The Growth-promoting Effects And The Mechanisms Of Maltose-binding Protein On Leukemia Cells Lines

Maltose-binding protein (MBP) is a high affinity protein responsible for the capture and transportation of maltose/maltodextrin through the periplasmic space in Gram-negative bacteria. In molecular biology research, MBP is known with inert or minimum bioactivity. Recent studies have shown that MBP induced human dendritic cell (DC) maturation and its proinflammatory cytokines production via TLR4activation. We found that fusion MUC1with MBP could enhance its immunogenicity and induce the cell immune response via TLR2activation. Toll-like receptors (TLRs) are widely expressed in a variety of immune cells, which could recognize pathogen-associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs), and act as sensors for the innate immune system. Recently, many reports have shown that hematologic tumor cells express a broad range of TLRs, thus implicating a role of TLRs in tumor biology. Increasing bodies of evidence have suggested that TLRs act as a double-edged sword in cancer cells.On one hand, TLRs can induce an antitumor immune response; On the other hand, TLRs are also necessary for tumor cells to proliferate and evade the immune response. The aim of present study is to explore the biological effects of MBP on leukemia cells lines in vitro, and on chemotherapy-induced apoptosis, and their underlying mechanisms.And to make sure the bioactivity of MBP, and to explore the value and application of TLRs on leukemia cells lines.This study was the following aspects: 1. Preparation of MBPThe pAML plasmid was transfected into E.coli DH5a. The MBP was expressed upon with IPTG. The MBP was purified by Amylose resin affinity chromatography.2. MBP promoted the proliferation of HL60and U937cellsWe used CCK8and blocking experiment to observe the impact of MBP in cells. The results showed that MBP promoted growth and enhanced the viability of HL60and U937cells, while shew no effects on NB4and K562cells. To determine whether HL60and U937cells viability was mediated by MBP, we used anti-MBP mAb to block MBP before its presence, and cell viability was inhibited by the blocker of MBP.3. MBP changed Cell cyclesDNA profile analysis revealed an accumulation of HL60and U937cells in an S-phase concomitant, and a decrease in G2/M events.4. HL60and U937cells modulated TLR expression in response to MBP stimulationWe further observed the TLR2and TLR4expression in leukemia cells lines lines in the protein level by flow cytometry. We found that there was no TLR2and TLR4expression in NB4and K562cells. TLR2was up-regulated with MBP stimulation in U937cells, and TLR4expression had no changes. However, TLR2and TLR4expression was up-regulated firstly, and then down-regulated in HL60cells. We further examined the effects of anti-TLR2and anti-TLR4treatment on MBP-induced viability of cells to characterize the membrane receptor of MBP. The MBP-mediated viability of HL60and U937cells was inhibited with anti-TLR2and anti-TLR4, which suggested that HL60and U937modulated TLR expression in response to MBP stimulation, and MBP mediates HL60and U937cells viability via TLR2and TLR4. 5. MBP changed cell sensitivity to chemotherapeutic drugsHL60and U937cells were cultured in the presence of1μg/ml Ara-c/Paclitaxel with or without MBP. After2days, we observed the viability of HL60and U937cells with CCK8, and cells was washed in PBS and survival was determined with fluorescein isothiocyanate (FITC)-conjugated Annexin-V labeling. Flow cytometry analysis was performed on a FAScan flow cytometer.The results showed that MBP significantly changed cell apoptosis induced by chemotherapeutic drugs.In summary, MBP promotes HL60and U937cells growth via TLR2and TLR4; while has no effect on the proliferation of NB4and K562cells which have no expression of TLR2and TLR4. MBP can change the sensitivities of cells to chemotherapeutic drugs. This experiment further studied the biological activity of MBP, suggesting that MBP still needs comprehensive evaluation. TLRs play an important role in leukemia, and may provide a new target for the treatment of leukemia. This experiment also points out the necessity of anti-infection and that chemotherapeutic drugs need more consideration when patients are in infection.