The Role Of Autophagy On Survival And Proliferation Of Multiple Myeloma Cells In Vitro And Its Possible Mechanism

Objective: To investigate the effects of rapamycin and3-MA on the survival andproliferation of multiple myeloma (MM) cells under serum-free and normal cultureconditions, and its correlation with autophagy.Methods: Multiple myeloma (MM) cell line U266was incubated under serum-freeand normal culture condition containing rapamycin or3-MA respectively. U266cells,lymphoma cell Jurkat and serum-free cultured Jurkat cell were used as control group. Theproliferation and apoptosis of cells were determined by CCK8assays and flow cytometryrespectively. MDC staining were employed to detect the autophagy. The expression of mtor,Beclin1and Atg5of U266cells were assayed by RT-PCR. In the meantime, western blotwas employed to detect the expression of autophage related protein LC3Ⅰ/ⅡandLAMP1.Results: The autophagy level in U266cells was higher than those with the Jurkat.Sera starvation increased the level of autophagy and promoted U266cells to proliferate forabout72h. U266cells apoptosis increased significantly after24h incubation in mediumwithout Sera as compared with normal cultured cells1.7±0%and16.1+0.35%(P <0.05).Rapamycin upregulated autophagy of the U266cells and stimulated their proliferation.3-MA had the same effect on U266cells, although it was on a short time. Both Rapamycinand3-MA could inhibited the serum-free induced apoptosis for24hour and the ratio ofcells apoptosis decreased to16.1±0.35%,6.2±0.76%and6.7±0.0.46%(P<0.01)respectively, but the effects elapsed with time. Cell apoptosis was at the same level after72hour incubation.30.0±1.87%,28.3±0.60%, and29.0±0.90%(P>0.05). However, the cellscould revive when they were incubated in nromal culture condition. Rapamycin or3-MA could induce cell apoptosis when incubated in nromal culture condition for24hour ascompared with normal cultured cells (1.7±0.2%,19.1±1.0%, and16.9+0.46%). Cellapoptosis has no difference for Rapamycin cultured cells (16.9±0.46%), but dead cell rateraised significantly after72hours incubation (14.0±0.98%and21.5±1.15%, P<0.01). Theapoptosis rate of3-MA cultured cells decrased after72hours incubation (9.0+0.70%)Conclusion: It is important for MM cells to maintain a level of autophagy which washigher than those with the Jurkat cells. Both Rapamycin and3-MA could induce the U266cells to apoptosis and decrease cell proliferation under normal culture condition. Up-regulated autophagy promoted survival and proliferation of U266cells under serastarvation. Rapamycin strengthened this effect through inhibition of mTOR protein, but theproliferation decreased when mTOR protein were excessive inhibited.3-MA had dualeffects on cell autophagy.