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It is believed that breast cancer stem cells (BCSCs), like normal stem cell counterparts, have the capacity of self-renewal and differentiation. Simultaneously, estrogen receptor (ER)-negative (-) BCSCs are affected by surrounding differentiated ER-positive (+) tumor cells by virtue of paracrine signaling within the tumor micro-environment. Genistein (GEN), as a sort of phytoestrogen, can act on ER+ breast cancer cells but the role of GEN in the differentiation of neighboring ER- BCSCs has not been defined. Transwell co-culture system was utilized so as to elaborate the interaction between well-differentiated ER+ breast cancer cells (MCF-7) and ER- breast cancer stem/progenitor cells (mammospheres derived from MDA-MB-231 cells). GEN-induced differentiation of BCSCs was analyzed by mammospheres formation assay, flow cytometry and RT-PCR after a 3 day solo-culture or co-culture. We find that GEN sized 2 µM, and 40 nM, effectively promotes morphological alteration of mammospheres, reduces the ratio of subset of CD44+/CD24-/ESA+ cells and upregulates the expression of differentiated cell markers of mammospheres in co-culture system, but not in solo-culture condition. Besides, we demonstrate that the differentiation-inducing effect of GEN on mammospheres is associated with PI3K/Akt and MEK/ERK signaling pathways which are activated by amphiregulin released from ER+ cancer cells. These results indicate that GEN was able to induce the differentiation of breast cancer stem/progenitor cells through interaction with ER+ cancer cells by a paracrine mechanism.

Cancer stem cells are enriched in triple-negative breast cancer (TNBC) tumor tissues, which present strong capacities of proliferation and tumorigenicity. The present study detected the distribution of cancer stem cell markers cluster of differentiation (CD)44/CD24 and analyzed the clinical outcomes of different CD44/CD24 phenotypes in patients with TNBC. Multivariate Cox regression analyses were performed with regard to the prognostic value of cancer stem cell markers CD44/CD24, aldehyde dehydrogenase 1 and other baseline clinical characteristics, including tumor size, lymph node involved, adjuvant chemotherapy, Ki-67, breast cancer susceptibility gene 1, cellular tumor antigen p53, vimentin and basal-like status. The multivariate analyses showed that three of these factors, CD44/CD24 phenotype, basal-like status and number of lymph nodes involved, had an impact on overall survival. Furthermore, patients with CD44+/CD24− phenotype, basal-like tumors and ≥4 lymph nodes involved had a significantly worse prognosis. The expression of CD44 and CD24 was detected by double-staining immunohistochemistry, which can locate cancer stem cells individually. Overall, the present results indicated that CD44/CD24 status evaluated by double-staining immunohistochemistry constitutes an independent prognostic factor for TNBC.

The aim of this study was to establish a prognostic risk model for patients with triple negative breast cancer (TNBC). A total of 278 specimens of human TNBC tissues were investigated by immunohistochemistry for growth-arrest specific protein 6 expression, infiltrations of stromal natural killer cells and tumor-associated macrophages. According to their prognostic risk scores based on the model, patients were divided into three groups (score 0, 1-2, 3). Correlations of prognostic risk scores, clinicopathologic features and overall survival (OS) were analyzed. To study the clinical value of this stratification model in early disease recurrence or metastasis, 177 patients were screened out for further analysis. Based on disease free survival (DFS), 90 patients fell within the DFS ≤3 years group and 87 patients within the DFS ≥5 years group. We analyzed the differences in prognostic risk scores between the two groups. The prognostic risk scores were negatively related to tumor size, lymph node metastasis and P53 status (P < 0.001 for all). Patients with low prognostic risk scores had longer OS (P = 0.001). Using multivariate analysis, it was determined that TNM stage (HR = 0.432, 95% confidence interval [CI] = 0.281-0.665, P = 0.003), FOXP3 positive lymphocytes (HR = 1.712, 95% CI = 1.085-2.702, P = 0.021) and prognostic risk scores (HR = 1.340, 95% CI = 1.192-1.644, P = 0.005) were independent prognostic factors for OS. Compared with the DFS ≥5 years group, the DFS ≤3 years group patients had significantly higher prognostic risk scores (P < 0.001). In conclusion, the prognostic risk score of the model was a significant indicator of prognosis for patients with TNBC.

An HPLC/MS/MS method was developed for the simultaneous determination of the following benzimidazole anthelmintics and metabolites in plasma: flubendazole, albendazole, fenbendazole, mebendazole, thiabendazole, hydrolyzed flubendazole, albendazole sulfoxide, albendazole sulfone, albendazole aminosulfone, oxfendazole, fenbendazole sulfone, aminomebendazole, hydroxymebendazole, and 5-hydroxythiabendazole. The sample preparation process involved a pH-dependent extraction of the analytes. Chromatographic separation was performed on a C18 column with a mobile phase gradient starting with methanol–water (20 + 80, v/v) containing 0.1% formic acid. The overall average recoveries of the analytes based on a matrix-matched calibration ranged from 75.0 to 120.0%, with RSD values of

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) method was established for the determination of methaqualone, chloropromazine, promethazine, diazepam, nitrazepam, oxazepam, temazepam, midazolam, triazolam and zolpidem residues in pork and kidney. After enzymolysis, the samples were extracted by ethyl acetate and tert-butyl methyl ether, separately. The separation of the ten sedatives was performed on a Waters Acquity UPLC system with a BEH C18 column. The mobile phases were acetonitrile (containing 0.1% formic acid) and water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. The electrospray was operated in the positive ionization mode and the ten sedatives were identified by multiple reaction monitoring (MRM) mode. The method of matrix-matched standard solution was adopted as the quantitative method. The calibration curves showed good linearity within the concentrations of 2 - 100 microg/L with the correlation coefficients r > 0. 998. The limits of detection of the ten sedatives were 0.5 microg/kg, and the limit of quantification was 1 microg/kg. The recoveries of the ten sedatives were 64.5% - 111.4% at the spiked levels of 2, 5 and 10 microg/kg. The relative standard deviations of intra- and inter-day coefficients of variation were both less than 15%. This method is simple, sensitive and accurate in the determination of sedative residues.

Early detection and treatment of human colorectal cancers remain a challenge. Identification of new potential markers may help in the diagnosis of colorectal cancer. By comparative two-dimensional gel electrophoresis using extracts from colorectal tumor and adjacent normal tissues, we identified a calcium-binding protein, S100A11, which was highly expressed in colorectal cancer compared with adjacent normal tissues. We expanded our study in 89 clinical colorectal tumor samples to validate this finding and correlates S100A11 expression in human colorectal cancer tissues with various stages of the tumor by Western blotting and immunohistochemical staining. We identified a calcium-binding protein, S100A11, which was highly expressed in colorectal cancer compared with adjacent normal tissues. S100A protein was expressed predominantly in the cytoplasm of normal tissue; however, it was expressed in both the nuclei and cytoplasm of colorectal cancer. S100A11 level in colorectal cancer tissue was increased following stage progression of the disease. These findings suggest S100A11 could be helpful in the pathological study of colorectal cancer, especially for the classification of different stages in colorectal cancer.

The nuclear factor of the activated T cell (NFAT) family was primarily recognized for its central role in T lymphocyte activation. Recent evidence showed that NFAT isoforms participate in the regulation of genes related to cell proliferation and differentiation in epithelial malignancies. Here, we investigated the expression and activation of the calcineurin/NFAT transcription pathway and its role in hepatocellular carcinoma (HCC) proliferation. Expression of NFATc1 and calcineurin proteins was examined by immunohistochemical analyses in 76 human HCC samples. The cellular NFAT activation and distribution in HepG2 cells were analyzed by immunofluorescence and western blot analyses. After NFATc1 expression was knocked down by NFATc1-specific siRNA, we analyzed its implications in cell cycle progression and growth by MTT and flow cytometry. The impact of calcineurin/NFAT signaling on protein expression of c-myc and cox-2 were performed by western blot analyses. NFATc1 is significantly overexpressed in HCC. The regulation of calcineurin activity by ionomycin or cyclosporin A caused rapid nuclear import or export of NFATc1 in HepG2 cells. NFATc1 knock-down led to a significant reduction in proliferation rates and cell cycle arrest at G1 phase. The expression of c-myc and cox-2 was decreased in the NFATc1 knock-down HepG2 cells. Ionomycin increased c-myc and cox-2 expression in HepG2 cells, but not in siNFATc1 HepG2 cells. The calcineurin/NFATc1 signal is overexpressed and active in HCC. It may enhance the proliferative potential of HepG2 cells through transcriptional activation of the c-myc and cox-2 oncogenes.

e11095 Background: The nuclear factors of activated T cells (NFATs) have been previously studied in the immune system as transcription factors during the activation of T lymphocyte. In the recent years, emerging evidences have showed that they may play an important role in the development and progression of human cancer. In this study we investigate NFATc1 protein expression and its relationship with clinical characteristics in breast cancer. Methods: NFATc1 protein expression was determined by immunohistochemical analysis in 86 surgically resected breast cancer samples between Jul 2004 and Jun 2006. The relationship between NFATc1 expression and clinical characteristics of breast cancer was analyzed by multivariate logistic regression and survival analyses. Results: NFATc1 protein was significantly overexpressed in breast cancer tumor cells compared with the matched normal tissues. High expression of NFATc1 proteins was associated with high tumor grade, lymph nodes involved and shorter disease free survival (P0.05). Conclusions: This study suggested that high NFATc1 expression was present in breast cancer and it might be involved in the process of cancer development and related to poor prognosis of breast cancer.