Yang Yong-Le, Gong Wei-Yao, Chen Fei-Fei, Chen Lu-Chao, Chen Yi-Tao
College of Life Sciences, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, P.R. China.
Oncol Lett. 2017 Jul;14(1):533-540. doi: 10.3892/ol.2017.6207. Epub 2017 May 19.
The aim of the present study was to investigate the effect of purified protein (pPep) extracted by polyvinylpyrrolidone from the fungus Schroet on the proliferation and cell cycle progression of MC-4 human gastric tumor cells. Using polyvinylpyrrolidone, pPep was extracted from . MC-4 cells were cultured with 30, 60 or 90 µg/ml pPep, with 5-fluorouracil used as a positive control. Survival rates of treated cells were significantly decreased compared with those of the untreated control group in a dose-dependent manner. Using flow cytometric analysis, cells treated with pPep were demonstrated to arrest in S phase and exhibit abnormal G0/G1 and G2/M phase cell cycle distribution. In addition, a wound healing assay demonstrated that pPep significantly inhibited the migration of MC-4 cells. The mRNA and protein expression levels of cyclin D1/cyclin-dependent kinase (CDK) 4, cyclin B/CDK1, cyclin A/CDK2, matrix metalloproteinase (MMP)-2 and MMP-9 were determined using reverse transcription-quantitative polymerase chain reaction analysis and western blotting. The mRNA expression level of CDK4 and cyclin A was significantly increased compared with the untreated control; however, cyclin D1, CDK1, CDK2, cyclin B, MMP-2, and MMP-9 exhibited a significantly decreased mRNA expression level, indicating that there is a negative association between concentration and cyclin D1 expression levels. The expression of the cycle arrest-associated proteins and migration-associated proteins examined were similar to the observed mRNA expression levels. In conclusion, pPep was identified to inhibit migration of and cause S phase cell cycle arrest in MC-4 cells.
本研究的目的是探讨用聚乙烯吡咯烷酮从真菌Schroet中提取的纯化蛋白（pPep）对MC-4人胃癌细胞增殖和细胞周期进程的影响。使用聚乙烯吡咯烷酮从……中提取pPep。将MC-4细胞与30、60或90μg/ml的pPep一起培养，以5-氟尿嘧啶作为阳性对照。与未处理的对照组相比，处理后的细胞存活率以剂量依赖的方式显著降低。通过流式细胞术分析表明，用pPep处理的细胞停滞在S期，并表现出异常的G0/G1和G2/M期细胞周期分布。此外，伤口愈合试验表明，pPep显著抑制MC-4细胞的迁移。使用逆转录定量聚合酶链反应分析和蛋白质印迹法测定细胞周期蛋白D1/细胞周期蛋白依赖性激酶（CDK）4、细胞周期蛋白B/CDK1、细胞周期蛋白A/CDK2、基质金属蛋白酶（MMP）-2和MMP-9的mRNA和蛋白质表达水平。与未处理的对照组相比，CDK4和细胞周期蛋白A的mRNA表达水平显著升高；然而，细胞周期蛋白D1、CDK1、CDK2、细胞周期蛋白B、MMP-2和MMP-9的mRNA表达水平显著降低，表明浓度与细胞周期蛋白D1表达水平之间存在负相关。所检测的细胞周期停滞相关蛋白和迁移相关蛋白的表达与观察到的mRNA表达水平相似。总之，已确定pPep可抑制MC-4细胞的迁移并导致其S期细胞周期停滞。
