Circulating MicroRNAs in Young Patients with Acute Coronary Syndrome

Circulating microRNAs (miRNAs) hold great potential as novel diagnostic markers for acute coronary syndrome (ACS). This study sought to identify plasma miRNAs that are differentially expressed in young ACS patients (mean age of 38.5 ± 4.3 years) and evaluate their diagnostic potentials. Small RNA sequencing (sRNA-seq) was used to profile plasma miRNAs. Discriminatory power of the miRNAs was determined using receiver operating characteristic (ROC) analysis. Thirteen up-regulated and 16 down-regulated miRNAs were identified in young ACS patients. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) validation showed miR-183-5p was significantly up-regulated (8-fold) in ACS patients with non-ST-segment elevated myocardial infarction (NSTEMI) whereas miR-134-5p, miR-15a-5p, and let-7i-5p were significantly down-regulated (5-fold, 7-fold and 3.5-fold, respectively) in patients with ST-segment elevated myocardial infarction (STEMI), compared to the healthy controls. MiR-183-5p had a high discriminatory power to differentiate NSTEMI patients from healthy controls (area under the curve (AUC) of ROC = 0.917). The discriminatory power for STEMI patients was highest with let-7i-5p (AUC = 0.833) followed by miR-134-5p and miR-15a-5p and this further improved (AUC = 0.935) with the three miRNAs combination. Plasma miR-183-5p, miR-134-5p, miR-15a-5p and let-7i-5p are deregulated in STEMI and NSTEMI and could be potentially used to discriminate the two ACS forms

The in vitro and in vivo anti-cancer activities of a standardized quassinoids composition from Eurycoma longifolia on LNCaP human prostate cancer cells.

Quassinoids are a group of diterpenoids found in plants from the Simaroubaceae family. They are also the major bioactive compounds found in Eurycoma longifolia which is commonly used as traditional medicine in South East Asia to treat various ailments including sexual dysfunction and infertility. These uses are attributed to its ability to improve testosterone level in men. Chronic consumption of E. longifolia extracts has been reported to increase testosterone level in men and animal model but its effect on prostate growth remains unknown. Therefore, the present study investigates the effects of a standardized total quassinoids composition (SQ40) containing 40% of the total quassinoids found in E. longifolia on LNCaP human prostate cancer cell line. SQ40 inhibited LNCaP cell growth at IC50 value of 5.97 μg/mL while the IC50 on RWPE-1 human prostate normal cells was 59.26 μg/mL. SQ40 also inhibited 5α-dihydrotestosterone-stimulated growth in LNCaP cells dose-dependently. The inhibitory effect of SQ40 in anchorage-independent growth of LNCaP cells was also demonstrated using soft agar assay. SQ40 suppressed LNCaP cell growth via G0/G1 phase arrest which was accompanied by the down-regulation of CDK4, CDK2, Cyclin D1 and Cyclin D3 and up-regulation of p21Waf1/Cip1 protein levels. SQ40 at higher concentrations or longer treatment duration can cause G2M growth arrest leading to apoptotic cell death as demonstrated by the detection of poly(ADP-ribose) polymerase cleavage in LNCaP cells. Moreover, SQ40 also inhibited androgen receptor translocation to nucleus which is important for the transactivation of its target gene, prostate-specific antigen (PSA) and resulted in a significant reduction of PSA secretion after the treatment. In addition, intraperitoneal injection of 5 and 10 mg/kg of SQ40 also significantly suppressed the LNCaP tumor growth on mouse xenograft model. Results from the present study suggest that the standardized total quassinoids composition from E. longifolia promotes anti-prostate cancer activities in LNCaP human prostate cancer cells

Senescent HUVECs-secreted exosomes trigger endothelial barrier dysfunction in young endothelial cells

Accumulation of senescent endothelial cells can cause endothelium dysfunction which eventually leads to agerelated vascular disorders. The senescent-associated secretory phenotype (SASP) cells secrete a plethora of soluble factors that negatively influence the surrounding tissue microenvironment. The present study sought to investigate the effects of exosomes, which are nano-sized extracellular vesicles known for intercellular communications secreted by SASP cells on young endothelial cells. Exosomes were isolated from the condition media of senescent human umbilical vein endothelial cells (HUVECs) and then confirmed by the detection of exosome specific CD63 and CD9 expressions, electron microscopy and acetylcholinesterase assay. The purified exosomes were used to treat young HUVECs. Exposure to exosomes repressed the expression of adherens junction proteins including vascular endothelial (VE)-cadherin and beta-catenin, decreased cell growth kinetics and impaired endothelial migration potential of young endothelial cells. These findings suggest that senescent HUVECs-secreted exosomes could disrupt barrier integrity that underpins endothelial barrier dysfunction in healthy young endothelial cells. © 2019, Leibniz Research Centre for Working Environment and Human Factors. All rights reserved

Quantitative measurement of the level of nuclear AR and PSA secretion in SQ40-treated LNCaP cells.

<p>LNCaP cells were first cultured in growth media supplemented with 5% CSS for 48 hours and then treated with 3, 6 and 12 μg/mL of SQ40 in the presence or absence of 100 nM DHT for 72 hours. Nuclear fraction of AR obtained from the cell lysate and concentrations of PSA secreted into culture medium were measured using commercial available ELISA kit. (<b>A</b>) AR level was normalized to the total nuclear protein level while (<b>B</b>) the concentration of PSA were normalised to the total cell number. Data were expressed as means ± SEM of three independent experiments and indicated as percentage of 100 nM DHT-stimulated cells set at 100%. ** indicates <i>p</i><0.01; ***, <i>p</i><0.001 versus unstimulated control. ^<b>###</b>, <i>p</i><0.001 versus 100 nM DHT-stimulated cells.</p

Dose-dependent cytotoxicity of SQ40 on human normal and prostate cancer cell lines.

<p>RWPE-1 normal prostate cells (black), WRL 68 normal liver cells (blue), LNCaP (red) and PC-3 prostate cancer cells (green) were treated with increasing concentrations (2.5–100 μg/mL) of SQ40 for 72 hours and cell viability was measured using MTT reduction assay. Data were expressed as mean ± SEM of four independent experiments. IC₅₀ values of SQ40 on RWPE-1, WRL 68, LNCaP and PC-3 cells at 72 hours treatment were 59.26 μg/mL, 27.69 μg/mL, 5.97 μg/mL and 87.94 μg/mL, respectively.</p

Growth profile of LNCaP and RWPE-1 cells upon treatment with SQ40.

<p>The growth kinetics of (<b>A</b>) LNCaP and (<b>B</b>) RWPE-1 cells were examined real-time using RTCA. The impedance values were recorded in real-time and were expressed as the Cell Index (CI). Cells treated with growth media alone were referred as vehicle control while 5 μM paclitaxel-treated cells were referred as positive control. (<b>C</b>) SQ40-treated LNCaP cells were stained with 0.4% trypan blue solutions in a ratio of 1:1 after 72 and 96 hours of treatment respectively. Cells treated with growth media alone were referred as vehicle control while 1 μM paclitaxel-treated cells were referred as positive control. Data were expressed as means ± SEM of three independent experiments. ** indicates <i>p</i><0.01 versus 72-hour treated vehicle control. ^## indicates <i>p</i><0.01; ^###, <i>p</i><0.001 versus 96-hour treated vehicle control.</p

A schematic representation describing the anti-proliferation activities of SQ40 in regulation of cell cycle proteins in LNCaP cells.

<p>A schematic representation describing the anti-proliferation activities of SQ40 in regulation of cell cycle proteins in LNCaP cells.</p

Protein expression of G₁/S regulatory proteins in LNCaP cells treated with SQ40.

<p>LNCaP cells were first cultured in growth media supplemented with 5% CSS for 48 hours and then treated with 3, 6 and 12 μg/mL of SQ40 with the presence of 100 nM DHT for 72 hours. Immunoblotting was performed on protein extracts to detect CDK4, CDK2, Cyclin D1, Cyclin D3, p21^Waf1/Cip1 and p27^Kip1. β-actin served as a loading control.</p

Anti-tumor activity of SQ40 against subcutaneous LNCaP cell tumors.

<p>LNCaP cells at 2 x 10⁶ were injected subcutaneously into right flank of NCr nude mice. SQ40 treatment was initiated when the tumor was palpable. Vehicle control (saline) and SQ40 were given intraperitoneally thrice a week for 6 weeks with a total of 18 doses. Graph of (<b>A</b>) mean body weight and (<b>B</b>) tumor volume for each treatment versus the number of days after initial injection of LNCaP cells. (<b>C</b>) Representative images of tumors isolated from vehicle control, 5 mg/kg and 10 mg/kg of SQ40-treated animals. Each point represents the mean ± SEM of data (n = 6). ** indicates <i>p</i><0.01 versus vehicle control.</p

Cell cycle distribution of LNCaP cells upon SQ40 treatment.

<p>LNCaP cells were treated with growth media (vehicle control), 3, 6 and 12 μg/mL of SQ40. Cell distribution in (<b>A</b>) G₀/G₁, (<b>B</b>) S and (<b>C</b>) G₂/M phase at 24, 48 and 72 hours treatment were analysed by FACSCanto II flow cytometry and evaluated using ModFit cell cycle analysis software. Data were expressed as means ± SEM of three independent experiments. * indicates <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001 versus vehicle control. (<b>D</b>) Protein expression of cleaved-PARP in LNCaP cells upon SQ40 treatment for 72 and 96 hours. β-actin served as a loading control.</p