Li-Rong Xiong1, Peng-Wei Jing1, Xiao-Ying Song1, Ya-Ping Wang1, Lu Wang2. 1. Department of Histology and Embryology, Laboratory of Stem Cells and Tissue Engineering, Chongqing Medical University, Chongqing 400016, China. 2. Department of Histology and Embryology, Laboratory of Stem Cells and Tissue Engineering, Chongqing Medical University, Chongqing 400016, China. E-mail: wanglu99@sina.com.
Abstract
OBJECTIVE: To investigate the damage effect of 5-fluorouracil(5-FU) with tumor inhibition concentration on human bone marrow mesenchymal stem cells (hBMSC) and influence of its effect on the hematopoietic cells. METHODS: The Cell Counting Kit-8 was used for determining the sensitivity of breast cancer cell line MCF-7, colon cancer cell line HCT-116 and HS-5 derived from human bone marrow stronal cell line to the different doses of 5-fluorouracil in vitro. After HS-5 was treated with 5-fluorouracil, crystal violet staining assay was used to count the number of colony forming unit-fibroblast, the distribution of cell cycle was analyzed by flow cytometry (FCM), apoptosis was assessed by Annexin V/PI double-stained method and Hoechest staining; DCFH-DA staining was used to analyse the level of reactive oxygen species (ROS), ELISA and immuofluorescence were used to detect cytokines KL, GM-CSF, RANTS and SDF. The hUCB-MNC was counted by trypan blue staining after co-culture with HS-5, FCM was used to detect the cell cycle distribution, ROS level and the ratio of CD34+ cells. The levels of glutathione peroxidase (GSH-Px) and total superoxide dismutase(T-SOD) were measured by enzymatic assay. The senescence associated-β-galactosidase (SA-β-Gal) staining was used to detect the senescent hUCB-MNC. RESULTS: 5-Fluorouracil of 12.5 µg/ml-100 µg/ml inhibited the proliferation of MCF-7, HCT-116 and HS-5 cells in dose-dependent and time-dependent manner, among them HS-5 was more sensitive to 5- fluorouracil. After treatment with 5-fluorouracil, the HS-5 cell cycle was blocked. The apoptosis rate and the intracellular ROS level of HS-5 significantly increased. Also HS-5-secreted hematopoietic growth factors decreased and inflammatory chemokines increased. After co-cultured with 5-fluorouracil-treated HS-5, the number of hUCB-MNC and the ratio of CD34+ cells were decreased. hUCB-MNC cell cycle blocked in G1 phase. The antioxidant capacity also decreased and the intracellular ROS level increased significantly. The senescent hUCB-MNC increased. CONCLUSION: 5-Fluorouracil can result in oxidative damage of bone marrow stromal cells and change of function secreting bioactivators, thus induce oxidative stress in hematopoietic cells to initiate stress-induced premature senescence (SIPS).
OBJECTIVE: To investigate the damage effect of 5-fluorouracil(5-FU) with tumor inhibition concentration on human bone marrow mesenchymal stem cells (hBMSC) and influence of its effect on the hematopoietic cells. METHODS: The Cell Counting Kit-8 was used for determining the sensitivity of breast cancer cell line MCF-7, colon cancer cell line HCT-116 and HS-5 derived from human bone marrow stronal cell line to the different doses of 5-fluorouracil in vitro. After HS-5 was treated with 5-fluorouracil, crystal violet staining assay was used to count the number of colony forming unit-fibroblast, the distribution of cell cycle was analyzed by flow cytometry (FCM), apoptosis was assessed by Annexin V/PI double-stained method and Hoechest staining; DCFH-DA staining was used to analyse the level of reactive oxygen species (ROS), ELISA and immuofluorescence were used to detect cytokines KL, GM-CSF, RANTS and SDF. The hUCB-MNC was counted by trypan blue staining after co-culture with HS-5, FCM was used to detect the cell cycle distribution, ROS level and the ratio of CD34+ cells. The levels of glutathione peroxidase (GSH-Px) and total superoxide dismutase(T-SOD) were measured by enzymatic assay. The senescence associated-β-galactosidase (SA-β-Gal) staining was used to detect the senescent hUCB-MNC. RESULTS:5-Fluorouracil of 12.5 µg/ml-100 µg/ml inhibited the proliferation of MCF-7, HCT-116 and HS-5 cells in dose-dependent and time-dependent manner, among them HS-5 was more sensitive to 5- fluorouracil. After treatment with 5-fluorouracil, the HS-5 cell cycle was blocked. The apoptosis rate and the intracellular ROS level of HS-5 significantly increased. Also HS-5-secreted hematopoietic growth factors decreased and inflammatory chemokines increased. After co-cultured with 5-fluorouracil-treated HS-5, the number of hUCB-MNC and the ratio of CD34+ cells were decreased. hUCB-MNC cell cycle blocked in G1 phase. The antioxidant capacity also decreased and the intracellular ROS level increased significantly. The senescent hUCB-MNC increased. CONCLUSION:5-Fluorouracil can result in oxidative damage of bone marrow stromal cells and change of function secreting bioactivators, thus induce oxidative stress in hematopoietic cells to initiate stress-induced premature senescence (SIPS).