The uncharacterized transcript KIAA0930 confers a cachexic phenotype on cancer cells.

Patients with cancer cachexia have a poor prognosis and impaired quality of life. Numerous studies using preclinical models have shown that inflammatory cytokines play an important role in the development of cancer cachexia; however, no clinical trial targeting cytokines has been successful. Therefore, it is essential to identify molecular mechanisms to develop anti-cachexia therapies. Here we identified the uncharacterized transcript KIAA0930 as a candidate cachexic factor based on analyses of microarray datasets and an in vitro muscle atrophy assay. While conditioned media from pancreatic, colorectal, gastric, and tongue cancer cells caused muscle atrophy in vitro, conditioned medium from KIAA0930 knockdown cells did not. The PANC-1 orthotopic xenograft study showed that the tibialis anterior muscle weight and cross-sectional area were increased in mice bearing KIAA0930 knockdown cells compared to control mice. Interestingly, KIAA0930 knockdown did not cause consistent changes in the secretion of inflammatory cytokines/chemokines from a variety of cancer cell lines. An initial characterization experiment showed that KIAA0930 is localized in the cytosol and not secreted from cells. These data suggest that the action of KIAA0930 is independent of the expression of cytokines/chemokines and that KIAA0930 could be a novel therapeutic target for cachexia.

Lcn2 mediates adipocyte-muscle-tumor communication and hypothermia in pancreatic cancer cachexia.

OBJECTIVE: Adipose tissue is the largest endocrine organ. When activated by cancer cells, adipocytes secrete adipocytokines and release fatty acids, which are then transferred to cancer cells and used for structural and biochemical support. How this metabolic symbiosis between cancer cells and adipocytes affects skeletal muscle and thermogenesis during cancer cachexia is unknown. Cancer cachexia is a multiorgan syndrome and how the communication between tissues is established has yet to be determined. We investigated adipose tissue secretory factors and explored their role in crosstalk of adipocytes, muscle, and tumor during pancreatic cancer cachexia. METHODS: We used a pancreatic cancer cachexia mouse model generated by syngenic implantation of pancreatic ductal adenocarcinoma (PDAC) cells (KPC) intraperitoneally into C57BL/6 mice and Lcn2-knockout mice. For in vitro studies, adipocytes (3T3-L1 and primary adipocytes), cachectic cancer cells (Panc0203), non-cachectic cancer cells (Du145 cells), and skeletal muscle cells (C2C12 myoblasts) were used. RESULTS: To identify molecules involved in the crosstalk of adipose tissue with muscle and tumors, we treated 3T3-L1 adipocytes with conditioned medium (CM) from cancer cells. Upon screening the secretomes from PDAC-induced adipocytes, several adipocytokines were identified, including lipocalin 2 (Lcn2). We investigated Lcn2 as a potential mediator of cachexia induced by adipocytes in response to PDAC. During tumor progression, mice exhibited a decline in body weight gain, which was accompanied by loss of adipose and muscle tissues. Tumor-harboring mice developed drastic hypothermia because of a dramatic loss of fat in brown adipose tissue (BAT) and suppression of the thermogenesis pathway. We inhibited Lcn2 with an anti-Lcn2 antibody neutralization or genomic ablation in mice. Lcn2 deficiency significantly improved body temperature in tumor-bearing mice, which was supported by the increased expression of Ucp1 and ß3-adrenergic receptor in BAT. In addition, Lcn2 inhibition abrogated the loss of fat and muscle in tumor-bearing mice. In contrast to tumor-bearing WT mice, the corresponding Lcn2-knockout mice showed reduced ATGL expression in iWAT and decreased the expression of muscle atrophy molecular markers MuRF-1 and Fbx32. CONCLUSIONS: This study showed that Lcn2 is causally involved in the dysregulation of adipose tissue-muscle-tumor crosstalk during pancreatic cancer cachexia. Therapeutic targets that suppress Lcn2 may minimize the progression of cachexia.

AT-rich interaction domain 5A regulates the transcription of interleukin-6 gene in prostate cancer cells.

BACKGROUND: Interleukin-6 (IL-6) is a pleiotropic cytokine that confers androgen-independence and aggressiveness in prostate cancer (PCa); however, the molecular mechanisms regulating IL-6 expression remain unclear. The expression of ARID5A, an AT-rich interaction domain (ARID) DNA-binding motif-containing transcription factor is positively correlated with IL-6 expression in human PCa. We, therefore, hypothesized that ARID5A could regulate IL-6 expression in PCa. METHODS: The relationship between ARID5A and IL-6 in PCa patients was analyzed using statistical analyses of multiple clinical microarray data sets. To investigate whether ARID5A regulates IL-6 expression, CRISPR-driven ARID5A knockout clones were established in DU145 and PC-3 cells. RESULTS: Analysis of three microarray data sets showed a positive correlation between ARID5A and IL-6 expression. The expression of IL-6 in ARID5A knockout clones was significantly reduced compared with control clones in both PCa cell lines. Knockout of ARID5A did not result in any loss of IL-6 mRNA stability. Instead, we observed a significant decrease in the occupancy of both active RNA Polymerase II and the active histone mark, H3K4me3 at the IL-6 transcriptional start site in ARID5A knockout PCa cells, suggesting a role for transcriptional regulation. CONCLUSIONS: Our study demonstrated that loss of ARID5A downregulates the expression of IL-6 at the transcriptional level.

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells.

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Characteristics of Biofilms Formed by Co-Culture of Listeria monocytogenes with Pseudomonas aeruginosa at Low Temperatures and Their Sensitivity to Antibacterial Substances.

We assessed the properties of biofilms (BFs) formed by mono- and co-cultures of Listeria monocytogenes and Pseudomonas aeruginosa (L+P-BF) at low temperatures and examined their sensitivity to several antibacterial substances. L. monocytogenes viable counts comprised only 1-10% of total L+P-BF viable counts at 10℃ and 15℃, indicating the significant prevalence of P. aeruginosa in co-cultures. L+P-BF formed at 10℃ and 15℃ showed very high resistance to antibiotics and NaClO. Examination of the effects of nattokinase and nisin, natural food additives with antibacterial properties, showed that their application alone failed to inhibit L+P-BF development at 10℃ and 15℃. However, a combined treatment with nisin and ethylenediaminetetraacetic acid, a food additive that can be used as a permeabilizing agent, suppressed the formation of L+P-BF at 10℃ and 15℃. Microscopy observations of L+P-BF did not reveal pronounced morphological changes in bacterial cell morphology. We also noted that P. aeruginosa resistance to the action of nisin during BF formation was higher when it was maintained in co-culture with L. monocytogenes. The results of the present study are an important step toward developing a safe formulation of acceptable food additives that could be used for suppression of BFs formed by pathogenic bacteria during food storage.

AT-rich interactive domain 5B regulates androgen receptor transcription in human prostate cancer cells.

BACKGROUND: The androgen receptor (AR) is one of the most important and dynamically regulated factors in prostate cancer (PCa) progression. Despite the importance of AR expression regulation, the precise mechanisms are not fully understood. ARID5B, an AT-rich interaction domain DNA-binding motif-containing transcription factor, is expressed higher in primary PCa than normal prostate, and correlated with AR expression. We therefore hypothesized that ARID5B could regulate AR expression. METHODS: Correlation between AR and ARID5B expression was analyzed using publicly and commercially available microarray data. To examine the role of ARID5B in AR expression, ARID5B was knocked down in VCaP and LNCaP cells, then mRNA and protein levels of AR were measured and an in vitro cell proliferation assay was performed. Chromatin immunoprecipitation was performed to further examine molecular mechanisms. RESULTS: Knockdown of ARID5B suppressed the AR mRNA and protein expression in VCaP and LNCaP cells and decreased in vitro cell proliferation. Suppression of ARID5B decreased the occupancy of active RNA polymerase II in the AR promoter, indicating that ARID5B regulates AR transcription. The active histone mark, H3K4me3, occupancy was decreased with ARID5B knockdown. CONCLUSION: Our study revealed that AR transcription is positively regulated by ARID5B through H3K4me3 recruitment in the AR promoter. Our findings reveal novel mechanisms of AR transcription, which is dynamically regulated in prostate tumor progression.

Heated scallop-shell powder treatment for deactivation and removal of Listeria sp. biofilm formed at a low temperature.

The ability of heated scallop-shell powder (HSSP) to work against Listeria sp. biofilm formed at a low temperature was investigated. A biofilm of L. innocua ATCC 33090 was grown on a glass plate at 15˚C for 15 days, then immersed in HSSP slurry. Following treatment, the disinfection ability of the HSSP against the biofilm was non-destructively quantified by conductimetric assay. The biofilm grown at 15˚C was less sensitive than that grown at 37˚C to HSSP treatment and alkaline treatment. The biofilm grown at 15˚C was completely deactivated by 30 min of HSSP treatment (10 mg/mL, pH 12.5). In contrast, after 30 min treatment with alkaline solution at pH 12.5 or sodium hypochlorite (100 ppm), the activity was reduced by only one order of magnitude. The disinfection efficacy of HSSP (10 mg/mL) against L. innocua is similar to or higher than that of sodium hypochlorite (200 ppm). Fluorescence microscopy validated the results of the conductimetric assay. Therefore, HSSP treatment is a potentially powerful alternative control agent against Listeria sp. biofilms that present hazards in the food industry.

Knockdown of AT-rich interaction domain (ARID) 5B gene expression induced AMPKα2 activation in cardiac myocytes.

This study demonstrated that ARID5B mRNA is present in mouse cardiomyocyte HL-1 cells, and that ARID5B siRNA constantly knocked down ARID5B gene expression to the 40% level of control. AMPKα2 protein was elevated in such ARID5B knockdown HL-1 cells, and this was accompanied by an increase in the level of phosphorylated AMPKα. Since AMPKα2 mRNA levels did not change in ARID5B knockdown cells, the stability of AMPKα2 protein was investigated using inhibitors for protein synthesis and proteasomal degradation. Treatment of HL-1 cells with either cycloheximide or MG132 caused an appreciable increase in the amount of AMPKα2 protein in ARID5B knockdown cells, which suggests that knockdown of ARID5B mRNA extends the half-life of AMPKα2 protein in HL-1 cells via yet unidentified mechanisms. As for the expected downstream consequences of AMPKα2 activation, we found thus far that glucose uptake, fatty acid uptake, or fatty acid oxidation remained unchanged in HL-1 cells after knockdown of ARID5B. Further studies are required to understand the mechanisms for ARID5B knockdown and resulting AMPKα2 activation, and also to identify which metabolic pathways are affected by AMPKα2 activation in these cells. In summary, this study provided the foundation for an in vitro cell culture system to study possible roles of ARID5B in cardiomyocytes.

Cucurbitane-type triterpenes from Citrullus lanatus (watermelon) seeds.

Two new cucurbitane-type triterpenes, 24-hydroperoxycucurbita-5,25-dien-3beta-ol (1) and 25-hydroperoxycucurbita-5,23-dien-3beta-ol (2), were isolated from a MeOH extract of Citrullus lanatus seeds. Compounds 1 and 2 exhibited moderate cytotoxic activities with IC50 values of 33.4-52.4 microM against HL-60 (human leukemia), P388 (murine leukemia), and L1210 (murine leukemia) cells. Compound 1 showed melanogenesis inhibitory activity (melanin content 80.0 %) with low cytotoxicity (cell viability 97.6%) at a low concentration (10 microM).

Modulator recognition factor-2 regulates triglyceride metabolism in adipocytes.

Mice lacking modulator recognition factor-2 (Mrf-2; ARID5B) have less fat in brown and white adipose tissues, partly because of a defect in adipocyte differentiation. We have also shown that knockdown of Mrf-2 decreases the expression of the adipogenic transcription factors C/EBPalpha and PPARgamma, and inhibits adipogenesis in 3T3-L1 preadipocytes. Since these transcription factors may also contribute to the maintenance of adipocyte function, we examined the effects of siRNA targeted to Mrf-2 on triglyceride metabolism in mature 3T3-L1-derived adipocytes. As it did in differentiating adipocytes, knockdown of Mrf-2 decreased the expression of both C/EBPalpha and PPARgamma. Knockdown of Mrf-2 also activated both lipolysis and triglyceride synthesis, and caused a significant increase in the ratio of glycerol release to free fatty acid release. This suggests that knockdown of Mrf-2 increases the rate of fatty acid recycling in 3T3-L1-derived adipocytes. Continual cycling of fatty acids through lipolysis and triglyceride synthesis could lead to dissipation of energy. Therefore, the activation of such a futile cycle via the suppression of Mrf-2 could be an effective treatment for obesity and diabetes.

Modulator recognition factor-2 is required for adipogenesis in mouse embryo fibroblasts and 3T3-L1 cells.

Previous study showed that mice lacking modulator recognition factor-2 (Mrf-2) were lean, with significant decreases in white adipose tissue. One postulated mechanism for the lean phenotype in Mrf-2 knockout mice is a defect in adipogenesis. In order to investigate this further, we examined the effects of Mrf-2 deficiency on adipogenesis in vitro. In mouse fibroblasts (MEFs) derived from Mrf-2(-/-) embryos, and in 3T3-L1 cells after knockdown of Mrf-2 by small interference RNA (siRNA) there was a potent inhibition of hormone-induced lipid accumulation, and significant decreases in the expression of the adipogenic transcription factors CCAAT/enhancer-binding protein (C/EBP) alpha and peroxisome proliferator-activated receptor-gamma and the mature adipocyte genes they control. Transduction of Mrf-2(-/-) MEFs with a retroviral vector expressing the longer Mrf-2 splice variant (Mrf-2B) stimulated both gene expression and lipid accumulation. Because 3T3-L1 cells are committed to the adipocyte lineage, we used this simpler model system to examine the effects of Mrf-2 deficiency on adipocyte maturation. Analyses of both mRNA and protein revealed that knockdown of Mrf-2 in 3T3-L1 cells prolonged the expression of C/EBP homologous protein-10, a dominant-negative form of C/EBP. Consistent with these findings, suppression of Mrf-2 also inhibited the DNA-binding activity of C/EBPbeta. These data suggest that Mrf-2 facilitates the induction of the two key adipogenic transcription factors C/EBPalpha and peroxisome proliferator-activated receptor-gamma indirectly by permitting hormone-mediated repression of the adipogenic repressor C/EBP homologous protein-10.