Identification of differentially expressed molecular functions associated with breast cancer using Gibbs sampling.

The aim of the present study was to identify differentially expressed molecular functions (DEMFs) for breast cancer using the Gibbs sampling approach. Molecular functions (MFs) were obtained on the basis of the Bayesian Approach for Geneset Selection package. Subsequently, MFs were converted into Markov chains (MCs) prior to calculating their probabilities, utilizing the MC Monte Carlo algorithm. DEMFs were identified with probabilities ≥0.8 and the gene compositions were studied. Finally, a co-expression network was constructed via the empirical Bayes method and a pathway enrichment analysis of genes in DEMFs was performed. A total of 396 MFs were identified and all transformed to MCs. With the threshold, 2 DEMFs (structural molecule activity and protein heterodimerization activity) were obtained. The DEMFs were comprised of 297 genes, 259 of which were mapped to the co-expression network. These 297 genes were identified to be enriched in 10 pathways, and ribosome was the most significant pathway. The results of the present study revealed 2 DEMFs (structural molecule activity and protein heterodimerization activity) which may be associated with the pathological molecular mechanisms underlying breast cancer, based on Gibbs sampling.

Effect of shRNA-Mediated Gene Silencing of Bmi-1 Expression on Chemosensitivity of CD44+ Nasopharyngeal Carcinoma Cancer Stem-Like Cells.

In this study, we investigate the effect of short hairpin RNA-mediated gene silencing of Bmi-1 expression on chemosensitivity of CD44(+) nasopharyngeal carcinoma cancer stem-like cells. The sequence-specific short hairpin RNA lentivirus targeting at human Bmi-1 was synthesized and used to infect CD44(+) nasopharyngeal cells that were sorted by flow cytometry. We also employed flow cytometry to detect transfection efficiency. Real-time polymerase chain reaction was used to detect Bmi-1 and its downstream repressor genes p16(INK4a) and p14(ARF) messenger RNA, while each protein expression level of Bmi-1, p16(INK4a), p14(ARF), and p53 was confirmed by Western blotting protocol. Tumor spheroid assay was used to evaluate the self-renewal capacity. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and colony formation assay were applied to detect proliferation capacity and colony-forming capacity under different concentrations of chemotherapeutic drugs 5-fluorouracil or cisplatin. Transwell cell migration and invasion assay were employed to observe migration and invasion capacity after cells were exposed to cisplatin for 24 hours. The constructed short hairpin RNA lentivirus targeting Bmi-1 gene successfully infected into the CD44(+) nasopharyngeal carcinoma cells and effectively inhibited the Bmi-1 messenger RNA and protein expression level, while the expression level of Bim-1 target genes, p16(INK4a), p14(ARF), and p53 was significantly increased (P < .05). Notably, the proliferation, colony formation, migration, and invasion capabilities of the sequence-specific short hairpin RNA lentivirus-infected CD44(+) nasopharyngeal carcinoma cells reduced significantly under chemotherapeutic treatments (P < .05). Our results indicated that Bmi-1 may play an important role in the chemosensitivity of CD44(+) nasopharyngeal carcinoma cancer stem-like cells. Bmi-1 may be a potential new target for the treatment of nasopharyngeal carcinoma displaying chemotherapy resistance.

[A Modified Procedure to Isolate Synchronous Cells from Yeasts with Continuous Percoll Density Gradient and Their Raman Discrimination].

A modified procedure of Percoll density gradient centrifugation was developed to isolate and fractionate synchronous cells from stationary phase (sp) cultures of different yeast strains, as well as Raman spectra discrimination of single yeast cells was reported. About 1.75 mL Percoll solution in 2 mL polypropylene centrifugal tube was centrifuged at 19,320 g, 20 °C with an angle rotor for 15 min to form continuous densities gradient (1.00~1.31 g · mL(-1)), approximately 100 µL sample was overlaid onto the preformed continuous density gradient carefully, subsequently, centrifuged at 400 g for 60 min in a tabletop centrifuge equipped with a angle rotor at 25 °C. Yeast samples could be observed that the suspensions were separated into two cell fractions obviously. Both fractions of different yeast strains were respectively determined by differential interference contrast (DIC), phase contrast microscope and synchronous culture to distinguish their morphological and growth trait. The results showed that the lower fraction cells were unbudded, mostly unicellular, highly refractive, homogeneous and uniform in size, and represented growth characteristic synchronously; Their protoplasm had relatively high density, and contained significant concentrations of glycogen; all of which were accordant with description of quiescent yeast cells and G0 cells in previously published paper. It was shown that lower fraction was quiescent cells, synchronous G0 cells as well. A Raman tweezers setup was used to investigate the differences between two fractions, G0 cells and non G0 cells, at a single cell level. The result showed that both G0 cells and the non G0 cells had the same characteristic peaks corresponding biological macromolecules including proteins, carbohydrates and nucleic acids, but all characteristic peak intensities of G0 cells were higher than that of non G0 cells, implied that the macromolecular substance content of G0 cells was more higher. Principal component analysis (PCA) was performed between G0 cells and non G0 cells, the results showed that the chemical composition content among the synchronization G0 cells has less difference, and G0 cells were homogeneous but non G0 cells were heterogeneous, indicating single cell optical tweezers Raman spectroscopy could identify the synchronous and asynchronous cells. The modified method is feasible, economical and efficient highly. G0 synchronous cells of most yeast strains could be isolated by a modification of Percoll density gradient centrifugation.

ShRNA targeting Bmi-1 sensitizes CD44⁺ nasopharyngeal cancer stem-like cells to radiotherapy.

Accumulating evidence indicates that cancer stem cells (CSCs) are involved in resistance to radiation therapy (RT). Bmi-1, a member of the Polycomb family of transcriptional repressors, is essential for maintaining the self-renewal abilities of stem cells and overexpression of Bmi-1 correlates with cancer therapy failure. Our previous study identified that the CD44+ nasopharyngeal cancer (NPC) cells may be assumed as one of markers of nasopharyngeal carcinoma cancer stem cell-like cells (CSC-LCs) and Bmi-1 is overexpressed in CD44+ NPC. In the present study, we used RNA interference technology to knock down the expression of Bmi-1 in CD44+ NPC cells, and then measured the radiation response by clonogenic cell survival assay. DNA repair was monitored by γH2AX foci formation. Bmi-1 downstream relative gene and protein expression of p16, p14, p53 were assessed by western blotting and real-time PCR. Cell cycle and apoptosis were detected by flow cytometry assays. We found that Bmi-1 knockdown prolonged G1 and enhanced the radiation-induced G2/M arrest, inhibited DNA damage repair, elevated protein p16, p14 and p53 expression, leading to increased apoptosis in the radiated CD44+ cells. These data suggest that Bmi-1 downregulation increases the radiosensitivity to CD44+ NPC CSC-LCs. Bmi-1 is a potential target for increasing the sensitivity of NPC CSCs to radiotherapy.

[Raman spectra of endospores of Bacillus subtilis by alkali stress].

To research the lethal mechanism of spores stressed by alkali, laser tweezers Raman spectroscopy (LTRS) combined with principal components analysis (PCA) was used to study the physiological process of single spore with alkali stress. The results showed that both spores and germinated spores had tolerance with alkali in a certain range, but the ability of spores was obviously lower than that of spores due to the release of their Ca2+ -DPA which plays a key role in spores resistance as well as spores resistance to many stresses; A small amount of Ca2+ -DPA of spores was observed to release after alkali stress, however, the behavior of release was different with the normal Ca2+ -DPA release behavior induced by L-alanine; The data before and after alkali stress of the spores and g. spores with PCA reflected that alkali mainly injured the membrane of spores, and alkali could be easily enter into the inner structure of spores to damage the structure of protein backbone and injure the nucleic acid of spores. We show that the alkali could result in the small amount of Ca2+ -DPA released by destroying the member channel of spores.

Identification of cancer stem-like CD44+ cells in human nasopharyngeal carcinoma cell line.

BACKGROUND AND AIMS: Recent studies suggest that cancer stem cells (CSC) may be responsible for tumorigenesis and contribute to some individuals' resistance to cancer therapy. Although research is rapidly advancing in this field, to our knowledge there are few published reports about the CSC in human nasopharyngeal carcinoma (NPC). We undertook this study to separate, expand, and explore the biological features of CD44+ stem-like cancer cells from the human NPC SUNE-1 5-8F cell line. METHODS: Immunocytochemistry and flow cytometry were used to detect the expression of CD44 in SUNE-1 5-8F. Fluorescence-activated cell sorting was applied to purify CD44+ cells. MTT assay or clone formation assay was used to detect the differences of CD44+ and CD44- cells in proliferation, differentiation, radiosensitivity and chemosensitivity in vitro. The expression of stem cell markers Oct-4 and Bmi-1 was examined by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: CD44 was positively expressed in ∼52.5% of NPC SUNE-1 5-8F cell line. Regardless of serum-free medium and serum medium culture conditions, freshly sorted CD44+ cells showed stronger proliferative capacity than CD44- and unsorted cells. The expression levels of Bmi-1 and Oct-4 mRNA in CD44+ cells were significantly higher than CD44- cells. After 2 Gy radiation, the average clone formation efficiency for CD44+ and CD44- cells was 22.17 ± 6.65% and 11.50 ± 5.00%, respectively (p <0.05). After cisplatin and docetaxel treatment with the same drug concentration, CD44+ cells showed a higher survival rate compared with CD44- cells. CONCLUSIONS: CD44+ cells have the biological characteristics of tumor stem cell and may be assumed as one of the markers of NPC tumor stem cells.

Clinical effect of erlotinib as first-line treatment for Asian elderly patients with advanced non-small-cell lung cancer.

PURPOSE: To evaluate the efficacy and toxicities of erlotinib as first-line treatment for Asian elderly patients with advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Untreated patients with advanced NSCLC were included in this study; erlotinib was orally administered at a dose of 150 mg daily until disease progression or intolerable toxicity or for other reasons. RESULTS: A total of 35 patients were enrolled. Patient characteristics were as follows: mean age 75.6 years (ranged 70-81 years), 24 (68.6%) male, 16 (45.7%) former or current smokers, 13 (37.1%) adenocarcinoma, 18 (51.4%) squamous cell carcinoma and 4 (11.4%) bronchioloalveolar carcinoma. Out of 35 patients, 1 CR, 16 PR and 10 SD, resulting in an overall response rate (CR + PR) of 48.6% and disease control rate (DCR = CR + PR + SD) of 77.1%. The median TTP was 6.4 months, and the median OS was 12.7 months. The CBR was 80%, and the 1-year survival rate was 48.6%. The most common adverse event (AE) was mild skin rash and diarrhea (CTC AE 1/2). Among them, the female never smokers with a non-squamous cell carcinoma histology was superior to the male smokers with a squamous cell carcinoma in disease control rate, with significant differences (P < 0.05). CONCLUSION: The results suggest that erlotinib monotherapy is an effective and well-tolerated treatment option for Asian elderly patients with advanced NSCLC.

Erlotinib in advanced non-small-cell lung cancer after gefitinib failure.

PURPOSE: To evaluate the efficacy and safety of erlotinib in advanced non-small-cell lung cancer after failure of gefitinib treatment. PATIENTS AND METHODS: Patients with advanced or metastatic NSCLC, who had progressed after gefitinib treatment, were included in this study; patients received erlotinib 150 mg/day until disease progression or intolerable toxicity. RESULTS: Twenty-one patients were included in this study. Among them, 14 (66.7%) were male and 7 (33.3%) were female; median age was 63 years; 10 (47.6%) patients were smokers; 9 (42.9%)patients had squamous cell carcinoma subtype; 8 (38.1%) patients had adenocarcinoma subtype and 4 (19%) patients had the other NSCLC subtype. Out of 21 patients, 2 (9.5%) had PR and 4 (19.0%) had SD, giving an overall response rate of 9.5% and a disease control rate of 28.5%. The median TTP were 55 days, the median OS were 135 days. Two patients with PR to erlotinib treatment were female never smokers with adenocarcinoma histology and both had partial response to prior gefitinib treatment. Three of four patients with a SD to erlotinib treatment also had SD from prior gefitinib therapy. Smoking history, histology and response to erlotinib were significantly correlated with survival. The most common toxic effects were skin rash. CONCLUSIONS: Erlotinib may be an option for a more highly selected subset of patients, especially those who had already benefited from prior gefitinib treatment.