Advantages of evaluating γH2AX induction in non-clinical drug development

Motoyama, Shigeki; Takeiri, Akira; Tanaka, Kenji; Harada, Asako; Matsuzaki, Kaori; Taketo, Junko; Matsuo, Saori; Fujii, Etsuko; Mishima, Masayuki

doi:10.1186/s41021-018-0098-z

Cited by 23 publications

(15 citation statements)

References 35 publications

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“…The typical mode of action of most anti-cancer agents is the induction of DNA damage. Nuclear γH 2 AX staining is a marker of DNA damage-induced cellular repair activity and is frequently used as very sensitive marker for the presence of DNA damage, especially DNA double strand breaks [27]. To assess if UFP induces DNA damage, non-immortalized human skin fibroblasts (HDF) and colon cancer cells (HCT-116) were exposed to 100 µg/mL UPF over 24 h. In untreated HDF, only about 10% of cells showed any γH 2 AX foci at all and these cells displayed typically less than four foci.…”

Section: Resultsmentioning

confidence: 99%

Pathway Analysis of Fucoidan Activity Using a Yeast Gene Deletion Library Screen

et al. 2019

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Fucoidan, the sulfated fucose-rich polysaccharide derived from brown macroalgae, was reported to display some anti-cancer effects in in vitro and in vivo models that included apoptosis and cell cycle arrest. The proposed mechanisms of action involve enhanced immune surveillance and direct pro-apoptotic effects via the activation of cell signaling pathways that remain largely uncharacterized. This study aimed to identify cellular pathways influenced by fucoidan using an unbiased genetic approach to generate additional insights into the anti-cancer effects of fucoidan. Drug–gene interactions of Undaria pinnatifida fucoidan were assessed by a systematic screen of the entire set of 4,733 halpoid Saccharomyces cerevsiae gene deletion strains. Some of the findings were confirmed using cell cycle analysis and DNA damage detection in non-immortalized human dermal fibroblasts and colon cancer cells. The yeast deletion library screen and subsequent pathway and interactome analysis identified global effects of fucoidan on a wide range of eukaryotic cellular processes, including RNA metabolism, protein synthesis, sorting, targeting and transport, carbohydrate metabolism, mitochondrial maintenance, cell cycle regulation, and DNA damage repair-related pathways. Fucoidan also reduced clonogenic survival, induced DNA damage and G1-arrest in colon cancer cells, while these effects were not observed in non-immortalized human fibroblasts. Our results demonstrate global effects of fucoidan in diverse cellular processes in eukaryotic cells and further our understanding about the inhibitory effect of Undaria pinnatifida fucoidan on the growth of human cancer cells.

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Section: Resultsmentioning

confidence: 99%

Pathway Analysis of Fucoidan Activity Using a Yeast Gene Deletion Library Screen

et al. 2019

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“…Furthermore, in their review, Siddiqui et al 101 mentioned that γH2AX persisted after exposure to IR under treatment with various radiosensitizing drugs, indicating that this sensor could be used to monitor cancer therapy and to tailor cancer treatments. Using anti-γH2AX monoclonal antibodies and immunofluorescence hybridization techniques to visualize γH2AX localization at sites of DNA damage 102,103 , even with a very low dose of IR exposure, γH2AX foci can be visualized as well, but once the DNA damage is repaired, the foci are eliminated 104,105 . Kuo and Yang 106 suggested that γH2AX foci represent DSBs in a 1:1 ratio and can be used as a biomarker for DNA damage 106 .…”

Section: Cellular Dna Damage Sensors and Early Signal Transducers In Response To Irmentioning

confidence: 99%

DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer

Huang

Zhou

2020

Sig Transduct Target Ther

614

457

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Radiotherapy is one of the most common countermeasures for treating a wide range of tumors. However, the radioresistance of cancer cells is still a major limitation for radiotherapy applications. Efforts are continuously ongoing to explore sensitizing targets and develop radiosensitizers for improving the outcomes of radiotherapy. DNA double-strand breaks are the most lethal lesions induced by ionizing radiation and can trigger a series of cellular DNA damage responses (DDRs), including those helping cells recover from radiation injuries, such as the activation of DNA damage sensing and early transduction pathways, cell cycle arrest, and DNA repair. Obviously, these protective DDRs confer tumor radioresistance. Targeting DDR signaling pathways has become an attractive strategy for overcoming tumor radioresistance, and some important advances and breakthroughs have already been achieved in recent years. On the basis of comprehensively reviewing the DDR signal pathways, we provide an update on the novel and promising druggable targets emerging from DDR pathways that can be exploited for radiosensitization. We further discuss recent advances identified from preclinical studies, current clinical trials, and clinical application of chemical inhibitors targeting key DDR proteins, including DNA-PKcs (DNA-dependent protein kinase, catalytic subunit), ATM/ATR (ataxia-telangiectasia mutated and Rad3-related), the MRN (MRE11-RAD50-NBS1) complex, the PARP (poly[ADP-ribose] polymerase) family, MDC1, Wee1, LIG4 (ligase IV), CDK1, BRCA1 (BRCA1 C terminal), CHK1, and HIF-1 (hypoxia-inducible factor-1). Challenges for ionizing radiation-induced signal transduction and targeted therapy are also discussed based on recent achievements in the biological field of radiotherapy.

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“…Some of the assays, such as the Vitotox™ assay measuring mutagenicity, the RadarScreen assay measuring clastogenicity, and the comet assay evaluating DNA breaks, have been optimized for medium to high-throughput formats [28][29][30][31]. Compared with these methods, HCS assays provide more insight into the mechanism of action of genotoxic compounds because of their ability to measure multiple parameters (e.g., γH2AX and micronucleus) [32]. In addition, HCS facilitates the filtering out of false positives.…”

Section: Hcs In Genotoxicitymentioning

confidence: 99%

Review of high-content screening applications in toxicology

Xia

2019

Arch Toxicol

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High content screening (HCS) technology combining automated microscopy and quantitative image analysis can address biological questions in academia and the pharmaceutical industry. Various HCS experimental applications have been utilized in the research field of in vitro toxicology. In this review, we describe several HCS application approaches used for studying the mechanism of compound toxicity, highlight some challenges faced in the toxicological community, and discuss the future directions of HCS in regards to new models, new reagents, data management, and informatics. Many specialized areas of toxicology including developmental toxicity, genotoxicity, developmental neurotoxicity/neurotoxicity, hepatotoxicity, cardiotoxicity, and nephrotoxicity will be examined. In addition, several newly developed cellular assay models including induced pluripotent stem cells (iPSCs), three-dimensional (3D) cell models, and tissueson-a-chip will be discussed. New genome editing technologies (e.g., CRISPR/Cas9), data analyzing tools for imaging, and coupling with high-content assays will be reviewed. Finally, the applications of machine learning to image processing will be explored. These new HCS approaches offer a huge step forward in dissecting biological processes, developing drugs, and making toxicology studies easier.

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Advantages of evaluating γH2AX induction in non-clinical drug development

Cited by 23 publications

References 35 publications

Pathway Analysis of Fucoidan Activity Using a Yeast Gene Deletion Library Screen

Pathway Analysis of Fucoidan Activity Using a Yeast Gene Deletion Library Screen

DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer

Review of high-content screening applications in toxicology

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