Ectopic expression of BIRC5-targeting miR-101-3p overcomes bone marrow stroma-mediated drug resistance in multiple myeloma cells

Background Multiple myeloma (MM) cells gain protection against drugs through interaction with bone marrow stromal cells (BMSCs). This form of resistance largely accounts for resistance to therapy in MM patients which warrants further exploration to identify more potential therapeutic targets. Methods We performed miRNA/mRNA qPCR arrays and western blotting to analyze transcriptional and translational changes in MM cells co-cultured with BMSCs. Drug cytotoxicity and apoptosis in MMGFP-BMSC co-cultures were measured using fluorescence plate reader and flowcytometry, respectively. miRNA was overexpressed in MM cell lines using Lentiviral transduction, miRNA-3’UTR binding was examined using luciferase assay. Results We found that BMSCs downregulated miR-101-3p and upregulated survivin (BIRC5) in MM cells. Survivin was downregulated by miR-101-3p overexpression and found to be a direct target of miR-101-3p using 3’UTR luciferase assay. Overexpression of survivin increased viability of MM cells in the presence of anti-myeloma drugs, and miR-101-3p inhibition by anti-miR against miR-101-3p upregulated survivin. Furthermore, overexpression of miR-101-3p or silencing of survivin triggered apoptosis in MM cells and sensitized them to anti-myeloma drugs in the presence of BMSCs overcoming the stroma-induced drug resistance. Conclusions Our study demonstrates that BMSC-induced resistance to drugs is associated with survivin upregulation which is a direct target of miR-101-3p. This study also identifies miR-101-3p-survivin interaction as a druggable target involved in stroma-mediated drug resistance in MM and suggests it for developing more efficient therapeutic strategies.

Development of two different formats of heterogeneous fluorescence immunoassay for bioanalysis of afatinib by employing fluorescence plate reader and KinExA 3200 immunosensor

Afatinib (AFT) is a potent and highly selective drug used to treat various solid tumors including non-small cell lung cancer (NSCLC). To ensure safe and effective treatment of cancer patients with AFT, its plasma concentrations should be monitored. Thus, sensitive immunoassays are required for measuring AFT concentrations in plasma samples. In this study, two different formats of heterogeneous fluorescent immunoassays were developed and validated for AFT bioanalysis. These assays were microwell-based fluorescence immunoassay (FIA) using fluorescence plate reader and kinetic exclusion assay (KinExA) using KinExA 3200 immunosensor. Both FIA and KinExA were developed using the same reagents: mouse anti-AFT antibody, solid-phase immobilized AFT conjugated with bovine serum albumin protein (AFT-BSA), and goat anti-mouse IgG labelled with fluorescein isothiocyanate (FITC-IgG) for signal generation. The analytical performances of both assays were comparatively evaluated, and the results revealed that although both assays had comparable accuracies, KinExA was superior to FIA in terms of sensitivity and precisions. Moreover, both FIA and KinExA were better alternatives to the existing chromatographic methods for bioanalysis of AFT. The proposed FIA and KinExA are anticipated to effectively contribute in ensuring safe and effective treatment with AFT in clinical settings.

Temperature-Dependent Expression of a CFP-YFP FRET Diacylglycerol Sensor Enables Multiple-Read Screening for Compounds That Affect C1 Domains

Intramolecular CFP-YFP fluorescence resonance energy transfer (FRET) sensors expressed in cells are powerful research tools but have seen relatively little use in screening. We exploited the discovery that the expression of a CFP-YFP FRET diacylglycerol sensor (DAGR) increases over time when cells are incubated at room temperature to assess requirements for robust measurements using a Molecular Devices Spectramax i3x fluorescence plate reader. Expression levels resulting in YFP fluorescence >10-fold higher than untransfected cells and phorbol ester-stimulated FRET ratio changes of 60% or more were required to consistently give robust Z′ > 0.5. As a means of confirming that these conditions are suitable for screening, we developed a novel multiple-read protocol to assay the NCI’s Mechanistic Set III for agonists and antagonists of C1 domain activation. Sixteen compounds prevented C1 domain translocation. However, none blocked phorbol ester-stimulated protein kinase C (PKC) activity assessed using a phospho-specific antibody—six actually stimulated PKC activity. Cytometry, which produces higher Z′ for a given FRET ratio change, might have been a better approach for discovering antagonists, as it would have allowed lower phorbol ester concentrations to be used. We conclude that CFP-YFP FRET measured in a Spectramax i3x plate reader can be used for screening under the conditions we defined. Our strategy of varying expression level and FRET ratio could be useful to others for determining conditions needed for robust cell-based intramolecular CFP-YFP FRET measurements on their instrumentation.

An Efficient and Simultaneous Analysis of Caffeine and Paracetamol in Pharmaceutical Formulations Using TLC with a Fluorescence Plate Reader

A simple, rapid, and effcient method using TLC with a fluorescence plate reader has been described for simultaneous determination of caffeine and paracetamol. Determination was carried out using the fluorescencequenching action of caffeine and paracetamol on a TLC plate with a fluorescent indicator at λ ex = 254 nm in the linear ranges of 0.2–1.9 and 0.03–1.5 µg/L, respectively. Separation of caffeine and paracetamol were performed on the TLC plate, and the best results were obtained using the optimized mobile phase n-hexane–ethyl acetate–ethanol (2.5 + 1.5 + 0.4, v/v). Some important parameters, such as solvent type and ratio of the mobile phase, the presence of other components, and instrumental parameters, were studied. Caffeine and paracetamol detection limits were 0.025 and 0.032 µg/L, and RSD values for 0.6 µg/L caffeine and 0.06 µg/L paracetamol (n = 5) were 1.93 and 2.06%, respectively. Using this technique, some pharmaceuticals containing caffeine and paracetamol were analyzed with satisfactory results.