Effects Of Brucine On Human Myeloid Leukemia Cell Line HL-60

ObjectiveTo investigate the inhibitory effect and the inducing apoptosis effect of brucine on human myeloid leukemia cell line HL-60cells.Method1.To Examine The Growth Inhibition of Brucine on HL-60Cells:HL-60cells were exposed to various dosages of brucine. MTT assay was used to examine the growth inhibition of different concentration brucine(40、80、80、160、320、640μg/ml) on HL-60cells, and to calculate the IC50of brucine.2.Cell Morphology Observation of Brucine on HL-60Cells:The apoptosis of HL-60cells was detected by Acridine Orange/Ethidium Bromide (AO/EB) double staining. Cells treated with the brucine observed under the490nm excitation wavelength of fluorescent microscope, and counting at least500cells..3. To Examine the Apoptosis Induction Effect of Brucine on HL-60Cells at the Cellular Level:Using Annexin V-FITC/PI double staining, the cell apoptosis was evaluated by flow cytometry before and after treatment with brucine of different concentration (40、80、160、320、640μg/ml) brucine.Results1. The Growth Inhibition of Brucine on HL-60Cells. The results showed that brucine could significantly inhibit the HL-60cell growth in a concentration-depedent and time-dependent manner at the range of40to640ug·ml-1, and the most prominent inhibition is at640μg/ml for48h. The difference of absorbance value in A570nm was not statistically significant between320and640μg/ml (P>0.05). The IC50value at24h、48h、72h of treatment were about211.82μg/ml,107μg/ml,83μg/ml2. AO/EB stains on HL-60cells showed that brucine could induce nuclear chromatin condensation. After treated with brucine of320μg/ml for48h, the Nuclear chromatin of about80%HL-60cells was stained orange and presented condensation-like or bead-like, showing apoptotic morphology. 3. The HL-60cells were treated with brucine of different concentration(4(40、80、160、320、640μg/ml) for48h, Using Annexin V-FITC/PI double staining,the results evaluated by flow cytometry showed brucine could induce apoptosis of HL-60cells. With the increase in the concentration of brucine,the apoptosis rate increase.The apoptosis rates were (2.1±1.1)%、(21.3±1.2)%、(38.6±1.3)%、(58.5±4.1)%、(75.3±0.87)%、(66.2±0.75)%. Early apoptotic rates were (0.9±0.14)%、(12.1±0.75)%、(34.9±0.42)%、(44.9±0.28)%、(50.9±1.34)%、(0.0±0)%.HL-60cells treated with brucine for48h showed morphological characteristics of apoptotic cells.Conclusion1. Brucine could remarkably inhibit the HL-60cell growth.The results displaied that brucine could significantly inhibit the HL-60cell growth in a concentration-depedent and time-dependent manner.2. Brucine could induce the HL-60cell apoptosis. The apoptosis of HL-60cells was detected by AO/EB double staining, Annexin-V/PI double labeling, and showed that brucine could induce the HL-60cell apoptosis.3. This research primarily showed that brucine could inhibit the growth of HL-60cell, and the inhibition may be stimulated by inducing cell apoptosis. It will provide experimental basis for further studying molecular mechanism of anti-tumor effection of brucine in the future.