Yang-Dan-Tang, Identified from 15 Chinese Herbal Formulae, Inhibits Human Lung Cancer Cell Proliferation via Cell Cycle Arrest

Lung cancer has long been one of the most deadly forms of cancer. The majority of lung cancers are of the non-small-cell lung cancer (NSCLC) type. Here we used the non-small-cell lung carcinoma cell line A549 to screen 15 different traditional Chinese herbal medicine (CHM) formulae to explore the possible mechanisms of alternative medicine in lung cancer therapy. We identified three formulae (Formulae 3, 5, and 14) that substantially decreased the survival of A549 cells but did not affect MRC5 normal lung tissue cells. Formula 14, Yang-Dan-Tang, a modified decoction of Ramulus Cinnamomi Cassiae, was chosen for further characterization. Flow cytometry analysis showed that treatment of Formula 14 induced cell cycle arrest in G1 and G2 phase without causing significant cell death. These results were also confirmed by Western blot analysis, with decreased expression of G1/S and G2/M promoting cell cycle machinery including cyclin D3, cyclin B1, CDK4, and CDK6. This study provides further insight into the possible working mechanism of Yang-Dan-Tang in patients

Targeting cancer addiction for SALL4 by shifting its transcriptome with a pharmacologic peptide

Sal-like 4 (SALL4) is a nuclear factor central to the maintenance of stem cell pluripotency and is a key component in hepatocellular carcinoma, a malignancy with no effective treatment. In cancer cells, SALL4 associates with nucleosome remodeling deacetylase (NuRD) to silence tumor-suppressor genes, such as PTEN. Here, we determined the crystal structure of an amino-terminal peptide of SALL4(1-12) complexed to RBBp4, the chaperone subunit of NuRD, at 2.7 Å, and subsequent design of a potent therapeutic SALL4 peptide (FFW) capable of antagonizing the SALL4-NURD interaction using systematic truncation and amino acid substitution studies. FFW peptide disruption of the SALL4-NuRD complex resulted in unidirectional up-regulation of transcripts, turning SALL4 from a dual transcription repressor-activator mode to singular transcription activator mode. We demonstrate that FFW has a target affinity of 23 nM, and displays significant antitumor effects, inhibiting tumor growth by 85% in xenograft mouse models. Using transcriptome and survival analysis, we discovered that the peptide inhibits the transcription-repressor function of SALL4 and causes massive up-regulation of transcripts that are beneficial to patient survival. This study supports the SALL4-NuRD complex as a drug target and FFW as a viable drug candidate, showcasing an effective strategy to accurately target oncogenes previously considered undruggable

Platelet physiology and function in neonates and children on extracorporeal membrane oxygenation (ECMO)

© 2019 Hui Ping YawExtracorporeal membrane oxygenation (ECMO) is a modified form of heart-lung machine that aims to provide short- to-medium length of support to patients with cardiac and/or respiratory dysfunction. Children represent the majority of the ECMO population. While increasing experience and technical improvements for ECMO over the years have seen some improvement in outcomes, the rates of morbidity and mortality remain high in this population and many complications are related to bleeding and thrombosis. Platelets are a key element of the coagulation system. Platelet dysfunction can cause coagulopathy in adults on ECMO, however, the association between modification of platelet function and coagulopathy remains unknown for children. This study hypothesised that there are platelet-specific differences: I.) for paediatric patients on ECMO according to their age, pathway onto ECMO and duration of ECMO that could be associated with the development of bleeding or thrombosis during ECMO and II.) at different sites in a paediatric ECMO system. This study aimed to characterize the molecular indices of circulating platelets in the paediatric ECMO population using whole blood flow cytometry approach: 1) To examine and compare the effect of a patient’s pathway onto ECMO on platelet phenotype and function and their associations with the development of bleeding or thrombosis during ECMO. 2) To examine and compare the effect of a patient’s age on platelet phenotype and function and their associations with the development of bleeding or thrombosis during ECMO. 3) To examine and compare the effect of a patient’s duration of ECMO on platelet phenotype and function and their associations with the development of bleeding or thrombosis during ECMO. 4) To examine and compare the site-specific differences for platelet phenotype and function in the paediatric ECMO system. A total of 22 paediatric patients [median (interquartile range): 0.34 (0.01 – 3.38) years] were included in this study. Citrated whole blood samples were collected and a whole blood flow cytometry method was developed for the evaluation of platelet phenotype and function in the setting of ECMO. The platelet assays were standardized to ensure minimal pre-analytical activation. By using multiple thrombin receptor activator peptide 6 (a thrombin mimic) concentrations, the platelet panels also showed sensitivity to detect subtle changes in platelet response. The multifaceted flow cytometry panels allowed simultaneous evaluation of platelets for their phenotype, function and interactions with monocytes and neutrophils. Such approach to investigate platelet-specific changes from different aspects suits well for the ECMO population, representing a complex group of patients. These results showed that the whole blood flow cytometry assay is a reliable and useful platelet function test for paediatric ECMO patients. Results from the analysis for platelet-specific markers within the first 24 hours showed no difference in platelet phenotype and function between patients from different pathways onto ECMO and different ages. However, the association of platelet-specific changes and the development of clinical events during ECMO were different according to a patient’s age and pathway onto ECMO. Patients who had cardiopulmonary bypass before coming onto ECMO and had bleeding had increased platelet integrin GPIIb/IIIa receptor expression and reduced circulating neutrophil-platelet aggregates level compared to patients who had no bleeding during ECMO. In contrast, patients who had no cardiopulmonary bypass before coming onto ECMO and developed bleeding had reduced platelet response compared to those who had no bleeding during ECMO. Conversely, increased lysosome release was observed for children with thrombosis and may indicate the presence of a protective mechanism against increased thrombus formation. Duration of ECMO had been recognized as an important factor affecting the outcome of paediatric ECMO patients. The results showed an increased level of von Willebrand factor (VWF) receptor and reduced platelet response for granule exocytosis with increasing number of days on ECMO (Day 2 vs. Day 5). Most importantly, such platelet-specific changes that involved GPIb/IX/V receptor and granule release with increasing duration of ECMO were only observed in patients who had bleeding but not in patients without bleeding after five days on ECMO. In addition, elevated circulating monocyte-platelet aggregates level was only observed in patients who had thrombosis but not for those without thrombosis. Together, these results suggested a link between pathway onto ECMO/age/duration of ECMO, bleeding/thrombosis and platelet dysfunction. Hence, markers relevant to the platelet-specific changes could be used as the indicators for increased bleeding or thrombosis risk for paediatric patients during ECMO. Platelet phenotype and function were also compared at different sites in the ECMO system to identify the site-specific differences of platelet-specific changes that have not previously been investigated in a paediatric ECMO system. In the setting of mechanical circulatory support, shear and oxidative stress are known to modify platelet phenotype via integrin GPIIb/IIIa and GPIb/IX/V receptors and increase platelet response via multiple platelet activation pathways. The results demonstrated that platelet phenotype and function were different at different sites in a paediatric ECMO system. The platelet-specific changes observed included the modification of platelet phenotype via increased VWF and integrin GPIIb/IIIa receptor expression, increased platelet activation through the activation of the integrin GPIIb/IIIa receptor and higher platelet responsiveness at the post-oxygenator site compared to the pre-oxygenator site. However, the exact cause of the site-specific differences of platelet phenotype and function remained to be identified. In summary, this study demonstrated the feasibility of using whole blood flow cytometry method with multifaceted platelet-specific panels as a reliable platelet function test in paediatric patients on ECMO. Platelet-specific changes could be associated with the development of bleeding or thrombosis during ECMO. In addition, platelet phenotype and function were different at different sites in a paediatric ECMO system. Together, this study provides new insights for the circuit-related platelet-specific changes and the understanding of how modifications of platelet phenotype and function that are dependent on patient’s factors may be associated with coagulopathy in children on ECMO

Glucose and lipid metabolism in rats supplemented with glycyrrhizic acid exposed to short- or long- term stress and fed on a high-calorie diet

Stress and consumption of high-calorie diet are well-recognized as the primary contributor to various metabolic diseases such as the metabolic syndrome. Glycyrrhizic acid (GA), an active compound in the root extract of the licorice plant, Glycyrrhiza glabra has been shown to improve hyperglycaemia and dyslipidaemia in rats fed on a high- calorie diet. However, the effect of GA on glucose and lipid metabolism in rats under stress in combination with high- calorie diet has yet to be explored. Hence, the objective of this study was to investigate the effect of short- and long- term stress on glucose and lipid metabolism and the role of GA in improving these. Forty-eight Sprague Dawley rats were exposed to constant light illumination (300-400 lux) for either 14 or 28 days for short- or long-term stress and fed on a high-calorie diet. The blood and tissues including liver, kidney, heart, pancreas, subcutaneous and visceral adipose tissues (SAT and VAT), abdominal muscle (AM) and quadriceps femoris (QF) were collected for analysis. The stress, glucose metabolism and lipid metabolism states in rats were examined using different parameters. The systolic blood pressure, stress hormones i.e. adrenaline and corticosterone were used as the stress parameters. Together with blood glucose level, serum insulin and homeostatic index of insulin resistance, the activities of gluconeogenic enzymes included glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK) and hexose-6-phosphate dehydrogenase (H6PDH) were determined for the glucose metabolism state. Lastly, the state of lipid metabolism was identified using lipid profile and serum free fatty acids, fatty acids profile and the expression levels of various genes involved in glucose and lipid metabolism which are the peroxisome proliferator-activated receptors (PPAR-α & PPAR-γ), lipoprotein lipase (LPL), elongases (ELOVL5 & ELOVL6) and desaturases (D5D, D6D & D9D). Continuous light exposure for 14 days induced stress response in the short-term exposure group while the long- term exposure group adapted to stress induced by continuous light exposure for 28 days. When comparisons were made within the 14- or 28-day exposure group, the short-term exposure group had significant elevated blood glucose concentrations but GA lowered the concentrations significantly. However, neither stress nor GA affected the blood glucose concentration of the long-term exposure group. With regards to adrenaline and corticosterone, rats given GA from the short-term exposure group had elevated adrenaline level while those from the long-term exposure group had reduced level of corticosterone. Gluconeogenesis is the process of synthesis of glucose from non- carbohydrate source. In response to stress, gluconeogenic enzymes activities are increased to increase fuel supply to the important organs. Specifically, GA was found to reduce H6PDH activities in the VAT of the short-term exposure group and QF of the long-term exposure group. Serum free fatty acids and lipid profile are the key indicators for diabetes and cardiovascular disease. The development of these diseases is closely associated with disrupted lipid metabolism. PPAR and LPL are the key genes for lipid metabolism while the elongases and desaturases determine the chemical properties of fatty acids and hence their metabolic fate. Recent studies have demonstrated the importance of these genes in the development of MetS. Within the 14-day exposure group, neither stress nor GA affected all the lipid profile parameters and serum free fatty acids. As for the 28-day exposure group, stress was found to only increase triacylglycerol concentration significantly only. GA did not affect the lipid profile parameters and serum free fatty acids. For different types of fatty acids, the long-term exposure group generally had higher fatty acids content in the liver, SAT and VAT and lower content in the pancreas. Stress did not affect PPAR-α expression in both the 14- and 28-day exposure groups. However, GA-treated rats from the former group had increased PPAR-α expression only in the kidney while all other tissues from the latter group were unaffected. Stress did not affect PPAR-γ expressions in the 14-day exposure group but GA elevated PPAR-γ expression in the kidney significantly. As for the 28-day exposure group, stress increased its expression in the heart. GA was found to increase PPAR-γ expression in both the AM and QF. With regards to LPL, stress did not affect LPL expressions in all the studied tissues from both the 14- and 28-day exposure groups except for significant up-regulation in the QF of the latter group. GA did not affect LPL expressions in both the 14- and 28-day exposure groups except for significant up-regulation in the heart of the latter group. As for the expression of elongases and desaturases in the liver, stress down-regulated ELOVL5 in the long-term exposure group while up-regulated ELOVL6 in the short- term exposure group. On the other hand, hepatic desaturases and elongases and desaturases in both the SAT and VAT were unaffected by stress. Neither elongases nor desaturases expressions in all the studied tissues were affected by GA except for significant down-regulation of ELOVL5 in the VAT of the 28-day exposure group. Thus, our results indicate that GA could improve blood glucose concentration in rats exposed to short-term stress and were on high-calorie diet via selective action on gluconeogenic enzymes activities and modifications of fatty acids via regulation of PPAR, elongases and desaturases in different tissues. This research is the first report of GA on glucose and lipid metabolism in rats under stress and on high-calorie diet. The results gave evidence supporting the role of GA in ameliorating MetS via site-specific regulation of gene expressions involved in glucose and lipid metabolism and modification of fatty acids

Glucose and lipid metabolism in rats supplemented with glycyrrhizic acid exposed to short- or long- term stress and fed on a high-calorie diet

Stress and consumption of high-calorie diet are well-recognized as the primary contributor to various metabolic diseases such as the metabolic syndrome. Glycyrrhizic acid (GA), an active compound in the root extract of the licorice plant, Glycyrrhiza glabra has been shown to improve hyperglycaemia and dyslipidaemia in rats fed on a high- calorie diet. However, the effect of GA on glucose and lipid metabolism in rats under stress in combination with high- calorie diet has yet to be explored. Hence, the objective of this study was to investigate the effect of short- and long- term stress on glucose and lipid metabolism and the role of GA in improving these. Forty-eight Sprague Dawley rats were exposed to constant light illumination (300-400 lux) for either 14 or 28 days for short- or long-term stress and fed on a high-calorie diet. The blood and tissues including liver, kidney, heart, pancreas, subcutaneous and visceral adipose tissues (SAT and VAT), abdominal muscle (AM) and quadriceps femoris (QF) were collected for analysis. The stress, glucose metabolism and lipid metabolism states in rats were examined using different parameters. The systolic blood pressure, stress hormones i.e. adrenaline and corticosterone were used as the stress parameters. Together with blood glucose level, serum insulin and homeostatic index of insulin resistance, the activities of gluconeogenic enzymes included glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK) and hexose-6-phosphate dehydrogenase (H6PDH) were determined for the glucose metabolism state. Lastly, the state of lipid metabolism was identified using lipid profile and serum free fatty acids, fatty acids profile and the expression levels of various genes involved in glucose and lipid metabolism which are the peroxisome proliferator-activated receptors (PPAR-α & PPAR-γ), lipoprotein lipase (LPL), elongases (ELOVL5 & ELOVL6) and desaturases (D5D, D6D & D9D). Continuous light exposure for 14 days induced stress response in the short-term exposure group while the long- term exposure group adapted to stress induced by continuous light exposure for 28 days. When comparisons were made within the 14- or 28-day exposure group, the short-term exposure group had significant elevated blood glucose concentrations but GA lowered the concentrations significantly. However, neither stress nor GA affected the blood glucose concentration of the long-term exposure group. With regards to adrenaline and corticosterone, rats given GA from the short-term exposure group had elevated adrenaline level while those from the long-term exposure group had reduced level of corticosterone. Gluconeogenesis is the process of synthesis of glucose from non- carbohydrate source. In response to stress, gluconeogenic enzymes activities are increased to increase fuel supply to the important organs. Specifically, GA was found to reduce H6PDH activities in the VAT of the short-term exposure group and QF of the long-term exposure group. Serum free fatty acids and lipid profile are the key indicators for diabetes and cardiovascular disease. The development of these diseases is closely associated with disrupted lipid metabolism. PPAR and LPL are the key genes for lipid metabolism while the elongases and desaturases determine the chemical properties of fatty acids and hence their metabolic fate. Recent studies have demonstrated the importance of these genes in the development of MetS. Within the 14-day exposure group, neither stress nor GA affected all the lipid profile parameters and serum free fatty acids. As for the 28-day exposure group, stress was found to only increase triacylglycerol concentration significantly only. GA did not affect the lipid profile parameters and serum free fatty acids. For different types of fatty acids, the long-term exposure group generally had higher fatty acids content in the liver, SAT and VAT and lower content in the pancreas. Stress did not affect PPAR-α expression in both the 14- and 28-day exposure groups. However, GA-treated rats from the former group had increased PPAR-α expression only in the kidney while all other tissues from the latter group were unaffected. Stress did not affect PPAR-γ expressions in the 14-day exposure group but GA elevated PPAR-γ expression in the kidney significantly. As for the 28-day exposure group, stress increased its expression in the heart. GA was found to increase PPAR-γ expression in both the AM and QF. With regards to LPL, stress did not affect LPL expressions in all the studied tissues from both the 14- and 28-day exposure groups except for significant up-regulation in the QF of the latter group. GA did not affect LPL expressions in both the 14- and 28-day exposure groups except for significant up-regulation in the heart of the latter group. As for the expression of elongases and desaturases in the liver, stress down-regulated ELOVL5 in the long-term exposure group while up-regulated ELOVL6 in the short- term exposure group. On the other hand, hepatic desaturases and elongases and desaturases in both the SAT and VAT were unaffected by stress. Neither elongases nor desaturases expressions in all the studied tissues were affected by GA except for significant down-regulation of ELOVL5 in the VAT of the 28-day exposure group. Thus, our results indicate that GA could improve blood glucose concentration in rats exposed to short-term stress and were on high-calorie diet via selective action on gluconeogenic enzymes activities and modifications of fatty acids via regulation of PPAR, elongases and desaturases in different tissues. This research is the first report of GA on glucose and lipid metabolism in rats under stress and on high-calorie diet. The results gave evidence supporting the role of GA in ameliorating MetS via site-specific regulation of gene expressions involved in glucose and lipid metabolism and modification of fatty acids

Whole blood flow cytometry protocol for the assessment of platelet phenotype, function, and cellular interactions

Platelets are a key component of the hemostatic system and their roles in inflammation via interactions with leukocytes have also gained attention in recent years. Changes in platelet phenotype and function can cause bleeding and/or thrombosis and, as such, monitoring platelet-specific changes is crucial to assessing hemostasis in the clinical setting. Currently, available platelet function tests such as platelet aggregometry and thromboelastography require a large volume of blood, which is a major limitation for the pediatric population. Whole blood flow cytometric analysis of platelets is increasingly utilized in recent years, primarily due to the sensitivity of this method, but also because it only requires a small amount of blood with minimal sample manipulation. We have developed a whole blood flow cytometry methodological approach that enables the assessment of platelet phenotype, function, and their interactions with monocytes and neutrophils

Guidelines for panel design, optimization, and performance of whole blood multi-color flow cytometry of platelet surface markers

Flow cytometry is a valuable tool in determining the phenotype and function of platelets accurately. The emergence of platelet flow cytometry in recent years provides an attractive alternative to other platelet analytical techniques, with advantages such as requiring small volumes and being highly sensitive to minimal changes in receptor function and expression. Here we present a methodical approach encompassing the stages in the development and optimization of platelet flow cytometry panels based on our extensive experience in this area

Eset partners with Oct4 to restrict extraembryonic trophoblast lineage potential in embryonic stem cells

The histone H3 Lys 9 (H3K9) methyltransferase Eset is an epigenetic regulator critical for the development of the inner cell mass (ICM). Although ICM-derived embryonic stem (ES) cells are normally unable to contribute to the trophectoderm (TE) in blastocysts, we find that depletion of Eset by shRNAs leads to differentiation with the formation of trophoblast-like cells and induction of trophoblast-associated gene expression. Using chromatin immmunoprecipitation (ChIP) and sequencing (ChIP-seq) analyses, we identified Eset target genes with Eset-dependent H3K9 trimethylation. We confirmed that genes that are preferentially expressed in the TE (Tcfap2a and Cdx2) are bound and repressed by Eset. Single-cell PCR analysis shows that the expression of Cdx2 and Tcfap2a is also induced in Eset-depleted morula cells. Importantly, Eset-depleted cells can incorporate into the TE of a blastocyst and, subsequently, placental tissues. Coimmunoprecipitation and ChIP assays further demonstrate that Eset interacts with Oct4, which in turn recruits Eset to silence these trophoblast-associated genes. Our results suggest that Eset restricts the extraembryonic trophoblast lineage potential of pluripotent cells and links an epigenetic regulator to key cell fate decision through a pluripotency factor

Irregularities in glucose metabolism induced by stress and high-calorie diet can be attenuated by glycyrrhizic acid

Stress and high-calorie diet increase the risk of developing metabolic syndrome. Glycyrrhizic acid (GA) has been shown to improve hyperglycaemia and dyslipidaemia under various physiological conditions. This study was aimed at examining the effects of stress and GA on glucose metabolism under short- or long-term stress. Forty-eight Sprague Dawley rats were divided into two groups with constant stress induced by light (300-400 lux) for either 14 days (short-term stress) or 28 days (long-term stress). Within each group, the rats were subdivided into three treatment groups i.e. Group A (control group): high-calorie diet (HCD) only; Group B: HCD + stress (14 or 28 days) and Group C: HCD + stress (14 or 28 days) + GA (100 mg/kg). The blood glucose concentrations of the rats exposed to 14-day stress were elevated significantly and GA lowered blood glucose concentration significantly in the 14-day exposure group. The 28-day exposure group adapted to stress as shown by the lower adrenaline level and gluconeogenic enzymes activities in most of the tissues than the 14-day exposure group. With regards to adrenaline and corticosterone, GA was found to increased adrenaline significantly in the short-term exposure group while lowering corticosterone in the long-term exposure group. GA-treated short- and long-term exposure groups had significant reduction in hexose-6-phosphate dehydrogenase activities in the visceral adipose tissues and quadriceps femoris respectively. The results may indicate the role of GA in improving blood glucose concentration in individuals exposed to short-term stress who are already on a high-calorie diet via selective action on gluconeogenic enzymes in different tissues