Up-regulation of Nucleophosmin 1 in Cisplatin-induced Death of Mouse Osteoblastic MC3T3-E1 Cells

Abstract

Cisplatin (cis-diamminedichloroplatinum II, Fig. 1A) is one of the most used chemotherapeutic agents in the management of malignant bone and soft tissue tumors, but its treatment can show the impairment of bone formation. Actually, during the administration of cisplatin, its effect has been reported to be targeted directly on the proliferation and differentiation of bone-forming cells. In addition, in the study to elucidate the effect of cisplatin on regenerate bone formation during the distraction and consolidation phases of bone transport osteogenesis, cisplatin-treated dogs had decreased mineralized bone volume, decreased percentage of woven bone volume, decreased percentage of osteoblastcovered bone and increased porosity, suggesting that bone formation and resorption may be uncoupled in cisplatintreated regenerate bone. Throughout adult life, bone tissue is subject to a continuous process of turnover, whereby old bone is removed and replaced by new bone by the coupled processes of bone resorption and bone formation. Osteoblasts are the bone lining cells responsible for the production of the matrix constituents such as a collagen and originate from a bone marrow stromal stem cells under the influence of local growth factors. These precursors undergo proliferation and differentiate into preosteoblasts and then into mature osteoblasts. Interestingly, several studies have clearly shown the deleterious effect of cytotoxic chemotherapy on protein synthesis and DNA replication resulting in diminished bone formation, but the precise mechanism has not been studied yet. Therefore, in this study, we evaluated the effect of cisplatin on the survival of osteoblasts and applied the proteomics technology for identifying protein(s) involved in the action of cisplatin in osteoblasts. First, the effect of cisplatin on the proliferation of MC3T3E1 cells was evaluated (Fig. 1B). In a dose-dependent manner, the proliferation of MC3T3-E1 cells was significantly inhibited ~90% and ~54% in 1-day and 4-day culture by the treatment of 100 μM cisplatin, respectively. Since 100 μM of cisplatin significantly inhibited the proliferation of MC3T3-E1 cells in 1-day culture, cells incubated with/ without 100 μM cisplatin for 1 day were used for identifying the differentially expressed protein(s). Using Phoretix 2D image analysis software, the altered spots were compared based on their volume percentages in the total spot volume over the whole gel images. Of over 500 protein spots resolved in a 2-DE gel with silver staining analyzed, 18 spots was above 2-fold increased, whereas 35 spots decreased in cisplatin-treated cells (data not shown). Among these spots, the circle-indicated spot in Figure 2A was increased 2.63fold in cisplatin-treated cells compared to control. The circle-indicated spot was digested in gel with trypsin and subjected to peptide mass fingerprinting (PMF). The representative MALDI-TOF MS spectrum for the spot was shown in Figure 2B. According to the PMF, the estimated pI and molecular weight (MW) by the 2-DE map, the circleindicated spot in Figure 2A was identified to nucleophosmin 1 (NPM, Fig. 2C). Cisplatin-induced protein expression of NPM was confirmed using Western blot analysis (Fig. 3). NPM was induced by cisplatin in a dose-dependent manner and its actin-normalized expression levels in 100 μM cisplatin was ~2-fold increased compared to control, which was similar with the result observed in 2-DE analysis. NPM is a nucleolar phosphoprotein and it appears to be a multi-func-