Heregulin-ß1-induced GPR30 upregulation promotes the migration and invasion potential of SkBr3 breast cancer cells via ErbB2/ErbB3-MAPK/ERK pathway.

Estrogen receptor (ER)-negative breast cancer cells are probably more aggressive with larger metastatic potential than ER-positive cells. Loss of ER in recurrent breast cancer is associated with poor response to endocrine therapy. G protein-coupled receptor 30 (GPR30) is expressed in half of ER-negative breast cancers. Tumor cell-derived heregulin-ß1 (HRG-ß1) is also found mainly in ER-negative cancer. In SkBr3 breast cancer cells that lack ER but express GPR30, HRG-ß1 upregulates mRNA and protein levels of GPR30 by promoting ErbB2-ErbB3 heterodimerization and activating the downstream MAPK-ERK signaling pathway. Moreover, GPR30 boosts HRG-ß1-induced migration and invasion of SkBr3 cells after combinative treatment with E2, 4-hydroxy-tamoxifen or the specific GPR30 agonist G-1, which are blocked by the specific GPR30 antagonist G-15 or the transfection with the small interfering RNA for GPR30. The ErbB2 inhibitor AG825 and the MEK1/2 inhibitor U0126 also partly inhibit the enhanced migration and invasion. Therefore, HRG-ß1-induced migration and invasion partly depend on the upregulation of GPR30 expression through activation of the ErbB2-ERK pathway in SkBr3 cells. The results of this study indicate that the crosstalk between GPR30 and HRGs signaling is important for endocrine therapy resistance and may provide a new therapeutic way to treat breast cancer.

Pathological significance of epidermal growth factor receptor expression and amplification in human gliomas.

AIMS: To investigate epidermal growth factor receptor (EGFR) expression and amplification in gliomas and to assess their association with survival. METHODS AND RESULTS: Immunohistochemistry and fluorescence in-situ hybridization were performed to analyse EGFR status in 158 cases of primary glioma. Kaplan-Meier survival and Cox regression analyses were performed to analyse the prognosis of patients. Overexpression of EGFR and expression of EGFR variant III (EGFRvIII) were found in 102 cases (64.6%) and 47 cases (29.7%), respectively. Overexpression of EGFR was significantly correlated with World Health Organization (WHO) grade and Karnofsky performance score (KPS) (both P < 0.05). Expression of EGFRvIII was significantly correlated with WHO grade, gender, age, and KPS (all P < 0.05). EGFR amplification was found in 46 cases (29.1%), and was significantly correlated with WHO grade, age, KPS and EGFR overexpression (all P < 0.05). Cox multifactor analysis showed that EGFR amplification was an independent unfavourable prognostic factor for human gliomas at all ages, and EGFRvIII was an independent prognostic factor in patients older than 60 years. CONCLUSION: EGFR amplification and EGFRvIII expression were associated with an unfavourable prognosis for patients of all ages, and for those older than 60 years, respectively. The differing significance of EGFR status in young and old glioma patients and its impact on prognosis needs further study.

Metal-organic frameworks for reverse-phase high-performance liquid chromatography.

Metal-organic framework MIL-53(Al) is explored for reverse-phase high-performance liquid chromatographic separation of a wide range of analytes from non-polar to polar, and acidic to basic solutes with high resolution, good selectivity, stability and reproducibility.

High-performance liquid chromatographic separation of position isomers using metal-organic framework MIL-53(Al) as the stationary phase.

Metal-organic framework MIL-53(Al) was explored as the stationary phase for high-performance liquid chromatographic separation of position isomers using a binary and/or polar mobile phase. Baseline separations of xylene, dichlorobenzene, chlorotoluene and nitrophenol isomers were achieved on the slurry-packed MIL-53(Al) column with high resolution and good precision. The effects of mobile phase composition, injected sample mass and temperature were investigated. The separation of xylene, dichlorobenzene, chlorotoluene and nitrophenol isomers on MIL-53(Al) were controlled by entropy change.

A strong inorganic acid-initiated methacrylate polymerization strategy for room temperature preparation of monolithic columns for capillary electrochromatography.

A facile strong inorganic acid-initiated methacrylate polymerization strategy was developed for fabricating monolithic columns at room temperature. The prepared monoliths were characterized by FTIR spectrometry, mercury intrusion porosimeter and SEM, while their performance was evaluated by CEC for the separation of various types of compounds including alkyl benzenes, polycyclic aromatic hydrocarbons, nonsteroidal anti-inflammatory drugs, anilines, and nitrophenol isomers. The column-to-column and batch-to-batch reproducibility for the prepared monoliths in terms of the RSD of EOF flow velocity, retention factor, and the minimum plate height of naphthalene ranged from 3.4 to 12.4%. The fabricated monoliths gave excellent performance for the separation of the test neutral compounds with the theoretical plates of 170,000-232,000 plates per meter for thiourea, and 77,400-112,300 plates per meter for naphthalene. The proposed strong inorganic acid-initiated methacrylate polymerization strategy is a promising alternative for fabricating organic polymer-based monoliths.

Exploration of coordination polymer as sorbent for flow injection solid-phase extraction on-line coupled with high-performance liquid chromatography for determination of polycyclic aromatic hydrocarbons in environmental materials.

The copper(II) isonicotinate (Cu(4-C5H4N-COO)2(H2O)4) coordination polymer was prepared, characterized and explored as sorbent for flow injection solid-phase extraction on-line coupled with high-performance liquid chromatography (HPLC) for determination of trace polycyclic aromatic hydrocarbons (PAHs) in environmental matrices. Naphthalene, phenanthrene, anthracene, fluoranthene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene and benzo(ghi)perylene with various shape, size and hydrophobicity were used as model analytes. The porosity of the coordination polymer allows these guest PAHs molecules to diffuse into the buck structure, and the shape and size of the pores lead to shape- and size-selectivity over the guests. The precolumn packed with the coordination polymer was shown to be promising for solid-phase extraction of PAHs in environmental samples with subsequent HPLC separation and UV detection. With extraction of 50 ml of sample solution, the enhancement factors for the PAHs studied ranged from 200 to 2337, depending on the shape, size and hydrophobic property of the PAHs. The detection limits (S/N = 3) of 2-14 ng l(-1) and the sample throughput of 3 samples h(-1) were obtained. The developed method was applied to the determination of trace PAHs in a certified reference material (coal fly ash) and local water samples.

On-line coupling of flow injection displacement sorption preconcentration to high-performance liquid chromatography for speciation analysis of mercury in seafood.

A simple and cost-effective method for speciation analysis of trace mercury in seafood was developed by on-line coupling flow injection microcolumn displacement sorption preconcentration to high-performance liquid chromatography (HPLC) with UV detection. The methodology involved the presorption of the Cu-PDC (pyrrolidine dithiocarbamate) chelate onto a microcolumn packed with a cigarette filter sorbent, simultaneous preconcentration of Hg(II), methylmercury (MeHg), ethylmercury (EtHg), and phenylmercury (PhHg) onto the microcolumn through a displacement reaction with the presorbed Cu-PDC, and their subsequent elution from the microcolumn for on-line HPLC separation. Interferences from heavy metal ions with lower stability of their PDC chelates relative to Cu-PDC were minimized without the need of any masking agents. With the consumption of 4.0 ml of sample solution, the enrichment factors were about 80. The detection limits were 10-25 ng g(-1) (as Hg) in fresh tissue. Precision (R.S.D. (%), n = 5) ranged from 2 to 3% at the 500 microg l(-1) (as Hg) level. The developed technique was validated by analyzing a certified reference material (DORM-2, dogfish-muscle), and was shown to be useful for mercury speciation in real seafood samples.

Cloud point extraction for high-performance liquid chromatographic speciation of Cr(III) and Cr(VI) in aqueous solutions.

Cloud point extraction (CPE) was applied as a preconcentration step for HPLC speciation of chromium in aqueous solutions. Simultaneous preconcentration of Cr(III) and Cr(VI) in aqueous solutions was achieved by CPE with diethyldithiocarbamate (DDTC) as the chelating agent and Triton X-114 as the extractant. Baseline separation of the DDTC chelates of Cr(III) and Cr(VI) was realized on a RP-C18 column with the use of a mixture of methanol-water-acetonitrile (65:21:14, v/v) buffered with 0.05 M NaAc-HAc solution (pH 3.6) as the mobile phase at a flow rate of 1.0 ml min(-1). The precision (R.S.D.) for eight replicate injections of a mixture of 100 microg l(-1) of Cr(III) and Cr(VI) were 0.6 and 0.5% for the retention time, 4.1 and 4.6% for the peak area measurement, respectively. The concentration factor, which is defined as the concentration ratio of the analyte in the final diluted surfactant-rich extract ready for HPLC separation and in the initial solution, was 65 for Cr(III) and 19 for Cr(VI). The linear concentration range was from 50 to 1000 microg l(-1) for Cr(III) and 50-2000 microg l(-1) for Cr(VI). The detection limits of Cr(III) and Cr(VI) were 3.4 and 5.2 microg l(-1), respectively. The developed method was applied to the speciation of Cr(III) and Cr(VI) in snow water, river water, seawater and wastewater samples.

Regulation on RhoA in vascular smooth muscle cells under inflammatory stimulation proposes a novel mechanism mediating the multiple-beneficial action of acetylsalicylic acid.

Recent studies have revealed the additional beneficial effects of acetylsalicylic acid (aspirin) in the medication of cardiovascular diseases. The small GTPase RhoA as an important signaling factor is implicated in a wide range of cell functions. This study aimed to investigate the regulatory effect of acetylsalicylic acid on RhoA in vascular smooth muscle cells (VSMCs). We found that aspirin at 300 µM suppressed VSMCs proliferation stimulated by LPS, and this inhibitory effect was partially mediated by inhibiting the iNOS/NO pathway. RhoA overexpression was downregulated by aspirin (both 30 and 300 µM) because of enhanced degradation of RhoA protein. The effect of LPS on increasing active RhoA level was significantly attenuated by aspirin (300 µM), which exerted no effect on RhoA translocation. The promoted RhoA phosphorylation under LPS stimulation, coupled with RhoA protein expression, was greatly decreased by aspirin treatment. No effect of aspirin was found on the expression, activation, and phosphorylation of RhoA in VSMCs devoid of inflammatory stimulation. Our investigation indicates that the regulation of RhoA by aspirin in VSMCs under inflammatory stimulus could be a novel mechanism via which aspirin, apart from the COX-dependent action, exerted the multiple beneficial effects.

Regulation of HRG-ß1-induced proliferation, migration and invasion of MCF-7 cells by upregulation of GPR30 expression.

The cooperation and communication between different cell signaling transduction pathways are considered critical in the development of various types of cancer as well as drug resistance. There is evidence of crosstalk between the G protein-coupled receptor 30 (GPR30), the newly discovered estrogen receptor (ER), and the ErbB family. Heregulin (HRG)-ß1, the ligand for ErbB3 and ErbB4, upregulates GPR30 expression in MCF-7, T-47D and BT-474 breast cancer cell lines that express ERα. In the present study, recombinant human HRG-ß1 was used to investigate the upregulation of GPR30 expression by HRGs in MCF-7 breast cancer cells which were ERα-positive. In MCF-7 cells, the ErbB2 inhibitor, AG825, the MAPK inhibitor, PD98059, and the MEK1/2 inhibitor, U0126, blocked the HRG-ß1-induced GPR30 expression. 17-ß-estradiol (E2) boosted the HRG-ß1-induced proliferation, migration and invasion of MCF-7 cells. Similar to E2, the specific GPR30 agonist, G-1, promoted HRG-ß1-induced migration and invasion, but inhibited growth. Using the specific GPR30 antagonist, G-15, or the small interfering RNA for GPR30, the functions of GPR30 after treatment with HRG-ß1 were further investegated. The results from our study indicate that the interruption between GPR30 signaling and the ErbB family system may serve as a promising therapeutic strategy for breast cancer.

Proteomic analysis of primary colon cancer-associated fibroblasts using the SELDI-ProteinChip platform.

OBJECTIVE: Cancer-associated fibroblasts (CAFs) are one of the hallmarks of the cancer microenvironment. Recent evidence has indicated that CAFs are more competent in enhancing cancer cell growth and migration than normal fibroblasts. However, the unique protein expression of CAFs has not been fully elucidated. This study aims to investigate the characterizations of colon CAFs by comparing the differential protein expression between CAFs and normal fibroblasts. METHODS: Primary fibroblasts were isolated from surgical specimen of human colon cancer and matched normal colonic tissue. Purity of the cell population was verified through immunostain analysis. Total cell lysates and conditioned media from each group of cells were extracted, and protein expression analysis was conducted using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip platform. RESULTS: Most primary cells showed typical fibroblast-like features after two weeks. Increased proportion of α-smooth muscle actin-positive myofibroblasts was detected within the CAFs in four of the six pairs of primary cells. Fibroblast activation protein was weakly expressed in most cells without differences. Using SELDI-TOF-MS ProteinChip platform, four protein peaks mass over charge ratio (m/z) 1142, 3011, 4035, and 4945 were detected in the total cell lysates, and two protein peaks m/z 1368 and 1389 were detected in the conditioned media. The potential candidate proteins found in the Swiss-Prot database include morphogenetic neuropeptides, FMRFamide-related peptides, insulin-like growth factor II, thymosin ß-4-like protein 3, and tight junction-associated protein 1. CONCLUSIONS: Using the SELDI-ProteinChip platform, differential protein expressions were identified in colon CAFs compared with normal colonic stromal fibroblasts. The complex proteomic alternations in colon CAFs may play important roles related to the colon cancer microenvironment.

Evaluation of expanded graphite as on-line solid-phase extraction sorbent for high performance liquid chromatographic determination of trace levels of DDTs in water samples.

Dichlorodiphenyltrichloroethane (DDT) and its metabolites are a typical kind of persistent organic pollutants (POPs). Development of a simple, cost-effective and sensitive methodology to monitor DDTs concentrations in water environment is of particular significance for understanding the fate and behavior of these pollutants. In this paper, a method on the basis of solid-phase extraction (SPE) using expanded graphite (EG) as sorbent coupled on-line with high performance liquid chromatography (HPLC) was developed for the determination of trace levels of p,p'-DDD (2,2-bis(4-chlorophenyl)-1,1-dichloroethane), p,p'-DDT, o,p'-DDT and p,p'-DDE (2,2-bis(4-chlorophenyl)-1,1-dichloroethene) in water. The analytes in water were preconcentrated onto the SPE column packed with expanded graphite, and subsequently eluted with methanol-water (90:10) mixed solvent. HPLC with a photodiode array detector was used for their separation and detection. The developed on-line solid-phase extraction protocol for HPLC permits the current HPLC separation and the next preconcentration proceeded in parallel, and thus allows one determination within 8min. The precision (R.S.D.) for 10 replicate injections of a mixture of 1mugl(-1) of each analyte was 3.2-6.2% for the peak area measurement. The detection limits (S/N=3) for preconcentrating 50ml of sample solution ranged from 10 to 25ngl(-1) at a sample throughput of 7.5samplesh(-1). The enhancement factors were about 700. The method was applied to the determination of trace p,p'-DDD, p,p'-DDT, o,p'-DDT and p,p'-DDE in local lake, river and tap water samples.

An amobarbital molecularly imprinted microsphere for selective solid-phase extraction of phenobarbital from human urine and medicines and their determination by high-performance liquid chromatography.

A 40-60 microm amobarbital molecularly imprinted microsphere, used as a solid-phase selective sorbent for extracting phenobarbital from human urine and medicines, was prepared by a suspension polymerization method. A series of binding studies was performed in order to find optimal loading, washing and eluting conditions for solid-phase extraction. Under optimal conditions, good recoveries of phenobarbital in samples were obtained. Normally, molecularly imprinted polymers, prepared in bulk, require laborious work. Significant losses occur during the procedure of grinding and washing. In this work all molecularly imprinted polymers made into microsphere could be utilized, and the cost of the template was reduced too (the price of phenobarbital is twice that of amobarbital). As the phenobarbital to be extracted was different from the template molecule amobarbital, the interference caused by template leaking could be avoided in the assay.