Spectrum of Histopathological, Immunohistochemical, Molecular and Radiological Features in 12 Cases of BCOR::CCNB3-positive Sarcomas With Literature Review.

Introduction BCOR::CCNB3-positive undifferentiated sarcomas are rare. Herein, we present clinicopathological features including immunohistochemical and molecular data, along with the radiological profile of 12 such tumors. Methods Tumors were tested for BCOR::CCNB3 fusion by reverse transcription polymerase chain reaction (RT-PCR) technique. Eight tumors were tested for EWSR1 and three for SS18 gene rearrangements by fluorescence in situ hybridization, and two for SS18::SSX fusion by fragment analysis. Results Ten of 12 patients were male with ages ranging between 4 and 17 years (median = 13, average = 14.4). Nine tumors occurred in bones and three in soft tissues (median size = 8 cm). Four of five tumors within the appendicular bones were metadiaphyseal and appeared as permeative lesions, invariably associated with cortical thickening. Three tumors displayed mineralization. Histopathologically, the tumors comprised round to epithelioid cells with round to oval to spindle-shaped nuclei, mostly diffusely arranged in a myxoid stroma with intervening thin-walled vessels. Immunohistochemically, tumor cells were positive for BCOR (10/11), SATB2 (8/9), TLE1 (5/6), cyclinD1 (4/4), and EMA (3/8). All tumors revealed BCOR::CCNB3 fusion transcript. Nine patients underwent neoadjuvant chemotherapy, including five who underwent surgical resection, with two patients, who received adjuvant radiation therapy. A single patient, each, underwent palliative chemotherapy and palliative radiotherapy, respectively. Four patients developed pulmonary metastasis and three developed local recurrences. Four patients were alive-with-disease and two were free-of-disease. Conclusions It is crucial to identify BCOR::CCNB3 fusion-positive sarcomas, given significant treatment-associated implications. Certain clinicoradiological, histopathological features, absent EWSR1 rearrangement and BCOR, SATB2, and TLE1 immunoexpression are useful for triaging these tumors for molecular testing. A review of the literature on these ultra-rare tumors, including their diagnostic mimics is presented.

Understanding the Molecular Mechanisms of Betel miRNAs on Human Health.

BACKGROUND: Since ancient times, "betel leaf" (Piper betle) has been revered for its religious, cultural, and medicinal properties. Phytochemicals from the Piper betle are effective in a variety of conditions, including cancer. To date, however, no genomic study or evidence has been found to elucidate the regulatory mechanism that underpins its therapeutic properties. This is the first study of its kind to predict Piper betle miRNAs and also the first genomics source representation of Piper betle. According to previous research, miRNAs from the plants we eat can regulate gene expression. In line with this, our in-silico study revealed that Piper betle and human cross-kingdom control occurs. METHODS: This study demonstrates the prediction and in-silico validation of Piper betle miRNAs from NGS-derived transcript sequences. The cross-kingdom regulation, which can also be understood as inter- species RNA regulation, was studied to identify human mRNA targets controlled by Piper betle miRNAs. Functional annotation and gene-disease association of human targets were performed to understand the role of Piper betle miRNAs in human health and disease. The protein-protein interaction and expression study of targets was further carried out to decipher their role in cancer development. RESULTS: Identified six Piper betle miRNAs belonging to miR156, miR164, miR172, and miR535 families were discovered to target 198 human mRNAs involved in various metabolic and disease processes. Angiogenesis and the cell surface signaling pathway were the most enriched gene ontology correlated with targets, both of which play a critical role in disease mechanisms, especially in the case of carcinoma. In an analysis of gene-disease interactions, 40 genes were found to be related to cancer. According to a protein-protein interaction, the CDK6 gene, which is thought to be a central regulator of cell cycle progression, was found as a hub protein, affecting the roles of CBFB, SAMD9, MDM4, AXIN2, and NOTCH2 oncogenes. Further investigation revealed that pbe-miRNA164a can be used as a regulator to minimise disease severity in Acute Myeloid Leukemia, where CDK6 expression is highest compared to normal cells. CONCLUSION: The predicted pbe-miRNA164a in this study can be a promising suppressor of CDK6 gene involved in tumour angiogenesis. In vivo validation of the pbe-miRNA164a mimic could pave the way for new opportunities to fight cancer and leverage the potential of Piper betle in the healthcare sector.

A phase 1/2 and biomarker study of preoperative short course chemoradiation with proton beam therapy and capecitabine followed by early surgery for resectable pancreatic ductal adenocarcinoma.

PURPOSE: To evaluate the safety, efficacy and biomarkers of short-course proton beam radiation and capecitabine, followed by pancreaticoduodenectomy in a phase 1/2 study in pancreatic ductal adenocarcinoma (PDAC) patients. METHODS AND MATERIALS: Patients with radiographically resectable, biopsy-proven PDAC were treated with neoadjuvant short-course (2-week) proton-based radiation with capecitabine, followed by surgery and adjuvant gemcitabine. The primary objective was to demonstrate a rate of toxicity grade ≥ 3 of <20%. Exploratory biomarker studies were performed using surgical specimen tissues and peripheral blood. RESULTS: The phase 2 dose was established at 5 daily doses of 5 GyE. Fifty patients were enrolled, of whom 35 patients were treated in the phase 2 portion. There were no grade 4 or 5 toxicities, and only 2 of 35 patients (4.1%) experienced a grade 3 toxicity event (chest wall pain grade 1, colitis grade 1). Of 48 patients eligible for analysis, 37 underwent pancreaticoduodenectomy. Thirty of 37 (81%) had positive nodes. Locoregional failure occurred in 6 of 37 resected patients (16.2%), and distant recurrence occurred in 35 of 48 patients (72.9%). With median follow-up of 38 months, the median progression-free survival for the entire group was 10 months, and overall survival was 17 months. Biomarker studies showed significant associations between worse survival outcomes and the KRAS point mutation change from glycine to aspartic acid at position 12, stromal CXCR7 expression, and circulating biomarkers CEA, CA19-9, and HGF (all, P<.05). CONCLUSIONS: This study met the primary endpoint by showing a rate of 4.1% grade 3 toxicity for neoadjuvant short-course proton-based chemoradiation. Treatment was associated with favorable local control. In exploratory analyses, KRAS(G12D) status and high CXCR7 expression and circulating CEA, CA19-9, and HGF levels were associated with poor survival.

Inhibition of beta-catenin and KRAS expressions by Piper betle in azoxymethane-induced colon cancer of male Fischer 344 rats.

OBJECTIVE: To investigate the chemopreventive effect of Piper betle (PB) on preneoplastic lesions (aberrant crypt foci [ACF]) induced by azoxymethane (AOM) in rats and its effect on colorectal cancer biomarkers (beta-catenin, KRAS, p53 and p21). STUDY DESIGN: A total of 32 male Fischer 344 rats were divided into phase 1 and phase 2 groups (8 and 24 weeks of AOM administration, respectively). Each phase was divided into 4 groups: control or normal saline (NS) (1 mL/kg), AOM (15 mg/kg body weight, once weekly for 2 weeks), PB (75 mg/kg body weight) and AOM + PB. PB was force-fed to rats a week after the second dose of AOM and NS. The colon was cut open longitudinally for methylene blue and immunohistochemistry staining. RESULTS: AOM administration showed formation of ACF at 8 and 24 weeks. PB, however, did not reduce ACF formation at either week, but it managed to reduce beta-catenin expression and KRAS found highly expressed in the AOM group of phase 1 rats. No immunoreactivities of p53 and p21 were detected in phase 2 rats, but instead inflammatory cells were visible in between the lesions. CONCLUSION: PB may act as a potential chemopreventive agent in the early stage of colon carcinogenesis by suppressing the expressions of beta-catenin and KRAS.

Hypericum androsaemum water extract inhibits proliferation in human colorectal cancer cells through effects on MAP kinases and PI3K/Akt pathway.

MAP kinase and PI3K/Akt signalling pathways are commonly altered in colorectal carcinoma (CRC) leading to tumor growth due to increased cell proliferation and inhibition of apoptosis. Several species of the genus Hypericum are used in Portugal to prepare herbal teas to which digestive tract effects are attributed. In the present study, the antiproliferative and proapoptotic effects of the water extracts of H. androsaemum (HA) and H. perforatum (HP) were investigated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which harbour activating mutations of KRAS and BRAF, respectively. Contrarily to HP, HA significantly inhibited cell proliferation and induced apoptosis in both cell lines. HA decreased BRAF and phospho-ERK expressions in CO115, but not in HCT15. HA also decreased Akt phosphorylation in CO115 and induced p38 and JNK in both cell lines. HA induced cell cycle arrest at S and G2/M phases as well as caspase-dependent apoptosis in both cell lines. Chlorogenic acid (CA), the main phenolic compound present in the HA extract and less represented in the HP water extract, did, however, not show any of those effects when used individually. In conclusion, water extract of HA, but not of HP, controlled CRC proliferation and specifically acted on mutant and not wild-type BRAF. The effect of HA was, however, not due to CA alone.

Time-resolved fluorescence resonance energy transfer as a versatile tool in the development of homogeneous cellular kinase assays.

Homogeneous cellular assays can streamline product detection in the drug discovery process. One commercially available assay employing time-resolved fluorescence resonance energy transfer (TR-FRET) that detects phosphorylated products was used to evaluate inhibitors of the receptor tyrosine kinase AXL in a cell line expressing an AXL-green fluorescent protein fusion protein. This TR-FRET assay was modified to evaluate the phosphorylation state of the AXL family member MER in a cell line expressing MER with a V5 tag by adding a fluorescein-labeled anti-V5 antibody. This homogeneous cellular assay was further modified to evaluate the nonreceptor tyrosine kinase focal adhesion kinase (FAK) in cell lines that expressed an untagged kinase by the inclusion of a commercially available anti-FAK antibody conjugated with an acceptor dye. The methods described here can be further adapted for TR-FRET detection of other cellular kinase activities.

Clinical metabolomics and urinary NGAL for the early prediction of chronic kidney disease in healthy adults born ELBW.

BACKGROUND: Clinical metabolomics is a recent "omic" technology which is defined as a global holistic overview of the personal metabolic status (fingerprinting). This technique allows to prove metabolic differences in different groups of people with the opportunity to explore interactions such as genotype-phenotype and genotype-environment type, whether normal or pathological. AIM: To study chronic kidney injury 1) using urine metabolomic profiles of young adults born extremely low-birth weight (ELBW) and 2) correlating a biomarker of kidney injury, urinary neutrophil gelatinase-associated lipocalin (NGAL), in order to confirm the metabolomic injury profile. METHOD: Urine samples were collected from a group of 18 people (mean: 24-year-old, std: 4.27) who were born with ELBW and a group of 13 who were born at term appropriate for gestational age (AGA) as control (mean 25-year-old, std: 5.15). Urine samples were analyzed by (1)H-nuclear magnetic resonance spectroscopy, and then submitted to unsupervised and supervised multivariate analysis. Urine NGAL (uNGAL) was measured using ARCHITECT (ABBOTT diagnostic NGAL kit). RESULTS: With a multivariate approach and using a supervised analysis method, PLS-DA, (partial least squares discriminant analysis) we could correlate ELBW metabolic profiles with uNGAL concentration. Conversely, uNGAL could not be correlated to AGA. CONCLUSIONS: This study demonstrates the relevance of the metabolomic technique as a predictive tool of the metabolic status of exELBW. This was confirmed by the use of uNGAL as a biomarker which may predict a subclinical pathological process in the kidney such as chronic kidney disease.

Low AZGP1 expression predicts for recurrence in margin-positive, localized prostate cancer.

BACKGROUND: Men with positive margins after radical prostatectomy (RP) for localized prostate cancer (PC) have a 40-50% biochemical relapse rate at 5 years. Adjuvant radiotherapy improves biochemical progression-free and overall survival in men with positive margins, but is associated with increased toxicity. There is an urgent need to identify new prognostic markers to define the group of patients who would benefit from multimodality therapy. METHODS: Nuclear ß-catenin, membranous secreted frizzled-related protein 4 (sFRP4), zinc-alpha 2-glycoprotein (AZGP1), and macrophage inhibitory cytokine-1 (MIC-1) have previously been identified as molecular markers of outcome in localized PC. From these published studies, we identified a subset of patients with positive margins. The aim of this study was to assess the association between these four molecular markers and outcome in men with margin-positive, localized PC. RESULTS: We identified 186 men with positive margins from 330 men with localized PC; 53% had preoperative PSA >10 ng/ml, 72% extraprostatic extension (EPE), 24% seminal vesicles involvement (SVI), and 57% RP Gleason score ≥ 7. AZGP1 (P = 0.009), membranous sFRP4 (P = 0.03) and MIC-1 (P = 0.04) expression predicted for biochemical relapse on univariate analysis. Only absent/low AZGP1 expression (P = 0.01) was an independent predictor of recurrence in margin-positive, localized PC when modeled with preoperative PSA (P = 0.2), EPE (P = 0.2), SVI (P = 0.4), Gleason score ≥ 7 (P = 0.5) and adjuvant treatment (P = 0.4). Furthermore, there was an association between absent/low AZGP1 expression and clinical recurrence (P = 0.007). CONCLUSIONS: AZGP1 is a potential molecular marker for biochemical relapse in men with margin-positive, localized PC. Routine assessment of this biomarker may lead to better selection of patients who will benefit from post-RP radiotherapy.

Combination of ionising irradiation and hyperthermia activates programmed apoptotic and necrotic cell death pathways in human colorectal carcinoma cells.

PURPOSE: The malignancy of tumor cells can be attenuated by interfering with cell death pathways. Since hyperthermia (HT) is a very potent radiosensitizer, the influence of HT (41.5 °C for 1 hour) alone and in combination with ionising irradiation (X-ray; 5 Gy or 10 Gy) on the form of cell death as well as on the expression of proteins known to be major components in tumor cells' apoptotic and necrotic pathways were examined in colorectal tumor cells. MATERIAL AND METHODS: The expression of proteins was analysed by western blot and the relative activity of caspases-3/7 by fluorescence- based assay. Colony formation was analysed using the clonogenic assay and cell death was determined with annexin V-FITC/propidium iodide staining. RESULTS: Combining X-ray with HT led to similar activation of caspase-3/7 and p53 expression in comparison to irradiation only while the amount of the pro-apoptotic proteins PUMA and Bax was increased in HCT15 and SW480 cells. HT alone or combinations with X-ray further resulted in a temporarily increased level of the anti-apoptotic protein Bcl-2. Irradiation plus HT further led to an up-regulation of IRF-5. The levels of RIP-1, a marker for programmed necrosis, increased in tumor cells which were treated with HT and/or X-ray. Combining 5 Gy irradiation with HT compared to irradiation resulted in a significantly increased number of necrotic tumor cells and in decreased colony formation. CONCLUSION: The combined treatment of colorectal tumor cells with X-ray and HT activates distinct tumor cell pathways and fosters the early appearance of a necrotic tumor cell phenotype.

E2A-positive gastric MALT lymphoma has weaker plasmacytoid infiltrates and stronger expression of the memory B-cell-associated miR-223: possible correlation with stage and treatment response.

Extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue of the stomach (gastric MALT lymphoma) is derived from memory B cells of the marginal zone. Normal memory B cells do not express markers of germinal-center B cells, such as E2A (immunoglobulin enhancer-binding factor E12/E47), B-cell chronic lymphocytic leukemia/lymphoma 6 (BCL6), or activation-induced cytidine deaminase (AID). E2A is a transcription factor that induces somatic hypermutations and blocks plasma cell differentiation. In 50 stage-I(E)/II(E1) gastric MALT lymphomas, we confirmed that all cases were BCL6(-)/AID(-), but a subset (50%, 25/50) was E2A(+). As E2A(-) and E2A(+) gastric MALT lymphomas had similar numbers of somatic hypermutations without intraclonal variations, which implied an origin from memory B cells, the expression of E2A was best regarded as a marker of aberrant follicular differentiation. Although the status of somatic hypermutation was not affected by E2A, E2A(+) gastric MALT lymphoma showed less plasmacytoid infiltrates and higher expressions of miRNA-223, a microRNA associated with memory B cells. Clinically, E2A(+) gastric MALT lymphomas were more likely to spread to perigastric lymph nodes and were less responsive to Helicobacter eradication therapy than were E2A(-) gastric MALT lymphomas. Taken together, aberrant E2A expression is a diagnostic feature of a subtype of gastric MALT lymphoma with weaker plasmacytoid infiltrates and stronger miR-223 expression. A prospective study would be necessary to verify the association between E2A expression and a poor response to Helicobacter eradication therapy.

Eugenol restricts DMBA croton oil induced skin carcinogenesis in mice: downregulation of c-Myc and H-ras, and activation of p53 dependent apoptotic pathway.

BACKGROUND: Eugenol is the active component of essential oil isolated from clove (Syzigium aromaticum). Eugenol has antimutagenic, antigenotoxic, anti-inflammatory properties. The anticarcinogenic effect of eugenol was evident in different types of cell lines. However, its anticarcinogenic effect in in vivo has not yet been fully explored. OBJECTIVE: The aim of this study is to evaluate the chemopreventive potential of eugenol in an experimental skin carcinogenesis mice model system. METHOD: Skin tumor was induced by topical application of DMBA croton oil in Swiss mice. To assess the chemopreventive potential of eugenol, it was orally administered 15 days prior carcinogen treatment. The development of skin carcinogenesis was confirmed by histopathological analysis. Cellular proliferation and apoptosis in the skin tumor were analyzed by in situ cellular proliferation and in situ cell death assay. Expression of some proliferation and apoptosis associated genes was analyzed by RT-PCR and protein expression was analyzed by Western blot. RESULTS: Reduction in incidence and sizes of skin tumors along with overall increase in survival of mice were seen due to eugenol treatment. Restriction of skin carcinogenesis at the dysplastic stage along with reduced rate of cellular proliferation and increase in apoptosis were evident in eugenol treated skin tumors. Eugenol treatment led to the downregulation of c-Myc, H-ras and Bcl2 expression along with upregulation of P53, Bax and active Caspase-3 expression in the skin lesions. CONCLUSION: Restriction of skin carcinogenesis at dysplastic stage by eugenol was due to attenuation of c-Myc, H-ras and modification of some p53 associated gene expression.

Evaluation of the ARCHITECT urine NGAL assay: assay performance, specimen handling requirements and biological variability.

OBJECTIVES: NGAL (Neutrophil Gelatinase-Associated Lipocalin) has emerged as a new biomarker for the identification of acute kidney injury. Reliable clinical evaluations require a simple, robust test method for NGAL, and knowledge of specimen handling and specimen stability characteristics. We evaluated the performance of a new urine NGAL assay on the ARCHITECT analyzer. METHODS: Assay performance characteristics were evaluated using standard protocols. Urine specimen storage requirements were determined and biological variability was assessed in a self-declared apparently healthy population. RESULTS: Assay performance data showed good precision, sensitivity and lot-to-lot reproducibility. There was good short term 2-8 degrees C sample stability, however, long term storage samples must be kept at -70 degrees C or colder. The largest variance component in a biological variance study was within-day. CONCLUSIONS: The ARCHITECT NGAL assay proved to be a precise and reproducible assay for the determination of urine NGAL.

FLT3 inhibition in t(4;11)+ adult acute lymphoid leukaemia.

The present study was designed to investigate, in t(4;11)+ adult lymphoid leukaemia (ALL) blast cells, the pathogenetic role of the FLT3 protein, its level of mRNA and protein expression, the degree of constitutive phosphorylation, the possible presence of mutations of the sequence, the capacity of signal transduction and the potential therapeutic role of specific inhibitors. We evaluated nine adult ALL patients carrying this translocation. The increased FLT3 mRNA levels, determined by oligonucleotide microarray analysis, was in agreement with the increased protein expression evaluated by Western blot. The protein was constitutively phosphorylated in all cases analysed. Polymerase chain reaction detected no internal tandem duplication or point mutations. The signal transduction apparatus, after stimulation with the specific ligand, was preserved. We then investigated the effect of specific FLT3 inhibition on signal transduction and survival. The PKC412 inhibitor specifically inhibited ligand-induced phosphorylation; the same inhibitor reduced the survival of leukaemic cells when compared with untreated cells. These data indicate that the FLT3 protein might play a role in this subgroup of ALL with a particularly poor prognosis. Specific inhibition of the kinase receptor must be hypothesised as an innovative therapeutic tool for t(4;11)+ ALL patients.

Intravascular large B-cell lymphoma involving hemangiomas: an unusual presentation of a rare neoplasm.

We report the clinicopathological features of two cases of intravascular large B-cell lymphoma involving cutaneous hemangiomas. The cases were identified from the consultation files of two of the authors. Both patients were women, 64 and 55 years of age, who presented with long-standing cutaneous hemangiomas of the posterior scalp and left shoulder, respectively. The lesions were brought to medical attention by an increase in size and change in color. Biopsies and immunohistochemical evaluation of the hemangiomas revealed extensive involvement by intravascular large B-cell lymphoma. The neoplastic cells were diffusely positive for CD20 in both cases and negative for CD3, pan-cytokeratin (AE1/AE3), epithelial membrane antigen, S-100, Factor VIII-related antigen, CD34 and CD31. Disease was limited to the hemangiomas in both patients. Treatment consisted of chemotherapy (both patients) and adjuvant radiation therapy (one patient). One patient had a recurrence of disease 33 months after initial diagnosis, leading to an autologous stem cell transplant. The other patient is without evidence of disease 27 months after initial diagnosis. Although this is a rare neoplasm, it is important to consider intravascular large B-cell lymphoma in the differential diagnosis of vascular lesions containing intravascular neoplastic cells.

Aggressive angiomyxoma: a clinicopathological and immunohistochemical study of 11 cases with long-term follow-up.

AIMS: To report the clinicopathological and immunohistochemical features and longer term biological behaviour of aggressive angiomyxoma, an uncommon mesenchymal neoplasm occurring predominantly in the pelvi-perineal region of adults. Using immunohistochemistry, possible overexpression of CDK4 and MDM2 was analysed, which might point to (cyto)genetic alteration(s) in chromosome region 12q13-15, an area reported to be altered in this tumour entity. METHODS AND RESULTS: Cases (n=11) of aggressive angiomyxoma were retrieved from the consultation files of the Comprehensive Cancer Centre of the Middle Netherlands (IKMN) panel for soft tissue tumours. Clinical and follow-up information were obtained, and immunohistochemical analysis was performed using antibodies directed against vimentin, cytokeratin AE1/AE3, desmin, alpha-smooth-muscle actin, CD34, S-100 protein, oestrogen receptors, CDK4 and MDM2. Five patients were female (age range 24-47 years; median 39 years), and six patients were male (age range 36-69 years; median 44.5 years). Of 11 cases, 10 arose in the pelvi-perineal area and 1 arose in the abdominal cavity in close relation to the bladder. Morphology was consistent with previous reports of this entity. Immunohistochemically, 8 of 11 cases were desmin positive (5 of 5 positive in females; 3 of 6 positive in males), 6 of 11 cases were positive for alpha-smooth-muscle actin, 5 of 11 cases were CD34 positive, 11 of 11 cases, irrespective of gender, were positive for oestrogen receptors and 3 of 11 cases were positive for cytokeratin AE1/AE3. Strong, diffuse nuclear positivity for CDK4 expression was present in all 6 cases tested, while only 1 of 11 cases tested for MDM2 showed weak focal positivity. Clinical follow-up in all cases (range 1-216 months; median 72 months) showed one local recurrence (9%) after 36 months. No metastases or tumour-related deaths were noted. CONCLUSIONS: The sex distribution of cases reported in this study was roughly equal, in contrast to previous reports emphasising the predominance of this tumour in females. Our study confirms the local aggressive nature of aggressive angiomyxoma, although our local recurrence rate is lower than previous reports (9% versus 36-72%); no metastases and/or disease-related patient deaths were documented. All cases arising in females were positive for desmin, while three of the six cases arising in males were negative for desmin, supporting previous findings and indicating that the lesion may be somewhat different in males. The strong diffuse positivity for CDK4 in all six cases tested goes some way in implicating CDK4, either directly or indirectly, in tumourigenesis. The negative immunostaining for MDM2 would argue against functional amplification of this gene.

Severe defects in dorsal thalamic development in low-density lipoprotein receptor-related protein-6 mutants.

Mice with mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein-6 (LRP6) have a smaller and severely disorganized dorsal thalamus and lack thalamocortical projections. Using molecular markers, we showed that most dorsal thalamic and epithalamic neurons were missing, and most of the major dorsal thalamic nuclei were not identifiable. However, the ventral thalamus was essentially unaffected, although the dorsal thalamic defect leads to rostral displacement of portions of the ventral thalamus. Analysis of younger embryos showed that epithalamic and dorsal thalamic neurons were not produced at early stages of development, whereas ventral thalamic neurons were still produced. These defects were accompanied by improper formation of the boundary between dorsal and ventral thalamus, the zona limitans interthalamica (ZLI). Furthermore, the expression of an early marker of posterior forebrain development that marks the compartment from the midbrain-hindbrain junction to the ZLI (including the future dorsal thalamus, pretectum, and midbrain) was disrupted, supporting the idea that diencephalic development is abnormal from very early in embryogenesis. This study provides compelling in vivo evidence that thalamic development requires normal activity of the LRP6-mediated canonical Wnt signaling pathway.

[Effect of melittin on apoptosis and necrosis of U2 OS cells].

OBJECTIVE: To study the effect of melittin on apoptsis and necrosis of osteosarcoma cell line U2 OS in vitro. METHODS: Osteosarcoma cell line U2 OS was treated with melittin. The growth and proliferation was observed by MTT assay and cell counting, and the necrosis was estimated by Trypan blue staining. The cell apoptsis, Fas and Apo2. 7 expression were detected by cytometer. RESULTS: The data showed that melittin could inhibit the proliferation of U2 OS dose-dependently at 16 and 64 mg/L. Cell apoptsis was detected by cytometer, when the cells were treated by 16 mg/L and 32 mg/L of melittin respectively, and the percentages of Fas and Apo2. 7 positive cells were increased. CONCLUSION: Melittin inhibits the proliferation of osterosarcoma cell line through up-regulating Fas expression and inducing apoptsis.

Modulations of the Bcl-2/Bax family were involved in the chemopreventive effects of licorice root (Glycyrrhiza uralensis Fisch) in MCF-7 human breast cancer cell.

Recently, cancer chemoprevention with strategies using foods and medicinal herbs has been regarded as one of the most visible fields for cancer control. Genistein in soy, American ginseng, and resveratrol are well-known to have antiproliferative properties in human breast cancer. Licorice root is a botanical, a shrub native to southern Europe and Asia, which primarily has desirable qualities in sweetening and herbal medicine. In this study, licorice (Glycyrrhiza uralensis Fisch) root also inhibits cell proliferation in human breast cancer cell. The cell proliferation study demonstrated that licorice root reduced the proliferation of MCF-7 cells in a dose- and time-dependent manner. The extracts were fractionated in CHCl(3), EtOAc, C(6)H(14), and CH(3)OH-H(2)O (70:30), and these extracts of licorice root (50 microg/mL) induced DNA fragmentation demonstrated by Hoechst 33258 staining. Apoptosis also determined the sub-G1 accumulation by flow cytometry analysis. These results were consistent with specific cleavage of PARP and antiapoptotic protein Bcl-2 and up-regulation of proapoptotic protein Bax demonstrated by Western blotting. Our findings suggest that licorice root may have chemopreventive effects against human breast cancer through the modulation of the expression of the Bcl-2/Bax family of apoptotic regulatory factors.

Isolation and characterization of lipid rafts with different properties from RBL-2H3 (rat basophilic leukaemia) cells.

Lipid rafts are plasma-membrane microdomains that are enriched in certain lipids (sphingolipids, glycosphingolipids and cholesterol), as well as in lipid-modified proteins. Rafts appear to exist in the liquid-ordered phase, which contributes to their partitioning from the surrounding liquid-disordered glycerophospholipid environment. DRM (detergent-resistant membrane) fractions isolated from cells are believed to represent coalesced lipid rafts. We have employed extraction using two different non-ionic detergents, Brij-96 and Triton X-100, to isolate detergent-resistant lipid rafts from rat basophilic leukaemia cell line RBL-2H3, and compared their properties with each other and with plasma-membrane vesicles. DRM fractions were isolated as sealed unilamellar vesicles of similar size (135-170 nm diameter), using either sucrose-density-gradient sedimentation or gel-filtration chromatography. Lipid rafts isolated using Brij-96 and Triton X-100 differed in density, protein content and the distribution between high- and low-density fractions of the known raft constituents, Thy-1, and the non-receptor protein tyrosine kinases, Yes and Lyn. Lyn was found in the raft microdomains in predominantly phosphorylated form. The level of enrichment of the protein constituents of the isolated lipid rafts seemed to depend on the ratio of cell lipid/protein to detergent. As indicated by reactivity with anti-Thy-1 antibodies, lipid rafts prepared using Brij-96 appeared to consist of vesicles with primarily right-side-out orientation. Both Brij-96 and Triton X-100 appear to isolate detergent-insoluble raft microdomains from the rat basophilic leukaemia cell line RBL-2H3, but the observed differences suggest that either the detergents themselves play a role in determining the physicochemical characteristics of the resulting DRM fractions, or different subsets of rafts are isolated by the two detergents.