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Polysaccharopeptides Derived from Trametes Versicolor Induce Cell Cycle Retardarce on Human Leukemia Molt-4 Cells

Author: LiuJia
Tutor: YangXiaoTong
School: Shanghai Normal University
Course: Biochemistry and Molecular Biology
Keywords: PSP cell cycle CyclinE CDK2 Molt-4
CLC: S567.31
Type: Master's thesis
Year: 2011
Downloads: 24
Quote: 0
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Abstract


Polysaccharide peptide (PSP) is an active substance of Yunzhi, which is isolated from the deep-layer cultivated mycelia of Trametes versicolor COV-1. As a new medicine, PSP is used to enhance immune functions and agonize side effect by tumor patients during the radiotherapy and chemotherapy. In vitro, PSP have inhibition effect on the proliferation of human leukemia Molt-4 cells and induce apoptosis.But researchers have different findings and views about cell cycle blocking effect of PSP on leukemia.This study was to investigate the mechanism of cell cycle progression inhibition by PSP on human leukemia Molt-4 cells.Molt-4 cells was short labeled with BrdU for cell cycle tracing then stained with DNA dyes (PI).After 5 and 25μg/ml PSP treatment for 3,6 and 9 h, flow cytometry analysis showed that BrdU labeled S phase cell have a time and dose dependent accumulation, while early and middle S phase cells increased from 17.45% (control) to 25.13% (25μg/mlPSP treated 6 h). Results of high concentration of PSP (0.1mg/ml) treatment for 12 and 24h also showed a time dependent retardation effect on S phase progression and the proportion of S phase cells increased from 56.02% (control)to 67.26%(0.1mg/mlPSP treated 24h). When Molt-4 cells were incubated with higher concentrations (0.5and 1mg/ml) and longer time (48h), the proportion of apoptotic cells was on the increase and reached 47.03%(1mg/ml PSP treated 48h). Compared to 500μg/ml group, apoptotic cells of 1000μg/ml group added 6.58% while S phase cells decreased 10.48%.It suggested that the inhibitory action of PSP was realized by cycle phase block in S phase and apoptosis inducement.After 25μg/ml PSP treatment by 6h, 12h and 24h, the expression of CDK2 which is S phase regulation signal molecules was analysed by western bolt. It confirmed that the short-term(6h) treatment of PSP can significantly increase the expression of CDK2 and the increment was 170% comparing with control. In the experiment of CyclinE and CDK2 bivariate immunostaining analysis by flow cytometry further analyzed the 25μg/ml PSP treated 24h group. It found that PSP can not only increase the expression level of CyclinE and CDK2, and the mean fluorescence intensity increased by 16.80% and 9.39%, but also enhance the percentage of CyclinE and CKD2 expressing cells, and the percentage of expressing cells were increased by 27.52% and 32.99%. In addition, the normal distribution of CyclinE and CDK2 in each cycle phase was disrupted.In late S phase and G2/M, the content of CDK2 CyclinE not declined dramatically, but have a significant improvement.These results demonstrate that PSP induce cell cycle retardarce in S phase on human leukemia Molt-4 cells,and relate to abnormal expression of S phase regulation signal molecules CyclinE and CDK2.

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