Doxorubicin (Adriamycin, ADM) is an antimitotic drug used in the treatment of a wide range of malignant tumors, including acute leukemia, lymphoma, osteosarcoma, breast cancer, and lung cancer. Multidrug resistance-associated proteins (MRPs) are members of a superfamily of ATP-binding cassette (ABC) transporters, which can transport various molecules across extra- and intra-cellular membranes. The aim of this study was to investigate whether there was a correlation between MRP4 and primary ADM resistance in osteosarcoma cells. In this paper, we chose the human osteosarcoma cell line MG63, ADM resistant cell line MG63/DOX, and the patient's primary cell GSF-0686. We checked the ADM sensitivity and cytotoxicity of all the three cells by cell proliferation assay. The intracellular drug concentrations were measured by using LC-MS/MS. We also examined MRP4 gene expression by RT-PCR and Western Blot. We found that the intracellular ADM concentration of the parent osteosarcoma cell line MG63 was higher than the ADM resistant osteosarcoma MG63/DOX cell line or the GSF-0686 cell after ADM treatment (P < 0.05). In addition, MRP4 mRNA and protein levels in ADM resistant osteosarcoma cells were higher than in MG63 cell (P < 0.05). Taking together, this work suggests that overexpression of MRP4 may confer ADM resistance in osteosarcoma cells.

β-Nicotinamide mononucleotide (NMN) has been widely used as a nutraceutical for self-medication. The one-step conversion of nicotinamide riboside (NR) to β-NMN has been considered to be the most promising synthetic route for β-NMN. Here, human nicotinamide riboside kinase 2 (NRK-2) was functionally displayed on the cell surface of Saccharomyces cerevisiae EBY100, forming a whole-cell biocatalyst (Whole-cell NRK-2). Whole-cell NRK-2 could convert nicotinamide riboside (NR) to β-NMN efficiently in the presence of ATP and Mg2+, with a maximal activity of 64 IU/g (dry weight) and a K m of 3.5 μM, similar to that of free NRK-2 reported previously. To get the best reaction conditions for β-NMN synthesis, the amounts of NR, ATP, and Mg2+ used, pH, and temperature for the synthetic reaction were optimized. Using Whole-cell NRK-2 as the catalyst under the optimized conditions, β-NMN synthesized from NR reached a maximal conversion rate of 98.2%, corresponding to 12.6 g/L of β-NMN in the reaction mixture, which was much higher than those of synthetic processes reported. Additionally, Whole-cell NRK-2 had good pH stability and thermostability, required no complicated treatments before or after use, and could be reused in sequential production. Therefore, this study provided a safe, stable, highly effective, and low-cost biocatalyst for the preparation of β-NMN, which has great potential in industrial production.

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