Synthesis of Nonsymmetrically Substituted 2,3-Dialkoxyphenazine Derivatives and Preliminary Examination of Their Cytotoxicity.

Fourteen new 2,3-dialkoxyphenazine derivatives with two different alkoxy groups bearing R1 and R2 alkyl chains, defined as -CH2CH(CH3)2 and -(CH2)n-1CH3 for n = 1, 2, 4, 6, 8, and 10, were prepared via regioselective synthesis. The applied synthetic protocol is based on the following reactions: the Buchwald-Hartwig coupling of a nonsymmetrically substituted 4,5-dialkoxy-2-nitroaniline with a 1-bromo-2-nitrobenzene derivative featuring additional tert-butyl, trifluoromethyl or two methoxy groups; the reduction of bis(2-nitrophenyl)amine; and a final step of tandem-like oxidation that leads to the preparation of a heterocyclic phenazine system. The regioselectivity of these steps and the molecular structure of the compounds under investigation were confirmed by nuclear magnetic resonance and additionally by single-crystal X-ray diffraction performed for some examples of 5 and 6 phenazine series. For 7-(tert-butyl)-3-isobutoxy-2-(octyloxy)phenazine (5f), 3-(hexyloxy)-2-isobutoxy-7-(trifluoromethyl)phenazine (6e), and 2,3-bis(hexyloxy)-7,8-dimethoxyphenazine (7), viability and cytotoxicity assays were performed on the LoVo human colon adenocarcinoma cell line, with 5f confirmed to exhibit cytotoxicity.

No differences in miRNA expression in the stroma of ERG+ and ERG- prostate cancers.

Prostate cancer (PC) is one of the most common cancers in males. A significant proportion of PCs bear TMPRSS2-ETS translocation and overexpress ERG transcription factor, allowing classification into ERG+ and ERG- groups, which differ in several features including the tumor microenvironment. The aim of the study was to verify whether they differ in expression of the miRNA in the microenvironment. The material consisted of 150 radical prostatectomies. Immunohistochemistry (IHC) for ERG was done using a routine method. FISH for TMPRSS2-ETS translocation was done with a ZytoLight SPEC ERG/TMPRSS2 TriCheck Probe. From each case, a representative section was selected, and tumor and non-tumor were microdissected with the LMD7000 device. RNA was isolated using the RNeasy Mini Kit system (Qiagen) and miRNA libraries were prepared with the NEBNext Multiplex Small RNA Library Prep Set for Illumina and their sequencing was performed on the NexSeq 500. Statistical analysis was done with Statistica and R software. When analyzing the expression of miRNAs some differences could be seen, but after correction for multiple comparisons was applied, these were found to be non- significant.

MicroRNA composition of plasma extracellular vesicles: a harbinger of late cardiotoxicity of doxorubicin.

BACKGROUND: The use of doxorubicin is associated with an increased risk of acute and long-term cardiomyopathy. Despite the constantly growing number of cancer survivors, little is known about the transcriptional mechanisms which progress in the time leading to a severe cardiac outcome. It is also unclear whether long-term transcriptomic alterations related to doxorubicin use are similar to transcriptomic patterns present in patients suffering from other cardiomyopathies. METHODS: We have sequenced miRNA from total plasma and extracellular vesicles (EVs) from 66 acute lymphoblastic leukemia (ALL) survivors and 61 healthy controls (254 samples in total). We then analyzed processes regulated by differentially expressed circulating miRNAs and cross-validated results with the data of patients with clinically manifested cardiomyopathies. RESULTS: We found that especially miRNAs contained within EVs may be informative in terms of cardiomyopathy development and may regulate pathways related to neurotrophin signaling, transforming growth factor beta (TGFß) or epidermal growth factor receptors (ErbB). We identified vesicular miR-144-3p and miR-423-3p as the most variable between groups and significantly correlated with echocardiographic parameters and, respectively, for plasma: let-7g-5p and miR-16-2-3p. Moreover, vesicular miR-144-3p correlates with the highest number of echocardiographic parameters and is differentially expressed in the circulation of patients with dilated cardiomyopathy. We also found that distribution of particular miRNAs between of plasma and EVs (proportion between compartments) e.g., miR-184 in ALL, is altered, suggesting changes within secretory and miRNA sorting mechanisms. CONCLUSIONS: Our results show that transcriptomic changes resulting from doxorubicin induced myocardial injury are reflected in circulating miRNA levels and precede development of the late onset cardiomyopathy phenotype. Among miRNAs related to cardiac function, we found vesicular miR-144-3p and miR-423-3p, as well as let-7g-5p and miR-16-2-3p contained in the total plasma. Selection of source for such studies (plasma or EVs) is of critical importance, as distribution of some miRNA between plasma and EVs is altered in ALL survivors, in comparison to healthy people, which suggests that doxorubicin-induced changes include miRNA sorting and export to extracellular space.

The relationship between myocardial fibrosis and myocardial microRNAs in dilated cardiomyopathy: A link between mir-133a and cardiovascular events.

It is unknown whether fibrosis-associated microRNAs: miR-21, miR-26, miR-29, miR-30 and miR-133a are linked to cardiovascular (CV) outcome. The study evaluated the levels of extracellular matrix (ECM) fibrosis and the prevalence of particular microRNAs in patients with dilated cardiomyopathy (DCM) to investigate any correlation with CV events. METHODS: Seventy DCM patients (48 ± 12 years, EF 24.4 ± 7.4%) underwent right ventricular biopsy. The control group was comprised of 7 patients with CAD who underwent CABG and intraoperative biopsy. MicroRNAs were measured in blood and myocardial tissue via qPCR. The end-point was a combination of CV death and urgent HF hospitalization at the end of 12 months. There were differential levels of circulating and myocardial miR-26 and miR-29 as well as myocardial miR-133a when the DCM and CABG groups were compared. Corresponding circulating and myocardial microRNAs did not correlate with one another. There was no correlation between microRNA and ECM fibrosis. By the end of the 12-month period of the study, CV death had occurred in 6 patients, and a further 19 patients required urgent HF hospitalization. None of the circulating microRNAs was a predictor of the combined end-point; however, myocardial miR-133a was an independent predictor in unadjusted models (HR 1.53; 95% CI 1.14-2.05; P < .004) and adjusted models (HR 1.57; 95% CI 1.14-2.17; P < .005). The best cut-off value for the miR-133a level for the prediction of the combined end-point was 0.74 ΔCq, with an AUC of 0.67. The absence of a correlation between the corresponding circulating and myocardial microRNAs calls into question their cellular source. This study sheds new light on the role of microRNAs in ECM fibrosis in DCM, which warrants further exploration.

Association of plasma miR-223 and platelet reactivity in patients with coronary artery disease on dual antiplatelet therapy: A preliminary report.

Decreased plasma levels of microRNA-223 (miR-223), predominantly of platelet origin, were proposed as a surrogate marker of efficacy of antiplatelet therapy. However, higher on-treatment platelet reactivity was associated with lower plasma miR-223 in patients with coronary artery disease (CAD) on dual antiplatelet therapy (DAPT) including clopidogrel and aspirin. Our aim was to compare plasma miR-223 and platelet reactivity in CAD patients on DAPT with newer P2Y12 antagonists vs. clopidogrel. We studied 21 men with CAD admitted to our centre owing to a non-ST-elevation acute coronary syndrome, and with an uncomplicated hospital course. From the day of admission, the patients were receiving either clopidogrel (n = 11) or prasugrel/ticagrelor (n = 10) in addition to aspirin. Before discharge, miR-223 expression in plasma was estimated by quantitative polymerase chain reaction using the comparative Ct method relative to miR-16 as an endogenous control. Multiple electrode aggregometry was used to assess platelet aggregation in response to adenosine diphosphate (ADP). ADP-induced platelet reactivity was decreased in the patients treated with prasugrel or ticagrelor compared with those on clopidogrel (mean ± SD: 139 ± 71 vs. 313 ± 162 arbitrary units [AU]*min, p = 0.006), due to a more potent antiplatelet activity of the novel P2Y12 antagonists. Consequently, six out of seven patients in the lower tertile of the ADP-induced platelet aggregation were treated with the newer P2Y12 blockers, whereas six out of seven patients in the upper tertile were on clopidogrel. Plasma miR-223 was elevated with decreasing platelet reactivity (Spearman's rho = -0.52; p = 0.015 for trend), being significantly higher in the lower tertile of the ADP-induced platelet aggregation (median [range]: 1.06 [0.25-2.31]) vs. the upper tertile (0.20 [0.13-2.30]) (p = 0.04). In conclusion, our preliminary results argue against the notion of low plasma miR-223 as a marker of platelet responsiveness to DAPT. On the contrary, more potent platelet inhibition associated mainly with newer P2Y12 antagonists appears to coincide with higher miR-223 relative to the subjects with attenuated responsiveness to DAPT.

Twelve-month kinetics of circulating fibrosis-linked microRNAs (miR-21, miR-29, miR-30, and miR-133a) and the relationship with extracellular matrix fibrosis in dilated cardiomyopathy.

Introduction: A single measurement of any biomarker may not reflect its full biological meaning. The kinetics of fibrosis-linked microRNAs and their relationship with extracellular matrix (ECM) fibrosis in dilated cardiomyopathy (DCM) have not been explored. Material and methods: We evaluated 70 consecutive DCM patients (48 ±12.1 years, left ventricular ejection fraction 24.4 ±7.4%). All patients underwent right ventricular endomyocardial biopsy in order to quantify ECM fibrosis and measure collagen volume fraction (CVF). Circulating microRNAs (miR-21-5p, miR-29b, miR-30c-5p, and miR-133a-3p) were measured with quantitative polymerase chain reaction (PCR) at baseline and at 3 and 12 months. Results: Based on the biopsy results, two groups of patients were identified: with (n = 24, 34.3%) and without (n = 46, 65.7%) ECM fibrosis. Except for a single measurement of miR-29b at 3 months (DCM with fibrosis: 6.03 ±0.72 vs. DCM without fibrosis: 6.4 ±0.75 ΔCq; p < 0.05), baseline, 3- and 12-month kinetics of microRNAs did not differ between the two groups. Moreover, 12-month microRNA kinetics did not differ in patients with new-onset DCM (duration < 6 months; n = 35) and chronic DCM (> 6 months; n = 35). Only miR-29 at 3 months correlated with CVF (r = -0.31; p < 0.05), whereas other microRNAs did not correlate with CVF either at 3 or at 12 months. Conclusions: Regardless of ECM fibrosis status or duration of the disease, 12-month patterns of circulating microRNAs are similar in DCM. Correlations between microRNAs, measured at 3 and 12 months, are lower than expected. In this study, regardless of the time point, circulating microRNAs were not able to differentiate between DCM patients with versus without fibrosis.

Relations between circulating and myocardial fibrosis-linked microRNAs with left ventricular reverse remodeling in dilated cardiomyopathy.

BACKGROUND: Left ventricular reverse remodeling (LVRR) determines clinical status and outcomes in dilated cardiomyopathy (DCM). The extent of myocardial fibrosis is connected to the systolic function of the heart. The recent discovery of the contribution of microRNAs (miRs) to the regulation of cardiac remodeling, LVRR and fibrosis warrants exploration. OBJECTIVES: The aim of the study was to examine the predictive value of circulating and myocardial miR expression for LVRR in DCM. MATERIAL AND METHODS: Seventy consecutive DCM patients (age 48 ±12.1 years, 90% male, ejection fraction (EF) 24.4% ±7.4%) were included in the study. At baseline, all patients underwent clinical assessment, echocardiography, venous blood sampling, and right ventricular endomyocardial biopsy. Circulating and myocardial miRs (miR-21, -26, -29, -30, -133a, and -423) were measured with quantitative real-time polymerase chain reaction (qRT-PCR). LVRR was defined as an increase in EF ≥ 10%, accompanied by a decrease in left ventricle end-diastolic diameter (LVEDd) ≥10% or LVEDd ≤ 33 mm/m2 between baseline and 3-month follow-up. RESULTS: At the 3-month follow-up, 4 patients had died and 3 patients had incomplete data. The remaining patients were divided according to the presence of LVRR into LVRR-present (n = 32, 51%) and LVRR-absent (n = 31, 49%) groups. Out of all the circulating and tissue miRs under study, only myocardial expression of miR-133a significantly differed between the LVRR-present and LVRR-absent group (1.22 (0.47-1.90) vs 0.61 (0.25-0.99) ΔCq, respectively, p < 0.01). miR-133a was found to be a significant LVRR predictor in unadjusted (odds ratio (OR) = 2.81 (1.23-6.40), p < 0.05) and adjusted for duration of disease, left ventricle end-diastolic (LVED) volume (LVEDvol), hs-troponin-T, and NT-proBNP (OR = 5.20 (1.13-24.050, p < 0.05) models. CONCLUSIONS: From all of the circulating and tissue miRs, only myocardial miR-133a showed increased expression in LVRR-present patients and was found an independent LVRR predictor. This indicates a link between miR-133 and cardiac remodeling in DCM.

Right ventricular morphology and function is not related with microRNAs and fibrosis markers in dilated cardiomyopathy.

BACKGROUND: The relationship between right ventricle (RV), extracellular matrix (ECM) fibrosis and fibrosis-linked, circulating microRNAs in dilated cardiomyopathy (DCM) is unknown. AIM: The aim of the study was to uncover the associations between serum markers of ECM metabolism and circulating microRNAs with RV morphological and functional parameters. METHODS: The study population consisted of 70 consecutive DCM patients (ejection fraction 24.4 ± ± 7.4%). Based on detailed echocardiographic assessment - 15 patients had normal RV, whereas 55 patients had RV dilatation (RVD) and/or RV systolic dysfunction (RVSD). Procollagens type I and III carboxy- and amino-terminal peptides, osteopontin (OPN), TGF1-b1, connective tissue growth factor (CTGF), MMP-2, MMP-9 and TIMP-1 were measured in serum as well as expression of miR-21, miR-26, miR-29, miR-30 and miR-133a. All patients underwent endomyocardial biopsy. RESULTS: Biopsy-proven fibrosis was evenly distributed in two groups. Serum levels of fibrosis markers did not differ between groups. OPN, CTGF, MMP-2, and TIMP-1 correlated with RV parameters. Only miR-133 a was differently expressed in both groups. MiR-21, miR-26, miR-30, and miR-133a cor-related with RV morphological but without functional parameters. Not a single marker of fibrosis was independently associated with RV. MiR-30 was associated with RV impairment in the logistic regression model adjusted for age, sex, body mass index, and disease duration; however, lost its significance in the more comprehensive model. CONCLUSIONS: Right ventricle structural and functional abnormalities are common in DCM. ECM fibrosis and serum markers are not associated with RV impairment. The prognostic value of studied microRNAs on RV is limited in DCM.