The Study Of Apoptosis On Multiple Myeloma U266 Cell By Phosphorylation C-Jun Of Brucine

Objective:Multiple myeloma is a malignant clone of plasma cells proliferated, synthesis and secretion of monoclonal immunoglobulin uniform structure and (or) the light chain of cancer. Active ingredient of strychnos alkaloids, brucine is one of its main components, has anti-tumor effect, can cause apoptosis in HepG2 cells, which may be related to mitochondrial depolarization. In this study, the role of brucine on human multiple myeloma U266 cells lines were observed on the growth and to explore its molecular mechanism of suppressing the multiple myeloma U266 cells line growth.Methods:Logarithmic growth phase of the U266 cells with different concentrations(0 mg/ml,0.05mg/ml,0.1mg/ml,0.2mg/ml,0.4mg/ml),the effect of brucine 24,48,72 hours was detected by MTT growth inhibition rate and calculate the IC50 of 48 hours. Cell morphology was also observed. Observing cell cycle and mitochondrial membrane potential by flow cytometry determine the manner of cell death after the role of brucine. Detecting Bcl-2,Bax,caspase-3,c-Jun,cycto-c expression changes.Results:1 The apoptotic effect of brucine show a dose and time dependent manner (p<0.05). 48 hour IC50 0.16mg/ml.2 Using RT-PCR, the role of brucine in the U266 cell line apoptosis gene Bax, Bcl-2 anti-apoptotic gene expression, the results show the expression of Bax increased with time, Bcl-2 expression decreased the time(P<0.05).3 Typical apoptotic bodies can be seen under the fluorescence microscope. By flow cytometry with different concentrations for 24 hours after the U266 cells, can see the typical sub-G0/G1 cell groups. The apoptosis rate increased with the concentration (P<0.05). So brucine play a role in the inhibition of U266 cells by inducing apoptosis.4 Mitochondrial membrane potential after different concentration of brucine had no significant decrease using flow cytometry.5 At the same time, detecting caspase-3,cyto-c gene expression changes combined with caspase-8 and caspase-9 inhibitor z-IETD-fmk,z-LETD-fmk show joins z-IETD-fmk, the caspase-3 expression quantity obvious drop, but joins z-LETD-fmk the caspase-3 expression gene expression quantity to drop not obviously. 6 Detecting caspse-3 and c-Jun gene expression respectively after joining brucine,brucine and JNK specific inhibitor SP600125 by RT-PCR show that brucine induce apoptosis in U266 cells by JNK signaling pathway with activating caspase-3. The result showed that JNK inhibitor significantly blocked by brucine on the caspase-3 and c-Jun actibvation their expression decreased significantly.Conclusion:1 Brucine inhibits proliferation of human multiple myeloma cells line U266.2 Brucine by inducing apoptosis to play its role in inhibiting the proliferation of U266 cells.3 Brucine can activate the expression of pro-apoptotic gene Bax, inhibition of anti-apoptotic Bcl-2 gene expression.4 Brucine by the death receptor pathway in turn activates caspase-8 activation of the apoptosis executor caspase-3 induced apoptosis in U266 cells.5 Brucine by JNK signaling pathway through phosphorylation of c-Jun induced apoptosis in U266 cells.