The Effect Of Lysosome On The Autophagy Induced By Bortezomib In MM Cells And Its Mechanism

Objective:To explore the effect of bortezomib on the proliferation and autophagy level of multiple myeloma cell lines,and further study on the effect of siramesine combined with bortezomib on the proliferation and autophagy of multiple myeloma cell lines.Methods:1 Cell culture: human multiple myeloma cell lines RPMI8226 and U266 were cultured in vitro.2 CCK-8 assay was used to detect cell activity:The proliferation inhibition rates of different concentrations of bortezomib(3.0,6.0,9.0,12.0nM)and different concentrations of siramesine(0.5,1.0?M)for RPMI8226 and U266 cell lines at different time(6h,12 h,24h)were detected.Combined effect of different concentration gradient of two drugs on the proliferation inhibition of multiple myeloma cell lines at 24 h were also detected.3 Real-time quantitative PCR(RT-qPCR)was used to detect the autophagy related factors LC3 B,Beclin-1,p62,LAMP1's mRNA expression.The experiment was divided into 4 groups: blank group,Bortezomib group(6nM),Siramesine group(1.0?M),Bortezomib combined with Siramesine(Bortezomib-Siramesine)group.The relative expression of autophagy related factors' mRNA were detected in each group after treatment of 24 h.4.Western blot(W-B)was used to detect the autophagy related factors LC3 B,Beclin-1,p62,LAMP1's protein expression.The experiment was divided into 4 groups: blank group,Bortezomib group(6nM),Siramesine(1.0?M),Bortezomib combined with Siramesine(Bortezomib-Siramesine)group.The relative expression of autophagy related factors' protein was detected in each group after treatment of 24 h.Results:1 Proliferation inhibition rates of multiple myeloma cell lines RPMI8226 and U266 were detected by CCK-8 methodThe proliferation inhibition rates of RPMI8226 and U266 cell lines treated with different concentrations of Bortezomib(3.0,6.0,9.0,12.0nM)for different time(6,12 and 24h)were shown in Fig 1,Fig 2 and Tab 1,Tab2.With the increase of bortezomib concentration and the prolongation of treatment time,the proliferation inhibition rates increased gradually(P<0.05).The inhibition rates of proliferation of MM cell lines in combination of two drugs at 24 h was shown in Tab 3 and Tab 4.We found a synergistic inhibitory effect(P<0.05)on the proliferation of these multiple myeloma cell lines by combination of bortezomib and siramesine.As a result of the observation of autophagy,we selected the concentration of bortezomib at 6.0nM,siramesine at 1.0?M and treated time at 24 h for subsequently experiments.2 RT-qPCR and Western Blot were used to analyze the autophagy related factors' mRNA and protein expression in multiple myeloma RPMI8226 and U266 cell lines2.1 The expression of autophagy related factors' mRNA and protein in multiple myeloma cell lines by bortezomib group: Bortezomib(6nM)treated multiple myeloma cell lines RPMI8226 and U266 after 24 h,compared with the control group,the expression of mRNA and protein of autophagy related factors LC3 B,Beclin-1,LAMP1 increased,p62 decreased,and the differences are statistically significant(P<0.05).Bortezomib can promote the increase of autophagy in multiple myeloma RPMI8226 and U266 cell lines.(All the results are shown in Fig 3,4,5,6 and Tab 7,8,9,10)2.2 The expression of autophagy related factors' mRNA and protein in multiple myeloma cell lines by bortezomib-siramesine group:Bortezomib-siramesine treated multiple myeloma cell lines RPMI8226 and U266 after 24 h,compared with the bortezomib group,the expression of mRNA and protein expression of autophagy related factors LC3 B,Beclin-1,LAMP1 increased,p62 decreased,and the difference between each group is meaningful(P<0.05).Bortezomib can increase the expression of autophagy in multiple myeloma RPMI8226 and U266 cell lines.Siramesine can promote the further increase of bortezomib autophagy.(All the results are shown in Fig 3,4,5,6 and Tab 7,8,9,10)Conclusion:1 Bortezomib has inhibitory effect on the proliferation of multiple myeloma cell lines RPMI8226 and U266,which is associated with the enhancement of the autophagy expression induced by bortezomib;2 Synergistic inhibitory effect of bortezomib combined with siramesine on the proliferation of cell lines RPMI8226 and U266 is related to the further enhancement of the expression of autophagy induced by siramesine.