The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis

<p>Abstract</p> <p>Background</p> <p>Radiation-induced DNA double-strand break (DSB) repair can be tested by using pulsed-field gel electrophoresis (PFGE) in agarose-encapsulated cells. However, previous studies have reported that this assay is impaired by the spontaneous DNA breakage in this medium. We investigated the mechanisms of this fragmentation with the principal aim of eliminating it in order to improve the estimation of radiation-induced DNA repair.</p> <p>Methods</p> <p>Samples from cancer cell cultures or xenografted tumours were encapsulated in agarose plugs. The cell plugs were then irradiated, incubated to allow them to repair, and evaluated by PFGE, caspase-3, and histone H2AX activation (γH2AX). In addition, apoptosis inhibition was evaluated through chemical caspase inhibitors.</p> <p>Results</p> <p>We confirmed that spontaneous DNA fragmentation was associated with the process of encapsulation, regardless of whether cells were irradiated or not. This DNA fragmentation was also correlated to apoptosis activation in a fraction of the cells encapsulated in agarose, while non-apoptotic cell fraction could rejoin DNA fragments as was measured by γH2AX decrease and PFGE data. We were able to eliminate interference of apoptosis by applying specific caspase inhibitors, and improve the estimation of DNA repair, and apoptosis itself.</p> <p>Conclusions</p> <p>The estimation of radiation-induced DNA repair by PFGE may be improved by the use of apoptosis inhibitors. The ability to simultaneously determine DNA repair and apoptosis, which are involved in cell fate, provides new insights for using the PFGE methodology as functional assay.</p

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Simvastatin Enhances the Effects of Radiotherapy and Cetuximab on a Cell Line (FaDu) Derived from a Squamous Cell Carcinoma of Head and Neck

Radiotherapy (XRT) delivered with the antibody cetuximab is a standard treatment option for squamous cell carcinomas of head and neck (SCCNH). Cetuximab acts by blocking epidermal growth factor receptor (EGFR) signaling to inhibit cancer progression. However, a significant percentage of patients will not respond to XRT and cetuximab. Statins reduce the synthesis of cholesterol and isoprenoid derivates that may be required for efficient EGFR signaling. We assessed whether the statin simvastatin could improve this combined therapy. In vitro, simvastatin enhanced the effects of XRT alone and in combination with cetuximab in wound healing, cell proliferation, and clonogenic assays in FaDu cells. These results were reflected in xenoimplanted tumors growing into subcutaneous tissue of athymic mice where concomitant treatment with simvastatin decreased tumor growth. Consistently, lower levels of phosphorylated extracellular signal–regulated kinases 1 and 2, phosphatidylinositol 3-kinase/AKT–protein kinase B, and signal transducer and activator of transcription 3 oncoproteins and higher levels of caspase-3 and apoptosis in cell cultures and xenografts were observed. The EGFR-overexpressing A431 cell line was used to reproduce these antitumor effects of simvastatin. Our findings suggest that simvastatin may improve the efficiency of concomitant XRT and cetuximab. Further investigation in the treatment of SCCNH is warranted

Simvastatin Enhances the Effects of Radiotherapy and Cetuximab on a Cell Line (FaDu) Derived from a Squamous Cell Carcinoma of Head and Neck

Radiotherapy (XRT) delivered with the antibody cetuximab is a standard treatment option for squamous cell carcinomas of head and neck (SCCNH). Cetuximab acts by blocking epidermal growth factor receptor (EGFR) signaling to inhibit cancer progression. However, a significant percentage of patients will not respond to XRT and cetuximab. Statins reduce the synthesis of cholesterol and isoprenoid derivates that may be required for efficient EGFR signaling. We assessed whether the statin simvastatin could improve this combined therapy. In vitro, simvastatin enhanced the effects of XRT alone and in combination with cetuximab in wound healing, cell proliferation, and clonogenic assays in FaDu cells. These results were reflected in xenoimplanted tumors growing into subcutaneous tissue of athymic mice where concomitant treatment with simvastatin decreased tumor growth. Consistently, lower levels of phosphorylated extracellular signal-regulated kinases 1 and 2, phosphatidylinositol 3-kinase/AKT-protein kinase B, and signal transducer and activator of transcription 3 oncoproteins and higher levels of caspase-3 and apoptosis in cell cultures and xenografts were observed. The EGFR-overexpressing A431 cell line was used to reproduce these antitumor effects of simvastatin. Our findings suggest that simvastatin may improve the efficiency of concomitant XRT and cetuximab. Further investigation in the treatment of SCCNH is warranted

Simvastatin Enhances the Effects of Radiotherapy and Cetuximab on a Cell Line (FaDu) Derived from a Squamous Cell Carcinoma of Head and Neck

Radiotherapy (XRT) delivered with the antibody cetuximab is a standard treatment option for squamous cell carcinomas of head and neck (SCCNH). Cetuximab acts by blocking epidermal growth factor receptor (EGFR) signaling to inhibit cancer progression. However, a significant percentage of patients will not respond to XRT and cetuximab. Statins reduce the synthesis of cholesterol and isoprenoid derivates that may be required for efficient EGFR signaling. We assessed whether the statin simvastatin could improve this combined therapy. In vitro, simvastatin enhanced the effects of XRT alone and in combination with cetuximab in wound healing, cell proliferation, and clonogenic assays in FaDu cells. These results were reflected in xenoimplanted tumors growing into subcutaneous tissue of athymic mice where concomitant treatment with simvastatin decreased tumor growth. Consistently, lower levels of phosphorylated extracellular signal-regulated kinases 1 and 2, phosphatidylinositol 3-kinase/AKT-protein kinase B, and signal transducer and activator of transcription 3 oncoproteins and higher levels of caspase-3 and apoptosis in cell cultures and xenografts were observed. The EGFR-overexpressing A431 cell line was used to reproduce these antitumor effects of simvastatin. Our findings suggest that simvastatin may improve the efficiency of concomitant XRT and cetuximab. Further investigation in the treatment of SCCNH is warranted

The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis

Background: Radiation-induced DNA double-strand break (DSB) repair can be tested by using pulsed-field gel electrophoresis (PFGE) in agarose-encapsulated cells. However, previous studies have reported that this assay is impaired by the spontaneous DNA breakage in this medium. We investigated the mechanisms of this fragmentation with the principal aim of eliminating it in order to improve the estimation of radiation-induced DNA repair. Methods: Samples from cancer cell cultures or xenografted tumours were encapsulated in agarose plugs. The cell plugs were then irradiated, incubated to allow them to repair, and evaluated by PFGE, caspase-3, and histone H2AX activation (gamma H2AX). In addition, apoptosis inhibition was evaluated through chemical caspase inhibitors. Results: We confirmed that spontaneous DNA fragmentation was associated with the process of encapsulation, regardless of whether cells were irradiated or not. This DNA fragmentation was also correlated to apoptosis activation in a fraction of the cells encapsulated in agarose, while non-apoptotic cell fraction could rejoin DNA fragments as was measured by gamma H2AX decrease and PFGE data. We were able to eliminate interference of apoptosis by applying specific caspase inhibitors, and improve the estimation of DNA repair, and apoptosis itself. Conclusions: The estimation of radiation-induced DNA repair by PFGE may be improved by the use of apoptosis inhibitors. The ability to simultaneously determine DNA repair and apoptosis, which are involved in cell fate, provides new insights for using the PFGE methodology as functional assay