scholarly journals The Comet Assay to Determine the Mode of Cell Death for the Ultrasonic Delivery of Doxorubicin to Human Leukemia (HL-60 Cells) from Pluronic P105 Micelles

2005 ◽  
Vol 4 (6) ◽  
pp. 707-711 ◽  
Author(s):  
Ghaleb A. Husseini ◽  
Kim L. O'Neill ◽  
William G. Pitt
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Comet Assay ◽  
Propidium Iodide ◽  
Cell Killing ◽  
Human Leukemia ◽  
Dna Profile ◽  
Fluorescent Microscope

This notes examines the mode of cell death of HL-60 cells exposed to 70 kHz and 1.3 W/cm2 ultrasound in the presence of 1% Pluronic P105 and 1.67 μg/ml doxorubicin (Dox). The cells were ultrasonicated for 30, 60, and 120 minutes. They were then lysed, electrophorised, stained using propidium iodide, and their DNA profile captured using a fluorescent microscope. The gradual DNA damage observed and the comet tails captured after one and two hours of insonation suggest that the mode of cell killing is apoptosis.

scholarly journals Transferrin-Bound Doxorubicin Enhances Apoptosis and DNA Damage through the Generation of Pro-Inflammatory Responses in Human Leukemia Cells

2020 ◽  
Vol 21 (24) ◽  
pp. 9390
Author(s):  
Monika Jedrzejczyk ◽  
Katarzyna Wisniewska ◽  
Katarzyna Dominika Kania ◽  
Agnieszka Marczak ◽  
Marzena Szwed
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Mononuclear Cells ◽  
Inflammatory Responses ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Morphological Observation ◽  
Human Leukemia ◽  
Dependent Manner ◽  
Normal Peripheral Blood

Doxorubicin (DOX) is an effective antineoplastic drug against many solid tumors and hematological malignancies. However, the clinical use of DOX is limited, because of its unspecific mode of action. Since leukemia cells overexpress transferrin (Tf) receptors on their surface, we proposed doxorubicin–transferrin (DOX–Tf) conjugate as a new vehicle to increase drug concentration directly in cancer cells. The data obtained after experiments performed on K562 and CCRF-CEM human leukemia cell lines clearly indicate severe cytotoxic and genotoxic properties of the conjugate drug. On the other hand, normal peripheral blood mononuclear cells (PBMCs) were more resistant to DOX–Tf than to DOX. In comparison to free drug, we observed that Tf-bound DOX induced apoptosis in a TRAIL-dependent manner and caused DNA damage typical of programmed cell death. These fatal hallmarks of cell death were confirmed upon morphological observation of cells incubated with DOX or DOX–Tf. Studies of expression of TNF-α, IL-4, and IL-6 at the mRNA and protein levels revealed that the pro-inflammatory response plays an important role in the toxicity of the conjugate. Altogether, the results demonstrated here describe a mechanism of the antitumor activity of the DOX–Tf conjugate.

scholarly journals Inhibition of the checkpoint kinase Chk1 induces DNA damage and cell death in human Leukemia and Lymphoma cells

2014 ◽  
Vol 13 (1) ◽  
pp. 147 ◽  
Author(s):  
Christopher Bryant ◽  
Kirsten Scriven ◽  
Andrew J Massey
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Human Leukemia ◽  
Lymphoma Cells ◽  
Checkpoint Kinase

scholarly journals DNA damage and cell death assessment in patients with severe multiple trauma using comet assay

Health ◽  
2010 ◽  
Vol 02 (05) ◽  
pp. 412-417 ◽  
Author(s):  
Aliy K. Zhanataev ◽  
Victor V. Moroz ◽  
Andrey D. Durnev ◽  
Maria Yu. Muravyeva ◽  
Vasiliy I. Reshetnyak
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Comet Assay ◽  
Multiple Trauma ◽  
Severe Multiple Trauma

scholarly journals EVALUATION OF CYTOTOXIC AND GENOTOXIC EFFECTS OF ZERUMBONE ON COLON ADENOCARCINOMA COLO205 CELLS AND HUMAN LYMPHOCYTES

2017 ◽  
Vol 9 (10) ◽  
pp. 92
Author(s):  
Rima Thiyam ◽  
Mangamoori Lakshmi Narasu
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Comet Assay ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Human Lymphocytes ◽  
Genotoxic Effect ◽  
Colorectal Cancer Cell Line ◽  
Dependent Manner ◽  
Ic50 Values

Objective: The objective of the present study was to investigate the growth inhibitory effect, apoptosis initiation and genotoxic effect of zerumbone (ZER), a phytochemical and cisplatin, a chemotherapeutic drug on human colorectal cancer cell line COLO205 and normal human lymphocytes.Methods: The antiproliferative activity of ZER and cisplatin (positive control) on COLO205 cells and lymphocytes was analysed by 3( 4, 5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay. Morphological analysis of the cells was studied by using inverted phase contrast microscope. Propidium iodide staining method was used to observe the apoptotic morphological changes in the treated cells. Finally comet assay was conducted to observe the extent of DNA damage induced by ZER and cisplatin on COLO205 and lymphocytes.Results: ZER and cisplatin exhibited growth inhibition in a dose and time dependent manner against COLO205 with no considerable effect on lymphocytes. The IC50 values of ZER on COLO205 for 24h, 48h and 72h were 19 µg/ml, 10 µg/ml and 5 µg/ml. Comparatively the IC50 values of cisplatin on COLO205 for 24h, 48h and 72h were 38 µg/ml, 24 µg/ml and 15 µg/ml.  Morphological changes such as cell shrinkage, membrane blebbing and nuclear condensation were observed in COLO205 while no significant change was observed in lymphocytes. Fluorescence imaging studies confirmed apoptotic cell death in treated COLO205 cells while no significant cell death was observed in treated lymphocytes. Comet assay revealed significant DNA damage in treated COLO205 cells.Conclusion: The present study demonstrated the cytotoxic and genotoxic effect of ZER and cisplatin on COLO205 cells. These drugs showed no significant effect on lymphocytes.

scholarly journals Cell survival after DNA damage in the comet assay

2021 ◽  
Author(s):  
Ezgi Eyluel Bankoglu ◽  
Carolin Schuele ◽  
Helga Stopper
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Comet Assay ◽  
Survival Data ◽  
Topoisomerase Ii ◽  
Basic Research ◽  
Human Biomonitoring ◽  
A Cell ◽  
Tk6 Cells ◽  
Genotoxicity Testing

AbstractThe comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H2O2) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20–30% DNA in tail caused the death of more than 50% of the cells, with etoposide causing slightly more cell death than H2O2 or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20% DNA in tail, survival data for the cells are provided.

scholarly journals Betulinic Acid Inhibits Growth of Cultured Vascular Smooth Muscle CellsIn Vitroby InducingG1Arrest and Apoptosis

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Raja Kumar Vadivelu ◽  
Swee Keong Yeap ◽  
Abdul Manaf Ali ◽  
Muhajir Hamid ◽  
Noorjahan Banu Alitheen
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Smooth Muscle ◽  
Comet Assay ◽  
Acridine Orange ◽  
Propidium Iodide ◽  
Betulinic Acid ◽  
Cycle Arrest ◽  
Dose Dependent

Betulinic acid is a widely available plant-derived triterpene which is reported to possess selective cytotoxic activity against cancer cells of neuroectodermal origin and leukemia. However, the potential of betulinic acid as an antiproliferative and cytotoxic agent on vascular smooth muscle (VSMC) is still unclear. This study was carried out to demonstrate the antiproliferative and cytotoxic effect of betulinic acid on VSMCs using 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry cell cycle assay, BrdU proliferation assay, acridine orange/propidium iodide staining, and comet assay. Result from MTT and BrdU assays indicated that betulinic acid was able to inhibit the growth and proliferation of VSMCs in a dose-dependent manner with IC50of 3.8 μg/mL significantly(P<0.05). Nevertheless, betulinic acid exhibited G1cell cycle arrest in flow cytometry cell cycle profiling and low level of DNA damage against VSMC in acridine orange/propidium iodide and comet assay after 24 h of treatment. In conclusion, betulinic acid induced G1cell cycle arrest and dose-dependent DNA damage on VSMC.

scholarly journals Stochastic multicellular modeling of x-ray irradiation, DNA damage induction, DNA free-end misrejoining and cell death

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jake C. Forster ◽  
Michael J. J. Douglass ◽  
Wendy M. Phillips ◽  
Eva Bezak
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Cell Killing ◽  
Track Structure ◽  
Linear Component ◽  
X Rays ◽  
Dna Double Strand Breaks ◽  
Cell Nuclei ◽  
Strand Breaks ◽  
X Ray

AbstractThe repair or misrepair of DNA double-strand breaks (DSBs) largely determines whether a cell will survive radiation insult or die. A new computational model of multicellular, track structure-based and pO2-dependent radiation-induced cell death was developed and used to investigate the contribution to cell killing by the mechanism of DNA free-end misrejoining for low-LET radiation. A simulated tumor of 1224 squamous cells was irradiated with 6 MV x-rays using the Monte Carlo toolkit Geant4 with low-energy Geant4-DNA physics and chemistry modules up to a uniform dose of 1 Gy. DNA damage including DSBs were simulated from ionizations, excitations and hydroxyl radical interactions along track segments through cell nuclei, with a higher cellular pO2 enhancing the conversion of DNA radicals to strand breaks. DNA free-ends produced by complex DSBs (cDSBs) were able to misrejoin and produce exchange-type chromosome aberrations, some of which were asymmetric and lethal. A sensitivity analysis was performed and conditions of full oxia and anoxia were simulated. The linear component of cell killing from misrejoining was consistently small compared to values in the literature for the linear component of cell killing for head and neck squamous cell carcinoma (HNSCC). This indicated that misrejoinings involving DSBs from the same x-ray (including all associated secondary electrons) were rare and that other mechanisms (e.g. unrejoined ends) may be important. Ignoring the contribution by the indirect effect toward DNA damage caused the DSB yield to drop to a third of its original value and the cDSB yield to drop to a tenth of its original value. Track structure-based cell killing was simulated in all 135306 viable cells of a 1 mm3 hypoxic HNSCC tumor for a uniform dose of 1 Gy.

scholarly journals DNA Damage and Cell Killing by Camptothecin and Its Derivative in Human Leukemia HL-60 Cells

1993 ◽  
Vol 84 (5) ◽  
pp. 566-573 ◽  
Author(s):  
Akira Yoshida ◽  
Takanori Ueda ◽  
Yuji Wano ◽  
Toru Nakamura
Keyword(s):  
Dna Damage ◽  
Cell Killing ◽  
Human Leukemia

Differential effects of selenium (Se) on normal and malignant cells treated with chemotherapy (CT) and radiation (RT).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e13583-e13583
Author(s):  
Michael B. Jameson ◽  
Richard J Lobb ◽  
Gregory M Jacobson ◽  
Ray T Cursons
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Stress Response ◽  
Comet Assay ◽  
Er Stress ◽  
Stress Responses ◽  
Mononuclear Cells ◽  
Malignant Cells ◽  
Er Stress Response ◽  
Therapeutic Selectivity

e13583 Background: Preclinical work has demonstrated that Se compounds potentiate anticancer effects of CT and RT while reducing normal tissue toxicities. The molecular basis for the therapeutic selectivity has yet to be fully elucidated but includes modulation of intracellular glutathione (GSH) concentrations, endoplasmic reticulum (ER) stress responses, DNA repair, induction of apoptosis and cellular resistance to CT and RT. Our aim was to evaluate the dose-response relationship of the Se compound methylseleninic acid (MSA) on molecular pathways involved in the response of normal and malignant cells to CT and RT. Methods: Peripheral blood mononuclear cells (PBMC) obtained from healthy blood donors and malignant THP-1 human monocytic leukaemia cells were exposed in vitro to MSA 2.5, 5 or 15 µM in varying combinations with MSA, RT, cisplatin (Pt), doxorubicin (Dox) and cytosine arabinoside (Ara-C). GSH concentration was measured by ELISA, DNA damage and repair by COMET assay, cell viability by MTT assay and ER stress response protein expression by western blotting. Results: MSA was selectively toxic to THP-1 cells and induced a protective increase in GSH in PBMC but a decrease in high concentrations within THP-1 cells. DNA damage induced by Ara-C or Dox in the COMET assay was significantly reduced by MSA in PBMC but increased in THP-1 cells. Cell death after 2 Gy RT was increased by all doses of MSA in THP-1 cells but only by the highest dose in PBMC. The cytotoxicity of Dox and Ara-C at sublethal doses was significantly enhanced by MSA in THP-1 cells and to a lesser extent in PBMC, but MSA increased cell death from Pt only in THP-1 cells. MSA induced a protective ER stress response in PBMC exposed to Ara-C but an apoptotic response in THP-1 cells. Conclusions: MSA at clinically-relevant concentrations had a differential effect on cell survival and death responses to RT and CT with relative protection of PBMC and enhanced death of THP-1 cells. Several mechanisms mediated this therapeutic selectivity and the dose-dependence of the Se effect varied between malignant and normal cells. These assays could potentially be used in clinical trials to evaluate pharmacodynamic markers of Se effects in conjunction with CT and/or RT.

Assessment of cell cytotoxicity and comet assay on HER2/neu positive cell line due to 111In Auger electrons as DNA-targeting radioimmunoconjugate

2021 ◽  
Vol 14 ◽  
Author(s):  
Behnaz Piroozfar ◽  
Behrouz Alirezapour ◽  
Farahnaz Motamedi Sedeh ◽  
Mohammad Mirzaii ◽  
Amir Reza Jalilian ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Comet Assay ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Nuclei ◽  
Auger Electrons ◽  
Oncology Research ◽  
Electron Therapy

Background: Breast cancer Auger electron therapy is a growing field of study in radioimmunotherapy and oncology research. Trastuzumab is a high affinity-binding monoclonal antibody against HER2/neu, which is overexpressed in breast tumors and used in radiopharmaceutical development. Objective: In this work, the lethal effects of 111In3+, 111In-DTPA-trastuzumab, and 111In-trastuzumab coupled-nuclear localizing sequence peptide (111In-DTPA-NLS-trastuzumab) on malignant cells were studied in vitro. Methods: DTPA-NLS-trastuzumab was prepared using sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) conjugation with NLS peptide in the first step, followed by conjugation with diethylenetriaminepentaacetic acid (DTPA). Both DTPA-trastuzumab and DTPA-NLS-trastuzumab were labeled with 111In, followed by purification and quality control techniques. Sk-Br-3 (a HER2/neu+ cell line) was used in the cell viability assessment assay for 11In, 111In-DTPA-trastuzumab, and 111In-DTPA-NLS-trastuzumab (3.7 MBq) at 37 ºC. The cytotoxicity of the three species was studied using MTT, and comet assay was utilized by DNA damage detection. Results: A significant radiochemical purity for 111In-DTPA-NLS-trastuzumab (99.36% ± 0.30%, ITLC) at the DTPA:antibody ratio of 6.90 ± 0.34:1 was obtained. Significant cell viability difference was found for 111In-DTPA-NLS-trastuzumab compared to the other treatments at two-time points. In addition, comet assay demonstrated significant DNA damage at 144 h using 111In-DTPA-NLS-trastuzumab. Conclusion: The results of cell viability and cell death using MTT assay and comet assay, respectively, demonstrate that the NLS-peptide effectively facilitates 111In-trastuzumab transport into the HER2/neu positive cancer cell nuclei to impose the radiotherapeutic effects of Auger electrons on DNA, leading to cell death.

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