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1-O-acetyl large flower Inula lactone (ABL) is one of the higher levels of active ingredient in the Eurasian Inula, sesquiterpene lactones, having anti-tumor activity, but its stability and dissolution is not very good, the drugs used in this experiment is the ABL derivatives, referred to as ABL-N. Objective: To observe the impact of ABL-N on murine leukemia L1210 cell proliferation and apoptosis, and to explore its role in the mechanism. Methods: (1) MTT method. Determination of ABL-N: Take proliferation of murine leukemia cells L1210 cells were cultured in 96-well culture plates, incubated for 24 h after administration groups with different concentrations of ABL-N solution, the control group plus with the highest concentration the group equal amount of DMSO. Were cultured for 24 h with Fluo-star for 48 h and 72 h after the detection of a wavelength of 570 nm was measured optical density (OD value) calculated the rate of inhibition of ABL-N concentration of L1210 cells. (2) the trypan blue exclusion staining ABL-N L1210 viable cell number. Take logarithmic growth phase cells, the administration group were treated with different concentrations ABL-N solution, plus the control group with the highest concentration group the same amount of DMSO. Culture, respectively, in the first 24 h, 48 h, 72 h sampling trypan blue staining to count the living cells, the growth curve of the time with the number of living cells. (3) by Wright - Giemsa staining and Hochest 33258 staining L1210 cell morphological changes observed after 48 h of ABL-N. Collect the ABL-N (20 mg / L) treated cells were stained, the morphological changes were observed. (4) the ABL-N was measured by flow cytometry L1210 cell apoptosis rate and cycle distribution. Take the logarithmic growth phase cells, given the ABL-N (10,20 mg / L) for 48 h, PI staining, flow cytometry determination each group rate of apoptosis and cell cycle distribution. (5) Western-blot assay the role of ABL-N the L1210 cells Bax and Bcl-2 protein expression changes. Immunofluorescence detection of ABL-N (6) changes in p53 protein expression in L1210 cells. Results: (1) with different concentrations of ABL-N (5,10,20 mg / L), respectively, deal with L1210 cells 24 h after inhibition rates were 14.6%, 38.5%, 47.4%. For 48 h, the inhibition rates were 28.8%, 63.2%, 74.8%. 72 h after the inhibition rates were 35.7%, 81.5%, 91.6%. That ABL-N significantly inhibited the proliferation of L1210 cells, and showed a concentration-and time-dependent manner. (2) with different concentrations of ABL-N (5, 10, 20, mg / L) acting on the L1210 after, it can be seen from the growth curve concentration-effect relationship, and the high concentration of group to dosing 1 d after concentration group plus drug after 2 d, the decrease in the number of living cells, strong inhibition and killing effect of ABL-N on cultured mouse leukemia L1210 cells. (3) In this study, Giemsa staining and Hoechst 33258 staining method were observed ABL-N in L1210 cells after 48 h of morphological changes, two kinds of staining methods showed dose group cells showed nuclear accumulation and fragmentation of apoptosis characteristic morphological changes. (4) different concentrations of ABL-N (10,20 mg / L) for 48 h, apoptosis rates were 13.6%, 29.9%. Apoptosis solvent control group was 3.57%. Visible role of ABL-N apoptosis rate increased significantly. (5) ABL-N (10,20 mg / L) for 48 h after the number of cells in G1 phase from 26.8% to 30.9%, 51.4%, the number of cells in S phase decreased from 73.2% to 69.1% and 48.6%. (6) with different concentrations of ABL-N (5,10,20 mg / L) for 48 h, Bax / β-actin ratio of 0.49 increased to 0.58,0.71 (P lt; 0.01), 0.82 (P lt; 0.01). Bcl-2/β-actin proportion decreased from 0.66 to 0.61,0.57 (P LT; 0.05), 0.28 (P LT; 0.01). Bcl-2/Bax ratio decreased from 1.36 to 1.17,0.80 (P lt; 0.01), 0.34 (P lt; 0.01). (7) The fluorescence intensity of p53 protein expression in the control group was 63.5. The fluorescence intensity of different concentrations of ABL-N (5,10,20 mg / L) for 48 h were 83.3 (P lt; 0.05), 188.0 (P lt; 0.01), 242.5 (P lt; 0.01), there significantly enhanced. Conclusion: (1) the ABL-N can significantly inhibit the proliferation of mouse leukemia cells L1210. (2) ABL-N L1210 cells have significant cytotoxicity. (3) ABL-N affect cell cycle kinetics, cell cycle arrest in G1 phase. (4) ABL-N inhibit L1210 cell proliferation may affect cell cycle kinetics. Arrest cells in G1 phase, affect the DNA synthesis. (5) ABL-N can induce apoptosis in L1210 cells, which may cut the Bcl-2 protein expression with upregulation of p53 and Bax. Regulation of p53 protein expression may be related to cell cycle arrest in G1 phase.
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