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.

scholarly journals Protein kinase Cδ regulates vaccinia-related kinase 1 in DNA damage–induced apoptosis

2011 ◽  
Vol 22 (8) ◽  
pp. 1398-1408 ◽  
Author(s):  
Choon-Ho Park ◽  
Bo-Hwa Choi ◽  
Min-Woo Jeong ◽  
Sangjune Kim ◽  
Wanil Kim ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Protein Kinase ◽  
Cell Viability ◽  
Apoptotic Cell ◽  
Critical Role ◽  
Regulatory Region ◽  
Dependent Manner ◽  
Protein Kinase Cδ

Vaccinia-related kinase 1 (VRK1) is a novel serine/threonine kinase that plays an important role in cell proliferation. However, little is known about the upstream regulators of VRK1 activity. Here we provide evidence for a role of protein kinase Cδ (PKCδ) in the regulation of murine VRK1. We show that PKCδ interacts with VRK1, phosphorylates the Ser-355 residue in the putative regulatory region, and negatively regulates its kinase activity in vitro. Intriguingly, PKCδ-induced cell death was facilitated by phosphorylation of VRK1 when cells were exposed to a DNA-damaging agent. In addition, p53 played a critical role in the regulation of DNA damage–induced cell death accompanied by PKCδ-mediated modulation of VRK1. In p53-deficient cells, PKCδ-mediated phosphorylation of VRK1 had no effect on cell viability. However, cells overexpressing p53 exhibited significant reduction of cell viability when cotransfected with both VRK1 and PKCδ. Taken together, these results indicate that PKCδ regulates phosphorylation and down-regulation of VRK1, thereby contributing to cell cycle arrest and apoptotic cell death in a p53-dependent manner.

Stew in its Own Juice: Protein Homeostasis Machinery Inhibition Reduces Cell Viability in Multiple Myeloma Cell Lines

2019 ◽  
Vol 19 (2) ◽  
pp. 112-119 ◽  
Author(s):  
Mariana B. de Oliveira ◽  
Luiz F.G. Sanson ◽  
Angela I.P. Eugenio ◽  
Rebecca S.S. Barbosa-Dantas ◽  
Gisele W.B. Colleoni
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Viability ◽  
Cell Lines ◽  
Treatment Options ◽  
Compensatory Mechanism ◽  
Protein Homeostasis ◽  
Protein Response ◽  
Rpmi 8226

Introduction:Multiple myeloma (MM) cells accumulate in the bone marrow and produce enormous quantities of immunoglobulins, causing endoplasmatic reticulum stress and activation of protein handling machinery, such as heat shock protein response, autophagy and unfolded protein response (UPR).Methods:We evaluated cell lines viability after treatment with bortezomib (B) in combination with HSP70 (VER-15508) and autophagy (SBI-0206965) or UPR (STF- 083010) inhibitors.Results:For RPMI-8226, after 72 hours of treatment with B+VER+STF or B+VER+SBI, we observed 15% of viable cells, but treatment with B alone was better (90% of cell death). For U266, treatment with B+VER+STF or with B+VER+SBI for 72 hours resulted in 20% of cell viability and both treatments were better than treatment with B alone (40% of cell death). After both triplet combinations, RPMI-8226 and U266 presented the overexpression of XBP-1 UPR protein, suggesting that it is acting as a compensatory mechanism, in an attempt of the cell to handle the otherwise lethal large amount of immunoglobulin overload.Conclusion:Our in vitro results provide additional evidence that combinations of protein homeostasis inhibitors might be explored as treatment options for MM.

scholarly journals Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

2020 ◽  
Vol 22 (1) ◽  
pp. 202
Author(s):  
Josephin Glück ◽  
Julia Waizenegger ◽  
Albert Braeuning ◽  
Stefanie Hessel-Pras
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Pyrrolizidine Alkaloids ◽  
Defense Mechanism ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Dependent Manner ◽  
Human Hepatoma Cell

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure–activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.

The Human Leukemic HL-60 Cell Line: An in Vitro Model for Cell Death Endpoint Identification

1996 ◽  
Vol 24 (4) ◽  
pp. 581-587
Author(s):  
Cristiana Zanetti ◽  
Arrnalaura Stammati ◽  
Orazio Sapora ◽  
Flavia Zucco
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
In Vitro Model ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Cell Type ◽  
Vitro Model ◽  
Promyelocytic Leukemia Cell Line

The aim of this study was to investigate the endpoints related to cell death, either necrosis or apoptosis, induced by four chemicals in the promyelocytic leukemia cell line, HL-60. Cell morphology, DNA fragmentation, cytofluorimetric analysis and oxygen consumption were used to classify the type of cell death observed. In our analysis, we found that not all the selected parameters reproduced the differences observed in the cell death caused by the four chemicals tested. As cell death is a very complex phenomenon, several factors should be taken into account (cell type, exposure time and chemical concentration), if chemicals are to be classified according to differences in the mechanisms more directly involved in cell death.

DNA damage induced by coal dust, fly and bottom ash from coal combustion evaluated using the micronucleus test and comet assay in vitro

2017 ◽  
Vol 324 ◽  
pp. 781-788 ◽  
Author(s):  
Cristina Araujo Matzenbacher ◽  
Ana Letícia Hilario Garcia ◽  
Marcela Silva dos Santos ◽  
Caroline Cardoso Nicolau ◽  
Suziane Premoli ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Coal Combustion ◽  
Micronucleus Test ◽  
Coal Dust ◽  
Bottom Ash

MO1048PHYTOTHERAPY ON RENAL HYPOXIA: THE PREVENTIVE EFFECTS OF HYDROXYTYROSOL - PRELIMINARY IN VITRO RESULTS

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Terézia Kamasová ◽  
Ana Sofia Abreu ◽  
Fátima Paiva-Martins ◽  
Luís Belo ◽  
Alice Santos-Silva ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Therapeutic Potential ◽  
Quantitative Polymerase Chain Reaction ◽  
Oxidative Status ◽  
Hypoxic Conditions ◽  
Thiazolyl Blue Tetrazolium Bromide ◽  
Anti Inflammatory ◽  
Preventive Effects

Abstract Background and Aims Renal hypoxia plays a key role in the pathophysiology of acute kidney injury and in the progression of chronic kidney disease, potentiating other important risk factors for renal disease, such as oxidative stress, renal fibrosis, and inflammation. Hydroxytyrosol (HT) is a phenolic compound extracted from olives and olive-derived products, that has been shown to detain potent in vitro antioxidant and anti-inflammatory activity. The aim of this study was to evaluate the preventive therapeutic potential of HT on a cellular model of renal hypoxia. Method A cell line of normal adult proximal tubular epithelium (HK-2 cell line) was used to determine the effects of the chemical induction of hypoxia with cobalt chloride (CoCl2), as well as the preventive potential of HT on the elicited effects. For this purpose, HK-2 cells were exposed for 24 h to 254 µM CoCl2, to mimic the hypoxic conditions, or pre-incubated for 1 h with 5 µM HT and further exposed to the CoCl2 for 24 h more. Cell viability was assessed by the thiazolyl blue tetrazolium bromide reduction assay. Oxidative status was evaluated by the measurement of reactive oxygen and nitrogen species (ROS and RNS) and reduced glutathione (GSH) levels, by using standardized fluorometric and colorimetric assays. The expression of several genes related to the hypoxic, inflammatory, and fibrotic responses was determined by quantitative polymerase chain reaction (PCR). Results CoCl2-exposed HK-2 cells (hypoxic conditions) showed a significant decrease in cell viability (p < 0.0001 vs. control), and a disruption of the oxidative status, characterized by an increase of ROS and RNS production of about 6-fold over control cells (p < 0.0001) and a decrease in GSH intracellular levels of nearly 50 % (p < 0.05). Although the pre-exposure to HT showed no significant effects on the loss of cell viability elicited by CoCl2, the presence of HT prior to induction of hypoxia reduced the generation of ROS and RNS (p < 0.05 for HT + CoCl2 vs. CoCl2) and prevented the GSH depletion (GSH levels for HT + CoCl2 were similar to those of control) elicited by CoCl2. When compared to control cells, CoCl2-exposed HK-2 cells also showed increased expression of genes related to hypoxia (HIF1A, p < 0.05; GAPDH, p < 0.0001), as well as of modulators of inflammation (IL6, p < 0.0001) and fibrosis (TGFB1, p < 0.05). Importantly, the expression of these genes was partially or even totally suppressed by the pre-exposure of cells to HT (GAPDH, p < 0.01 for HT + CoCl2 vs. CoCl2; expression of HIF1A, IL6 and TGFB1 for HT + CoCl2 was similar to that of control). Conclusion Our data supports the potential for a multiplicity of preventive effects of HT, providing antioxidant, anti-inflammatory and anti-fibrotic defenses to renal cells under hypoxic conditions. Importantly, the development of safe and effective therapeutic approaches based on phytochemicals such as HT, may present substantial advantages for renal patients over synthetic drugs, including fewer side effects, significantly lower price, and ease of administration in the form of dietary supplements. Acknowledgments This work was supported by Applied Molecular Biosciences Unit (UCIBIO), financed by national funds from FCT/MCTES (UIDB/04378/2020), by North Portugal Regional Coordination and Development Commission (CCDR-N)/NORTE2020/Portugal 2020 (Norte-01-0145-FEDER-000024), and co-financed by FCT/MCTES (PTDC/OCE-ETA/32492/2017) and FEDER/COMPETE 2020 (POCI-01-0145-FEDER-032492).

scholarly journals Effects of acetylsalicylic acid and acetic acid solutions in VX2 carcinoma cells: In vitro analysis

2006 ◽  
Vol 21 (3) ◽  
pp. 151-154 ◽  
Author(s):  
Rogério Saad-Hossne ◽  
René Gamberini Prado ◽  
William Saad Hossne
Keyword(s):  
Cell Death ◽  
Acetic Acid ◽  
Cell Viability ◽  
Acetylsalicylic Acid ◽  
Neoplastic Cell ◽  
Carcinoma Cells ◽  
Acid Solutions ◽  
Vx2 Carcinoma ◽  
Vitro Analysis

PURPOSE: To analyze, in vitro, the effects of acetylsalicylic acid (aspirin) and acetic acid solutions on VX2 carcinoma cells in suspension and to examine the correlation between these effects and neoplastic cell death. METHODS: The VX2 tumor cells (10(7) cells/ml) were incubated in solutions containing differing concentrations (2.5% and 5%) of either acetylsalicylic acid or acetic acid, or in saline solution (controls). Every five minutes, cell viability was tested (using the trypan blue test) and analyzed under light microscopy. RESULTS: Tumor cell viability (in %) decreased progressively and, by 30 minutes, neoplastic cell death had occurred in all solutions. CONCLUSION: Based on this experimental model and the methodology employed, we conclude that these solutions cause neoplastic cell death in vitro.

scholarly journals FORMULATION AND CHARACTERIZATION OF PAPAIN LOADED SOLID LIPID NANOPARTICLES AGAINST HUMAN COLORECTAL ADENOCARCINOMA CELL LINE

2018 ◽  
Vol 11 (10) ◽  
pp. 393
Author(s):  
Suriyakala Perumal Chandran ◽  
Kannikaparameswari Nachimuthu
Keyword(s):  
Particle Size ◽  
Drug Release ◽  
Zeta Potential ◽  
Cell Viability ◽  
Cell Line ◽  
Solid Lipid Nanoparticles ◽  
Colorectal Adenocarcinoma ◽  
Lipid Nanoparticles ◽  
In Vitro Drug Release

Objective: Colorectal cancer is one of the most commonly diagnosed cancer and also most common gastrointestinal malignancy with high prevalence rate in the younger population. Usually, cancer cells are surrounded by a fibrin coat which is resistant to fibrinolytic degradation. This fibrin coat is act as self-protective against natural killing mechanism. The main objective was to prepare papain-loaded solid lipid nanoparticles (P-SLN) by melt dispersion-ultrasonication method and investigated the cytotoxic efficacy against colorectal adenocarcinoma (human colorectal adenocarcinoma [HCT 15]) cells.Methods: Optimized polymer ratio was characterized by differential scanning calorimetry, Fourier-transform infrared, X-ray diffraction, scanning electron microscopy, entrapment efficiency, particle size and zeta potential analysis, in vitro drug release, and in vitro cytotoxicity studies on HCT-15 colorectal adenocarcinoma cells.Results: The results showed that the particle size, morphological character and zeta potential value of optimized batch P-SLN were 265 nm, spherical and −26.5 Mv, respectively. The in vitro drug profile of P-SLN exhibited that it produced sustain drug release, and the cell viability of HCT-15 against P-SLN shown better efficacy than pure papain enzyme.Conclusion: P-SLNs were successfully prepared and investigated the in vitro drug release and in vitro cell viability against HCT-15 cell line.

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