Cell apoptosis and necrosis studied by flow cytometry

Background: The search for novel metallic chemical compounds with toxicogenic effects have been of great importance for more efficient cancer treatment. Objective: The study evaluated the cytotoxic, genotoxic and mutagenic activity of organoteluran RF07 in S-180 cell line. Methods: The bioassays used were cell viability with 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test, evaluation of apoptosis and necrosis using fluorescence and flow cytometry, cytokinesis-block micronucleus test and comet assay. The compound was tested at 1; 2.5 and 5 µM. Results: The results showed the cytotoxicity of RF07 at concentrations of 2.5, 5, 10 and 20 µM when compared to the negative control. For genotoxicity tests, RF07 showed effects in all concentrations assessed by increased index and frequencies of damage and mutagenic alterations. The compound was also cytotoxic due to the significant decrease in nuclear division index, with significant values of apoptosis and necrosis. The results of fluorescence and flow cytometry showed apoptosis as the main type of cell death caused by RF07 at 5 µM, which is thought to avoid an aggressive immune response of the organism. Conclusion: In addition to cytotoxic and genotoxic effects, RF07 creates good perspectives for future antitumor formulations.

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Rsf-1 Influences the Sensitivity of Non-Small Cell Lung Cancer to Paclitaxel by Regulating NF-κB Pathway and Its Downstream Proteins

Cellular Physiology and Biochemistry ◽

10.1159/000486116 ◽

2017 ◽

Vol 44 (6) ◽

pp. 2322-2336 ◽

Cited By ~ 18

Author(s):

Xitao Chen ◽

Xiaodi Sun ◽

Jingqian Guan ◽

Junda Gai ◽

Jilin Xing ◽

...

Keyword(s):

Lung Cancer ◽

Flow Cytometry ◽

Cell Proliferation ◽

Small Cell Lung Cancer ◽

Cell Apoptosis ◽

Small Cell ◽

Small Cell Lung ◽

Protein Levels ◽

H460 Cell ◽

Xenograft Mice

Background/Aims: The therapeutic efficacy of paclitaxel is hampered by chemotherapeutic resistance in non-small cell lung cancer (NSCLC). Rsf-1 enhanced paclitaxel resistance via nuclear factor-κB (NF-κB) in ovarian cancer cells and nasopharyngeal carcinoma. This study assessed the function of Rsf-1 in the modulation of the sensitivity of NSCLC to paclitaxel via the NF-κB pathway. Methods: The mRNA and protein levels of the related genes were quantified by RT-PCR and Western blotting. Rsf-1 silencing was achieved with CRISPR/Cas9 gene editing. Cell cycle, migration and proliferation were tested with flow cytometry, transwell test and CCK8 test. Cell apoptosis was analyzed with flow cytometry and quantification of C-capase3. The parameters of the tumors were measured in H460 cell xenograft mice. Results: Rsf-1 was highly expressed in H460 and H1299 cells. Rsf-1 knockout caused cell arrest at the G1 phase, increased cell apoptosis, and decreased migration and cell proliferation. Rsf-1 knockout increased the inhibition of cell proliferation, the reduction in cell migration and the augment in cell apoptosis in paclitaxel treated H460 and H1299 cells. Rsf-1 knockout further enhanced the paclitaxel-mediated decrease in the volume and weight of the tumors in H460 cell xenograft mice. Helenalin and Rsf-1 knockout decreased the protein levels of p-P65, BcL2, CFLAR, and XIAP; hSNF2H knockout decreased the protein level of NF-κB p-P65 without altering Rsf-1 and p65 protein levels, while Rsf-1 and hSNF2H double knockout decreased the level of NF-κB p-P65, in H1299 and H460 cells. Conclusion: These results demonstrate that Rsf-1 influences the sensitivity of NSCLC to paclitaxel via regulation of the NF-κB pathway and its downstream genes.

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Feasibility study of stain-free classification of cell apoptosis based on diffraction imaging flow cytometry and supervised machine learning techniques

APOPTOSIS ◽

10.1007/s10495-018-1454-y ◽

2018 ◽

Vol 23 (5-6) ◽

pp. 290-298 ◽

Cited By ~ 4

Author(s):

Jingwen Feng ◽

Tong Feng ◽

Chengwen Yang ◽

Wei Wang ◽

Yu Sa ◽

...

Keyword(s):

Machine Learning ◽

Flow Cytometry ◽

Cell Apoptosis ◽

Supervised Machine Learning ◽

Machine Learning Techniques ◽

Imaging Flow Cytometry ◽

Learning Techniques ◽

Diffraction Imaging ◽

Free Classification

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Overexpression of TEL-MN1 Fusion Enhances Resistance of HL-60 Cells to Idarubicin

Chemotherapy ◽

10.1159/000495073 ◽

2018 ◽

Vol 63 (6) ◽

pp. 308-314 ◽

Cited By ~ 1

Author(s):

Shenglan Gong ◽

Mengqiao Guo ◽

Gusheng Tang ◽

Jianmin Yang ◽

Huiying Qiu

Keyword(s):

Flow Cytometry ◽

Drug Resistance ◽

Cell Viability ◽

Cell Apoptosis ◽

Chromosome Abnormality ◽

Chemotherapy Resistance ◽

Retroviral Vector ◽

Promote Cell Proliferation ◽

Enhanced Resistance

Background: The translocation t(12; 22) (p13;q12) is a recurrent but infrequent chromosome abnormality in human myeloid malignancies. To date, the role of TEL-MN1 fusion in leukemogenic process and drug resistance is still largely unknown. Methods: In the present study, the TEL-MN1 fusion was transfected into HL-60 cells to upregulate TEL-MN1 expression via a retroviral vector. MTT assay was employed to examine cell viability and flow cytometry was performed to evaluate cell apoptosis. Idarubicin was used to treat HL-60 cells for estimating the effect of TEL-MN1 fusion on the chemotherapy resistance. Results: The results showed that overexpression of TEL-MN1 in HL-60 cells could promote cell proliferation, suggesting that TEL-MN1 may be involved in the leukemogenesis process. HL-60 cells treated with idarubicin showed a weakened cell viability, whereas TEL-MN1 overexpression attenuated the idarubicin-induced inhibition of cell viability and acceleration of cell apoptosis of HL-60 cells. Conclusion: Taken together, our results indicated that TEL-MN1 fusion is an oncogene involved in the leukemogenesis process and TEL-MN1 overexpression enhanced resistance of HL-60 cells to idarubicin, which may provide a useful tool for studying the mechanism of leukemogenesis and drug resistance.

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Intracameral Toxicity of Bacterial Components Muramyl Dipeptide and Staurosporine: Ciliary Cyst Formation, Epithelial Cell Apoptosis and Necrosis

Cutaneous and Ocular Toxicology ◽

10.1080/15569520600695538 ◽

2006 ◽

Vol 25 (2) ◽

pp. 85-101 ◽

Cited By ~ 6

Author(s):

Marlyn P. Langford ◽

Dequan Chen ◽

Jeffrey Gosslee ◽

Raghunath P. Misra ◽

Thomas B. Redens ◽

...

Keyword(s):

Epithelial Cell ◽

Cell Apoptosis ◽

Muramyl Dipeptide ◽

Cyst Formation ◽

Epithelial Cell Apoptosis ◽

Apoptosis And Necrosis

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miR-20b-5p attenuates hypoxia-induced apoptosis in cardiomyocytes via the HIF-1α/NF-κB pathway

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmaa056 ◽

2020 ◽

Vol 52 (9) ◽

pp. 927-934 ◽

Cited By ~ 1

Author(s):

Zhongquan Zhou ◽

Songwen Chen ◽

Zhiming Tian ◽

Shibing Deng ◽

Xuying Yi ◽

...

Keyword(s):

Flow Cytometry ◽

Signaling Pathway ◽

Cell Apoptosis ◽

Target Genes ◽

Annexin V ◽

Pcr Analysis ◽

H9c2 Cell ◽

H9c2 Cells ◽

Flow Cytometry Analysis ◽

Low Oxygen

Abstract Chronic hypoxia is a common inducer of end-stage cardiovascular disease. In cells under hypoxia, the hypoxia-inducible factor-1 (HIF-1) plays a vital role in regulating downstream target genes. However, the mechanism of hypoxia in cardiomyocytes is still unclear. In this study, we aimed to identify novel downstream epigenetic targets of HIF-1α in cardiomyocytes under hypoxia. H9c2 cells were exposed to hypoxia condition, and quantitative real-time PCR analysis was performed to evaluate the expression of miR-20b-5p. The results indicated that the expression of miR-20b-5p was down-regulated in H9c2 cells under low oxygen condition. Meanwhile, HIF-1α overexpression further down-regulated the miR-20b-5p expression in H9c2 cells transfected with HIF-1α plasmids. In addition, Annexin-V-FITC/PI flow cytometry analysis suggested that overexpression of miR-20b-5p attenuated cell apoptosis under hypoxia condition in H9c2 cells. Western blot analysis showed that the hypoxia apparently increased Bax and cleaved-caspase-3, but decreased Bcl-2 expression in H9c2 cells, indicating that hypoxia-induced NF-κB signaling pathway activation is mediated by miR-20b-5p. Hypoxia-induced H9c2 cell apoptosis was reduced after HIF-1α knockdown as shown by the flow cytometry analysis. In conclusion, we identified that miR-20b-5p plays an important role in mediating cardiomyocytes apoptosis under hypoxia, which is mediated by the HIF-1/NF-κB signaling pathway.

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Study on the antitumor activity and mechanism of novel targeted nanodrugs based on miRNA in cervical cancer

Materials Express ◽

10.1166/mex.2020.1797 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1218-1223

Author(s):

Xinping Chen ◽

Zhichao Ma ◽

Juan Zhu ◽

Weihua Xu ◽

Junjie Hu ◽

...

Keyword(s):

Gene Expression ◽

Flow Cytometry ◽

Antitumor Activity ◽

A549 Cell ◽

Cell Apoptosis ◽

Caspase 3 ◽

A549 Cells ◽

Dependent Manner ◽

Double Staining ◽

Dose Dependent

The aim of this study was to investigate the effect of different concentrations of novel targeted nanodrugs based on miRNA on the antitumor activity and mechanism in cervical carcinoma A549 cells. The MTT method was used to determine the effect of different concentrations of novel targeted nanodrugs based on miRNA on A549 cell proliferation, and annexin V FITC/PI double staining flow cytometry was performed to analyze the effect of these nanodrugs on A549 cell apoptosis. Western blotting was performed to observe the effect of these nanodrugs on the expression of Bax, Bcl-2, and caspase-3-related genes involved in A549 cell apoptosis. Compared with the control group, the novel targeted nanodrugs based on miRNA significantly inhibited the proliferation of A549 cells in a time- and dose-dependent manner. Results of double staining flow cytometry demonstrated that these nanodrugs could increase the apoptotic rate of A549 cells in a dose-dependent manner 48 h later. Western blotting revealed that these nanodrugs could upregulate the expression of Bax and caspase3 genes and downregulate the expression of Bcl-2 gene. Nanodrugs display an obvious antitumor activity in vitro, and the underlying mechanism may be associated with the upregulation of Bax and caspase-3 gene expression and the downregulation of Bcl-2 gene expression.

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The effects of photodynamic therapy on leukemia cells mediated by KillerRed, a genetically encoded fluorescent protein photosensitizer

BMC Cancer ◽

10.1186/s12885-019-6124-0 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Meng Yuan ◽

Chengcheng Liu ◽

Jiao Li ◽

Wenpeng Ma ◽

Xiaozhuo Yu ◽

...

Keyword(s):

Flow Cytometry ◽

Photodynamic Therapy ◽

Western Blotting ◽

Cell Apoptosis ◽

Fluorescent Protein ◽

Laser Scanning Microscopy ◽

Myelogenous Leukemia ◽

Leukemia Cells ◽

Acute Monocytic Leukemia ◽

Monocytic Leukemia

Abstract Background Leukemia is a cancer of blood and bone marrow cells, causing about 300,000 deaths worldwide. Photodynamic therapy (PDT) is a promising alternative for the treatment of malignant tumors. KillerRed is a genetically encoded red fluorescent protein photosensitizer (PS). In this study, we aimed to investigate the effects of KillerRed-mediated PDT on chronic myelogenous leukemia K562 cells, acute monocytic leukemia NB4 cells, and acute monocytic leukemia THP1 cells. Methods KillerRed was expressed in Escherichia coli cells, purified by Q-Sepharose column, and confirmed by western-blotting. The PDT effect on cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8). Cell apoptosis was determined by PE Annexin V/7-AAD staining and flow cytometry. The distribution of KillerRed in leukemia cells was detected by confocal laser scanning microscopy (CLSM) and western-blotting. The ROS generation was measured by flow cytometry. Results Pure KillerRed was obtained with a yield of about 37 mg per liter of bacterial cells. KillerRed photodynamic inactivated the leukemia cells in a concentration-dependent manner, but exhibited no obvious dark toxicity. PDT mediated by KillerRed could also induce apoptotic response (mainly early apoptosis) in the three cell lines. The CLSM imaging indicated that KillerRed was distributed within the cytoplasm and nuclei of leukemia cells, causing damages to the cytoplasm and leaving the nuclear envelope intact during light irradiation. KillerRed distributed both in the cytosol and nuclei was confirmed by western blotting, and ROS significantly increased in PDT treated cells compared to the cells treated with KillerRed alone. Conclusions Our studies demonstrated that KillerRed-mediated PDT could effectively inactivate K562, NB4, and THP1 leukemia cells and trigger cell apoptosis, and it has potential to be used individually or complementally, in the treatment of leukemia.

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Evaluation of apoptosis and necrosis induced by statins using fluorescence-enhanced flow cytometry

Journal of Pharmaceutical and Biomedical Analysis ◽

10.1016/j.jpba.2005.04.022 ◽

2005 ◽

Vol 39 (3-4) ◽

pp. 712-717 ◽

Cited By ~ 21

Author(s):

Noriko Yasuda ◽

Sumio Matzno ◽

Chihiro Iwano ◽

Mayumi Nishikata ◽

Kenji Matsuyama

Keyword(s):

Flow Cytometry ◽

Apoptosis And Necrosis

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The Study of Bortezomib Alone or in Combination with Arsenic Trioxide on Multidrug Resistance of HL60/ADR cells.

Blood ◽

10.1182/blood.v114.22.4823.4823 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4823-4823

Author(s):

Fanyi Meng ◽

Ming Huang

Keyword(s):

Flow Cytometry ◽

Multidrug Resistance ◽

Cell Line ◽

Arsenic Trioxide ◽

Cell Apoptosis ◽

Conflicts Of Interest ◽

Synergistic Effects ◽

Caspase 9 ◽

Reduce Cell Survival ◽

Apoptosis Proteins

Abstract Abstract 4823 INTRODUCTION Proteĉ¢msome inhibitor bortezomib has used in treatment of hematology malignancies widely. We found it may reduce cell survival rate of HL60/ADR cell line and induce cell apoptosis in previous study. Now we are to investigate the effect of bortezomib alone or combined with arsenic trioxide (As2O3) on reversing multidrug resistance of HL60/ADR cell line and the possible machines. METHODS HL60/ADR cells were incubated with bortezomib at different doses alone and in combination with As2O3. The proliferation ratio was observed by MTT assay. Cell apoptosis was studied by fluorescence microscopy and flow cytometry. Intracellular concentration of daunorubicin (DNR) and multidrug resistance related protein-1 (MRP1) was determined by flow cytometry. P65Ap-p65Abcl-2AbaxAcaspase-3Acaspase-9APARP proteins were determined by western blot. RESULTS In bortezomib-treated tumor cells, inhibition rate increased in time- and dose-dependently, as well as apoptotic cells. Compared to bortezomib alone, combination with As2O3 inhibited the proliferation and induced the apoptosis of HL60/ADR cells more evident. Bortezomib can enhance the intraceflular accumulation of DNR and decrease MRP1 in HL60/ADM cells. The dual combination of As2O3 with bortezomib presents a superior anticancer and MRP1-decreased efficiency to either one of the drugs alone. Bortezomib can also elevate the expression of bax, caspase-3, caspase-9, PARP, and decelerate the expression of bcl-2, NF-κB p65, p-p65. CONCLUSIONS Bortezomib can reverse multidrug resistance of HL60/ADR cells and decrease the expression of MRP1 in cells. When combined with As2O3, it appears to synergistic effects. Its mechanisms might be associated with the inhibition of NF-κB activation, also with inhibiting anti-apoptosis proteins, boosting pro-apoptosis proteins, with followed activating caspase pathway. Disclosures: No relevant conflicts of interest to declare.

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