Involvement of POMC neurons in LEAP2 regulation of food intake and body weight.

Liver-expressed antimicrobial peptide 2 (LEAP2) is a newly discovered antagonist of the growth hormone secretagogue receptor (GHSR) and is considered the first endogenous peptide that can antagonize the metabolic actions of ghrelin. The effects of ghrelin administration on feeding behavior, body weight, and energy metabolism involve the activation of orexigenic neurons in the arcuate nucleus (ARC) of the hypothalamus. It is unclear, however, if LEAP2 applied directly to the ARC of the hypothalamus affects these metabolic processes. Here, we show that overexpression of LEAP2 in the ARC through adeno-associated virus (AAV) reduced food intake and body weight in wild-type (WT) mice fed chow and a high-fat diet (HFD) and improved metabolic disorders. LEAP2 overexpression in the ARC overrides both central and peripheral ghrelin action on a chow diet. Interestingly, this AAV-LEAP2 treatment increased proopiomelanocortin (POMC) expression while agouti-related peptide (AGRP)/neuropeptide Y (NPY) and GHSR levels remained unchanged in the hypothalamus. Additionally, intracerebroventricular (i.c.v.) administration of LEAP2 decreased food intake, increased POMC neuronal activity, and repeated LEAP2 administration to mice induced body weight loss. Using chemogenetic manipulations, we found that inhibition of POMC neurons abolished the anorexigenic effect of LEAP2. These results demonstrate that central delivery of LEAP2 leads to appetite-suppressing and body weight reduction, which might require activation of POMC neurons in the ARC.

Development of fluorescence/MR dual-modal manganese-nitrogen-doped carbon nanosheets as an efficient contrast agent for targeted ovarian carcinoma imaging.

BACKGROUND: Development of sensitive and specific imaging approaches for the detection of ovarian cancer holds great promise for improving the therapeutic efficacy and the lifespan of the patients. RESULTS: In this study, manganese-nitrogen doped carbon nanosheets (Mn-N-CNSs) coupled with Anti-HE4 monoclonal antibody (Mn-N-CNSs@Anti-HE4) were synthesized for the specific and targeted fluorescence/MR dual-modal imaging of ovarian carcinoma. The prepared Mn-N-CNSs revealed excellent aqueous dispersity, good colloidal stability, great optical properties and high longtudinal relaxivity rate (r1 = 10.30 mM-1 s-1). Encouraged by the tunable photoluminiscence of the nanoprobe and Anti-HE4 targeting ligand, the ovarian carcinoma cells were specifically labeled by the Mn-N-CNSs@Anti-HE4 nanoprobe with multi-color fluorescences. Benefiting from the high r1 relaxivity, the nanoprobe exhibited targeted and enhanced MR contrast effect in the ovarian carcinoma cells and tumor bearing mice model. Besides, the high biocompatibility and easy excretion from the body of the nanoprobe were further confirmed in vivo. CONCLUSION: The prepared Mn-N-CNSs@Anti-HE4 with excellent biocompatibility, high-performance and superior tumor-targeting ability provides a novel fluorescence/MR dual-modal nanoprobe for specific labeling and detection of ovarian carcinoma cells in vitro and in vivo.

Multifunctional iron oxide-carbon hybrid nanoparticles for targeted fluorescent/MR dual-modal imaging and detection of breast cancer cells.

An efficient method for the highly sensitive and specific detection of cancer cells is crucial for the early diagnosis of cancer. In this work, we propose a one-pot approach to fabricating magnetic-fluorescent iron oxide-carbon hybrid nanomaterials (MCNP) with excellent stable, high quantum yield and excellent magnetic properties for breast cancer cells recognition and detection via magnetic resonance and multicolour fluorescence imaging. MCNPs were efficiently synthesised via one-pot, multi-component reactions of FeCl3, FeCl2•4H2O, citric acid and ethylenediamine in diethylene glycol. The MCNPs showed strong excitation wavelength-dependent fluorescence in the blue-red region with a high quantum yield of 58.4%, and they presented higher stability and T2 relaxivity than pure iron oxide nanoparticles. After conjugating with CD44 monoclonal antibodies, the fabricated targeting nanoprobe, MCNPs-CD44, demonstrated a specific fluorescence/MRI dual imaging contrast effect in 4T1 breast cancer cells. Biological transmission electron microscope imaging showed a significant preferential uptake of the nanoparticle conjugates by the 4T1 cells. By taking advantage of the high binding affinity and specificity of the CD44 antibodies to the overexpressed CD44 on the cancer cell surface, the developed MCNPs-CD44 probe distinguished 4T1 breast cancer cells from normal cells and detected as low as a few hundred cancer cells, thus indicating the potential application of multifunctional nanocomposites in the MR diagnosis and fluorescence positioning of breast cancer at cellular-level resolution.

Preparation of gadolinium doped carbon dots for enhanced MR imaging and cell fluorescence labeling.

Gadolinium doped carbon dots (Gd-CDs) were prepared as a dual-modal imaging agent for enhanced MR imaging and cell fluorescence imaging. The Gd-CDs were synthesized via one-step solvent free technique with Gd-DTPA and l-arginine as the Gd and carbon sources with a quantum yield of 57.78%. The Gd-CDs exhibited good crystal structure, excellent aqueous dispersity, high colloidal stability, intense fluorescence and low cytotoxicity. The bio-TEM images revealed that the Gd-CDs could be easily internalized by cancer cells and escape from the endosomes. Furthermore, the Gd-CDs demonstrated wonderful multi-color fluoresence cell labeling ability at various excitation wavelength and much better MR contrast effect compared with commercial Gd-DTPA with a high r1 relaxivity value 6.27 mM-1s-1. In addition, Gd-CDs exhibited brighter MR signal than Gd-DTPA in the animal MR imaging test. Finally, the Gd-CDs also indicated low long-term toxicity by the serum biochemistry analysis. Thus, these results indicated that Gd-CDs would be an excellent dual-modal imaging probe for enhanced MR imaging and fluorescence imaging.

Stimuli-sensitive hollow spheres from chitosan-graft-ß-cyclodextrin for controlled drug release.

In this paper, sensitive polymeric hollow spheres self-assembled from chitosan-grafted-ß-cyclodextrin (CS-g-CD) and sodium tripolyphosphate (TPP) were prepared for controlled release of doxorubicin (DOX). The assemblies were formed by electrostatic interactions between positively charged amino group in CS-g-CD and negatively charged phosphate in TPP. The hollow spheres with diameters about 100nm were confirmed by transmission electron microscopy (TEM) and laser particle analyzer. The microspheres with hollow cavity were beneficial to improve the drug loading capacity for DOX with entrapment efficiency above 60%. The cumulative release of DOX from CS-g-CD/TPP hollow microspheres increased with the decrease of pH and the increase of temperature or ionic strength. At 37 °C and pH 5.2, the maximum drug release was above 90% with a continuous release rate. In-vitro cytotoxicity tests indicate that drug loaded hollow spheres exhibited evidently inhibition against cancer cells. These sensitive polymeric hollow spheres are expected to be used in biomedical field as potential carrier.

Cytochrome b5 reductase 2 is a novel candidate tumor suppressor gene frequently inactivated by promoter hypermethylation in human nasopharyngeal carcinoma.

Cytochrome b5 reductase 2 (CYB5R2), a member of the flavoprotein pyridine nucleotide cytochrome reductase family, is associated with a number of physiological reactions. However, its role in cancer, especially nasopharyngeal carcinoma (NPC), has not been addressed. Here, we investigate the transcript levels and promoter methylation status of CYB5R2 in NPC derived cell lines and tumor biopsies and experimentally address its role as a tumor suppressor gene. We find that CYB5R2 transcript levels are decreased in NPC cell lines and tumor biopsies. Promoter hypermethylation of CYB5R2 was detected in all six tested NPC cell lines and in 84% of primary NPC tumor biopsies but not in normal nasopharyngeal epithelium. Clinically, CYB5R2 methylation was associated with lymph node metastasis in NPC patients (P < 0.05). The endogenous expression of CYB5R2 could be restored in vitro by the methyltransferase inhibitor 5-aza-2'-deoxycytidine in NPC cell lines. Ectopic expression of CYB5R2 had an inhibitory effect on proliferation, clonogenicity and migration of NPC cells. Moreover, in vivo tests in nude mice indicated that ectopic expression of CYB5R2 reduces the tumorigenicity of CYB5R2-negative NPC cells. Collectively, these findings suggest that CYB5R2 may be a functional tumor suppressor gene, frequently inactivated by hypermethylation of its promoter in NPC. We report here the first instance of epigenetic downregulation in NPC tumor biopsies of a key enzyme, CYB5R2, which is responsible for the detoxification of environmental carcinogens. We propose the possibility of utilizing CYB5R2 promoter methylation as a diagnostic biomarker of NPC in the future.

A preliminary study of apparent diffusion coefficient in chemotherapy-induced liver damage.

OBJECTIVES: To investigate changes in the hepatic apparent diffusion coefficient (ADC) in patients undergoing chemotherapy. METHODS: We enrolled 54 patients (25 women; mean age 57.0±13.1 years, range 29-89 years) undergoing chemotherapy for tumor and 10 controls (7 women; mean age 55.1±17.5 years, range 23-81 years). The patients were tested for serum alanine aminotransferase (ALT) activity (abnormal, normal) and fatty liver. Hepatic ADC values were compared among controls, patients and subgroups. Pearson correlation coefficient was used to assess the correlation between ADC and ALT activity. RESULTS: Hepatic ADC0,850 (×10(-3) mm2/s) was lower for patients than controls (1.14±0.18 vs. 1.28±0.12, P=0.02) and was lower for patients with than without fatty liver and controls (1.01±0.06 vs. 1.18±0.18 and 1.28±0.12, respectively, all P<0.01), with no significant difference between patients without fatty liver and controls (P=0.07). ADC0,850 was lower for patients with abnormal ALT than normal ALT activity and controls (0.99±0.06 vs. 1.17±0.18 and 1.28±0.12, respectively, all P<0.05), with a significant difference also being seen between patients with normal ALT activity and controls (P=0.04). Hepatic ADC0,850 was not correlated with ALT activity in patients (r=-0.24, P=0.08). CONCLUSIONS: Although ADC did not correlate with ALT values, it did distinguish patient likely to have chemotherapy-induced liver damage as indicated by abnormal ALT values or fatty liver. These mechanisms need to be disentangled.

[Aberrant promoter hypermethylation of CHFR in nasopharyngeal carcinoma].

OBJECTIVE: To discover the relationship of transcriptional levels and promoter methylation status of CHFR gene in human nasopharyngeal carcinoma,to discuss the significance and epigenetic mechanism of CHFR inactivation in NPC, and to evaluate the feasibility of detecting methylated CHFR in nasopharyngeal swab as a means for diagnosis of NPC. METHOD: Transcriptional levels of CHFR was evaluated by RT-PCR. Methylation specific PCR was used to detect the methylation status of CHFR in NPC cells, normal nasopharyngeal epithelia, primary tumors and their paired nasopharyngeal swabs. Detailed methylation status was confirmed by bisulfite sequencing. NPC cells were treated by the methyltransferase inhibitor 5-aza-dC and the reactivation of CHFR was evaluated by RT-PCR. RESULT: CHFR transcription was inactivated in NPC. The methylation frequency in NPC primary tumors and their paired swabs were 65.5% and 63.8%, respectively, with a 86.2% concordance. Bisulfite sequencing revealed a dense methylation in NPC cells and primary tumors, but all the normal nasopharyngeal epithelia were unmethylated. CHFR expression were restored after 5-aza-dC treatment. CONCLUSION: CHFR is epigenetically inactivated by promoter methylation in NPC. Detecting methylated CHFR can be served as a useful non-invasive means for diagnosis of NPC.

CDH4 as a novel putative tumor suppressor gene epigenetically silenced by promoter hypermethylation in nasopharyngeal carcinoma.

We investigated the transcription levels, promoter methylation status and role as a tumor suppressor gene (TSG) of the cadherin CDH4 in nasopharyngeal carcinoma (NPC). The expression of CDH4 was decreased in NPC cell lines, xenografts and primary tumor biopsies. Promoter hypermethylation of CDH4 was detected in all five NPC cell lines, both NPC xenograft lines and 94.3% of primary tumors but not in any of the 12 normal epithelial samples. Loss of CDH4 expression could be restored by the methyltransferase inhibitor 5-aza-2'-deoxycytidine in NPC cell lines. Ectopic expression of CDH4 in the NPC cell lines inhibits cell proliferation, colony formation, migration and elicit cell communication. CDH4 may be a novel putative TSG that can be frequently and tumor-specifically inactivated by its promoter methylation in NPC.

TFPI-2 is a putative tumor suppressor gene frequently inactivated by promoter hypermethylation in nasopharyngeal carcinoma.

BACKGROUND: Epigenetic silencing of tumor suppressor genes play important roles in NPC tumorgenesis. Tissue factor pathway inhibitor-2 (TFPI-2), is a protease inhibitor. Recently, TFPI-2 was suggested to be a tumor suppressor gene involved in tumorigenesis and metastasis in some cancers. In this study, we investigated whether TFPI-2 was inactivated epigenetically in nasopharyngeal carcinoma (NPC). METHODS: Transcriptional expression levels of TFPI-2 was evaluated by RT-PCR. Methylation status were investigated by methylation specific PCR and bisulfate genomic sequencing. The role of TFPI-2 as a tumor suppressor gene in NPC was addressed by re-introducing TFPI-2 expression into the NPC cell line CNE2. RESULTS: TFPI-2 mRNA transcription was inactivated in NPC cell lines. TFPI-2 was aberrantly methylated in 66.7% (4/6) NPC cell lines and 88.6% (62/70) of NPC primary tumors, but not in normal nasopharyngeal epithelia. TFPI-2 expression could be restored in NPC cells after demethylation treatment. Ectopic expression of TFPI-2 in NPC cells induced apoptosis and inhibited cell proliferation, colony formation and cell migration. CONCLUSIONS: Epigenetic inactivation of TFPI-2 by promoter hypermethylation is a frequent and tumor specific event in NPC. TFPI-2 might be considering as a putative tumor suppressor gene in NPC.