| BackgroundMultiple myeloma(MM)is a malignant tumor of the blood system.It is still incurable and has a poor overall prognosis.It belongs to plasma cell abnormal malignancyclonal disease.Although new western medicines and autologous hematopoietic stem cell transplantation(ASCT)have improved the prognosis of MM patients.However,great economic pressure and adverse drug reactions affect the prognosis of many MM patients.Ultimately,they still face the risk of relapse and refractory.Therefore,it is urgent to develop new drugs to improve the prognosis and quality of life of myeloma patients.Natural products such as arsenic trioxide,homoharringtonine and so on play a crucial role in the treatment of hematological tumors.Formosanin C(FC)is an anti-tumor natural compound,which is extracted from the traditional Chinese medicine Chonglou.The phosphoinositide 3-kinase/protein kinase B(PI3K/AKT)signaling pathway has cross-activation with other several pathways.And it is related to MM drug resistance.So,in many clinical or basic experimental studies,the PI3K/AKT pathway is usually regarded as an entry point to explore the therapeutic mechanism of MM.The downstream target gene-mammalian target of rapamycin(mTOR)can inhibit autophagy,which is related to the incidence of myeloma.ObjectiveBased on the proven anti-tumor activity of FC and the important role of the PI3K/AKT pathway in MM,this study investigated the effects of certain concentration FC on the proliferation,autophagy and apoptosis of myeloma cell lines(H929 and ARP1)in vitro,and explored the the role of PI3K/AKT/mTOR signaling pathway in these effects.Methods1.CCK-8 was taken to detect the influences of 0,0.5,1.0,1.5,2.0,2.5,3.0,3.5 μM FC on the proliferation of myeloma cells H929 and ARP1 after treatment for 24 h.Statistic software was used to calculate the IC50 concentration of MM cells after FC intervention.2.CCK-8 was used to calculate the OD value at 450 nm or viability in H929 and ARP1 cell,after they were treated with 2μM FC for 12,24,36,48 h or IGF-1(100 ng/mL)pretreatment for 1 h,and then 2 μM FC treatment for 24 h.3.Colony formation experiment was applied to evaluate the colony-forming ability of H929 and ARP 1 cells,after they were treated with 2 μM FC for 24 h.4.Flow cytometry(FCM)was used to detect the apoptosis rate of H929 and ARP1 cells,after they were dealed with 2 μM FC for 24 h.5.H929 and ARP1 cells were pretreated with 2 μM FC or 3-MA for 2 h/IGF-1 for 1h and then treated with 2 μM FC for 24 h.WB was used to detect apoptosis-related proteins,autophagy-related proteins and PI3K/AKT/mTOR signaling pathway-related proteins in MM cells.6.The fluorescence microscope was used to count the number of LC3 puncta structures in each MM cell to evaluate the effect of FC on autophagy in H929 and ARP1 cells,after MM cells infected with Ad-GFP-LC3 were treated with 2 μM FC for 24 h.7.Network pharmacologic analysis was used to preliminarily explore the anti-MM mechanism of FC.8.AmpliteTM fluorescence assay was used to measure the chymotrypsin-like proteasome activity in MM cells,after H929 and ARP1 cells were treated with 2 μM FC,MG-132 or bortezomib(BTZ)for 24 h.Results1.The proliferation of H929 and ARP1 cells was effectively inhibited after MM cells were dealed with 0.5,1.0,1.5,2.0,2.5,3.0,3.5 μM FC for 24 h,and the inhibitory effect of FC on MM cells was concentration-dependent.The IC50 value of FC was 1.64 μM in H929 cells while it was 1.68 μM in ARP1 cells.Therefore,2 μM was chosen as the FC concentration in following studies.2.The increase of OD value of MM cells at 450 nm was significantly inhibited,after H929 and ARP1 cells were treated with 2 μM FC alone for 12,24,36 and 48 h(P<0.05).IGF-1(100 ng/ml)could weaken the inhibitory effect of FC on the viability of H929 and ARP1 cells to a certain extent.3.After 2 μM FC interventing H929 and ARP1 cells for 24 h,the number of clone formation of FC group was significantly reduced compared with the control group(Con group)(P<0.05).4.FCM showed that:after 2 FC interventing H929 and ARP1 cells for 24 h,the apoptosis rate of FC group was apparently increased compared with Con group(P<0.05).5.The WB result shows:after the intervention of 2 μM FC alone in H929 and ARP1 cells for 24 h,the expression-of Bax and Cleaved caspase-3 in FC group was increased,while the expression of anti-apoptosis protein(Bcl-2)was greatly decreased(P<0.05),compared with the Con group.But 3-MA(10 μM)effectively reduced the the promotive effect of FC on LC3-II,Beclin 1,Bax and Cleaved caspase-3 and the inhibitory effect of FC on anti-apoptosis protein(Bcl-2).6.When H929 and ARP1 cells infected with Ad-GFP-LC3 were exposed to 2 μM FC for 24 h,the number of LC3 puncta structures in FC group was apparently elevated compared with the Con group(P<0.05).Meanwhile,WB showed that the expression of LC3-Ⅱ and Beclin 1 in FC group was elevated compared with that in Con group,after the intervention of 2 μM FC in H929 and ARP 1 cells for 24 h(P<0.05).7.The KEGG pathway enrichment analysis showed that there were 16 intersection genes between the targets of Chonglou and myeloma,and PI3K/AKT signaling pathway was an important anti-MM pathway of Chonglou.8.After H929 and ARP1 cells were treated with 2 μM FC alone for 24 h,WB showed that:compared with the Con group,the expression of p-PI3K,p-AKT and p-mTOR in FC group was inhibited(P<0.05).While PI3K/AKT/mTOR pathway was activated,IGF-1(100 ng/mL)effectively attenuated the promotive effect of FC on LC3-II,Beclin 1,Bax,Cleaved caspase-3 and the inhibitory effect of FC on anti-apoptosis protein(Bcl-2)(P<0.05).9.FCM and CCK-8 showed that after 24 h intervention with 2 μM FC in H929 and ARP1 cells,the apoptosis rate of FC group was apparently elevated and cell viability was greatly inhibited compared with the Con group.Compared with FC group,the apoptosis rate of FC+IGF-1 group was declined,and cell viability was brought back(P<0.05).10.After 2 μM FC intervention in H929 and ARP1 cells for 24 h,the results of AmpliteTM fluorescence showed that compared with the Con group,the cellular chymotrypsin-like proteasome activity in FC group was not greatly inhibited,while in MG-132 group and BTZ group,it was greatly inhibited(P<0.05).ConclusionFC of a certain concentration can effectively inhibit the viability and proliferation of myeloma cell lines(H929 and ARP1)in vitro,and induce the apoptosis of cells.The inhibition of PI3K/AKT/mTOR pathway triggers excessive autophagy,which is an important anti-MM mechanism of FC.Furthermore,the inbibition of FC on MM cells does not involve its effect on cellular chymotrypsin-like proteasome activity.This study provides a scientific basis for FC as a potential therapeutic drug against MM,and explores a new strategy to treat MM from-the effective ingredients of Chinese herbal medicine. |
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