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  • br Our novel D MBR platform using MCF cells with

    2019-10-07


    Our novel 3D 40-MBR platform using MCF-7 Acetylcysteine with a human survivin promoter sequence to identify new cancer drug candidates can meet this need. In the present work, we successfully cloned a 303 bp of the core sequence of human survivin promoter and used it to develop a survivin promoter assay with EGFP as the reporter. We first demon-strated the correlation between survivin expression and EGFP expres-sion in 2D cultures with RT-PCR (Fig. 2) and fluorescence microscopy (Fig. 3), and then validated the survivin promoter-EGFP reporter assay in 3D culture with YM155 (Fig. 5). YM155, a survivin inhibitor, was first discovered by Astellas Pharma in 2007 and is currently in clinical trials for tumor therapeutics (Rauch et al., 2014). In addition to YM155, doxorubicin and cisplatin were also tested in our survivin promoter assay. Doxorubicin and cisplatin are widely applied chemotherapeutic agents and their mechanisms of antitumor action occur mainly through DNA damage, disruption of DNA repair, and induction of the apoptosis pathways in tumor cells (Thorn et al., 2011; Dasari and Tchounwou, 2014). Our survivin promoter assay showed that doxorubicin down-regulated survivin while cisplatin did not, demonstrating its ability to screen for drugs actively inhibiting survivin expression. Such a reporter gene assay (RGA) would play an important role in cell culture systems for biological research and drug screening (Nierode et al., 2016).  Journal of Biotechnology 289 (2019) 80–87
    treatment). B: 10 nM YM155 treatment. Survivin promoter activity was in-dicated by the specific survivin expression or the ratio of fluorescence intensity of MCF-7-SP-EGFP cells to that of MCF-7-CMV-EGFP cells. Each data point re-presents the average from triplicate samples with the error bar indicating the standard deviation.
    It should be noted that some of the small-molecule survivin in-hibitors (e.g. YM155 and FL118) were identified by using survivin promoter luciferase-reporter assays (Nakahara et al., 2007; Ling et al., 2012). Luciferase-based methods have been widely applied to the mo-lecular promoter assays, in which luciferase genes (such as firefly lu-ciferase) were cloned with the promoter sequence of interest to eval-uate its activity under experimental modulation (Smale, 2010). In addition, cell viability and drug cytotoxicity were also evaluated by luciferase-based assays in a number of in vitro systems (Lanz et al., 2017; Howes et al., 2014). However, luciferase-based technologies re-quire cell lysis for data capture and thus intensive labor consumption in the screening and yet can only provide endpoint data. In contrast, our assay utilizing EGFP as the reporter was more sensitive and enabled non-invasive monitoring of survivin gene expression in real time. With the strong linearity between cell density (number) and EGFP fluores-cence intensity, the EGFP signals can be used directly to represent the viable cell density (number) in the culture systems.
    It should be mentioned that the 3D cell culture in the 40-MBR system has also been used for high throughput assessment of drug cy-totoxicity with better prediction of in vivo cancer drug efficacy com-pared to conventional 2D culture (Zang et al., 2016). MCF-7 cells dis-played a tissue-like morphology in the fibrous scaffold (see Figure S1) while maintained a similar doubling time to that for cells in 2D monolayer culture. Cell-based drug screening has been predominantly
    Fig. 6. Cytotoxicity and survivin promoter assays for doxorubicin in 40-MBR. Drug was added at 48 h post cell inoculation. A: Fluorescence kinetics of MCF-7-CMV-EGFP cells under different concentrations of doxorubicin. Each data point was normalized by the initial fluorescence value (0 h). B: Survivin promoter activity in MCF-7 cells treated with doxorubicin (100 nM). Each data point represents the average from triplicate samples with the error bar indicating the standard deviation.