Effect Of Inducing Apoptosis Of Betulinic Acid On Human Multiple Myeloma Cell Line Rpmi-8226

Objective To investigate the apoptosis of RPMI-8226 which is a cell line of human multiple myeloma induced by betulinic acid .Methods The inhibiting effect on proliferation, apoptosis inducing effect , influence on cell cycle and induced morphological changes of betulinic acid on RPMI-8226 was individually evaluated by MTT, Annexin-V/PI double staining flow cytometry., PI staining flow cytometry and Hoechst33258 staining under fluorescence microscope. The transcription level changes of Bcl-xl gene and Caspase3 gene which are two kinds of apoptosis related protein gene were determined by RT-PCR.Results Within a certain range of concentration(0,5,10,15,20μg/ml), MTT method showed that 24h IC50 of betulinic acid to RPMI-8226 is (10.156±0.659)μg/ml while 48h IC50 is (5.434±0.212)μg/ml,and its inhibiting effect to proliferation of RPMI-8226 is in a time and dose dependent manner. Annexin-V/PI double staining flow cytometry revealed that apoptotic rate of RPMI-8226 cells increased as betulinic acid concentration increased. PI staining flow cytometry showed rate of cells in G0/G1 phase increased while in S phase decreased, rate of cells in G2/M phase did not present a significant change. Morphological differences were typical and significant between cells in treatment group and control group under fluorescence microscope using Hoechst33258 staining. Caspase3 gene was detected that its transcription level showed a increasing trend as the betulinic acid concentration increased while the Bcl-xl showed the opposite trend.Conclusion Betulinic acid can induce apoptosis of RPMI-8226 within a certain range of concentration in a time and dose dependent manner. This phenomenon may be relevant to the transcriptional level increase of Caspase3 gene and decrease of Bcl-xl gene which both are apoptosis related protein genes. Betulinic acid can also affect the G1/S cell cycle detecting point which arrest cells at G0/G1 phase.