Homoharringtonine Induced Apoptosis In Multiple Myeloma Cells And Reduced Mcl-1 Expression

Objective: To investigate the effect of HHT on killing of Multiple myeloma cells and the expression of Mcl-1.Methods:The inhibitory effect of HHT(10,20,40 ng/ml)on proliferation of RPMI8226 cells was detected by CCK-8 method,trypan blue staining and colony formation assay.Annexin V-PE / 7AAD double staining flow cytometry was used to detect the apoptosis of co-culture RPMI8226 cells treated with 40ng/ml HHT 12 h or24h.The changes of mitochondrial membrane potential after 40ng/ml HHT on RPMI8226 cells was detected by flow cytometry.The cell cycle distribution of RPMI8226 cells before and after co-culture with HS-5 cells was detected by flow cytometry.The expression of p-AktT308,p-Akt S473,Mcl-1,Bcl-2,PARP,Caspase3 and Caspase9 in cells which treated with 40ng/ml HHT for 24 h were evaluated by Western blot.RT-qPCR was used to detect the level of mRNA in mono-culture and co-culture RPMI8226 cells and which treated with 40ng/ml HHT.The effect of40ng/ml HHT on the degradation of Mcl-1 protein in RPMI8226 cells was studied by actinomycin test and proteasome inhibitor.Results:1)The cycle distribution and the growth rate were changed in RPMI8226 cells adhesion co-cultured with human stromal cell line HS-5 cells.The G1-phase increased 9% and the S-phase decreased 12% and the growth rate was slowed down in co-cultured RPMI8226 cells compared with mono-cultured cells.2)HHT significantly inhibited the proliferation of mono-culture RPMI8226 cells in a dose-and time-dependent manner.However,the sensitivity to HHT decreased significantly after co-culturing.Treated with 20ng/ml HHT for 24 h,the inhibition rates of mono-cultured and co-cultured RPMI8226 cells were 63% and 31%,respectively.The colony formation capacity of RPMI8226 cells decreased after HHT treatment also in dose-dependent manner.3)HHT could induce the apoptosis of RPMI8226 cells.Treated with HHT for12 h and 24 h,the apoptotic rate of co-culture RPMI8226 cells were(11.16 ±0.78)% and(23.39±0.86)%,respectively.After treated for 24 h,the percentage of mitochondrial membrane potential decreased in co-cultured RPMI8226 cells was(19.20 ± 0.91)%.The cleavage of PARP,Caspase3 and Caspase9 and the down-regulated Mcl-1 protein were observed in RPMI8226 cells treated with HHT.However,there is no impact on the protein level of Bcl-2.4)HHT could also inhibit the proliferation of human primary MM cells.HHT inhibited the proliferation of primary MM in a dose-and time-dependent manner.The apoptosis was significant in the primary MM cells treated with HHT based on flow cytometry.The protein level of Mcl-1 were significantly decreased while the level of Bcl-2 was not changed.5)The expression of p-AktT308 and p-AktS473 were up-regulated in co-culture RPMI8226 cells.The expression of p-Akt S473 in RPMI8226 cells began to weaken after being treated with Akt-specific inhibitor LY294002 for 6 h.Compared with HHT treated alone,LY294002 combined with HHT significantly increased the inhibition rate and down-regulated the protein in co-cultured RPMI8226 cells.These data indicated that the PI3 K / Akt signaling pathway play an important role in reducing the sensitivity to HHT in co-cultured RPMI8226 cells.6)The protein level of Mcl-1 was significantly up-regulated in co-cultured RPMI8226 cells,and down-regulated in RPMI8226 cells after HHT treatment,however the mRNA level did not change significantly.The data suggests that the up-regulation and down-regulated of Mcl-1 is related to the regulation of translation.Both cycloheximide test and the proteasome inhibition assay showed that HHT could accelerate the degradation of Mcl-1 protein.Conclusion:HHT can induce apoptosis in Multiple myeloma cells and down-regulate Mcl-1 protein.