Study On The ART Induced Apoptosis And Autophagy Of H929 And U266 Cells Via PI3K/Akt/mTOR Pathway In Vitro

Background and ObjectiveMultiple myeloma(MM)is a malignant tumor characterized by abnormal proliferation of monoclonal plasma cells,which secretes a large number of monoclonal immunoglobulins.Targeting PI3K/Akt/mTOR signaling pathway has attracted wide attention in MM.It has been confirmed that the sensitivity of different phenotypes of Akt myeloma cell lines to specific targeted drugs is different.Previous studies have confirmed that compared with U266 cells,the baseline p-Akt level of H929 cells is higher,while Oridonin(ORl)can significantly reduce the phosphorylation level of PI3K upstream of Akt.And the expression of p-Akt(s).Artesunate(ART)is a kind of highly effective and lower toxic antimalarial drug.It has also been found that ART has anti-tumor activity.Therefore,this study investigated the effects of certain concentration of ART on the proliferation,apoptosis and autophagy of human myeloma cell lines H929 and U266,and explored its possible molecular mechanism and the effect of baseline Akt level on the proliferation of human myeloma cell lines.Methods1.MTT assay was used to detect the inhibitory effect of 0,1,2,5,10,20,40?g/ml ART on the proliferation of H929 and U266 cells after treatment for 12,24,36,48,72 hours.2.Flow cytometry and TUNNEL+DAPI staining was used to detect the apoptosis rate of H929 and U266 cells after 0,2,10,20?g/ml ART treatment for 48 hours.3.Western blot assay was used to detect the effect of 0,2,10,20?g/ml ART on the expression of apoptosis,autophagy,PI3K/AKT/mTOR pathway associated protein in multiple myeloma cell lines.4.Western blot assay was used to detect the effect of 1?g/ml 3-MA.1?g/ml?RAPA?20?g/ml ART?1?g/ml 3-MA+20?g/ml ART on the expression of apoptosis,autophagy,PI3K/AKT/mTOR pathway associated protein in multiple myeloma cell lines.Results1.The proliferation of H929 and U266 was significantly inhibited after treatment with ART,and the effect was proportional to time and concentration.The inhibition rates of H929 cells were 20.82%±1.64%,31.75%±1.70%,38.92%±2.15%,and that of U266 cells were 53.95%±1.58%,61.79%±1.40%,72.21%±1.83%respectively after treated with 2,10 and 20?g/ml ART for 48 hours.2.Compared with blank group,nuclear shrinkage and disintegration of H929 and U266 cells were observed after 2,1 0,20?g/ml ART treatment for 48h and showed a significant increase.The percent of TUNEL positive cells were 4.50%±0.26%,20.46%±1.49%,44.56%±1.27%in H929 cells and 10.25%±0.79%,40.67%±2.89%,70.79%±2.11%in U266 cells,respectively.The early aopototic rate were 12.2%±0.75%,19.46%±0.68%,44.30%±0.81%in H929 cells and 19.50±0.80%,21.60%±0.88%,56.67%±11.46%in U266 cells,respectively.3.2,10,20?g/ml ART up-regulated cleaved caspase-3,cleaved PRAP,p62,LC3B,Beclinl expression and down-regulated p-Akt?p-mTOR expression in two cell lines,while there is no significant change in PI3K p110?,suggesting that ART could inhibit the phosphorylation of Akt and mTOR and enhance the level of autophagy.4.Compared with ART group,ART+RAPA group up-regulated the expression of cleaved caspase-3,cleaved PRAP,Beclinl and LC3B and the expression of p-AKT and p-mTOR was completely inhibited,suggesting that ART could induce autophagic cell death.Conclusion2?10?20?g/ml ART can effectively inhibit the proliferation and induce apoptosis of H929 and U266 cells in vitro.The possible molecular mechanism is related to down-regulation of PI3K/Akt/mTOR signaling pathway.ART can also enhance the autophagy of the two cells promoting apoptisis.