The Apoptosis Induction Effect Of Brucine On Human Leukemia Cell Line THP-1Cells And Its Possible Mechanism

ObjectiveTo explore the apoptosis-inducing effect of brucine on human leukemia cell lineTHP-1cells and its possible mechanism.Method1. To Evaluate The Growth Inhibition of Brucine on THP-1Cells: CCK-8assaywas used to measure the growth inhibition of different concentration of brucine(50、100、200、400μg/ml) on THP-1cells.2. Morphological Changes of THP-1Cells Treated With Brucine:Morphological observation of THP-1cells which were treated with400μg/ml brucinefor48hours was detected by Acridine Orange/Ethidium Bromide (AO/EB) doublestaining.A fluorescent microscope was used to observe THP-1cells under the490nmexcitation wavelength.3. To Examine the Apoptosis Induction Effect of Brucine on THP-1Cells.Annexin-Ⅴ/PI double staining flow cytometry was used to detect the apoptosis ofTHP-1cells after treated with brucine of different concentration (50、100、200、400μg/ml) for48hours.4. To Observe The Cell Cycle Distribution of THP-1Cells: THP-1cells wastreated with brucine of different concentration (50、100、200、400μg/ml) for48hours,then the cell cycle distribution was detected by PI staining flowcytometry.5.Reverse transcription-PCR(RT-PCR) was used to determine the transcription level changes of BCL-2gene and BAX gene which are two kinds of apoptosisrelated protein genes.Results1. The Growth Inhibition of Brucine on THP-1Cells. CCK-8method showedthat brucine within a certain range of concertration(50、100、200、400μg/ml) couldremarkably inhibit the growth of THP-1cells,and its inhibiting efffect to proliferationof THP-1cells was in a concentration-depedent and time-dependent manner. Thedifference of inhibition rate was statistically significant between brucine treated groupand control group (P<0.05).2. Staining cells with AO-EB revealed that brucine induced nuclear chromatincondensation. After the THP-1cells were treated with the brucine of400μg/ml for48h, more orange dense stain material appeared in the nuclei of THP-1cells treatedwith brucine,whereas the cell nucleus and cytoplasm of the untreated THP-1cellsappear bright green flourscence.3.The Apoptosis of Brucine-treated THP-1Cells.Annexin-V/PI double stainingdetection indicated that brucine could induce apoptosis of THP-1cells. The apoptosisrate of THP-1cells treated with brucine increased gradually in aconcentration-dependent manner. The difference of apoptosis rate was statisticallysignificant between brucine treated group and control group (P<0.05).4.Effect of Different Concentration of Brucine on the Cell Cycle Distribution ofTHP-1Cells.PI staining flow cytometry showed that the rate of cells in G0/G1phaseincreased while in S phase decreased in a concentration-dependent manner.There wasstatistically significant difference between brucine treated group and control group(P<0.05).5.The mRNA expression of BCL-2and BAX in THP-1cells treated withbrucine.RT-PCR detection revealed that the expression of BCL-2was down-regulatedstrikingly while BAX was up-regulated in THP-1cells after treated with brucine(50、100、200、400μg/ml) for48hours.Statistically significant difference could be foundbetween brucine-treated group and control group (P<0.01) Conclusion1. Brucine can remarkably inhibit the growth of THP-1cells.Within a certainrange of concertration(50、100、200、400μg/ml),the inhibition rate of brucineincreased in a concentration-depedent and time-dependent manner.2. Brucine can induce the apoptosis of THP-1cells. Both the AO/EB doublestaining method and Annexin-V/PI double labeling method revealed that brucinecould induce the apoptosis of THP-1cells in a dose and time dependent way.3.Brucine can affect the cell cycle distribution of THP-1cells. PI stainingrevealed that brucine can also affect the G0/S cell cycle detecting point with arrestcells at G0/G1phase.4.The mechanism by which brucine induced apoptosis in THP-1cells may berelated to the inhibition of BCL-2gene and activation of BAX gene.