Obestatin and growth hormone reveal the interaction of central obesity and other cardiometabolic risk factors of metabolic syndrome.

Metabolic syndrome (MetS) is a multi-factorial disorder including central obesity (CO), insulin resistance, hyperglycemia, dyslipidemia and hypertension which increases the risk of diabetes mellitus and cardiovascular diseases. CO is considered as an essential component of MetS according to International Diabetes Federation (IDF), which may further modulate distinct signalling pathways compared with the other four MetS risk factors. Given that ghrelin signalling and the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis regulates energy balance and metabolic homeostasis, this study examined the changes in various ghrelin products and circulating hormones in response to the interaction between CO and other MetS components including blood pressure, fasting blood glucose, triglycerides, and high-density lipoprotein cholesterol in 133 Hong Kong Chinese adults. Circulating obestatin and GH were increased and reduced, respectively, by either CO or the other 4-risk factor cluster. These changes were further augmented by the presence of all MetS risk factors. However, changes of ghrelin levels were not mediated by CO but the other MetS risk factors. Our findings suggest that CO does not predict all the dysregulation of signalling pathways in individuals with MetS. Although CO and other MetS may share common signalling targets (i.e., obestatin and GH), CO does not contribute to the perturbation of ghrelin signalling.

One Year of Yoga Training Alters Ghrelin Axis in Centrally Obese Adults With Metabolic Syndrome.

Introduction: Metabolic syndrome (MetS) is a multiplex cardiometabolic manifestation associated with type 2 diabetes mellitus and cardiovascular diseases. Yoga training has been shown to alleviate MetS. Recently, circulatory ghrelin profile was demonstrated to be associated with MetS. This study examined the effects of 1 year of yoga training on ß-cell function and insulin resistance, and the involvement of metabolic peptides, including unacylated ghrelin (UnAG), acylated ghrelin (AG), obestatin, growth hormone (GH), and insulin, in the beneficial effects of yoga training in centrally obese adults with MetS. Methods: This was a follow up study, in which data of risk factors of MetS, physical performance tests [resting heart rate (HR), chair stand test (CS), chair sit and reach test (CSR), back scratch test (BS), and single leg stand tests (SLS)] and serum samples of 79 centrally obese MetS subjects aged 58 ± 8 years (39 subjects received 1-year yoga training and 40 subjects received no training) were retrieved for analyses. ß-cell function and insulin resistance were examined by Homeostasis Model Assessment (HOMA). Circulating levels of UnAG, AG, obestatin, GH, and insulin were determined by enzyme-linked immunosorbent assay using fasting serum samples. Generalized estimating equation analysis and Mann-Whitney U-test were used to detect statistically significant differences between groups. Results: Waist circumference (WC) was significantly decreased after yoga intervention (control: +2%; yoga: -4%). Significant improvements in HR (control: +2%; yoga: -5%), CS (control: -1%; yoga: +24%), CSR left (control: worsen by 0.90 cm; yoga: improved by 4.21 cm), CSR right (control: worsen by 0.75 cm; yoga: improved by 4.28 cm), right side of BS (control: improved by 0.19 cm; yoga: improved by 4.31 cm), SLS left (control: -10%; yoga: +86%), and SLS right (control: -6%; yoga: +47%) were observed after 1-year yoga training. No significant difference was found between the two groups in insulin, HOMA indices, and disposition index. Yoga training significantly increased circulating GH (control: -3%; yoga: +22%), total circulating ghrelin (control: -26%; yoga: +13%), and UnAG (control: -27%; yoga: +14%), whereas decreased AG (control: -7%; yoga: -33%) and obestatin (control: +24%; yoga: -29%). Conclusion: One-year of yoga training modulated total ghrelin, UnAG, AG, obestatin, and GH while exerting beneficial effects on physical functions and central obesity in adults with MetS. The beneficial effects of yoga may be associated with the alteration of ghrelin gene product and GH.

Ghrelin Axis Reveals the Interacting Influence of Central Obesity and Hypertension.

Objective: This study aimed to investigate how central obesity and hypertension modulate unacylated ghrelin (UnAG), acylated ghrelin (AG), obestatin, growth hormone (GH), and the ratios of UnAG/obestatin, AG/obestatin, and total ghrelin/obestatin. Methods: Circulatory abundances of UnAG, AG, obestatin and GH were determined in 387 Hong Kong Chinese female adults with age between 24 to 86 years based on a 2 × 2 factorial design of hypertension (blood pressure ≥140/90 mmHg) and central obesity (waist circumference or WC ≥80 cm). Participants were categorized as neither hypertensive nor centrally obese (NHNO; n = 105), hypertensive but not centrally obese (HNO; n = 102), centrally obese but not hypertensive (NHO; n = 74) and hypertensive and centrally obese (NO; n = 106). Pearson's correlation analyses were performed to detect the association between the peptides examined with WC and blood pressure. The main and interaction effects of hypertension and central obesity were examined by generalized estimating equations analyses. Results: Correlation analyses revealed that systolic blood pressure was negatively correlated with AG/obestatin, UnAG/obestatin and total ghrelin/obestatin ratios, AG, total ghrelin, and GH, while diastolic blood pressure was negatively correlated with UnAG/obestatin, total ghrelin/obestatin ratios, and GH. WC was negatively correlated with AG/obestatin, UnAG/obestatin, and total ghrelin/obestatin ratios, UnAG, AG, total ghrelin, GH, and obestatin. Interaction effects of hypertension and central obesity were observed on UnAG/obestatin, AG/obestatin and total ghrelin/obestatin ratios, and obestatin. Obestatin in NHO group was significantly higher compared to NHNO and HO groups. UnAG/obestatin, AG/obestatin, and total ghrelin/obestatin ratios were higher in NHNO group compared to HNO and HO groups. Main effects of central obesity and hypertension were observed in UnAG, total ghrelin and GH. The HO group manifested the lowest level of UnAG, total ghrelin and GH among all the groups studied. Main effect of hypertension was observed on AG, suggesting that hypertensive individuals exhibited lower levels of AG regardless of central obesity. Conclusion: Circulatory ghrelin gene products and GH exhibit different modes of modulation in response to the co-manifestation of multiple cardiovascular risk factors compared with a single risk factor alone.

S100A8 and S100A9 Are Associated with Doxorubicin-Induced Cardiotoxicity in the Heart of Diabetic Mice.

Cardiomyopathy is a clinical problem that occurs in the hearts of type 2 diabetic patients as well as cancer patients undergoing doxorubicin chemotherapy. The number of diabetic cancer patients is increasing but surprisingly the cardiac damaging effects of doxorubicin, a commonly used chemotherapeutic drug, on diabetic hearts have not been well-examined. As the signaling mechanisms of the doxorubicin-induced cardiomyopathy in type 2 diabetic heart are largely unknown, this study examined the molecular signaling pathways that are responsible for the doxorubicin-induced cardiotoxicity in type 2 diabetic hearts. Male 14- to 18-week-old db/db mice were used as the type 2 diabetic model, and age-matched non-diabetic db/+ mice served as controls. The db/+ non-diabetic and db/db diabetic mice were randomly assigned to the following groups: db/+CON, db/+DOX-5d, db/+DOX-7d, db/dbCON, db/dbDOX-5d, and db/dbDOX-7d. Mice assigned to doxorubicin (DOX) group were exposed to an intraperitoneal (i.p.) injection of DOX at a dose of 15 mg/kg to induce cardiomyopathy. Mice in control (CON) groups were i.p. injected with the same volume of saline instead of DOX. Mice were euthanized by overdose of ketamine and xylazine 5 or 7 days after the DOX injection. Microarray analysis was adopted to examine the changes of the whole transcriptional profile in response to doxorubicin exposure in diabetic hearts. Ventricular fractional shortening was examined as an indicator of cardiac function by transthoracic echocardiography. The presence of diabetic cardiomyopathy in db/db mice was evident by the reduction of fractional shortening. There was a further impairment of cardiac contractile function 7 days after the DOX administration in db/db diabetic mice. According to our microarray analysis, we identified a panel of regulatory genes associated with cardiac remodeling, inflammatory response, oxidative stress, and metabolism in the DOX-induced cardiac injury in diabetic heart. The microarray results of selected genes were confirmed by real time PCR. Notably, S100A8 and S100A9 were found to have a unique specific expression pattern that was coincident with the DOX-induced cardiomyopathy in diabetic hearts. Correspondingly, NF-κB expression in diabetic hearts was increased together with the elevation of S100A8/9 and activation of p38 MAPK signaling after DOX administration, which induced cardiac inflammation as demonstrated by the elevation of cardiac IL-6 level. These findings provide novel pre-clinical information for revealing the S100A8/A9-associated molecular signaling pathways that mediate the doxorubicin-induced cardiotoxicity in diabetic hearts.

Acute Treatment of Resveratrol Alleviates Doxorubicin-Induced Myotoxicity in Aged Skeletal Muscle Through SIRT1-Dependent Mechanisms.

Study of the exacerbating effects of chemotherapeutics, such as doxorubicin, on the impairment of insulin metabolic signaling in aged skeletal muscle is very limited. Here, we tested the hypothesis that activation of sirtuin 1 deacetylase activity by resveratrol would prevent the disruption of insulin signaling and augmentation of catabolic markers induced by doxorubicin in aged skeletal muscle. Two- and 10-month-old senescence-accelerated mice (prone 8) were randomized to receive saline, doxorubicin, doxorubicin and resveratrol, or a combination of doxorubicin, resveratrol, and sirtinol or EX527. Doxorubicin reduced the sirtuin 1 activity without affecting the phosphorylation levels of IRS1(Ser307), mTOR(Ser2481), Akt(Thr308/Ser473), membranous glucose transporter 4, protein abundance of PDK4, and enzymatic activity of pyruvate dehydrogenase in aged muscles. Intriguingly, resveratrol attenuated the doxorubicin-induced elevations of apoptotic and catabolic markers measured as Bax, caspase 3 activity, apoptotic DNA fragmentation, MuRF-1, ubiquitinated proteins, and proteasomal activity in aged muscles, whereas these beneficial effects were abolished on inhibition of sirtuin 1 by sirtinol or EX527. Markers of insulin signaling were not affected by doxorubicin or resveratrol in the senescent skeletal muscle. Nevertheless, the antiapoptotic and anticatabolic effects of resveratrol in aged skeletal muscle treated with doxorubicin were mediated in a sirtuin 1-dependent signaling manner.

SIRT1-dependent myoprotective effects of resveratrol on muscle injury induced by compression.

Our current understanding on the molecular mechanisms by which sustained compression induces skeletal muscle injury is very limited. This study aimed to test the hypothesis that activation of SIRT1 by the natural antioxidant resveratrol could deactivate apoptotic and catabolic signaling in skeletal muscle exposed to moderate compression. Two cycles of 6-h constant pressure at 100 mmHg was applied to the tibialis region of right, but not left hindlimbs of Sprague Dawley rats pre-treated with DMSO (vehicle control) or resveratrol with/without sirtinol. Skeletal muscle tissues lying underneath and spatially corresponding to the compressed sites were collected for analyses. Resveratrol prevented the compression-induced manifestations of pathohistological damages including elevations of the number of interstitial nuclei and area of interstitial space and ameliorated oxidative damages measured as 4-hydroxy-2-nonenal (4HNE) and nitrotyrosine in skeletal muscle. In parallel, resveratrol augmented the expression level and activity of SIRT1 and phosphorylation levels of Foxo3a and Akt while suppressed the increases in protein abundances of p53, Bax, MAFbx, and ubiquitin, enzymatic activities of caspase 3 and 20S proteasome, and apoptotic DNA fragmentation in the compressed muscle. These favorable myoprotective effects of resveratrol were diminished upon pharmacological blockade of SIRT1 by using sirtinol. These novel data support the hypothesis that the anti-apoptotic and anti-catabolic effects of resveratrol on compression injury in skeletal muscle required the action of SIRT1.

Unacylated ghrelin restores insulin and autophagic signaling in skeletal muscle of diabetic mice.

Impairment of insulin signaling in skeletal muscle detrimentally affects insulin-stimulated disposal of glucose. Restoration of insulin signaling in skeletal muscle is important as muscle is one of the major sites for disposal of blood glucose. Recently, unacylated ghrelin (UnAG) has received attention in diabetic research due to its favorable actions on improving glucose tolerance, glycemic control, and insulin sensitivity. The investigation of UnAG has entered phase Ib clinical trial in type 2 diabetes and phase II clinical trial in hyperphagia in Prader-Willi syndrome. Nonetheless, the precise mechanisms responsible for the anti-diabetic actions of UnAG remain incompletely understood. In this study, we examined the effects of UnAG on restoring the impaired insulin signaling in skeletal muscle of db/db diabetic mice. Our results demonstrated that UnAG effectively restored the impaired insulin signaling in diabetic muscle. UnAG decreased insulin receptor substrate (IRS) phosphorylation, increased protein kinase B (Akt) phosphorylation, and, hence, suppressed mTOR signaling. Consequently, UnAG enhanced Glut4 localization and increased PDH activity in the diabetic skeletal muscle. Intriguingly, our data indicated that UnAG normalized the suppressed autophagic signaling in diabetic muscle. In conclusion, our findings illustrated that UnAG restored the impaired insulin and autophagic signaling in skeletal muscle of diabetic mice, which are valuable to understand the underlying mechanisms of the anti-diabetic action of UnAG at peripheral skeletal muscle level.

DECODE: an integrated differential co-expression and differential expression analysis of gene expression data.

BACKGROUND: Both differential expression (DE) and differential co-expression (DC) analyses are appreciated as useful tools in understanding gene regulation related to complex diseases. The performance of integrating DE and DC, however, remains unexplored. RESULTS: In this study, we proposed a novel analytical approach called DECODE (Differential Co-expression and Differential Expression) to integrate DC and DE analyses of gene expression data. DECODE allows one to study the combined features of DC and DE of each transcript between two conditions. By incorporating information of the dependency between DC and DE variables, two optimal thresholds for defining substantial change in expression and co-expression are systematically defined for each gene based on chi-square maximization. By using these thresholds, genes can be categorized into four groups with either high or low DC and DE characteristics. In this study, DECODE was applied to a large breast cancer microarray data set consisted of two thousand tumor samples. By identifying genes with high DE and high DC, we demonstrated that DECODE could improve the detection of some functional gene sets such as those related to immune system, metastasis, lipid and glucose metabolism. Further investigation on the identified genes and the associated functional pathways would provide an additional level of understanding of complex disease mechanism. CONCLUSIONS: By complementing the recent DC and the traditional DE analyses, DECODE is a valuable methodology for investigating biological functions of genes exhibiting disease-associated DE and DC combined characteristics, which may not be easily revealed through DC or DE approach alone. DECODE is available at the Comprehensive R Archive Network (CRAN): http://cran.r-project.org/web/packages/decode/index.html .

Effects of long-term resveratrol-induced SIRT1 activation on insulin and apoptotic signalling in aged skeletal muscle.

AIMS: Activation of Foxo1 is known to promote apoptosis and disturbances to insulin signalling. However, their modulating roles in aged skeletal muscle are not clear. The present study tested the hypothesis that long-term (i.e. 8 month) resveratrol supplementation would improve physical traits including exercise capacity and basal voluntary activity of aged mice and modulate insulin/apoptotic signalling in aged skeletal muscle. This study also examined whether these resveratrol-associated alterations would involve orchestration of the SIRT1-Foxo1 signalling axis. METHODS: Two-month-old SAMP8 mice were randomly assigned to young, aged and aged with resveratrol treatment (AR) groups. The AR mice were supplemented with 4.9 mg(-1) kg(-1) day(-1) resveratrol for 8 months. All animals were subject to endurance capacity test and voluntary motor behaviour assessment. The lateral gastrocnemius muscle tissues were harvested for further analyses. RESULTS: Long-term resveratrol treatment significantly alleviated the age-associated reductions in exercise capacity and voluntary motor behaviour. The protein content, but not the deacetylase activity of SIRT1 was increased with concomitant elevations of p300 acetylase and acetylation of Foxo1 in aged muscle. The aged muscle also manifested signs of impaired insulin signalling including attenuated phosphorylation of Akt, activity of pyruvate dehydrogenase and membrane trafficking of GLUT4 and elevated levels of phosphorylated IRS1 and iNOS and apoptotic activation measured as Bim, p53 and apoptotic DNA fragmentation. Intriguingly, all these age-related adverse changes were mitigated with the activation of SIRT1 deacetylase activity after long-term resveratrol treatment. CONCLUSIONS: These data suggest that modulation of the SIRT1-Foxo1 axis by long-term resveratrol treatment enhances physical traits and alleviates the unfavourable changes in insulin and apoptotic signalling in aged muscle.

Clinical utility of plasma Epstein-Barr virus DNA and ERCC1 single nucleotide polymorphism in nasopharyngeal carcinoma.

BACKGROUND: Single nucleotide polymorphism (SNP) of the excision repair cross-complementing group 1 (ERCC1) gene has been linked with sensitivity to platinum and radiation. The authors hypothesized that the ERCC1 genotype for the SNPs cytosine-to-thymine substitution at codon 118 (C118T) and cytosine-to-adenine substitution at codon 8092 (C8092A) is prognostic in patients with nasopharyngeal carcinoma (NPC) who receive either radiotherapy (RT) or cisplatin plus RT. METHODS: The authors tested their hypothesis using biomarker screening samples from the Hong Kong NPC Study Group 0502 trial, which was a prospective, multicenter clinical trial that used post-RT plasma Epstein-Bar virus (EBV) DNA (pEBV) levels to screen patients with high-risk NPC for adjuvant chemotherapy. RESULTS: ERCC1 SNPs were analyzed in 576 consecutive patients who were screened by pEBV. In the total biomarker population, there was no significant association of ERCC1 C118T or C8092A genotype with relapse-free survival (RFS) or overall survival (OS). There also was no correlation between ERCC1 genotype and ERCC1 protein or messenger RNA expression in a subset of patients who had available paired biopsies. Post-RT pEBV status was the only independent prognosticator for RFS and OS in multivariate analyses. However, there was a significant interaction between ERCC1 C118T genotype and post-RT pEBV status (RFS, P = .0106; OS, P = .0067). The ERCC1 C118T genotype was significantly associated with both RFS (hazard ratio, 1.67; 95% confidence interval, 1.07-2.61; P = .024) and OS (hazard ratio, 2.31; 95% confidence interval, 1.22-4.40; P = .0106) in the post-RT pEBV-negative population, but not in the pEBV-positive population. CONCLUSIONS: The current results prospectively validate pEBV as the most significant prognostic biomarker in NPC that can be used to select high-risk patients for adjuvant therapy. The ERCC1 C118T genotype may help to identify a favorable subgroup (approximately 7%) of pEBV-negative patients with NPC who have an excellent prognosis and can be spared the toxicities of further therapy.

Resveratrol protects against doxorubicin-induced cardiotoxicity in aged hearts through the SIRT1-USP7 axis.

KEY POINTS: Doxorubicin induced functional deteriorations and elevations of USP7-related apoptotic/catabolic signalling in the senescent heart Resveratrol protects against doxorubicin-induced alterations through the restoration of SIRT1 deacetylase activity ABSTRACT: A compromised cardiac function is often seen in elderly cancer patients receiving doxorubicin therapy. The present study tested the hypothesis that acute intervention with resveratrol, a natural anti-oxidant found in grapes and red wine, reduces the cardiotoxicity of doxorubicin through restoration of sirtuin 1 (SIRT1) deacetylase activity, and attenuation of the catabolic/apoptotic pathways orchestrated by USP7, a p53 deubiquitinating protein, using young (aged 2 months) and old (aged 10 months) senescence-accelerated mice prone 8 (SAMP8). Animals were randomised to receive saline, doxorubicin, and doxorubicin in combination with resveratrol, in the presence or absence of SIRT1 inhibitors, sirtinol or EX527. Resveratrol alone, but not in combination with either of the SIRT1 inhibitors, suppressed the doxorubicin-induced impairment of cardiac systolic function in aged animals. Doxorubicin reduced SIRT1 deacetylase activity, and elevated proteasomal activity and USP7; it also increased the protein level of p300 and ubiquitinated proteins in hearts from aged SAMP8. These doxorubicin-induced alterations were prevented by resveratrol, whereas the protective action of resveratrol was antagonised by sirtinol and EX527. In young SAMP8 hearts, resveratrol attenuated the doxorubicin-induced increases in acetylation of Foxo1 and transactivation of MuRF-1, whereas these mitigations were not found after treatment with SIRT1 inhibitors. However, the protein contents of acetylated Foxo1 and MuRF-1 were not affected by any of the drugs studied in aged SAMP8 hearts. Resveratrol also ameliorated the augmentation of pro-apoptotic markers including p53, Bax, caspase 3 activity and apoptotic DNA fragmentation induced by doxorubicin in hearts from aged animals, whereas these reductions were diminished by combined treatment with SIRT1 inhibitors. These data demonstrate that resveratrol ameliorates doxorubicin-induced cardiotoxicity in aged hearts through the restoration of SIRT1 activity to attenuate USP7-related catabolic/pro-apoptotic signalling.

[D-Lys3]-GHRP-6 exhibits pro-autophagic effects on skeletal muscle.

[D-Lys3]-GHRP-6 is regarded as a highly selective growth-hormone secretagogue receptor (GHSR) antagonist and has been widely used to investigate the dependency of GHSR-1a signalling mediated by acylated ghrelin. However, [D-Lys3]-GHRP-6 has been reported to influence other cellular processes which are unrelated to GHSR-1a. This study aimed to examine the effects of [D-Lys3]-GHRP-6 on autophagic and apoptotic cellular signalling in skeletal muscle. [D-Lys3]-GHRP-6 enhanced the autophagic signalling demonstrated by the increases in protein abundances of beclin-1 and LC3 II-to-LC3 1 ratio in both normal muscle and doxorubicin-injured muscle. [D-Lys3]-GHRP-6 reduced the activation of muscle apoptosis induced by doxorubicin. No histological abnormalities were observed in the [D-Lys3]-GHRP-6-treated muscle. Intriguingly, the doxorubicin-induced increase in centronucleated muscle fibres was not observed in muscle treated with [D-Lys3]-GHRP-6, suggesting the myoprotective effects of [D-Lys3]-GHRP-6 against doxorubicin injury. The [D-Lys3]-GHRP-6-induced activation of autophagy was found to be abolished by the co-treatment of CXCR4 antagonist, suggesting that the pro-autophagic effects of [D-Lys3]-GHRP-6 might be mediated through CXCR4. In conclusion, [D-Lys3]-GHRP-6 exhibits pro-autophagic effects on skeletal muscle under both normal and doxorubicin-injured conditions.

Protective effects of desacyl ghrelin on diabetic cardiomyopathy.

AIM: Diabetic cardiomyopathy is a specific complication of type 2 diabetes mellitus, which causes progressive cardiac dysfunction. Desacyl ghrelin has been preliminarily demonstrated to have beneficial effects on cardiovascular system and glucose metabolism, which are both related to diabetic cardiomyopathy. The aim of this study was to investigate the protective effects of desacyl ghrelin on cardiac dysfunction, cardiac fibrosis, and cellular autophagy in a type 2 diabetic mouse model. MATERIALS AND METHODS: Fourteen- to eighteen-week-old db/db diabetic and db/+ non-diabetic mice were intraperitoneally treated with desacyl ghrelin at a dosage of 100 µg/kg for ten consecutive days. Ventricular fractional shortening was examined as an indicator of cardiac function by transthoracic echocardiography. RESULTS: The presence of diabetic cardiomyopathy was evident by the reduction in fractional shortening shown in our examined db/db mice. Intriguingly, this reduction in fractional shortening was not observed in the hearts of db/db mice treated with desacyl ghrelin. Cardiac fibrosis (indicated by excessive collagen deposition, decreased by Adiponectin and Mmp13 expression, and up-regulated by Mmp8 expression) and impairment of autophagic signalling (indicated by decreases in Foxo3 and LC3 II-to-LC3 I ratio) were shown in the hearts of diabetic mice. All these cellular and molecular alterations were alleviated by desacyl ghrelin treatment. The key cardiac pro-survival cellular signals including AMPK, Akt, ERK1/2, and GSK3α/ß were impaired in the diabetic hearts, but the administration of desacyl ghrelin attenuated these signalling impairments. CONCLUSIONS: These results collectively demonstrate that desacyl ghrelin protects the heart against cardiac dysfunction in type 2 diabetic mice by inhibiting excessive collagen deposition and enhancing cardiac autophagic signalling via the pro-survival cellular AMPK/ERK1/2 signalling pathways.

Targeting angiogenic genes as a therapeutic approach for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a complex liver disease with limited treatment options and often resulting in a poor prognosis. The development of HCC depends on the formation of new blood vessels and it demonstrates hypervascularity and invasive property to the surrounding vasculature clinically. A complex network of growth factors acting on both tumor cells and endothelial cells mediates the angiogenesis in HCC. It is an attractive approach to inhibit the angiogenic processes as the treatment of HCC and therefore, anti-angiogenic TKIs were developed to inhibit the vessel formation in the tumors. However, it is currently perceived that the efficacy of these anti-angiogenic TKIs has reached plateau, and it is necessary to develop novel agents with non-TKI mechanism to inhibit the angiogenic targets. With the better understanding of molecular mechanisms that govern angiogenesis, as well as the advancement in biomedical engineering, new approaches of gene therapy have brought hopes for therapeutic intervention in HCC. Gene therapy is based on the transfer of genetic material to the patients with the aim to modify or correct the malignancy from its molecular basis. In this article, we will discuss the conventional anti-angiogenic therapies and the gene therapy approaches in HCC. The therapeutic potential of gene therapy for HCC treatment has been demonstrated and further development of anti-angiogenic may result in new treatment option for HCC patients.

Label-free enumeration of colorectal cancer cells from lymphocytes performed at a high cell-loading density by using interdigitated ring-array microelectrodes.

We report the label-free enumeration of human colorectal-carcinoma cells from blood lymphocytes by using interdigitated ring-array microelectrodes; this enumeration was based on the dielectrophoretic selection of cells. Because of the novel design of the device, a continuous flow of cells is uniformly distributed into parallel streams through 300 rings (~40 µm in diameter each) that are integrated into the electrode digits. Using this array, 82% of cancer cells were recovered and 99% of blood lymphocytes were removed. Most of the cancer cells recovered were viable (94%) and could be cultivated for >8 days, during which period they retained their normal cell morphology and proliferation rates. The recovery rate correlated closely with cancer-cell loadings in spiked samples and this relationship was linear over a range of at least 2 orders of magnitude. Importantly, because of the 3D structure of the rings, these results were obtained at a high cell-loading concentration (10(7)cells/mL). The rings could be further optimized for use in accurate label-free identification and measurement of circulating tumor cells in cancer research and disease management.

Modulating effect of SIRT1 activation induced by resveratrol on Foxo1-associated apoptotic signalling in senescent heart.

Elevations of cardiomyocyte apoptosis and fibrotic deposition are major characteristics of the ageing heart. Resveratrol, a polyphenol in grapes and red wine, is known to improve insulin resistance and increase mitochondrial biogenesis through the SIRT1-PGC-1α signalling axis. Recent studies attempted to relate SIRT1 activation by resveratrol to the regulation of apoptosis in various disease models of cardiac muscle. In the present study, we tested the hypothesis that long-term (8-month) treatment of resveratrol would activate SIRT1 and improve the cardiac function of senescent mice through suppression of Foxo1-associated pro-apoptotic signalling. Our echocardiographic measurements indicated that the cardiac systolic function measured as fractional shortening and ejection fraction was significantly reduced in aged mice when compared with the young mice. These reductions, however, were not observed in resveratrol-treated hearts. Ageing significantly reduced the deacetylase activity, but not the protein abundance of SIRT1 in the heart. This reduction was accompanied by increased acetylation of the Foxo1 transcription factor and transactivation of its target, pro-apoptotic Bim. Subsequent analyses indicated that pro-apoptotic signalling measured as p53, Bax and apoptotic DNA fragmentation was up-regulated in the heart of aged mice. In contrast, resveratrol restored SIRT1 activity and suppressed elevations of Foxo1 acetylation, Bim and pro-apoptotic signalling in the aged heart. In parallel, resveratrol also attenuated the ageing-induced elevations of fibrotic collagen deposition and markers of oxidative damage including 4HNE and nitrotyrosine. In conclusion, these novel data demonstrate that resveratrol mitigates pro-apoptotic signalling in senescent heart through a deacetylation mechanism of SIRT1 that represses the Foxo1-Bim-associated pro-apoptotic signalling axis.

Investigation of biological factors influencing the placental mRNA profile in maternal plasma.

OBJECTIVE: Circulating placental-derived RNA is useful for noninvasive prenatal investigation. However, in addition to placental gene expression, there are limited investigations on other biological parameters that may affect the circulating placental RNA profile. In this study, we explored two of these potential parameters. METHODS: We first demonstrated the existence of such biological parameters by comparing the relative levels of a panel of placental-derived transcripts between the placentas and maternal plasma by digital PCRs. We then compared the post-delivery clearance of the transcripts by serial plasma samples collected from pregnant women after delivery. We also studied the placental in vivo localization of the transcripts by in situ hybridization. RESULTS: There was an imperfect correlation of the transcript levels between the placentas and maternal plasma, with placenta-specific 4 (PLAC4) mRNA showing the largest discrepancy. Although PLAC4 mRNA showed a similar clearance half-life with other transcripts, we observed a preferential localization of PLAC4 mRNA around the villous surface. We speculated that this phenomenon might play a role in favoring the release of PLAC4 mRNA molecules into maternal plasma. CONCLUSION: We revealed that in addition to expression levels in the placenta, other biological factors might interplay to determine the maternal plasma profile of placental-derived RNAs.

Noninvasive detection of cancer-associated genome-wide hypomethylation and copy number aberrations by plasma DNA bisulfite sequencing.

We explored the detection of genome-wide hypomethylation in plasma using shotgun massively parallel bisulfite sequencing as a marker for cancer. Tumor-associated copy number aberrations (CNAs) could also be observed from the bisulfite DNA sequencing data. Hypomethylation and CNAs were detected in the plasma DNA of patients with hepatocellular carcinoma, breast cancer, lung cancer, nasopharyngeal cancer, smooth muscle sarcoma, and neuroendocrine tumor. For the detection of nonmetastatic cancer cases, plasma hypomethylation gave a sensitivity and specificity of 74% and 94%, respectively, when a mean of 93 million reads per case were obtained. Reducing the sequencing depth to 10 million reads per case was found to have no adverse effect on the sensitivity and specificity for cancer detection, giving respective figures of 68% and 94%. This characteristic thus indicates that analysis of plasma hypomethylation by this sequencing-based method may be a relatively cost-effective approach for cancer detection. We also demonstrated that plasma hypomethylation had utility for monitoring hepatocellular carcinoma patients following tumor resection and for detecting residual disease. Plasma hypomethylation can be combined with plasma CNA analysis for further enhancement of the detection sensitivity or specificity using different diagnostic algorithms. Using the detection of at least one type of aberration to define an abnormality, a sensitivity of 87% could be achieved with a specificity of 88%. These developments have thus expanded the applications of plasma DNA analysis for cancer detection and monitoring.

A study of circulating interleukin 10 in prognostication of unresectable hepatocellular carcinoma.

BACKGROUND: The level of circulating interleukin 10 (IL-10) is elevated in a proportion of patients with hepatocellular carcinoma (HCC). The objective of the current study was to evaluate the prognostic significance of serum the IL-10 level in patients with unresectable HCC. METHODS: Patients with unresectable HCC who provided serum at the time of diagnosis were enrolled prospectively in the study. The level of circulating IL-10 in serum samples was determined by enzyme-linked immunosorbent assay. The association of the IL-10 level with overall survival was evaluated in relation to sociodemographics, liver function, hepatitis B viral load, and tumor staging. RESULTS: In total, 222 patients were recruited; of these, 82.4% were positive for hepatitis B virus surface antigen, and 65.8% had Barcelona Clinic Liver Cancer stage C disease. The mean log IL-10 level was 1.1 pg/mL, and 146 patients had an IL-10 level >1 pg/mL (high IL-10 group). The high IL-10 group had worse overall survival than the low IL-10 group (5.0 months vs 14.9 months; hazard ratio, 2.192; P < .0001). The IL-10 level was associated with worse hepatic function and with a high alanine transaminase (ALT) level. The IL-10 level remained an independent prognostic factor (hazard ratio, 1.824; P = .0005) after adjustment for sociodemographics, tumor staging, treatment, Child-Pugh stage, and ALT level. The IL-10 level also subdivided patients into 2 populations with distinct survival (10.2 months vs 3.5 months; P = .0027). CONCLUSIONS: The serum IL-10 level was identified as an independent prognostic factor for unresectable HCC. The current findings suggested that an elevated IL-10 level may be related to hepatic injury caused by cirrhotic processes rather than tumor load. The authors concluded that the IL-10 level offers additional prognostic value to the existing tumor staging systems.