Celastrol Induce Apoptosis Of Human Multiple Myeloma Cells Involving Inhibition Of Proteasome Activity

Multiple Myeloma(MM)is a hematological malignant tumor with abnormal proliferation of plasma cells.With the rapid production of immunoglobulins,more protein misfolding events occurs,and the endoplasmic reticulum is under stress.To relieve this stress,MM cells are highly dependent on the proteasome to remove misfolded proteins and to maintain cellular homeostasis.At the same time,this feature makes MM cells a therapeutic target.For example,when the proteasome is inhibited by bortezomib,the misfolded proteins,which cannot be degraded,remain in the cytoplasm,leading to excessive endoplasmic reticulum stress and inducing apoptosis of MM cells.This is why MM cells are particularly sensitive to proteasome inhibitors.Although proteasome inhibitors are revolutionary drugs for the treatment of MM,they still have some limitations.For example,some patients are accompanied by primary drug resistance,or secondary drug resistance during drug treatment;the treatment window is narrow,and there are dose-limiting adverse reactions.Therefore,it is important but challenging to study on the mechanism of proteasome inhibitor resistance or to continue to discover new proteasome inhibitors in the field of MM.As a quinone methyl triterpenoid compound,Celestrol(Cel)has attracted wide attention due to its remarkable anticancer activity.One study found that Cel can inhibit proteasome activity in rabbit,which may be related to the inhibition of human prostate cancer cells.However,the above study lacks evidence directly based on human proteasome activity inhibition.Moreover,it is still unknown whether Cel can exert the same inhibition effect on human pr oteasome in tumor cells and transplanted tumor tissue.ObjectiveTo explain the anti-mm effect mechanism of Cel based on proteasome,so as to provide theoretical basis for future study on Cel.MethodsFirst,based on the AMC release value of the fluorescent substrates(Z)?LLE?AMC,Bz?VGR?AMC and Suc?Leu?Leu-Val?Tyr?AMC in the reaction system,the molecular and cellular inhibition activities of Cel on caspase-like(?1),trypsin-like(?2)and chymotrypsin-like(?5)subunits of 20S proteasome were measured.Further,the CCK-8method was used to evaluate the ability of Cel to inhibit growth of MM cells;Annexin V-PI double staining was used to detect the apoptosis-inducing activity of Cel on MM cells;Analysis of the blockage of cell cycle of Cel on MM based on PI staining;Finally,the MM xenograft model was used to detect the proteasome inhibitory activity of Cel on tumor tissues,and the apoptosis-inducing activity of Cel on tumor tissues was further detected by Annexin V-PI double staining,Western-Blot,H&E and TUNEL staining.The growth inhibitory activity of Cel on MM.1S and RPMI8226 xenografts was confirmed by continuous administration.Results1.The results of proteasome activity on molecular level showed that Cel inhibited the activity of purified human proteasome?1,?2,?5 subunits,and the median inhibition concentration(IC50)was 7.1,6.3 and 9.3?M,respectively.The detection of proteasome activity at cellular level showed that Cel inhibited the activity of proteasome?1,?2,?5 subunits on human MM cells with IC50 values of 2.3,2.1 and 0.9?M,respectively.2.Cel can significantly inhibit the proliferation of four MM cells,MM.1S,MM.1R,RPMI8226 and U266 with GI50 of 0.63,0.52,1.14 and 1.32?M,respectively.3.Cel significantly induced the apoptosis of MM cells.The apoptosis rates of MM.1S,MM.1R,RPMI8226 and U266 cells after Cel(500 nM)treatment for 8 hours were 35.5%,35.1%,25.6%and 40.9%,respectively;For the negative cells,the apoptosis rates were 4.6%,5.3%,12.8%,and 12.5%,respectively.At the same time,Western-Blot results showed that Cel(1000 nM)induced the formation of PARP apoptosis-activated fragment-the cleaved PARP in MM.1S and MM.1R cells.4.Cel blocked cell cycle of MM.1S,MM.1R in G0/G1 phase.MM.1S cells were treated with 250nM and 500 nM of Cel for 24 hours,and the percentage of G0/G1 phase were 49.7±1.9%and 64.6±1.3%,respectively,while for the negative control,G0/G1phase accounted for 40.8±0.4%;MM.1R cells were treated with 250 nM and 500 nM of Cel for 24 hours,the percentage of G0/G1 phase were 57.1±3.0%and 64.2±2.9%,respectively,while the percentage of the G0/G1 phase of the negative control was 39.2±1.8%.5.The results on MM xenograft model showed that Cel could inhibit the activity of?1,?2 and?5 subunits in the 20S proteasome;Cel could induce apoptosis in tumor tissues,which was accompanied by production of the cleaved caspase-3 and the cleaved PARP.The volume of MM.1S and RPMI8226 xenograft tumors in treatment groups were 1296.65±568.02 mm~3 and 789.10±117.51 mm~3,respectively.The control groups were 1831.88±949.24 mm~3 and 1522.15±569.04 mm~3,respectively.The tumor mass of treatment groups were 640.9±415.4 mg and 457.0±91.3 mg,respectively,while the control groups were 1046.0±570.0 mg and 918.4±289.3 mg,respectively.Conclusion1.The proteasome activity of?1,?2 and?5 subunits were detected on molecular and cellular levels,and it has confirmed that Cel inhibited the activities of the proteasome?1,?2 and?5 subunits simultaneously.2.Cel significantly inhibited the proliferation of the four MM cells(MM.1S,MM.1R,RPMI8226 and U266)in a dose-dependent manner.3.Celastrol significantly induced apoptosis in MM.1S,MM.1R,RPMI8226 and U266 cells,and induced the formation of PARP apoptosis-activated fragment-the cleaved PARP in MM.1S and MM.1R cells.4.Cel blocked cell cycle of MM.1S,MM.1R in G0/G1 phase.5.Cel inhibited the activity of the?1,?2 and?5 subunits of 20S proteasome in tumor tissues;Cel induced apoptosis in tumor tissues,which was accompanied by the formation of the cleaved Caspase-3 and the cleaved PARP;Cel inhibited the growth of MM.1S and RPMI8226 xenograft tumors.