The Effect And Mechanism Of Compound Huangdai Tablet Combination On NB4 Apoptosis And Mutant PML-RAR?

BackgroundAcute promyelocytic leukemia(APL)is a subtype of acute myeloid leukemia,and 90%of patients with APL have a promyelocytic leukemia protein(PML)-retinoic acid receptor?(RAR?)fusion gene is positive and drives the development of APL.All-trans retinoic acid and arsenic trioxide can cure 90%of APL patients by targeting PML-RARa,which is an advantageous disease in traditional Chinese medicine(TCM).According to TCM,"poisonous evil" plays a key role in the development of APL,and "poison damage to bone marrow" is the key pathogenesis of APL.Realgar-Indigo naturalis Formula(RIF)was developed by Prof.Huang Shilin of the 967th Hospital of the People's Liberation Army(PLA)according to the characteristics of APL pathogenesis,with the treatment principle of "expelling evil and restoring righteousness" and "detoxifying The formula contains four drugs,namely realgar,natural indigo,radix salviae miltiorrhizae and heterophylly falsestarwort root.Clinical studies have confirmed the efficacy of RIF in APL,and it is not inferior to arsenic trioxide.However,the basic study of RIF is not sufficient and further research is needed.In addition,it was found that patients with relapsed APL have missense mutations in PML-RARa that lead to arsenic resistance,and it is important to study how to overcome the problem of APL resistance caused by missense mutations to further improve the clinical efficacy of APL.Objectives1.To observe the effects of RIF components and their combinations on the cell viability and apoptosis rate of human acute promyelocytic leukemia cells NB4,as well as the changes of apoptosis-related regulatory factors,and to understand the mechanism of the synergistic antiAPL effects of RIF components in vitro.2.To observe the effects of RIF components in combination with realgar alone on the general condition,tumor and important organs of APL-bearing mice,and to evaluate the antiAPL effect of RIF components in vivo.3.Seamless cloning was used to construct a missense mutant PML-RARa high expression lentiviral plasmid for subsequent studies.4.To observe the effect of RIF component pairing on the level of missense mutation PMLRARa and the possible mechanism to explore the potential of RIF in reversing the problem of missense mutation-associated arsenic resistance in APL.Methods1.In vitro observation of the effect and mechanism of RIF components in combination with anti-APLThe effects of As4S4,indirubin,tanshinone ?a and radix pseudostellariae saponin on the viability of NB4 cells and the IC50 of each component were observed by CCK-8 method,andNB4 cells were treated with different concentrations of each component and their combinations according to the IC50 results.Western blotting was performed to detect the changes of Caspase9,Caspase-3,Cleaved-Caspase-3,PARP-1,p-Akt,p-mTOR and LC3B levels.2.In vivo observation of the effect and mechanism of RIF components paired with antiAPLThe transplantation tumor model of APL in nude mice was established by subcutaneous injection of NB4 cells,and randomly divided into model group,AS4S4 group,and RIF group.The AS4S44 group was treated with 10 mg/kg AS4S4 intraperitoneally daily,the RIF group was treated with 10 mg/kg As4S4,20 mg/kg indirubin,20 mg/kg tanshinone ?a and 20mg/kg radix pseudostellariae saponin intraperitoneally daily,and the model group was treated with equal volume of 0.9%NaCl solution daily for 14 days.During this period,the general status changes were observed daily,and the volume of transplanted tumor was weighed and measured every other day.At the end of the administration,the tumors were removed from the necks and killed,and the tumor bodies were peeled and weighed to calculate the tumor suppression rate,and the changes of caspase-3,Cleaved-Caspase-3 and PARP-1 were detected by Western blotting.3.Construction of missense mutant PML-RARa lentiviral overexpression plasmidThe mRNA sequences of PML and RARa and their expression in each tissue were obtained by searching the database and literature,designing primers,selecting suitable cell line mRNA,reverse transcribing into cDNA,PCR to obtain the target fragment,inserting the missense mutation by seamless cloning,and then linking PML and RARa to the vector plasmid,transforming,picking single clones,sequencing,and testing the eukaryotic expression efficiency of the plasmid.4.Effect of RIF component conjugation on the level of missense mutant PML-RARa and mechanismA lentivirus was used to construct a stable HL-60 stably transformed strain expressing PML-RARa(HL-60 is an acute myeloid leukemia cell not expressing PML-RARa can exclude wild-type PML-RAR? interference).The transfected HL-60 cells were treated with the combination of tetra-arsenic sulfide,indirubin and tanshinone ?a,and the changes of PMLRAR?,p-mTOR and LC3B levels were observed by Western blotting.The changes of PMLRARa levels after blocking proteasome pathway or autophagy pathway were observed by combining proteasome inhibitor carfilzomib and autophagy inhibitor bafilomycin A1 on the basis of component pairing.Results1.In vitro observation of the effect and mechanism of the RIF components paired against APLThe four components As4S4,tanshinone ?a,indirubin and radix pseudostellariae saponin inhibited the viability of NB4 cells in a dose-dependent manner,with IC50s of As4S4:19.76?mol/L,tanshinone ?a:31.25 ?mol/L,indirubin:180.7 ?mol/L and radix pseudostellariae saponin:382.1 mg/L.Gimsa staining showed that As4S4 The effect of As4S4 and tanshinone ?a on the morphology of NB4 cells was shown to be dose-dependent,while the effect of indirubin at high concentrations caused apoptotic changes in NB4 cells.The flow-through Annexin V/PI assay showed that As4S4,tanshinone ?a,indirubin and radix pseudostellariae saponin induced apoptosis of NB4 cells in a dose-dependent manner,with As4S4 and tanshinone ?a inducing apoptosis of NB4 cells,followed by indirubin,and radix pseudostellariae saponin induced apoptosis in an insignificant manner.Western blotting showed that As4S4,tanshinone ?a and indirubin all down-regulated Caspas-9,Caspase-3,p-Akt and p-mTOR and up-regulated Cleaved-Caspase-3 and Cleaved-PARP-1 in a dose-dependent manner,while radix pseudostellariae saponin up-regulated Cleaved-Caspase-3 and Cleaved-PARP-1 in a dosedependent manner.Cleaved-Caspase-3 was up-regulated by radix pseudostellariae saponin in a dose-dependent manner,and the remaining factors were not significantly changed.The combination of different components of arsenic tetrasulfide,tanshinone ?a and indirubin treated NB4 cells showed that the combination of components synergistically promoted apoptosis in NB4 cells,and Caspase-9,Caspase-3,p-Akt,p-mTOR,upregulated CleavedCaspase-3,Cleaved-PARP-1 and LC3B.The combination of As4S4,tanshinone ?a and indirubin showed the most obvious trend.2.In vivo observation of the anti-APL effect and mechanism of the RIF components in combinationThe general condition of the nude rats in all groups was normal and no mortality occurred during the administration treatment.The body weight of the nude rats in all three groups maintained an increase from d 0-8 after administration,while the body weight of the nude rats in the model group decreased on average from d 10-14 after administration,with an increase in variance,but the difference was not statistically significant when compared with the other two groups.The tumor growth rate in the RIF group was smaller than that in the tetra-arsenic tetrasulfide group,and the tetra-arsenic tetrasulfide group was smaller than that in the model group.The RIF group showed an increase in Cleaved-PARP-1 and Cleaved-Caspase-3 in comparison with the control group.No significant morphological changes were observed in the HE staining of the heart,liver and kidney of the nude mice in each group.3.Construction of a missense mutant PML-RARa overexpression plasmidThe PMLWT-RAR?,PMLS214L-RAR?,PMLA216T-RAR?,PMLA216V-RAR?,PMLL217FRAR? and PMLS220G-RAR? plasmids were successfully constructed by seamless cloning,and the sequencing results showed that the sequences and mutation sites were as expected,and all of them could express the corresponding PML-RAR? protein in eukaryotic cells.4.Effect of RIF component pairing on the level of missense mutant PML-RAR? and its mechanismA HL-60 cell line was constructed to stably express the missense mutation PML-RAR?.After treatment with a certain concentration of As4S4(0-8 ?mol/L)for 24 h,the PML-RARa protein in HL-60 cells all showed different degrees of decrease,and the decrease was PMLS214LRAR??PMLA216T-RAR??PMLA216V-RAR?<PMLL217F-RAR?<PMLS220G-RAR? The combination of AS4S4,tanshinone ?a and indirubin was seen to significantly decrease PMLA216T-RARa levels,accompanied by a decrease in p-mTOR and an increase in LC3B.The combination of carfilzomib did not block the effect of the combination of AS4S4,tanshinone ?a and indirubin in decreasing PMLA216T-RAR? levels,but the combination of bafilomycin A1 melted the effect of the component combination.Conclusion1.As4S4,tanshinone ?a and indirubin in the components of compound huangdai tablets have strong anti-APL activity,while the direct anti-APL activity of radix pseudostellariae saponins is not strong.The combination of As4S4,tanshinone ?a and indigo red exerted a synergistic antiAPL effect by inhibiting Akt/mTOR-induced mitochondrial apoptosis in NB4 cells.2.Treatment with both RIF components and As4S4 alone could inhibit tumor growth in APLbearing nude mice,but the effect of RIF components in combination was stronger than that of As4S4 alone,indicating that RIF components in combination exerted synergistic anti-APL effects in vivo.3.Seamless cloning technology can efficiently construct missense mutant PML-RAR?overexpression plasmids.4.The sensitivity of missense mutant PML-RARa to tetrasulfuration arsenic was decreased,and the down-regulation of missense mutant PML-RAR? by tetrasulfuration arsenic could be enhanced by the combination of tanshinone ?a and indirubin,and the mechanism was related to the activation of autophagy.