Increased Extracellular Nucleosome Levels and Microparticles in Atrial Fibrillation Patients Compared to the Age-Matched Normal Population

Introduction : Atrial fibrillation (AF) is currently the most common cardiac arrhythmia encountered in clinical practice and a major cause of morbidity and mortality among adults. It is estimated that atrial fibrillation affects approximately 2.7 million people in the United States. Extracellular plasma nucleosomes (PNs) are complexes of DNA and histones that are released during cell death. Histones have been shown to function as DAMPs when they are translocated from the nucleus to the extracellular space. It is hypothesized that these extracellular nucleosomes contribute to the observed hypercoagulable state and embolic complications in AF. Methods : The concentration of PNs in 48 plasma samples of atrial fibrillation patients and 38 aged-matched controls were measured using the Cell Death Detection ELISA PLUS assay (Roche Diagnostics, Mannheim, Germany) and functional microparticle levels were measured using an annexin binding method (Hyphen Biomedical, Paris, France). Results : When comparing the concentration of nucleosomes in plasma, the atrial fibrillation patients (306.1 ± 328.86 μg/mL) had much higher levels than the aged-matched controls (177.9 ± 146.7 μg/mL), (p = 0.0015). Functional microparticles were also markedly elevated in the AF patients (18.6 ± 4.1 nM) in comparison to the aged-matched controls (4.6 ± 3.1 nM), (p=0.001). A significant correlation was observed between the PN's and microparticles, (p < 0.05). Conclusion : Both the microparticles and PNs were elevated in atrial fibrillation when compared to aged-matched controls, suggesting increased cell death resulting in the generation of these biomarkers. Extracellular nucleosomes function as DAMPs by binding to receptors and triggering the activation of multiple signal pathways which may contribute to the elevation of microparticles. Due to the involvement of inflammation and thrombosis in the pathology of atrial fibrillation, the simultaneously increased circulating nucleosome and microparticle levels in patients with AF implies their role in the pathogenesis of this syndrome. Disclosures No relevant conflicts of interest to declare.

Effect of inhibition of nuclear protein export using a novel CRM1 inhibitor on the apoptotic response to SN38 in preclinical colon cancer models.

609 Background: Resistance to conventional chemotherapy remains a major challenge in Stage IV colon cancer. CRM1 inhibition leads to nuclear sequestration of proteins such as tumor suppressor p53, growth regulatory proteins, and chemotherapy targets such as topoisomerase I/II. We examined the effects of combination use of KPT185 (a novel CRM1 inhibitor) with SN38 (active metabolite of irinotecan) and the effect of drug administration sequence in human colon cancer cell lines to determine if CRM1 inhibition enhances the cytotoxic effect of chemotherapy. Methods: We evaluated the combination effect of KPT185 with SN-38 on both Lovo (KPT-sensitive, IC50 = ∼ 500nM) and HT29 cells (KPT-resistant, IC50 = 1000 ∼ 3000nM) by the Chou-Talalay method, an MTT-based assay that interrogates response across a spectrum of drug dosages: KPT185 (0, 1, 10, 100, 1000, 10000 nM) and SN38 (0, 100, 500, 1000 nM). Cell cycle analysis by FACS with propidium iodide (PI) staining was performed. Effects on apoptosis were determined by FACS (Annexin V/PI staining) and Cell Death Detection ELISA assay. Results: The Chou-Talalay method determined that there is a synergistic effect when KPT185 is combined with SN38 in both Lovo and HT29 cells (combination index > 1). FACS analysis demonstrated combination use of KPT185 and SN38 induced an increase in the apoptotic sub-G1 fraction and a shift toward G2/M arrest. Combination treatment also significantly increased the Annexin V/PI-positive fraction compared with SN38 alone case (P < 0.05). Treatment sequence studies demonstrated that pretreatment of SN38 followed by KPT185 (KPT-post) produced the maximum synergistic effect compared with pretreatment of KPT185 followed by SN38 (KPT-pre) or concurrent use (KPT-con); Cell Death Detection ELISA assay showed KPT-post increased apoptosis most (4.3-fold) compared with KPT-pre (4.2-fold), KPT-con (3.8-fold) and SN38 alone (1-fold). Conclusions: Our results show KPT185, a novel CRM1 inhibitor, sensitizes the response to SN38 in KPT-sensitive as well as KPT-resistant colon cancer cells. This method of sensitizing colon cancer cells warrants further evaluation in preclinical models of colon cancer.