HDAC5-mediated exosomal Maspin and miR-151a-3p as biomarkers for enhancing radiation treatment sensitivity in hepatocellular carcinoma.

BACKGROUND: Tumor-derived exosomes are critical elements of the cell-cell communication response to various stimuli. This study aims to reveal that the histone deacetylase 5 (HDAC5) and p53 interaction upon radiation in hepatocellular carcinoma intricately regulates the secretion and composition of exosomes. METHODS: We observed that HDAC5 and p53 expression were significantly increased by 2 Gy and 4 Gy radiation exposure in HCC. Normal- and radiation-derived exosomes released by HepG2 were purified to investigate the exosomal components. RESULTS: We found that in the radiation-derived exosome, exosomal Maspin was notably increased. Maspin is known as an anti-angiogenic gene. The expression of Maspin was regulated at the cellular level by HDAC5, and it was elaborately regulated and released in the exosome. Radiation-derived exosome treatment caused significant inhibition of angiogenesis in HUVECs and mouse aortic tissues. Meanwhile, we confirmed that miR-151a-3p was significantly reduced in the radiation-derived exosome through exosomal miRNA sequencing, and three HCC-specific exosomal miRNAs were also decreased. In particular, miR-151a-3p induced an anti-apoptotic response by inhibiting p53, and it was shown to induce EMT and promote tumor growth by regulating p53-related tumor progression genes. In the HCC xenograft model, radiation-induced exosome injection significantly reduced angiogenesis and tumor size. CONCLUSIONS: Our present findings demonstrated HDAC5 is a vital gene of the p53-mediated release of exosomes resulting in tumor suppression through anti-cancer exosomal components in response to radiation. Finally, we highlight the important role of exosomal Maspin and mi-151a-3p as a biomarker in enhancing radiation treatment sensitivity. Therapeutic potential of HDAC5 through p53-mediated exosome modulation in radiation treatment of hepatocellular carcinoma.

Radiation-induced C-reactive protein triggers apoptosis of vascular smooth muscle cells through ROS interfering with the STAT3/Ref-1 complex.

Damage to normal tissue can occur over a long period after cancer radiotherapy. Free radical by radiation can initiate or accelerate chronic inflammation, which can lead to atherosclerosis. However, the underlying mechanisms remain unclear. Vascular smooth muscle cells (VSMCs) proliferate in response to JAK/STAT3 signalling. C-reactive protein (CRP) can induce VSMCs apoptosis via triggering NADPH oxidase (NOX). Apoptotic VSMCs promote instability and inflammation of atherosclerotic lesions. Herein, we identified a VSMCs that switched from proliferation to apoptosis through was enhanced by radiation-induced CRP. NOX inhibition using lentiviral sh-p22phox prevented apoptosis upon radiation-induced CRP. CRP overexpression reduced the amount of STAT3/Ref-1 complex, decreased JAK/STAT phosphorylation and formed a new complex of Ref-1/CRP in VSMC. Apoptosis of VSMCs was further increased by CRP co-overexpressed with Ref-1. Functional inhibition of NOX or p53 also prevented apoptotic activity of the CRP-Ref-1 complex. Immunofluorescence showed co-localization of CRP, Ref-1 and p53 with α-actin-positive VSMC in human atherosclerotic plaques. In conclusion, radiation-induced CRP increased the VSMCs apoptosis through Ref-1, which dissociated the STAT3/Ref-1 complex, interfered with JAK/STAT3 activity, and interacted with CRP-Ref-1, thus resulting in transcription-independent cell death via p53. Targeting CRP as a vascular side effect of radiotherapy could be exploited to improve curability.

PIBF1 suppresses the ATR/CHK1 signaling pathway and promotes proliferation and motility of triple-negative breast cancer cells.

PURPOSE: This study evaluates the oncogenic role of PIBF1 in triple-negative breast cancer (TNBC). TNBC is considered to have a poorer prognosis than other types of breast cancer and is associated with high risk of recurrence and distant metastasis. Currently, there are no effective therapies for the TNBC patients with distant metastasis due to the lack of targeted therapeutic options. METHODS: The effects of PIBF1 knockdown on the cell viability and motility of TNBC cell lines were investigated. Effects of PIBF1 overexpression on tumorigenicity and cell motility were confirmed using Ba/F3 cell line and xenograft study on BALB/c nude mice. RESULTS: In TNBC cell lines that highly express PIBF1, knockdown of PIBF1 induces apoptosis and suppresses cell viability and motility with activation of the ATR/CHK1 signaling pathway. Moreover, the oncogenic function of PIBF1 was confirmed using the Ba/F3 cell line. CONCLUSION: For the first time, these findings clarify the role of PIBF1 in regulating ATR/CHK1 signaling pathway and inhibiting the proliferation and migration of TNBC cell lines. These results demonstrate the oncogenic roles of PIBF1 and provide new insights into the function and the molecular mechanism of PIBF1 in malignant TNBC.

Radiation-Induced Phosphorylation of Serine 360 of SMC1 in Human Peripheral Blood Mononuclear Cells.

In the event of a mass casualty radiation scenario, biodosimetry has the potential to quantify individual exposures for triaging and providing dose-appropriate medical intervention. Structural maintenance of chromosomes 1 (SMC1) is phosphorylated in response to ionizing radiation. The goal of this study was to develop a new biodosimetry method using SMC1 phosphorylation as a measure of exposure to radiation. In the initial experiments, two normal human cell lines (WI-38VA-13 and HaCaT) and four lymphoblastoid cell lines were irradiated, and the levels of SMC1 phosphorylation at Ser-360 and Ser-957 were assessed using Western blotting. Subsequently, similar experiments were performed using peripheral blood mononuclear cells (PBMCs) obtained from 20 healthy adults. Phosphorylation of SMC1 at Ser-957 and Ser-360 was increased by exposure in a dose-dependent manner, peaked at 1-3 h postirradiation and then decreased gradually. Ser-360 was identified as a new phosphorylation site and was more sensitive to radiation than Ser-957, especially at doses below 1 Gy. Our results demonstrate a robust ex vivo response of phospho-SMC1-(Ser-360) to ionizing radiation in human PBMCs. Detection of phosphorylation at Ser-360 in SMC1 could be used as a marker of radiation exposure. Our findings suggest that it is feasible to measure blood cell-based changes in the phosphorylation level of a protein as an ex vivo radiation exposure detection method, even after low-dose exposure.

Selumetinib Inhibits Melanoma Metastasis to Mouse Liver via Suppression of EMT-targeted Genes.

AIM: We investigated the therapeutic effects of a mitogen-activated protein (MEK) inhibitor, selumetinib, in a hepatic melanoma metastasis model and studied its possible mechanism of action. MATERIALS AND METHODS: Melanoma cell lines were exposed to selumetinib under different experimental conditions. We established a mouse model of liver metastasis and treated mice orally with vehicle or selumetinib and then evaluated metastasis progress. RESULTS: Growth inhibition was observed in melanoma cells as a consequence of G1-phase cell-cycle arrest and the subsequent induction of apoptosis in a dose- and time-dependent manner. Mice with established liver metastases that were treated with selumetinib exhibited significantly less tumor progression than vehicle-treated mice. c-Myc expression in metastasized liver tissues were suppressed by selumetinib. Moreover, oral treatment with selumetinib modulated expression of epithelial-to-mesenchymal transition- and metastasis-related genes, including integrin alpha-5 (ITGA5), jagged 1 (JAG1), zinc finger E-box-binding homeobox 1 (ZEB1), NOTCH, and serpin peptidase inhibitor clade E (SERPINE1). CONCLUSION: We established a mouse model of hepatic metastasis using a human melanoma cell line, such models are essential in elucidating the therapeutic effects of anti-metastatic drugs. Our data suggest the possibility that selumetinib presents a new strategy to treat liver metastasis in patients with melanoma by suppressing epithelial-to-mesenchymal transition-related genes.

Paradoxical induction of growth arrest and apoptosis by EGF via the up-regulation of PTEN by activating Redox factor-1/Egr-1 in human lung cancer cells.

Epidermal growth factor (EGF) signaling promotes cell proliferation and survival in several types of cancer. Here, however, we showed that EGF inhibits proliferation and promotes programmed cell death in non-small cell lung cancer (NSCLC) cells. In A549 cells, EGF increased redox factor-1 (Ref-1) expression and the association of Ref-1 with zinc finger-containing transcriptional regulator (EGR1) via activation of p22phox, RAC1, and an NADPH oxidase subunit. EGF increased p22phox and RAC1 expression through activation of purinergic receptors (P2Y). Elevated Ref-1/EGR1 levels increased phosphatase and tensin homolog (PTEN) levels, leading to inhibition of the Akt pathway. EGF-induced PTEN upregulation increased apoptosis and autophagy-induced damage in A549 cells, whereas Ref-1 knockdown blocked EGF-induced PTEN upregulation in an NADPH oxidase p22phox subunit-independent manner. In addition, p22phox knockdown restored EGF-induced effects, implying that changes in P2Y activity caused by EGF, which activates NADPH oxidase via RAC1, influenced Ref-1-mediated redox regulation. Finally, EGF similarly attenuated cell proliferation and promoted autophagy and apoptosis in vivo in a xenograft model using A549 cells. These findings reveal that EGF-induced redox signaling is linked to Ref-1-induced death in NSCLC cells.

Glucose-6-Phosphate Dehydrogenase Deficiency Improves Insulin Resistance With Reduced Adipose Tissue Inflammation in Obesity.

Glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway, plays important roles in redox regulation and de novo lipogenesis. It was recently demonstrated that aberrant upregulation of G6PD in obese adipose tissue mediates insulin resistance as a result of imbalanced energy metabolism and oxidative stress. It remains elusive, however, whether inhibition of G6PD in vivo may relieve obesity-induced insulin resistance. In this study we showed that a hematopoietic G6PD defect alleviates insulin resistance in obesity, accompanied by reduced adipose tissue inflammation. Compared with wild-type littermates, G6PD-deficient mutant (G6PD(mut)) mice were glucose tolerant upon high-fat-diet (HFD) feeding. Intriguingly, the expression of NADPH oxidase genes to produce reactive oxygen species was alleviated, whereas that of antioxidant genes was enhanced in the adipose tissue of HFD-fed G6PD(mut) mice. In diet-induced obesity (DIO), the adipose tissue of G6PD(mut) mice decreased the expression of inflammatory cytokines, accompanied by downregulated proinflammatory macrophages. Accordingly, macrophages from G6PD(mut) mice greatly suppressed lipopolysaccharide-induced proinflammatory signaling cascades, leading to enhanced insulin sensitivity in adipocytes and hepatocytes. Furthermore, adoptive transfer of G6PD(mut) bone marrow to wild-type mice attenuated adipose tissue inflammation and improved glucose tolerance in DIO. Collectively, these data suggest that inhibition of macrophage G6PD would ameliorate insulin resistance in obesity through suppression of proinflammatory responses.

Protective effect of α-lipoic acid against radiation-induced fibrosis in mice.

Radiation-induced fibrosis (RIF) is one of the most common late complications of radiation therapy. We found that α-lipoic acid (α-LA) effectively prevents RIF. In RIF a mouse model, leg contracture assay was used to test the in vivo efficacy of α-LA. α-LA suppressed the expression of pro-fibrotic genes after irradiation, both in vivo and in vitro, and inhibited the up-regulation of TGF-ß1-mediated p300/CBP activity. Thus, α-LA prevents radiation-induced fibrosis (RIF) by inhibiting the transcriptional activity of NF-κB through inhibition of histone acetyltransferase activity. α-LA is a new therapeutic methods that can be used in the prevention-treatment of RIF.

In vivo MR evaluation of the effect of the CCR2 antagonist on macrophage migration.

The CCR2 antagonist is a receptor antagonist for monocyte chemoattractant protein-1 and is known to be a potential anti-inflammatory therapeutic agent. Recently used optimized labeling techniques for superparamagnetic iron oxide, macrophage homing, and recruitment toward the infection site can be observed on in vivo MRI. This study details the effect of the CCR2 antagonist on the macrophage migration and the feasibility of in vivo MRI for assessing the inhibition of chemotactic activity by the CCR2 antagonist. On binding assay, the CCR2 antagonist inhibits the binding affinity of monocyte chemoattractant protein-1 to CCR2. Increased expression of messenger ribonucleic acid (mRNA) and expression of CCR2 and CD11b on the cellular surface, as induced by monocyte chemoattractant protein-1, was shown, and the effect of monocyte chemoattractant protein-1 on CCR2 and CD11b was restricted by the CCR2 antagonist. In a migration test using the transwell system, macrophages treated with the CCR2 antagonist showed significantly decreased chemotactic migration compared to that of wild-type macrophages. MR images of infected left calf muscles in 12 mice were obtained 24 h after administration of macrophages labeled with superparamagnetic iron oxide. MRI successfully demonstrated the effect of the CCR2 antagonist on the directional migration of macrophages.

Essential role of mitochondrial function in adiponectin synthesis in adipocytes.

OBJECTIVE: Adiponectin is an important adipocytokine that improves insulin action and reduces atherosclerotic processes. The plasma adiponectin level is paradoxically reduced in obese individuals, but the underlying mechanism is unknown. This study was undertaken to test the hypothesis that mitochondrial function is linked to adiponectin synthesis in adipocytes. RESEARCH DESIGN AND METHODS: We examined the effects of rosiglitazone and the measures that increase or decrease mitochondrial function on adiponectin synthesis. We also examined the molecular mechanism by which changes in mitochondrial function affect adiponectin synthesis. RESULTS: Adiponectin expression and mitochondrial content in adipose tissue were reduced in obese db/db mice, and these changes were reversed by the administration of rosiglitazone. In cultured adipocytes, induction of increased mitochondrial biogenesis (via adenoviral overexpression of nuclear respiratory factor-1) increased adiponectin synthesis, whereas impairment in mitochondrial function decreased it. Impaired mitochondrial function increased endoplasmic reticulum (ER) stress, and agents causing mitochondrial or ER stress reduced adiponectin transcription via activation of c-Jun NH(2)-terminal kinase (JNK) and consequent induction of activating transcription factor (ATF)3. Increased mitochondrial biogenesis reversed all of these changes. CONCLUSIONS: Mitochondrial function is linked to adiponectin synthesis in adipocytes, and mitochondrial dysfunction in adipose tissue may explain decreased plasma adiponectin levels in obesity. Impaired mitochondrial function activates a series of mechanisms involving ER stress, JNK, and ATF3 to decrease adiponectin synthesis.

The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes.

Ghrelin, a stomach-derived hormone, induces adiposity when administered to rodents. Because ghrelin receptor is abundantly expressed in adipose tissue, we investigated the role of ghrelin in adipocyte biology. We observed ghrelin receptor expression in 3T3-L1 preadipocytes and adipocytes. Treatment of preadipocytes with ghrelin induced cellular proliferation and differentiation to mature adipocytes, as well as basal and insulin-stimulated glucose transport, but it inhibited adipocyte apoptosis induced by serum deprivation. Exposure of 3T3-L1 cells to ghrelin caused a rapid activation of MAPKs, especially ERK1/2. Chemical inhibition of MAPK blocked the mitogenic and antiapoptotic effects of ghrelin. Ghrelin also stimulated the insulin receptor substrate-associated phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 preadipocytes and adipocytes, whereas inhibition of this pathway blocked the effects of ghrelin on cell proliferation, antiapoptosis and glucose uptake. These findings suggest that the direct effects of ghrelin on proliferation, differentiation, and apoptosis in adipocytes may play a role in regulating fat cell number. These effects may be mediated via activation of the MAPK and phosphatidylinositol 3-kinase/Akt pathways.

Anti-obesity effects of alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) functions as a fuel sensor in the cell and is activated when cellular energy is depleted. Here we report that alpha-lipoic acid (alpha-LA), a cofactor of mitochondrial enzymes, decreases hypothalamic AMPK activity and causes profound weight loss in rodents by reducing food intake and enhancing energy expenditure. Activation of hypothalamic AMPK reverses the effects of alpha-LA on food intake and energy expenditure. Intracerebroventricular (i.c.v.) administration of glucose decreases hypothalamic AMPK activity, whereas inhibition of intracellular glucose utilization through the administration of 2-deoxyglucose increases hypothalamic AMPK activity and food intake. The 2-deoxyglucose-induced hyperphagia is reversed by inhibiting hypothalamic AMPK. Our findings indicate that hypothalamic AMPK is important in the central regulation of food intake and energy expenditure and that alpha-LA exerts anti-obesity effects by suppressing hypothalamic AMPK activity.

Overexpression of uncoupling protein 2 in THP1 monocytes inhibits beta2 integrin-mediated firm adhesion and transendothelial migration.

OBJECTIVE: Uncoupling protein 2 (UCP2) belongs to the mitochondrial anion carrier family and regulates production of reactive oxygen species in macrophages. Previous studies have shown that selective genetic disruption of UCP2 in bone marrow cells results in excess accumulation of monocytes/macrophages in the vascular wall of hypercholesterolemic low-density lipoprotein receptor-deficient (LDLR-/-) mice. Here we investigated whether UCP2 regulates expression of genes involved in monocyte recruitment. METHODS AND RESULTS: UCP2 overexpression in THP1 monocytes, which induced a 10-fold increase in mitochondrial UCP2 protein levels, reduced steady-state level of intracellular reactive oxygen species (ROS) and H2O2-induced ROS production. THP1 monocytes with UCP2 overexpression showed lower intracellular calcium levels and less H2O2-triggered intracellular calcium mobilization, and less protein and mRNA levels of beta2 integrins, most notably CD11b. UCP2 overexpression reduced beta2 integrin-mediated firm adhesion of monocytes to either tumor necrosis factor-alpha (TNF-alpha)-stimulated human aortic endothelial cell (HAEC) monolayers or to plates coated with intercellular adhesion molecule-1, not vascular cell adhesion molecule-1. UCP2 overexpression also inhibited cell spreading and actin polymerization in monocytes treated with TNF-alpha and monocyte chemoattractant protein-1 (MCP-1), and reduced MCP-1-induced transmigration of monocytes through HAEC monolayers. CONCLUSIONS: Mitochondrial UCP2 in circulating monocytes may prevent excessive accumulation of monocytes/macrophages in the arterial wall, thereby reducing atherosclerotic plaque formation.

DHEA administration increases brown fat uncoupling protein 1 levels in obese OLETF rats.

We found that UCP-1 and UCP-3 mRNA expression levels and the UCP-1 protein content in brown adipose tissue (BAT) were reduced in prediabetic OLETF rats than the lean LETO rats. Administration of dehydroepiandrosterone (DHEA) for 17 days induced remarkable weight loss, which was in part attributed to an enhanced utilization of ingested energy. DHEA administration significantly increased the levels of BAT UCP-1 and UCP-3 mRNA expression. Among the upstream signals for UCP-1 regulation, expression levels of the beta 3 adrenergic receptor (beta(3)AR) and PPAR gamma coactivator-1 (PGC-1) were significantly decreased in the OLETF rats and increased by DHEA administration. The decreased expression levels of UCP-1 and its upstream regulators, beta(3)AR and PGC-1, in BAT may contribute to inefficient energy utilization and obesity in OLETF rats, which was corrected by DHEA treatment.