| Chronic myelocytic leukemia(CML) is a malignant disease derived from bone marrow pluripotent stem cells proliferation disorder.95% Of CML patients are with the specific Ph chromosome t(9; 22) (q34; qll) c-abl translocats from chromosome 9 to the broken cluster region of chromosome 22, with bcrabl fusion gene and BCR/ABL fusion protein expression. As the conventional therapies are limitedly effective, while the novel tyrosine kinase inhibitor, one kind of targeting drug, GleevecTM (STI 571) is expensive, with primary or secondary drug resistance, it is very important to develope new, highly efficient and low-cost drug to reduce the drug resistance of CML.6-Gingerol is the main active ingredient of ginger. Overseas studies have reported that 6-gingerol displayed growth inhibitor and apoptosis inducer of a variety of tumor cell lines, including leukemia HL-60 cells. However, the precise mechanisms of 6-gingerol have not been completely elucidated.AIMS:To observe the effects of 6-gingerol on the chronic myelocytic leukemia cell line K562 cells proliferation, cell cycle, apoptosis, reactive oxygen free radicals and other biological characteristics. To identify and analyze differentially expressed proteins of K562 cells before and after treatment of 6-gingerol by proteomics and bioinformatics, to explore its anti-leukemia mechanisms, then to afford the theory evidence of searching the targeted proteins for 6-gingerol treating K562 cells.METHODS:1.1) Morphology changes of Hochest33258 staining K562 cells were observed by fluorescence microscope.2) Cell proliferation activity of K562 cell line treated with 6-gingerol were measured by CCK-8 assay.3) Inhibitory effects of cell cycles and apoptosis rates on K562 cells treated with different concentrations of 6-gingerol were detected by flow cytometry.4) Mitochondrial membrane potential and reactive oxygen species levels of K562 cells were detected by flow cytometry.2. Half inhibitory amount of 6-gingerol treated K562 cells for 48 hours. Then 6-ginger treated and untreated K562 cells were collected. Total proteins of two groups were extracted, respectively. The two-dimensional gel electrophoresis map of K562 cell line before and after treated by 6-gingerol was established. Then ImageMaster 2-D Platinum image analysis software was used to compare and identify differentially expressed proteins which have multiple differences more than two times of the protein spots as a significant difference in protein expression screening criteria. Peptide mass fingerprint (PMF) and MS/MS-MS identification of right candidate differentially expressed proteins were identified by tandem time of flight mass spectrometry MALDI-TOF/TOF-MS. The differentially expressed proteins were analyzed preliminarily and sorted on the functional classification by bioinformatics and database searching.RESULTS:1.1) 6-Gingerol treated K562 cells desplayed nuclear condensation, margination and apoptotic body formation, while the control and DMSO groups had no significant changes.2) 6-Gingerol could significantly inhibit the proliferation of K562 cells after treated for 24,48,72 hours. The half inhibitory (IC50) concentrations of 24,48,72 hours was 22.86μg/ml,15.75μg/ml,11.18μg/ml, respectively. The inhibitory rates were dosage and time dependent.3) The cell proportions in cell cycle of blank control group were 43.2%±0.50% in G1 stage,49.5%±0.30% in S stage,7.3%±0.20% in G2 stage, respectively. The cell proportions in 6-gingerol treated group 157μg/ml and 20μg/ml were 50.5%±0.36%,61.6%±0.38% in G1 stage, respectivel,45.7%±0.30%,35.7%±0.35% in S stage, respectively, and 3.8%±0.06%, 2.5%±0.10% in G2 stage, respectively.6-Gingerol 15μg/ml and 20μg/ml treated groups had statistically difference as compared with control groups(P<0.01). (4) Early apoptosis rates in control group and DMSO group were 4.5%±0.5%,5.8%±0.6%, respectively. Early apoptosis rates of 6-gingerol with lOμg/ml,15μg/ml,20μg/ml concentration treated group were 12.3%±0.3%,14.5%±0.5%,19.5%±1% respectively, significantly higher than the two control groups(P<0.01). (5) With Rho-123 as the mitochondrial membrane potential indicator, the mean fluorscence intensity (MFI)of blank control group and DMSO control group were 100±0.8, 98.5±0.7, respectively. MFI of 6-gingerol 15μg/ml and 20μg/ml concentration treated group were 68.48±3.5 and 64.87±4.5, respectively.(P<0.01). (6) 6-Gingerol 15μg/ml treated K562 cells for different time (0.5h, 1h,2h,3h), and the intracellular ROS levels increased gradually with time (P<0.05). With different concentrations of 6-gingerol acting on K562 cells, ROS levels of K562 cells gradually increased when concentration increased (P<0.05).2. The two-dimensional gel electrophoresis map of K562 cell line was established.42 differentially expressed proteins were identified. A total of 35 were non-recurring differences,19 of which were highly-expression proteins, while 16 of which lowly-expression proteins.CONCLUSIONS:1.6-Gingerol could significantly inhibit the proliferation of K562 cells, arrest cells in G1 phase, decline mitochondrial membrane potential, induce cell apoptosis and induce producing reactive oxygen free radicals.2. The differentially expressed proteins after treatment of 6-gingerol involved oxidative stress response, cell cycle regulation, apoptosis signal transduction, protein biosynthesis and carbohydrate metabolism and other important proteins and protease. The efficacy of 6-gingerol on the proliferation inhibition and apoptosis induction of K562 cells involved multiple signal transduction pathways.
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