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  • br However it should be noted

    2019-10-07


    However, it should be noted that the reduction in culture EGFP fluorescence could be attributed to the down-regulation of survivin promoter driving EGFP expression as well as the cytotoxicity effect of drug on cell growth. Since the CMV promoter is constitutive and gen-erally not affected by drug or external stimuli (Hunt et al., 1999), EGFP  Journal of Biotechnology 289 (2019) 80–87
    production in MCF-CMV-EGFP ABT-263 (Navitoclax) is growth-associated and can be used to quantify cell proliferation and assess cytotoxicity of drugs (Li et al., 2013b). The survivin promoter-EGFP reporter assay thus requires using both MCF-7-SP-EGFP and MCF-7-CMV-EGFP cells to distinguish between drug effects on the survivin promoter activity and cell pro-liferation.
    To validate the survivin promoter assay, the cytotoxicity of YM155 was evaluated at various concentrations (0, 10, 20, 50, 100, 200, 500 and 1000 nM) with MCF-CMV-EGFP cells cultured in 3D 40-MBR, and the results are shown in Fig. 4. As indicated by the normalized EGFP fluorescence intensity, cells grew well and multiplied ∼10.4-fold in 7 days in the medium without YM155 (only 0.002% w/v DMSO). The corresponding doubling time of 49.6 h was in line with the reported specific growth rate of 0.015 h−1 for MCF-7 cells (Li et al., 2013b), indicating that the 3D PET scaffold did not inhibit cell growth. Mean-while, cell growth was significantly reduced by the addition of YM155 at a dose-dependent manner. The IC50 of YM155 based on 50% re-duction in cell growth was 14.31 nM at 72 h and 12.57 nM at 96 h, both were comparable to the previously reported IC50 (∼13 nM) obtained in the 2D MTT assay (Cheng et al., 2015). These results further validated that the EGFP expression driven by the CMV promoter was growth-associated and can be used as a dynamic reporter to track cell pro-liferation and assess drug cytotoxicity. As can be seen in Fig. 4B, the specific growth rate decreased with increasing the YM155 concentra-tion and became negative or specific death rate when the concentration was more than 20 nM. The IC50 of YM155 based on 50% reduction in the specific growth rate, which can minimize the time sensitivity of IC50 estimation and provide a more consistent evaluation of cytotoxicity (Zhang and Yang, 2011), was 10.57 nM. The result was consistent with a previous study showing that YM155 was able to induce lethal effects on various breast cancer cell subtypes regardless of the expression le-vels of ER, HER2 and caspase-3 (Cheng et al., 2015).
    3.4. Effect of YM155 on survivin promoter activity
    YM155, a small-molecule survivin promoter suppressant, disrupts the binding of different transcription factors (e.g., Sp1 and ILF3/p54nrb) to the region of survivin core promoter and thus causes survivin tran-scriptional silencing (Yamauchi et al., 2012; Cheng et al., 2012). Its down-regulation effects are highly survivin gene-selective, with minimal activity against the expression of other genes driven by similar genetic promoters (Wang et al., 2016; Cheng et al., 2012). YM155 at 10 nM (approx. the IC50 value determined in the cytotoxicity assay) was thus used to study its down-regulation effect on survivin promoter in MCF-7 cells. The fluorescence kinetics of MCF-7-SP-EGFP and MCF-7-CMV-EGFP cells cultured in their respective micro-bioreactors on the 40-MBR were monitored. As can be seen in Fig. 5, the specific survivin expression or the ratio of the fluorescence intensity from MCF-7-SP-EGFP to that of MCF-7-CMV-EGFP decreased continuously after adding YM155 at 48 h (Fig. 5B), while the control (without the drug addition) remained almost unchanged throughout the culture period (Fig. 5A). In the assay, the EGFP signals driven by CMV promoter and human sur-vivin promoter were used to quantify cell proliferation and survivin expression, respectively. The combination between cytotoxicity and survivin promoter assays in one system accounted for the effect of cell number on survivin expression level. The down-regulation of survivin promoter by YM155 in MCF-7 cells was thus successfully captured and quantified in the 3D survivin promoter-EGFP reporter assay.