17 Cell Cycle Synchronization of Bovine Fibroblasts by Azadirachta Indica Extracts

Abstract

One requirement for somatic cell nuclear transfer (NT) is the coordination between donor cell cycle and recipient cytoplasm. As an alternative to commercially available substances to synchronize the cell cycle in G0/G1, we tested 2 extracts, aqueous and hexane obtained from the plant Azadirachta indica A. Juss (popularly called Neem). Extracts from this plant have shown antiviral, antibacterial and anticancer activities, widely described in the literature (Kumar et al. 2009 Invest. New Drugs 27, 246–252). The hexane extract was prepared in the Soxhlet apparatus until total collapse and then submitted to rotary evaporation. The aqueous extract was prepared by dynamic maceration and was subsequently lyophilized. Bovine fibroblasts collected from Gyr cows were cultured in DMEM (Sigma) supplemented with 10% fetal cow serum (FCS) and incubated at 37°C, 5% CO2 and 95% humidity. After obtaining 70% of cell confluence, the extract was added to cells at the following concentrations: 0 μg mL–1 (negative control), 50 μg mL–1, 100 μg mL–1, 200 μg mL–1 and 300 μg mL–1, for 12 and 24 h. Simultaneously, a group with serum starvation (positive control; cells cultured in DMEM plus 0.5% FBS for 3 days) was prepared. Three repetitions were performed in triplicate for each concentration and control groups. Cell cycle readings were performed by flow cytometry (Facs Callibur, Becton Dickinson, San Jose, CA, USA) and DNA histograms were analysed by WinMDI software to determine the percentage of cells in G0/G1 phase, S and G2 cell cycle, so that 10 000 cells were analysed in each reading on a flow cytometer. Data were analysed by analysis of variance and means were compared by Student-Newman-Keuls test. The percentages of cells at G0/G1 phase for aqueous extracts were lower (P < 0.05), regardless of the concentration and exposure time, than the 0 μg mL–1 (83.73 ± 1.14%) and serum starvation (86.64 ± 1.44%). In contrast, the percentages of cells synchronized at G0/G1 with 50 μg mL–1 for 12 h (84.23 ± 0.56%), 50 μg mL–1 for 24 h (85.66 ± 0.57%), 100 μg mL–1 for 12 h (87.85 ± 0.51%) and 200 μg mL–1 for 12 h (85.87 ± 0.45%) using hexane extracts were higher (P < 0.05) than with 0 μg mL–1 (81.44 ± 0.29%), but lower (P < 0.05) than the serum starvation (91.33 ± 0.31%). In conclusion, hexane extracts from the plant Azadirachta indica A. Juss can synchronize mammalian cell cycle at G0/G1 despite the low proportion when compared with serum starvation. Studies to evaluate efficiency of cell cycle resuming and viability after somatic cell nuclear transfer are ongoing. Financial support: FAPEMIG, CNPq and Project 01.07.01.002.