The androgen receptor-lncRNASAT1-AKT-p15 axis mediates androgen-induced cellular senescence in prostate cancer cells.

The bipolar androgen therapy (BAT) to treat prostate cancer (PCa) includes cycles of supraphysiological androgen levels (SAL) under androgen-deprivation therapy (ADT). We showed previously that SAL induces cellular senescence in androgen-sensitive PCa cells and in ex vivo-treated patient PCa tumor samples. Here, we analyzed the underlying molecular pathway and reveal that SAL induces cellular senescence in both, castration-sensitive (CSPC) LNCaP and castration-resistant PCa (CRPC) C4-2 cells through the cell cycle inhibitor p15INK4b and increased phosphorylation of AKT. Treatment with the AKT inhibitor (AKTi) potently inhibited SAL-induced expression of p15INK4b and cellular senescence in both cell lines. Proximity-ligation assays (PLA) combined with high-resolution laser-scanning microscopy indicate that SAL promotes interaction of endogenous androgen receptor (AR) with AKT in the cytoplasm as well as in the nucleus detectable after three days. Transcriptome sequencing (RNA-seq) comparing the SAL-induced transcriptomes of LNCaP with C4-2 cells as well as with AKTi-treated cell transcriptomes revealed landscapes for cell senescence. Interestingly, one of the identified genes is the lncRNASAT1. SAL treatment of native patient tumor samples ex vivo upregulates lncRNASAT1. In PCa tumor tissues, lncRNASAT1 is downregulated compared with nontumor tissues of the same patients. Knockdown indicates that the lncRNASAT1 is crucial for SAL-induced cancer-cell senescence as an upstream factor for pAKT and for p15INK4b. Further, knockdown of lncRNASAT1 enhances cell proliferation by SAL, suggesting that lncRNASAT1 serves as a tumor suppressor at SAL. Interestingly, immunoprecipitation of AR detected lncRNASAT1 as an AR-interacting partner that regulates AR target-gene expression. Similarly, RNA-ChIP experiments revealed the interaction of AR with lncRNASAT1 on chromatin. Thus, we identified a novel AR-lncRNASAT1-AKT-p15INK4b signaling axis to mediate SAL-induced cellular senescence.

Genome-wide identification, phylogenetic and expression analysis of the maize HECT E3 ubiquitin ligase genes.

HECT (homologous to the E6AP carboxyl terminus) ubiquitin ligase genes (E3s) are enzymes with diverse functions influencing plant growth, development, and responses to abiotic stresses. However, there is relatively little information available regarding the maize HECT E3 gene family. In the present study, 12 maize HECT E3 genes (ZmUPL1 to ZmUPL12) were identified at the whole-genome level. The phylogenetic relationships, structures, and expression levels of the maize HECT E3 genes were then analyzed. On the basis of the constructed maximum likelihood phylogenetic tree, the HECT E3 genes were divided into six groups. The quantitative real-time polymerase chain reaction assay results revealed that all of the maize ZmUPL genes were expressed in most of the examined tissues and were responsive to three abiotic stresses. Considered together, the study results may provide a useful foundation for future investigations of maize stress-tolerance genes as well as functional analyses of the E3 enzymes in diverse agriculturally important crop species.

Does Notch play a tumor suppressor role across diverse squamous cell carcinomas?

The role of Notch pathway in tumorigenesis is highly variable. It can be tumor suppressive or pro-oncogenic, typically depending on the cellular context. Squamous cell carcinoma (SCC) is a cancer of the squamous cell, which can occur in diverse human tissues. SCCs are one of the most frequent human malignancies for which the pathologic mechanisms remain elusive. Recent genomic analysis of diverse SCCs identified marked levels of mutations in NOTCH1, implicating Notch signaling pathways in the pathogenesis of SCCs. In this review, evidences highlighting NOTCH's role in different types of SCCs are summarized. Moreover, based on accumulating structural information of the NOTCH receptor, the functional consequences of NOTCH1 gene mutations identified from diverse SCCs are analyzed, emphasizing loss of function of Notch in these cancers. Finally, we discuss the convergent view on an intriguing possibility that Notch may function as tumor suppressor in SCCs across different tissues. These mechanistic insights into Notch signaling pathways will help to guide the research of SCCs and development of therapeutic strategies for these cancers.

Cellulose/soy protein isolate composite membranes: evaluations of in vitro cytocompatibility with Schwann cells and in vivo toxicity to animals.

OBJECTIVE: To evaluate the in vitro cytocompatibility of cellulose/soy protein isolate composite membranes (CSM) with Schwann cells and in vivo toxicity to animals. METHODS: A series of cellulose/soy protein isolate composite membranes (CSM) were prepared by blending, solution casting and coagulation process. The cytocompatibility of the CSM to Schwann cells were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by direct cells culture of Schwann cells on the surfaces of the CSM, respectively. The in vivo toxicity of the CSM to animals were also evaluated by acute toxicity testing, skin sensitization testing, pyrogen testing and intracutaneous stimulation testing, respectively, according to the ISO 10993 standard. RESULTS: The MTT assay showed that the cell viability of Schwann cells cultured in extracts from the CSM was higher than that from the neat cellulose membrane without containing SPI component. The direct cells culture indicated that the Schwann cells could attach and grow well on the surface of the CSM and the incorporation of SPI into cellulose contributed to improvement of cell adhesion and proliferation. The evaluations of in vivo biological safety suggested that the CSM showed no acute toxicity, no skin sensitization and no intracutaneous stimulation to the experimental animals. CONCLUSION: The CSM had in vitro cytocompatibility with Schwann cells and biological safety to animals, suggesting potential for the applications as nerve conduit for the repair of nerve defect.

Induction of B7-H1 expression by human cytomegalovirus in extravillous cytotrophoblast cells and role of MAPK pathway.

OBJECTIVE: This paper is aimed at to evaluate B7-H1 expression as induced by human cytomegalovirus (HCMV) in extravillous cytotrophoblast cell line HPT-8 and possible underlying mechanism. METHOD: Real time PCR and flow cytometry were used to determine B7-H1 mRNA and protein before and after HCMV infection in HPT-8 cells. Western blot analysis was used to determine the level of MAPK phosphorylation in HPT-8 cell lines infected with HCMV. RESULTS: 100TCID50 was found to be the most effective dose, capable of stimulating B7-H1 mRNA and protein expression in HPT-8 cells. When empty control group was considered to have a B7-H1 mRNA value of 1, B7-H1 mRNA was 4.32 in 100TCID50 group. In flow cytometry study, mean fluorescence intensity (MFI) of 100TCID50 group was 16.14, while empty control group was 1.34. Both mRNA and protein expression were found to be significantly increased (P<0.05) in 100TCID50 group compared to empty control group. The result of Western blot analysis showed increase in B7-H1 expression caused by the extracellular signaling that was related to ERK activation and the ERK inhibitor U0126 was found to reverse this increase. CONCLUSION: HCMV upregulates B7-H1 expression in human extravillous cytotrophoblast cell line HPT-8, which is related to MAPK activation. Our result would be helpful in finding better therapies against intrauterine HCMV infection.

Inhibition of miR-92b suppresses nonsmall cell lung cancer cells growth and motility by targeting RECK.

microRNAs play critical roles in the progression and metastasis of nonsmall cell lung cancer (NSCLC). miR-92b acts as an oncogene in some malignancies; however, its role in NSCLC remains poorly understood. Here, we found that miR-92b was significantly increased in human NSCLC tissues and cell lines. Inhibition of miR-92b remarkably suppressed cell proliferation, migration, and invasion of NSCLC cells. Reversion-inducing-cysteine-rich protein with kazal motifs (RECK) was identified to be a target of miR-92b. Expression of miR-92b was negatively correlated with RECK in NSCLC tissues. Collectively, miR-92b might promote NSCLC cell growth and motility partially by inhibiting RECK.

The Tumor Cytosol miRNAs, Fluid miRNAs, and Exosome miRNAs in Lung Cancer.

The focus of this review is to provide an update on the progress of microRNAs (miRNAs) as potential biomarkers for lung cancer. miRNAs are single-stranded, small non-coding RNAs that regulate gene expression and show tissue-specific signatures. Accumulating evidence indicates that miRNA expression patterns represent the in vivo status in physiology and disease. Moreover, miRNAs are stable in serum and other clinically convenient and available tissue sources, so they are being developed as biomarkers for cancer and other diseases. Cancer is currently the primary driver of the field, but miRNA biomarkers are being developed for many other diseases such as cardiovascular and central nervous system diseases. Here, we examine the framework and scope of the miRNA landscape as it specifically relates to the translation of miRNA expression patterns/signatures into biomarkers for developing diagnostics for lung cancer. We focus on examining tumor cytosol miRNAs, fluid miRNAs, and exosome miRNAs in lung cancer, the connections among these miRNAs, and the potential of miRNA biomarkers for the development of diagnostics. In lung cancer, miRNAs have been studied in both cell populations and in the circulation. However, a major challenge is to develop biomarkers to monitor cancer development and to identify circulating miRNAs that are linked to cancer stage. Importantly, the fact that miRNAs can be successfully harvested from biological fluids allows for the development of biofluid biopsies, in which miRNAs as circulating biomarkers can be captured and analyzed ex vivo. Our hope is that these minimally invasive entities provide a window to the in vivo milieu of the patients without the need for costly, complex invasive procedures, rapidly moving miRNAs from research to the clinic.

miR-205 promotes the growth, metastasis and chemoresistance of NSCLC cells by targeting PTEN.

Non-small cell lung cancer (NSCLC) is one of the most common causes of cancer-related mortality worldwide. microRNAs (miRNAs) play critical roles in carcinogenesis. miR-205 has been shown to be upregulated in NSCLC. In the present study, we identified the promotive effects of miR-205 on various significant biological properties of NSCLC cells, and confirmed the regulation of PTEN by miR-205. The expression of miR-205 was examined by quantitative real-time PCR both in NSCLC cell lines and tissues. The effect of miR-205 on PTEN expression was assessed in NSCLC cell lines with miR-205 mimics/inhibitor to elevate/decrease miR-205 expression. Furthermore, the roles of miR-205 in regulating the biological properties of NSCLC cells, including growth, invasion and chemoresistance, were assayed using miR-205 mimic/inhibitor-transfected cells. The 3'-untranslated region (3'-UTR) of PTEN combined with miR-205 and this was confirmed by luciferase reporter assay and western blotting. miR-205 expression was increased in NSCLC cell lines as well as in tissues. Overexpression of miR-205 promoted growth, migration and invasion, and enhanced the chemoresistance of NSCLC cells. Luciferase activity and western blotting demonstrated that miR-205 negatively regulated PTEN at a posttranscriptional level. However, miR-205 knockdown suppressed these processes in A549 cells and increased the expression of PTEN protein. Furthermore, overexpression of PTEN exhibited effects identical with those of the miR-205 inhibitor in NSCLC cells. Our results demonstrated that miR-205 is involved in the tumorigenesis of NSCLC through modulation of the PTEN signaling pathway.

Stem cell models for drug discovery and toxicology studies.

Human stem cells and their derivatives could provide virtually unlimited sources of tissue for a wide range of toxicity models that could complement conventional animal models with more relevant, humanized versions. Human embryonic stem cells (hESCs) have already been proven valuable for drug/toxicity screens and mechanistic studies including analysis of disease pathway and developmental toxicity. Human-induced pluripotent stem cells (iPSCs), which are generated by reprogramming somatic cells back to become hESC-like cells, allow assays to be designed where the contribution of an individual's genetic background or environmental exposure history to toxicity response can be determined. Comprehensive profiling of hESC/iPSCs via genomics, proteomics, transcriptomics, and metabolomics could be used to elucidate pathway perturbations that underlie toxicity and disease, enabling the development of predictive assays for toxicity. While technological hurdles still exist for widespread use and implementation, incorporation of human stem cell based assays into drug discovery and toxicity testing offers the potential for safer, more customized medicines and more accurate risk assessment for environmental toxicants, as well as reduced costs and decreased use of animal models. We examine limitations and deficiencies of current toxicology approaches and how human stem cell based in vitro assays may overcome them. We describe how human stem cells are used for predictive toxicology. We also identify technological limitations that prevent stem cells from being integrated into standard practice, as well as new tools and technologies that may overcome them. We discuss research priorities that are most useful for transforming cell-based toxicology models into reality, and research areas in which stem cell technology could make substantial contributions to the development and implementation of stem cell based models for toxicity testing. Increased use of human in vitro models of toxicity could reduce the use of animals in safety and risk assessment studies and offers the potential to dramatically enhance our understanding of the molecular basis of toxicity, leading to improved human models and assays for predicting biological response to drugs and environmental hazards.

Paclitaxel induced B7-H1 expression in cancer cells via the MAPK pathway.

In this study, we investigated the mechanisms by which the chemotherapeutic agent paclitaxel (PTX) induced the expression of B7-H1 immunosuppressive molecules in the human colorectal adenocarcinoma cell line SW480 and the hepatocellular carcinoma cell line HepG2. We found ptX induced B7-H1 protein expression in SW480 and HepG2 cells as demonstrated by immunofluorescence and flow cytometry and mRNA expression by using real-time quantitative polymerase chain reaction (PCR). Moreover, PTX treatment induced Erk½ phosphorylation in both cell lines. PTX-increased B7-H1 mRNA expression was significantly blocked by MEK inhibitor U0126. However, the protein expression caused by PTX was only partially blocked by U0126. Our results suggest that PTX upregulated B7-H1 expression in cultured SW480 and HepG2 cells via both transcriptional and post-transcriptional mechanisms. This may help us better understand PTX-related tumor immune evasion.

c-Jun N-terminal kinase is involved in the regulation of proliferation and apoptosis by integrin-linked kinase in human retinoblastoma cells.

PURPOSE: To determine the presence of integrin-linked kinase (ILK) in tissue samples of retinoblastoma patients, and to explore the function of ILK in human Y79 retinoblastoma cells. METHODS: The expression of ILK was studied in samples of retinoblastoma patients by immunohistochemistry. In vitro, specific small interfering RNA (siRNA) targeting ILK was transfected into Y79 retinoblastoma cells using liposome. Silencing of ILK expression was measured by reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR and Western blotting assays. Then the regulation of cell proliferation and apoptosis was assessed by Cell Counting Kit-8(CCK-8), Annexin V-FITC/ propidium iodide (PI) immunofluorescence, and flow cytometry assays. Furthermore, the involvement of c-Jun N-terminal kinase signal pathway was tested by JNK signal transduction inhibitor assay. RESULTS: Positive staining for ILK was detected in 15 of the 17 retinoblastoma tissue samples. Specific siRNA targeting ILK significantly silenced ILK expression in Y79 retinoblastoma cells, as confirmed by RT-PCR, real-time PCR and Western blotting assays (P < 0.01). This was accompanied by decreased cell proliferation (P < 0.05) and enhanced apoptosis (P < 0.01). The phosphorylation status of JNK and c-Jun was constitutively activated by ILK siRNA (P < 0.01), and JNK inhibitor simultaneously reversed the effects on cell proliferation and apoptosis induced by ILK siRNA. CONCLUSION: Our results demonstrated that ILK promoted proliferation and suppressed apoptosis via repressing phosphorylations of the JNK signal pathway in human retinoblastoma cells. This might provide a potential therapeutic target in the treatment of this deadly disease.

Integrin-alpha5 mediates epidermal growth factor-induced retinal pigment epithelial cell proliferation and migration.

Proliferation and migration of retinal pigment epithelial (RPE) cells play a crucial role in proliferative vitreoretinopathy (PVR)-related pathology. Cytokines, including EGF, can result in RPE cell activation and cause PVR. In this study, integrin-alpha(5) expression was first studied in PVR membranes by immunofluorescence. Then the effect of EGF on integrin-alpha(5) expression was determined by flow cytometry, Western blot analysis and the reverse-transcription polymerase chain reaction (RT-PCR) in the ARPE-19 cell line. Proliferation and migration of ARPE-19 cells were measured by the methylthiazolyldiphenyl-tetrazolium bromide and Boyden chamber assays. We found that a higher level integrin-alpha(5) was present at the RPE cell surface in PVR compared with normal retina. EGF could dose dependently increase integrin-alpha(5) mRNA and protein levels in vitro. EGF promoted ARPE-19 cell proliferation and migration. Neutralizing integrin-alpha(5) by specific anti-integrin-alpha(5) antibody abolished most of the effects of EGF. The study provided evidence that EGF might influence PVR by promoting integrin-alpha(5) expression and subsequent proliferation and migration of RPE.

Cutting edge: IFN-gamma enables APC to promote memory Th17 and abate Th1 cell development.

Th1-derived IFN-gamma targets naive T cells and inhibits Th17 development. However, Th1, Th17, and memory but not naive T cells are colocalized in an inflammatory environment. To demonstrate the kinetic relationship between these T cell subsets, we investigated the role of IFN-gamma in regulating the development and balance between Th17 and Th1 in humans. We show that IFN-gamma stimulates B7-H1 expression on APC subsets and abates their Th1 polarization capacity in a B7-H1-dependent manner. Interestingly, IFN-gamma triggers APCs to produce IL-1 and IL-23 and enables them to induce memory Th17 expansion via IL-1 and IL-23 in a B7-H1-independent manner. We propose a novel dynamic between Th1 and Th17 in the course of inflammation as follows: Th1-mediated inflammation is attenuated by IFN-gamma-induced B7-H1 on APCs and is evolved toward Th17-mediated chronic inflammation by IFN-gamma-induced, APC-derived IL-1 and IL-23. Our study challenges the dogma that IFN-gamma suppresses Th17 and enhances Th1 development.

Hypothermia reduces sulphur mustard toxicity.

The effect of temperature on the development of sulphur mustard (HD)-induced toxicity was investigated in first passage cultures of human skin keratinocytes and on hairless guinea pig skin. When cells exposed to HD were incubated at 37 degrees C, a concentration-dependent decline in viability was observed that was maximal by 2 days. In contrast, no significant HD-induced toxicity was evident up to 4 days posttreatment when the cells were incubated at 25 degrees C. However, these protective effects were lost by 24 h when the cells were switched back to 37 degrees C. The protective effects of hypothermia were also demonstrated when apoptotic endpoints were examined. The HD concentration-dependent induction of fragmented DNA (as quantitated using soluble DNA and the TUNEL reaction), morphology, and p53 expression were all significantly depressed when cell cultures were incubated at 25 degrees C compared to 37 degrees C. When animals were exposed to HD vapour for 2, 4, and 6 min and left at room temperature, lesions were produced whose severity was dependent on exposure time and that were maximal by 72 h posttreatment. Moderate cooling (5-10 degrees C) of HD exposure sites posttreatment (4-6 h) significantly reduced the severity of the resultant lesions. However, in contrast to the in vitro results, these effects were permanent. It appears that the early and noninvasive act of cooling HD-exposed skin may provide a facile means of reducing the severity of HD-induced cutaneous lesions.

Stimulation of Ca(2+) influx through ATP receptors on rat brain synaptosomes: identification of functional P2X(7) receptor subtypes.

1. ATP receptors of the P2X class have previously been identified on autonomic nerve endings and on a limited population of CNS neurons. 2. In the present study P2X receptors on mammalian cortical synaptosomes have been identified by a variety of functional and biochemical studies. In choline buffer ATP analogues caused concentration/time dependent Ca(2+) influx. Relative to the effects caused by ATP, benzoylbenzoyl ATP (BzATP) was about seven times more active than ATP while 2-me-S-ATP and ATPgammaS were much less active. alpha,beta-me- ATP and beta,gamma-me-ATP were virtually inactive. In sucrose buffer, relative to choline buffer, the activity of BzATP was more than doubled while activity in sodium buffer was reduced. Moreover, the P2X antagonists PPADS or Brilliant Blue G both significantly attenuated influx. These observations suggest the presence of P2X receptors on synaptosomes which subserve Ca(2+) influx. This activity profile of the ATP analogues and the response to blocking agents are characteristic of responses of P2X(7) receptors. 3. Influx was unaffected by the VSCC inhibitors omega-CTx-MVIIC and (-) 202 - 791, indicating that ATP induced Ca(2+) influx occurred primarily through P2X receptors. 4. P2X(7) receptor protein was identified by Western blotting and immunohistochemical staining. Purified preparations were devoid of significant concentrations of GFAP or the microglial marker OX-42 but contained greatly enriched amounts of syntaxin and SNAP 25. 5. The various pharmacological and biochemical studies were all consistent with the presence of functional P2X(7) receptors.