An Anti-EGFR/anti- HER2 Bispecific Antibody with Enhanced Antitumor Activity Against Acquired Gefitinib-Resistant NSCLC Cells.

BACKGROUND: Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is a recurrent phenomenon during clinical therapy of non-small-cell lung cancer (NSCLC). Studies have shown that HER2 is a key factor contributing to drug resistance in a variety of cancers. Furthermore, we have observed that HER2 is overexpressed in PC-9 NSCLC cells with acquired gefitinib-resistance (PC-9/GR) as compared to that in PC-9 cells. OBJECTIVE: We hypothesized that blocking both EGFR and HER2 may serve as a potential strategy for the treatment of NSCLC with acquired gefitinib-resistance. METHODS: To target both EGFR and HER2 simultaneously, we developed a bispecific antibody HECrossMAb, which was derived from a humanized Cetuximab and Trastuzumab. The binding affinity of HECrossMAb for EGFR and HER2 was measured using an enzyme-linked immunosorbent assay. The MTT assay was used to determine the effect of HECrossMAb on the proliferation of PC-9 and PC-9/GR cells in vitro. Finally, the effect of HECrossMAb on PI3K/AKT signaling and associated transcription factors was measured using western blot analysis. RESULTS: Our results showed that HECrossMAb exerts enhanced cytotoxicity in both PC-9 and PC-9/GR cells by inhibiting the activation of PI3K/AKT signaling and expression of relevant transcription factors such as AEG-1, c-Myc, and c-Fos. CONCLUSION: Our results suggest that HECrossMAb may function as a potential therapeutic agent for treating NSCLC overexpressing EGFR and HER2.

Nicotinic modulation of Ca2+ oscillations in rat cortical neurons in vitro.

The roles of nicotine on Ca(2+) oscillations [intracellular Ca(2+) ([Ca(2+)]i) oscillation] in rat primary cultured cortical neurons were studied. The spontaneous [Ca(2+)]i oscillations (SCO) were recorded in a portion of the neurons (65%) cultured for 7-10 days in vitro. Application of nicotine enhanced [Ca(2+)]i oscillation frequency and amplitude, which were reduced by the selective α4ß2-nicotinic acetylcholine receptors (nAChRs) antagonist dihydro-ß-erythroidine (DHßE) hydrobromide, and the selective α7-nAChRs antagonist methyllycaconitine citrate (MLA, 20 nM). DHßE reduced SCO frequency and prevented the nicotinic increase in the frequency. DHßE somewhat enhanced SCO amplitude and prevented nicotinic increase in the amplitude. MLA (20 nM) itself reduced SCO frequency without affecting the amplitude but blocked nicotinic increase in [Ca(2+)]i oscillation frequency and amplitude. Furthermore, coadministration of both α4ß2- and α7-nAChRs antagonists completely prevented nicotinic increment in [Ca(2+)]i oscillation frequency and amplitude. Thus, our results indicate that both α4ß2- and α7-nAChRs mediated nicotine-induced [Ca(2+)]i oscillations, and two nAChR subtypes differentially regulated SCO.

Up-regulation of CYLD enhances Listeria monocytogenes induced apoptosis in THP-1 cells.

Listeria monocytogenes (Lm), a facultative anaerobic gram-positive bacterium, causes listeriosis. Immune cell apoptosis is considered to be one pathogenic factor for listeriosis. As a deubiquitinase, CYLD is an important regulator both in innate immune response and apoptosis by negatively modulating NF-κB pathway. However the role of CYLD in Lm induced apoptosis remains unclear. Here we found that CYLD is significantly up-regulated in macrophages upon its infection. There is a moderate decrease in Lm proliferation and apoptotic cells in siRNA-induced CYLD knockdown THP-1 cells. Thereby CYLD may be involved in cell apoptosis mediated by Lm infection and its proliferation.

Directly suspended droplet microextraction for the analysis of fungicides.

In the proposed method, a free microdroplet of extraction solvent was placed on the top center of the aqueous sample while the solution was agitated with a stirring bar on the bottom of the sample vial. After extraction, the sample vial was cooled in an ice bath for 2 min. The solidified droplet was transferred into a conical vial and melted quickly at room temperature. Based on preliminary studies, 1-dodecanol was selected as the extraction solvent. Effective parameters such as the type and volume of organic solvent, stirring speed, extraction time, sample temperature and salt effect were optimized. Under the optimized conditions, the enrichment factors of fungicides varied between 103 and 175. Based on a signal-to-noise ratio of 3, the limits of detection in this method ranged between 1.01 and 1.49 µg L(-1). Linearity was obtained in the concentration range of 5-2000 µg L(-1) yielding correlation coefficients (r) >0.9985. Good reproducibility and recovery were also obtained.

Development of an improved single-drop microextraction method and its application for the analysis of carbamate and organophosphorus pesticides in water samples.

An improved single-drop microextraction (SDME) method combined with high performance liquid chromatography has been developed for the detection of trace carbamate and organophosphorus pesticides in water samples. The most fascinating feature of the proposed method is the use of an oval-shaped polychloroprene rubber (PCR) tube to load the extraction solvent, which efficiently loads more solvent and improves the stability of extraction microdrop. Furthermore, this device provides a larger contact surface between the extraction solvent and the inner surface of the oval-shaped PCR tube than that between the extraction solvent and the tip of a microsyringe needle in the conventional SDME. It thereby avoids the problem of the drop floating upwards or dislodging from the tip of the microsyringe needle as observed in the traditional SDME. This method is significant for the great improvement it can offer in extraction efficiency. A series of extraction parameters were investigated systematically using carbamate and organophosphorus as the model analytes. Under the optimal conditions, the enrichment factors for analysis were between 117 and 177, and the limits of detection were ≦0.63 µg L(-1) (S/N = 3). The repeatability study was carried out by extracting the spiked water samples. Here the relative standard deviations varied between 4.0 and 5.8% (n = 5). Additionally, the proposed method was successfully applied to the determination of pesticides in real water samples, and good recoveries were obtained from 79% to 112%. The proposed method was demonstrated to hold advantages of low cost, simplicity of operation, and successful application to in real water samples.

In-syringe demulsified dispersive liquid-liquid microextraction and high performance liquid chromatography-mass spectrometry for the determination of trace fungicides in environmental water samples.

An in-syringe demulsified dispersive liquid-liquid microextraction (ISD-DLLME) technique was developed using low-density extraction solvents for the highly sensitive determination of the three trace fungicides (azoxystrobin, diethofencarb and pyrimethanil) in water samples by high performance liquid chromatography-mass spectrometry chromatography-diode array detector/electrospray ionisation mass spectrometry. In the proposed technique, a 5-mL syringe was used as an extraction, separation and preconcentration container. The emulsion was obtained after the mixture of toluene (extraction solvent) and methanol (dispersive solvent) was injected into the aqueous bulk of the syringe. The obtained emulsion cleared into two phases without centrifugation, when an aliquot of methanol was introduced as a demulsifier. The separated floating organic extraction solvent was impelled and collected into a pipette tip fitted to the tip of the syringe. Under the optimal conditions, the enrichment factors for azoxystrobin, diethofencarb and pyrimethanil were 239, 200, 195, respectively. The limits of detection, calculated as three times the signal-to-noise ratio (SN(-1)), were 0.026 µg L(-1) for azoxystrobin, 0.071 µg L(-1) for diethofencarb and 0.040 µg L(-1) for pyrimethanil. The repeatability study was carried out by extracting the spiked water samples at concentration levels of 0.02 µg mL(-1) for all the three fungicides. The relative standard deviations varied between 4.9 and 8.2% (n=5). The recoveries of all the three fungicides from tap, lake and rain water samples at spiking levels of 0.2, 1, 5 µg L(-1) were in the range of 90.0-105.0%, 86.0-114.0% and 88.6-110.0%, respectively. The proposed ISD-DLLME technique was demonstrated to be simple, practical and efficient for the determination of different kinds of fungicide residues in real water samples.