Real time monitoring and quantification of reactive oxygen species in breast cancer cell line MCF-7 by 2′,7′–dichlorofluorescin diacetate (DCFDA) assay

Figueroa, Diego; Asaduzzaman, Mohammad; Young, Fiona

doi:10.1016/j.vascn.2018.03.007

Cited by 72 publications

(44 citation statements)

References 60 publications

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“…One of the internal stressors linked to cell apoptosis is damaged DNA, which is particularly important in senescent cells chronically exposed to endogenously generated ROS 28 . To determine whether ROS can cause DNA damage and cell apoptosis during viral infection, we measured the ROS levels in naïve CD4 T cells isolated from HCV and HS by flow cytometry using the DCFDA, a fluorogenic dye that measures ROS within the cells 29 . Indeed, the mean fluorescence intensity (MFI) of DCFDA was significantly increased in naïve (Fig.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of TRF2 accelerates telomere attrition and DNA damage in naïve CD4 T cells during HCV infection

et al. 2018

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T cells play a crucial role in viral clearance and vaccine responses; however, the mechanisms that regulate their homeostasis during viral infections remain unclear. In this study, we investigated the machineries of T-cell homeostasis and telomeric DNA damage using a human model of hepatitis C virus (HCV) infection. We found that naïve CD4 T cells in chronically HCV-infected patients (HCV T cells) were significantly reduced due to apoptosis compared with age-matched healthy subjects (HSs). These HCV T cells were not only senescent, as demonstrated by overexpression of aging markers and particularly shortened telomeres; but also DNA damaged, as evidenced by increased dysfunctional telomere-induced foci (TIF). Mechanistically, the telomere shelterin protein, in particular telomeric repeat binding factor 2 (TRF2) that functions to protect telomeres from DNA damage, was significantly inhibited posttranscriptionally via the p53-dependent Siah-1a ubiquitination. Importantly, knockdown of TRF2 in healthy T cells resulted in increases in telomeric DNA damage and T-cell apoptosis, whereas overexpression of TRF2 in HCV T cells alleviated telomeric DNA damage and T-cell apoptosis. To the best of our knowledge, this is the first report revealing that inhibition of TRF2 promotes T-cell telomere attrition and telomeric DNA damage that accelerates T-cell senescent and apoptotic programs, which contribute to naïve T-cell loss during viral infection. Thus, restoring the impaired T-cell telomeric shelterin machinery may offer a new strategy to improve immunotherapy and vaccine response against human viral diseases.

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Section: Resultsmentioning

confidence: 99%

Inhibition of TRF2 accelerates telomere attrition and DNA damage in naïve CD4 T cells during HCV infection

et al. 2018

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“…COV434 cells exposed to 50 µM 4-Cyc for 2 h, or 1 µM for 6 h, significantly increased the production of ROS [52]. Although the concentration of DCFDA used in this study was 100 times higher than the one used in our experiments, and higher DCFDA concentrations can be toxic and contribute to ROS generation [38], the amount of ROS generated by COV434 cells after 24 h exposure to 21 µM 4-Cyc in our study was in broad agreement with the previous study [52].…”

Section: Discussionmentioning

confidence: 57%

“…Controls were cells in medium only (background negative control), and cells exposed to low (12.5 µM) or high (50 µM) concentrations of TBHP (positive controls) [38], or 0.8% DMSO as a vehicle control for the tocopherols. Each experiment was repeated on three separate occasions (n = 3).…”

Section: Effect Of Dox 4-hydroperoxycyclophosphamide (4-cyc) α or γmentioning

confidence: 99%

“…The ROS production was detected by recording fluorescence immediately after addition of test agents (time 0), every hour for a 3 h incubation period, and after 24 h continuous incubation. Fluorescence was measured according to protocol described by Figueroa et al, [38]. Fluorescence readings were made using a plate spectrofluorometer (GloMax ® Explorer, Promega, Sydney, Australia).…”

Section: Ros Measurement By Dcfda Assaymentioning

confidence: 99%

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Gamma Tocopherol Reduced Chemotherapeutic-Induced ROS in an Ovarian Granulosa Cell Line, But Not in Breast Cancer Cell Lines In Vitro

Gonzalez

Young

2020

Antioxidants

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Doxorubicin and cyclophosphamide are used to treat breast cancer, but they also cause infertility through off-target cytotoxicity towards proliferating granulosa cells that surround eggs. Each chemotherapeutic generates reactive oxygen species (ROS) but the effects of the combination, or the antioxidants alpha (αToc) and gamma tocopherol (γToc) on ROS in breast cancer or ovarian cells are unknown. Human breast cancer (MCF7, T47D) and ovarian cancer (OVCAR, COV434) cells were loaded with DCDFA and exposed (1, 2, 3, 24 h) to the MCF7-derived EC25 values of individual agents, or to combinations of these. ROS were quantified and viable cells enumerated using crystal violet or DAPI. Each chemotherapeutic killed ~25% of MCF7, T47D and OVCAR cells, but 57 ± 2% (doxorubicin) and 66 ± 2% (cyclophosphamide) of the COV434 granulosa cells. The combined chemotherapeutics decreased COV434 cell viability to 34 ± 5% of control whereas doxorubicin + cyclophosphamide + γToc reduced ROS within 3 h (p < 0.01) and reduced cytotoxicity to 54 ± 4% (p < 0.05). αToc was not cytotoxic, whereas γToc killed ~25% of the breast cancer but none of the ovarian cells. Adding γToc to the combined chemotherapeutics did not change ROS or cytotoxicity in MCF7, T47D or OVCAR cells. The protection γToc afforded COV434 granulosa cells against chemotherapy-induced ROS and cytotoxicity suggests potential for fertility preservation.

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“…For example, sperm function tests such as SDF are time consuming and time sensitive, and require large financial investments due to their reliance on the use of sperm chromatin structure assays and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) [16][17][18]. Many common ROS measurement techniques only capture the ROS concentration at a single point in time and do not provide a comprehensive evaluation of the relation between oxidant and antioxidant levels, therefore failing to provide a complete picture of the true oxidative stress (OS) environment [16,19]. To manage patients with infertility related to elevated OS, it is important to identify a test that includes all constituents of OS and measures the real-time redox status in a given semen sample.…”

Section: Introductionmentioning

confidence: 99%

A Novel Approach to Improving the Reliability of Manual Semen Analysis: A Paradigm Shift in the Workup of Infertile Men

Douglas

Parekh

Kahn

et al. 2021

World J Mens Health

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Conventional semen analysis (SA) is an essential component of the male infertility workup, but requires laboratories to rigorously train and monitor technicians as well as regularly perform quality assurance assessments. Without such measures there is room for error and, consequently, unreliable results. Furthermore, clinicians often rely heavily on SA results when making diagnostic and treatment decisions, however conventional SA is only a surrogate marker of male fecundity and does not guarantee fertility. Considering these challenges, the last several decades have seen the development of many advances in SA methodology, including tests for sperm DNA fragmentation, acrosome reaction, and capacitation. While these new diagnostic tests have improved the scope of information available to clinicians, they are expensive, time-consuming, and require specialized training. The latest advance in laboratory diagnostics is the measurement of seminal oxidation-reduction potential (ORP). The measurement of ORP in an easy, reproducible manner using a new tool called the Male Infertility Oxidative Stress System (MiOXSYS) has demonstrated ORP's potential as a feasible adjunct test to conventional SA. Additionally, the measurement of ORP by this device has been shown to be predictive of both poor semen quality and male infertility. Assessing ORP is a novel approach to both validating manual SA results and identifying patients who may benefit from treatment of male oxidative stress infertility.

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Real time monitoring and quantification of reactive oxygen species in breast cancer cell line MCF-7 by 2′,7′–dichlorofluorescin diacetate (DCFDA) assay

Cited by 72 publications

References 60 publications

Inhibition of TRF2 accelerates telomere attrition and DNA damage in naïve CD4 T cells during HCV infection

Inhibition of TRF2 accelerates telomere attrition and DNA damage in naïve CD4 T cells during HCV infection

Gamma Tocopherol Reduced Chemotherapeutic-Induced ROS in an Ovarian Granulosa Cell Line, But Not in Breast Cancer Cell Lines In Vitro

A Novel Approach to Improving the Reliability of Manual Semen Analysis: A Paradigm Shift in the Workup of Infertile Men

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