSDME), caspases, and related proteins associated with pyroptosis in cancer remain controversial. This study aimed to explore whether the expression levels of caspase-3 and GSDME affect the clinical stage, pathological grade, and survival prognosis of patients with lung cancer. Methods We examined the protein levels of GSDME, caspase-3, caspase-8, and caspase-9 in lung tissues from 100 patients with lung cancer by using immunohistochemistry. Results We found that GSDME, caspase-3, and caspase-8 were more highly expressed in the tumor tissues than in the adjacent normal tissues. Moreover, we found that GSDME could serve as a prognostic factor because there was a positive correlation between its expression level and the postoperative survival rate of patients with lung cancer. Conclusions GSDME may be an independent factor affecting the prognosis of patients with lung cancer. However, the role of GSDME and its related proteins in cancer requires further research.

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.

SMAC-Mimetic BV-6 Sensitizes Therapeutic Agents-Induced Apoptosis In AML Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2177-2177
Author(s):  
Duncan H Mak ◽  
Christa Manton ◽  
Michael Andreeff ◽  
Bing Z Carter
Keyword(s):  
Cell Death ◽  
Vector Control ◽  
Caspase 3 ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Smac Mimetic ◽  
P53 Activation ◽  
Induced Apoptosis

Abstract Abstract 2177 The antiapoptotic function of the inhibitors of apoptosis family of proteins (IAPs) is antagonized by mitochondria-released SMAC protein. The IAP-member XIAP suppresses apoptosis by directly binding and inhibiting caspase-9 and caspase-3, while cIAP1, a component of the cytoplasmic signaling complex containing TNF receptor associated factors, suppresses apoptosis via the caspase-8-mediated pathway. BV-6 (Genentech) is a bivalent SMAC-mimetic and has been shown to promote cell death by inducing cIAP autoubiquitination, NF-κB activation, and TNFα-dependent apoptosis. We examined its effect on leukemic cells and found that BV-6 only moderately induced apoptosis. The EC50 was found to be 15.3±5.1 μM at 48 hours in OCI-AML3 cells which are relatively sensitive. We then determined whether BV-6 sensitizes leukemic cells to the HDM2-inhibitor nutlin-3a and to Ara-C. p53 modulates the expression and activity of Bcl-2 family proteins and promotes the mitochondrial-mediated apoptosis. We showed previously that activation of p53 by nutlin-3a sensitizes AML cells to XIAP inhibition induced-death in part by promoting the release of SMAC from mitochondrion (Carter BZ et al., Blood 2010). We treated OCI-AML3 cells with BV-6, nutlin-3a or Ara-C, and BV-6+nutlin-3a or BV-6+Ara-C and found that the combination of BV-6 and nutlin-3a or BV-6 and Ara-C synergistically induced cell death in OCI-AML3 cells with a combination index (CI) of 0.27±0.11 and 0.22±0.05 (48 hours), respectively. To demonstrate that p53 activation is essential for the synergism of BV-6+nutlin-3a combination, we treated OCI-AML3 vector control and p53 knockdown cells with these two agents and found that the combination synergistically promoted cell death in the vector control (CI=0.47±0.15) but not in the p53 knockdown cells, as expected, while BV6+Ara-C was synergistic in both vector control and p53 knockdown cells (CI=0.15±0.03 and 0.08±0.03, respectively, 48 hours). BV-6 induced activation of caspase-8, caspase-9, and caspase-3 and decreased XIAP levels, but did not cause rapid cIAP1 degradation, as reported by others. To assess the contribution of death receptor-mediated apoptosis in BV-6-induced cell death, we treated Jurkat and caspase-8 mutated Jurkat cells (JurkatI9.2) with BV-6 and found that BV-6 induced cell death and significantly potentiated TRAIL-induced apoptosis in Jurkat cells (CI=0.14±0.08, 48 hours). Caspase-8 mutated JurkatI9.2 cells were significantly less sensitive to BV-6 than Jurkat cells and as expected, JurkatI9.2 was completely resistant to TRAIL. Collectively, we showed that the bivalent SMAC-mimetic BV-6 potentiates p53 activation-, chemotherapy-, and TRAIL-induced cell death, but has only minimal activity by itself in leukemic cells. SMAC-mimetics could be useful in enhancing the efficacy of different classes of therapeutic agents used in AML therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Exendin-4 Protects MIN6 Cells from t-BHP-Induced Apoptosis via IRE1-JNK-Caspase-3 Signaling

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Wen-Jia Chen ◽  
Lin-Xi Wang ◽  
Yan-Ping Wang ◽  
Zhou Chen ◽  
Xiao-Ying Liu ◽  
...  
Keyword(s):  
Cell Apoptosis ◽  
Caspase 3 ◽  
Transmembrane Protein ◽  
Annexin V ◽  
Flow Cytometric Assay ◽  
Flow Cytometric ◽  
Tert Butyl Hydroperoxide ◽  
Microscopy Analysis ◽  
Induced Apoptosis ◽  
Fluorescence Microscopy Analysis

Objectives.This study aimed to explore the effect of exendin-4 on t-BHP-induced apoptosis in pancreaticβcells and the mechanism of action.Methods.Murine MIN6 pancreaticβcells were treated with exendin-4 in the presence or absence of tert-butyl hydroperoxide (t-BHP). Cell survival was assessed by MTT staining. The percentage of apoptotic cells was determined by fluorescence microscopy analysis after Hoechst/PI staining and flow cytometric assay after Annexin V-FITC/PI staining. The activity of caspase-3 was determined using a caspase-3 activity kit. Expression of P-IRE1α, IRE1α, C-Jun N-terminal kinase (JNK), P-JNK, C-JUN, and P-C-JUN was detected by western blotting.Results.Exendin-4 was found to inhibit t-BHP-induced apoptosis in pancreaticβ-cells by downregulating caspase-3 activity. Exendin-4 also inhibited the endoplasmic reticulum transmembrane protein IRE1, the apoptosis-related signaling molecule JNK, and c-Jun activation.Conclusions.Our findings suggest that exendin-4 ultimately reduces t-BHP-inducedβ-cell apoptosis. IRE1-JNK-c-Jun signaling is involved in the exendin-4-mediated modulation ofβ-cell apoptosis.

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