Crif1 deficiency reduces adipose OXPHOS capacity and triggers inflammation and insulin resistance in mice.

Impaired mitochondrial oxidative phosphorylation (OXPHOS) has been proposed as an etiological mechanism underlying insulin resistance. However, the initiating organ of OXPHOS dysfunction during the development of systemic insulin resistance has yet to be identified. To determine whether adipose OXPHOS deficiency plays an etiological role in systemic insulin resistance, the metabolic phenotype of mice with OXPHOS-deficient adipose tissue was examined. Crif1 is a protein required for the intramitochondrial production of mtDNA-encoded OXPHOS subunits; therefore, Crif1 haploinsufficient deficiency in mice results in a mild, but specific, failure of OXPHOS capacity in vivo. Although adipose-specific Crif1-haploinsufficient mice showed normal growth and development, they became insulin-resistant. Crif1-silenced adipocytes showed higher expression of chemokines, the expression of which is dependent upon stress kinases and antioxidant. Accordingly, examination of adipose tissue from Crif1-haploinsufficient mice revealed increased secretion of MCP1 and TNFα, as well as marked infiltration by macrophages. These findings indicate that the OXPHOS status of adipose tissue determines its metabolic and inflammatory responses, and may cause systemic inflammation and insulin resistance.

Akt and Cks1 are related with lymph node metastasis in gastric adenocarcinoma.

BACKGROUND/AIMS: There is increasing evidence that the PI3K/Akt signal pathway plays a crucial role in tumorigenesis and tumor progression. Akt protein modulates the function of numerous substrates related to the regulation of cell proliferation, such as cyclin dependent kinase inhibitors, including p27 and Cks1. The aim of this study was to investigate the immunohistochemical expressions of Akt, p27 and Cks1 proteins according to clinicopathological parameters such as gender, lymph node metastases, tumor invasion depth, the TNM stage and Lauren classification in gastric adenocarcinoma. METHODOLOGY: Immunohistochemical staining with monoclonal Akt, p27 and Cks1 antibodies was performed on the paraffin embedded specimens of 43 gastric adenocarcinomas. RESULTS: The expression of Akt was increased more in the adenocarcinomas that showed lymph node involvement (53.5%) than in the adenocarcinomas that did not (9.3%) (p=0.007). The expression of Cks1 was increased more in the adenocarcinomas that showed lymph node involvement (39.5%) than in the adenocarcinomas that did not (7%) (p<0.043). CONCLUSIONS: Akt and Cks1 are associated with lymph node metastasis in gastric adenocarcinoma. We suggest that Akt and Cks1 proteins might be related with poor prognosis in gastric adenocarcinoma.

Promoter hypermethylation of the CFTR gene and clinical/pathological features associated with non-small cell lung cancer.

BACKGROUND AND OBJECTIVE: The exact role of the cystic fibrosis transmembrane conductance regulator (CFTR) in pathophysiology, and the mechanisms regulating its expression are poorly understood. The CFTR gene is known to be genetically or epigenetically associated with several cancers. In the present study, the methylation status of the promoter region of the CFTR gene and its expression in primary non-small cell lung cancer (NSCLC) were investigated. METHODS: The methylation status of the promoter region of the CFTR gene in NSCLC tissue was assessed by pyrosequencing and methylation-specific PCR. Expression of the CFTR gene was analysed by real-time PCR, and CFTR gene reactivation was investigated using 5-aza-2'-deoxycytidine. The correlation between methylation of the CFTR gene and the clinical features of the patients was assessed. RESULTS: Methylation of the CFTR gene in NSCLC was quantitatively high by pyrosequencing analysis and qualitatively frequent by methylation-specific PCR analysis. Expression of the CFTR gene was significantly lower in NSCLC compared with normal lung tissue. In addition, the demethylating agent 5-aza-2'-deoxycytidine increased CFTR gene expression. Methylation of the CFTR gene was significantly greater in squamous cell carcinomas than in adenocarcinomas. CFTR gene methylation was associated with significantly poorer survival in young patients, but not in elderly patients. CONCLUSIONS: These findings suggest that DNA methylation may be important for downregulation of CFTR gene expression in lung cancer. Promoter hypermethylation of the CFTR gene may be an important prognostic factor in younger patients with NSCLC.

Intraneural chondroid lipoma on the common peroneal nerve.

Both intraneural lipoma and chondroid lipoma have been reported in the previous literature as a separate disease entity on rare occasions. But intraneural chondroid lipoma has not been ever reported till now. So we present such a patient with an intraneural chondroid lipoma localized to common peroneal nerve.

Thyroid dysfunction associated with follicular cell steatosis in obese male mice and humans.

Adult thyroid dysfunction is a common endocrine disorder associated with an increased risk of cardiovascular disease and mortality. A recent epidemiologic study revealed a link between obesity and increased prevalence of hypothyroidism. It is conceivable that excessive adiposity in obesity might lead to expansion of the interfollicular adipose (IFA) depot or steatosis in thyroid follicular cells (thyroid steatosis, TS). In this study, we investigated the morphological and functional changes in thyroid glands of obese humans and animal models, diet-induced obese (DIO), ob/ob, and db/db mice. Expanded IFA depot and TS were observed in obese patients. Furthermore, DIO mice showed increased expression of lipogenesis-regulation genes, such as sterol regulatory element binding protein 1 (SREBP-1), peroxisome proliferator-activated receptor γ (PPARγ), acetyl coenzyme A carboxylase (ACC), and fatty acid synthetase (FASN) in the thyroid gland. Steatosis and ultrastructural changes, including distension of the endoplasmic reticulum (ER) and mitochondrial distortion in thyroid follicular cells, were uniformly observed in DIO mice and genetically obese mouse models, ob/ob and db/db mice. Obese mice displayed a variable degree of primary thyroid hypofunction, which was not corrected by PPARγ agonist administration. We propose that systemically increased adiposity is associated with characteristic IFA depots and TS and may cause or influence the development of primary thyroid failure.

Mig-6 gene knockout induces neointimal hyperplasia in the vascular smooth muscle cell.

Although advances in vascular interventions can reduce the mortality associated with cardiovascular disease, neointimal hyperplasia remains a clinically significant obstacle limiting the success of current interventions. Identification of signaling pathways involved in migration and proliferation of vascular smooth muscle cells (SMCs) is an important approach for the development of modalities to combat this disease. Herein we investigate the role of an immediate early response gene, mitogen-inducible gene-6 (Mig-6), in the development of neointimal hyperplasia using vascular smooth muscle specific Mig-6 knockout mice. We induced endoluminal injury to one side of femoral artery by balloon dilatation in both Mig-6 knockout and control mice. Four weeks following injury, the artery of Mig-6 knockout mice demonstrated a 5.3-fold increase in the neointima/media ratio compared with control mice (P = 0.04). In addition, Mig-6 knockout vascular SMCs displayed an increase in both cell migration and proliferation compared with wild-type SMCs. Taken together, our data suggest that Mig-6 plays a critical role in the development of atherosclerosis. This finding provides new insight into the development of more effective ways to treat and prevent neointimal hyperplasia, particularly in-stent restenosis after percutaneous vascular intervention.

A SIRT7-dependent acetylation switch of GABPß1 controls mitochondrial function.

Mitochondrial activity is controlled by proteins encoded by both nuclear and mitochondrial DNA. Here, we identify Sirt7 as a crucial regulator of mitochondrial homeostasis. Sirt7 deficiency in mice induces multisystemic mitochondrial dysfunction, which is reflected by increased blood lactate levels, reduced exercise performance, cardiac dysfunction, hepatic microvesicular steatosis, and age-related hearing loss. This link between SIRT7 and mitochondrial function is translatable in humans, where SIRT7 overexpression rescues the mitochondrial functional defect in fibroblasts with a mutation in NDUFSI. These wide-ranging effects of SIRT7 on mitochondrial homeostasis are the consequence of the deacetylation of distinct lysine residues located in the hetero- and homodimerization domains of GABPß1, a master regulator of nuclear-encoded mitochondrial genes. SIRT7-mediated deacetylation of GABPß1 facilitates complex formation with GABPα and the transcriptional activation of the GABPα/GABPß heterotetramer. Altogether, these data suggest that SIRT7 is a dynamic nuclear regulator of mitochondrial function through its impact on GABPß1 function.

Transurethral resection of bladder tumour (TURBT) as an optional treatment method on pheochromocytoma of the urinary bladder.

Pheochromocytoma of the urinary bladder is rare. We have experienced a case of unexpected pheochromocytoma of the urinary bladder in a 45-year-old female. An ultrasonographic, computed tomography scan and cystoscopic examination showed a submucosal bladder mass. After transurethral resection of bladder tumour was performed, the bladder mass was confirmed as pheochromocytoma by a pathologist. After surgery, the patient underwent a subsequent pelvic magnetic resonance imaging, positron emission tomography and I(131)-methyliodobenzylguanidine (I(131)-MIBG). An image study showed no residual tumour sites and no lymphatic metastasis. The patient has had no tumour recurrence and no voiding symptoms 3 years after the surgery.

An indole derivative protects against acetaminophen-induced liver injury by directly binding to N-acetyl-p-benzoquinone imine in mice.

AIMS: Acetaminophen (APAP)-induced liver injury is mainly due to the excessive formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) through the formation of a reactive intermediate, N-acetyl-p-benzoquinone imine (NAPQI), in both humans and rodents. Here, we show that the indole-derived synthetic compound has a protective effect against APAP-induced liver injury in C57Bl/6 mice model. RESULTS: NecroX-7 decreased tert-butylhydroperoxide (t-BHP)- and APAP-induced cell death and ROS/RNS formation in HepG2 human hepatocarcinoma and primary mouse hepatocytes. In mice, NecroX-7 decreased APAP-induced phosphorylation of c-Jun N-terminal kinase (JNK) and 3-nitrotyrosine (3-NT) formation, and also protected mice from APAP-induced liver injury and lethality by binding directly to NAPQI. The binding of NecroX-7 to NAPQI did not require any of cofactors or proteins. NecroX-7 could only scavenge NAPQI when hepatocellular GSH levels were very low. INNOVATION: NecroX-7 is an indole-derived potent antioxidant molecule, which can be bound to some types of radicals and especially NAPQI. It is well known that the NAPQI is a major intermediate of APAP, which causes necrosis of hepatocytes in rodents and humans. Thus, blocking NAPQI formation or eliminating NAPQI are novel strategies for the treatment or prevention of APAP-induced liver injury instead of GSH replenishment. CONCLUSION: Our data suggest that the indole-derivative, NecroX-7, directly binds to NAPQI when hepatic GSH levels are very low and the NAPQI-NecroX-7 complex is secreted to the blood from the liver. NecroX-7 shows more preventive and similar therapeutic effects against APAP-induced liver injury when compared to the effect of N-acetylcysteine in C57Bl/6 mice.

Promoter Methylation of CDKN2A, RARß, and RASSF1A in Non-Small Cell Lung Carcinoma: Quantitative Evaluation Using Pyrosequencing.

BACKGROUND: While qualitative analysis of methylation has been reviewed, the quantitative analysis of methylation has rarely been studied. We evaluated the methylation status of CDKN2A, RARß, and RASSF1A promoter regions in non-small cell lung carcinomas (NSCLCs) by using pyrosequencing. Then, we evaluated the association between methylation at the promoter regions of these tumor suppressor genes and the clinicopathological parameters of the NSCLCs. METHODS: We collected tumor tissues from a total of 53 patients with NSCLCs and analyzed the methylation level of the CDKN2A, RARß, and RASSF1A promoter regions by using pyrosequencing. In addition, we investigated the correlation between the hypermethylation of CDKN2A and the loss of p16(INK4A) immunoexpression. RESULTS: Hypermethylation of CDKN2A, RARß, and RASSF1A promoter regions were 16 (30.2%), 22 (41.5%), and 21 tumors (39.6%), respectively. The incidence of hypermethylation at the CDKN2A promoter in the tumors was higher in undifferentiated large cell carcinomas than in other subtypes (p=0.002). Hyperrmethylation of CDKN2A was significantly associated with p16(INK4A) immunoexpression loss (p=0.045). With regard to the clinicopathological characteristics of NSCLC, certain histopathological subtypes were found to be strongly associated with the loss of p16(INK4A) immunoexpression (p=0.016). Squamous cell carcinoma and undifferentiated large cell carcinoma showed p16(INK4A) immunoexpression loss more frequently. The Kaplan-Meier survival curves analysis showed that methylation level and patient survival were barely related to one another. CONCLUSION: We quantitatively analyzed the promoter methylation status by using pyrosequencing. We showed a significant correlation between CDKN2A hypermethylation and p16(INK4A) immunoexpression loss.

Dual specificity phosphatase 6 as a predictor of invasiveness in papillary thyroid cancer.

OBJECTIVE: The genetic mutations causing the constitutive activation of MEK/ERK have been regarded as an initiating factor in papillary thyroid carcinoma (PTC). The ERK-specific dual specificity phosphatase 6 (DUSP6) is part of the ERK-dependent transcriptional output. Therefore, the coordinated regulation of the activities of ERK kinases and DUSP6 may need to be reestablished to make new balances in PTC. METHODS: To investigate the role of DUSP6 in the regulation of ERK1/2 (MAPK3/1)-dependent transcription, 42 benign neoplasms and 167 PTCs were retrospectively analyzed by immunohistochemistry with dideoxy sequencing to detect BRAF(V600E) mutation. RESULTS: The expressions of total ERK1/2, DUSP6, c-Fos (FOS), c-Myc (MYC), cyclin D1, and PCNA were markedly increased in PTC compared with those in benign neoplasms. However, phospho-ERK1/2 was detected in only eight (4.8%) cases out of 167 PTC samples. Unexpectedly, the staining intensity and nuclear localization of ERK1/2 were not affected by the presence or absence of the BRAF(V600E) mutation. However, the expressions of c-Fos and PCNA were elevated in BRAF(V600E)-positive PTC compared with those in BRAF(V600E)-negative PTC. Interestingly, the higher staining intensities of DUSP6 were associated with the level of total ERK1/2 expression (P=0.04) and with high-risk biological features such as age (P=0.05), tumor size (P=0.01), and extrathyroidal extension (linear by linear association, P=0.02). In addition, DUSP6 silencing significantly decreased the cell viability and migration rate of FRO cells. CONCLUSIONS: The coordinated upregulation of total ERK1/2 and its phosphatase, DUSP6, is related to bare detection of phospho-ERK1/2 in PTC regardless of BRAF(V)(600E) mutation status. A link between DUSP6 expression and high-risk features of PTC suggested that DUSP6 is an important independent factor affecting the signaling pathways in established PTC.

Aberrant l1 cell adhesion molecule affects tumor behavior and chemosensitivity in anaplastic thyroid carcinoma.

PURPOSE: Anaplastic thyroid carcinoma (ATC) is one of the most invasive human cancers and has a poor prognosis. Molecular targets of ATC that determine its highly aggressive nature remain unidentified. This study investigated L1 cell adhesion molecule (L1CAM) expression and its role in tumorigenesis of ATCs. EXPERIMENTAL DESIGN: Expression of L1CAM in thyroid cancer was evaluated by immunohistochemical analyses of tumor samples from patients with thyroid cancer. We investigated the role of L1CAM in proliferation, migration, invasion, and chemoresistance using short hairpin RNA (shRNA) knockdown experiments in human ATC cell lines. Finally, we evaluated the role of L1CAM on tumorigenesis with ATC xenograft assay in a nude mouse model. RESULTS: L1CAM expression was not detectable in normal follicular epithelial cells of the thyroid or in differentiated thyroid carcinoma. In contrast, analysis of ATC samples showed specifically higher expression of L1CAM in the invasive area of the tumor. Specific knockdown of L1CAM in the ATC cell lines, FRO and 8505C, caused a significant decrease in the proliferative, migratory, and invasive capabilities of the cells. Suppression of L1CAM expression in ATC cell lines increased chemosensitivity to gemcitabine or paclitaxel. Finally, in an ATC xenograft model, depletion of L1CAM markedly reduced tumor growth and increased the survival of tumor-bearing mice. CONCLUSIONS: We report that L1CAM is highly expressed in the samples taken from patients with ATCs. L1CAM plays an important role in determining tumor behavior and chemosensitivity in cell lines derived from ATCs. Therefore, we suggest that L1CAM may be an important therapeutic target in patients with ATCs.

MRI of a subcutaneous myolipoma in the ankle: a case report.

Myolipoma is a rare benign tumor, composed of irregularly admixed adipose tissue and smooth muscle fibers. Few literature studies have described the radiologic appearance of myolipoma, especially in the soft tissue. No MRI findings in subcutaneous myolipoma of an extremity have been reported. Here, we report on the case of a 34-year-old woman with myolipoma in the subcutaneous tissue of the ankle and describe MRI features of the lesion.

Therapeutic potential of adipose tissue-derived stem cells for liver failure according to the transplantation routes.

PURPOSE: Even though adipose tissue-derived stem cells (ADSCs) have been spotlighted as a possible alternative for liver transplantation in an experimental setting, the mechanism by which ADSCs improve liver dysfunction remains poorly characterized. The objective of this study was to evaluate the therapeutic ability of undifferentiated ADSCs, and find a few clues on how ADSCs alleviate liver damage by comparing the transplantation routes. METHODS: In vitro generated human ADSCs were checked for surface markers and stage-specific genes for characterization. Afterwards, they were transplanted into C57BL/6 mice with CCl4-induced liver injury. The transplantations were made via tail vein, portal vein, and direct liver parenchymal injection. At 1 and 3 post-transplantation days, serum biochemical parameters and/or liver specimens were evaluated. RESULTS: We have shown here that ADSCs have the characteristics of mesenchymal stem cells, and belong to endodermal and/or early hepatic differentiation stage. After transplantation into the mice with acute liver failure, markers of liver injury, such as alanineaminotransferase, aspartateaminotransferase, as well as ammonia, decreased. Of these transplantation routes, transplantation via tail vein rendered the most prominent reduction in the biochemical parameters. CONCLUSION: Undifferentiated ADSCs have the ability to improve hepatic function in mice with acute liver injury. Moreover, our transplantation route study supports the theory that ADSCs in systemic circulation can exert endocrine or paracrine effects to ameliorate the injured liver.

microRNA-101 inhibits lung cancer invasion through the regulation of enhancer of zeste homolog 2.

microRNAs (miRNAs) may function as oncogenes or tumor-suppressor genes depending on the targets that are regulated. Enhancer of zeste homolog 2 (EZH2) is the target of miR-101 and a member of the polycomb repressive complex 2, which is involved in the methylation of histone H3 at lysine 27 (H3K27). Therefore, we aimed to ascertain whether or not the overexpression of miR-101 inhibits invasion of lung cancer through regulation of EZH2. In this study, the expression of miR-101 was down-regulated and the expression of EZH2 was up-regulated in lung cancer. Global methylation of H3K27 was higher in metastatic lung cancer than in early lung cancer lesions. Overexpression of miR-101 induced a marked reduction in EZH2 mRNA levels in several lung cancer cell lines. A reduction in the trimethyl H3K27 histone mark was detected at the CDH1 promoter in miR-101 precursor-transfected cells. Moreover, the expression of CDH1 and MMP-2 was reversed by miR-101 transfection. Therefore, the overexpression of miR-101 inhibits the invasiveness of lung cancer. miR-101 may be a potent tumor suppressor by altering chromatin structure through repression of EZH2 and may be a potential therapeutic tool for patients with lung cancer.

Mitochondrial localization and regulation of BRAFV600E in thyroid cancer: a clinically used RAF inhibitor is unable to block the mitochondrial activities of BRAFV600E.

CONTEXT: The oncogenic BRAF(V600E) mutation results in an active structural conformation characterized by greatly elevated ERK activity. However, additional cellular effects caused by subcellular action of BRAF(V600E) remain to be identified. OBJECTIVE: To explore these effects, differences in the subcellular localization of wild-type and mutant BRAF in thyroid cancer were investigated. RESULTS: A significant proportion of endogenous and exogenous BRAF(V600E), but not wild-type BRAF, was detected in the mitochondrial fraction, similar to other BRAF mutants including BRAF(V600D), BRAF(V600K), BRAF(V600R), and BRAF(G469A), which showed elevated kinase activity and mitochondrial localization. Induced expression of BRAF(V600E) suppressed the apoptotic responses against staurosporine and TNFα/cycloheximide. Interestingly, the mitochondrial localization and antiapoptotic activities of BRAF(V600E) were unaffected by sorafenib and U0126 suppression of MAPK kinase (MEK) and ERK activities. Similarly, although the RAF inhibitor sorafenib effectively inhibited MEK/ERK activation, it did not block the mitochondrial localization of BRAF(V600E). In addition, inducible expression of BRAF(V600E) increased the glucose uptake rate and decreased O(2) consumption, suggesting that BRAF(V600E) reduces mitochondrial oxidative phosphorylation, a signature feature of cancer cells. Again, these metabolic alterations resulted by BRAF(V600E) expression were not affected by the treatment of thyroid cells by sorafenib. Therefore, RAF and MEK inhibitors are unable to block the antiapoptotic activity of BRAF(V600E) or correct the high glucose uptake rate and glycolytic activity and suppressed mitochondrial oxidative phosphorylation induced by BRAF(V600E). CONCLUSIONS: The mitochondrial localization observed in oncogenic BRAF mutants might be related to their altered responses to apoptotic stimuli and characteristic metabolic phenotypes found in thyroid cancer. The inability of MEK and RAF inhibitors, U0126 and sorafenib, respectively, to block the mitochondrial localization of BRAF(V600E) has additional therapeutic implications for BRAF(V600E)-positive thyroid cancers.