| Objective: To observe the effect on quercetin, investigated the possible mechanism of quercetin on the proliferation and apoptosis of THP-1 cells by testing the expression of three telomere-binding protein, for the drug targeted in acute single nuclear leukemia, which seek to supports for experimrnts. In addition, providing in a hope of an advantage of the clinical efficacy of leukemia and new ideas and methods.Methods: 1. In vitro:(1) Cultured the human acute single nuclear leukemia THP-1 cells.(2)Cellular viability was measured by CCK-8 assay, and the cells were incubated in the absence or presence of increasing concentration of QIN for different time, which determined the IC50 of the experimental group.(3) Apoptosis and cell cycle distribution were monitored by Flow cytometry(FCM).(4) The protein expression levels of each group of POT1, TRF1, TRF2 were assessed by Western-blot analysis.(5) The levels of POT1, TRF1, TRF2 mRNA were detected by real-time fluorescent quantitative PCR(RT-PCR) analysis. 2 In vivo:(1) Establishment and evaluation of human acute single nuclear leukemia THP-1 Model in NOD/SCID Mice, and mice were treated of QIN.(2)We observe the survival status, then monitoring the changes of peripheral blood before and after treatment with daily i.g. of QIN, in addition, we evaluated the dying mouse organization by microscopy.(3) At the end of the experiment, the mice were killed by institutionally approved method after 28 days. The apoptosis and the cell cycle progression of spleen cells were examined using FCM. And the changes of POT1, TRF1, TRF2 protein expression were detected using immunohistochemisty.Results: 1 In vitro:(1) CCK-8 assay showed that inhibited cell growth and decreased cell viability in a dose- and time- dependent manner, the experience group of IC50 was 84μmol/L. Therefore, in our study, we choose with the medium concentration 80μmol/L(lower than IC50) at 48 h.(2) The study demonstrated that after treatment for 48 h, the experimental group apoptosis and the cell cycle progression of THP-1 cells were compared with control group. The experimental group were significantly higher than those in the control group, and the difference was statistically significant(p<0.05).(3) As shown by western blot analysis, as depicted showed that compared with controls, the experimental group elevated POT1, TRF1 protein level and degraded TRF2 protein level(p<0.01), there were significant differences. In addition, the findings demonstrated that POT1 and TRF1 were positively correlated(r=0.88), TRF2 and POT1, TRF1 were negatively correlated(r1=-0.90、r2=-0.93).(4) We evaluated the level of each gene mRNA expression by real-time fluorescent quantitative PCR analysis, respectively, and found that the level of POT1, TRF1, TRF2 mRNA were concordant with the protein levels. Overall, our results implied that after drugs treatments, the expression of the protein at the levels of gene transcription. 2 In vivo: Transplantation was always successful,and results showed that the experimental group treatments increased the average survival time(p<0.01). After drugs treatments, immunohistochemistry showed that cleaved of POT1, TRF1 level were increased and TRF2 level was decreased compared with controls, the protein expressions of the experimental groups were concordant with the vitro.Conclusion: These findings indicate that the study on quercetin in vitro and in vivo have some degrees of anti-cancer activity, its anticancer mechanism may be related to elevated POT1, TRF1 protein level and degraded TRF2 protein level in order to induce the apoptosis of leukemia cells, which as a target of cancer treatment for experimental and theoretical basis. |
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