Study On The Mechanism Of Action Of The Combination Of Compound Huangdai Tablets In Reversing Arsenic Resistance In AP

BackgroundAcute promyelocytic leukemia(APL)is a subtype of acute myeloid leukemia,and 90%of APL patients have positive promyelocytic leukemia protein(PML)-retinoic acid receptor a(RARα)fusion gene,which drives the development of APL.All-trans retinoic acid and arsenic trioxide can cure 90%of APL patients by targeting PML-RARα,but 5%-10%of patients still relapse due to drug resistance.APL belongs to the category of "urgent labor" in traditional Chinese medicine(TCM).TCM believes that "toxic evil" plays a key role in the occurrence and development of APL,and "toxic injury to the bone marrow" is the key pathogenesis of APL.Compound Realgar-Indigo naturalis Formula(RIF)was developed by Professor 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 qi".The formula contains four drugs,namely realgar,natural indigo,radix salviae miltiorrhizae and heterophylly falsestarwort root.Clinical studies have confirmed that RIF has an exact effect on APL and is not inferior to arsenic trioxide.However,there is no relevant research on reversing arsenic resistance.The treatment plan of RIF combined with alltrans retinoic acid is already the first-line treatment plan of the domestic APL guidelines.Based on clinical problems and preliminary research,this study will further clarify the efficacy of RIF on arsenic-resistant APL,and explore its mechanism,in order to further improve the clinical efficacy of APL,and lay foundations for researches.Objectives1.The HL60-PMLA216V-RARα cell line stably expressing the arsenic-resistant plasmid PMLA216V-RARα was established by lentivirus transfection.2.To observe the effect and mechanism of different combinations of RIF:tetraarsenic tetrasulfide,indirubin+tanshinone Ⅱa+total saponins of Radix Pseudostellariae and the above four components on the level of PMLA216V-RARα in arsenic-resistant HL60-PMLA216V-RARαcells,to clarify the mechanism by which RIF reverses missense mutation-related APL arsenic resistance.3.To observe the different combinations of RIF components:tetraarsenic tetrasulfide,indirubin+tanshinone Ⅱa+total saponins of Radix Pseudostellariae and the above four components on the general condition,tumor and important organs of arsenic-resistant APL tumor-bearing mice,to clarify the mechanism of RIF reversing arsenic resistanceMethodsThe active ingredient of realgar tetraarsenic tetrasulfide(A),natural indigo’s active ingredient indirubin(Ⅰ),danshen root’s active ingredient tanshinone Ⅱa(T),and total saponins’s active ingredient total saponins of Radix Pseudostellariae(S),were selected for experiments.And the components were combined to investigate the mechanism of RIF reverses missense mutations in the treatment of arsenic-resistant APL.1.Establish of the drug-resistant APL cell line HL60-PMLA216V-RARαThe arsenic-resistant PMLA216V-RARα plasmid was transformed,extracted,PCR and verified by sequencing.The arsenic-resistant cell line HL60-PMLA216V-RARα stably expressing PMLA216V-RARα was established by lentiviral transfection.The transfection efficiency was verified by fluorescence,and the drug resistance efficiency was verified by comparing the IC50 values of each active ingredient against HL60-PMLA216V-RARα and HL60 cells.2.Study on the mechanism of anti-HL60-PMLA216v-RARα in the combination of different components of RIF to reverse arsenic resistanceThe study was divided into tetraarsenic tetrasulfide group(A group),indirubin+tanshinone Ⅱa+total saponins of Radix Pseudostellariae(ITS group),tetraarsenic tetrasulfide+indirubin+tanshinone Ⅱa+total saponins of Radix Pseudostellariae(AITS group)and blank control group(control group)treated with HL60-PMLA216V-RARα,the cell viability at 0,24,48,72,96 h was detected by CCK-8 method,the cell morphology was detected by Hoechst,the mitochondrial membrane potential(early apoptosis)was detected by JC-1,Annexin V/PI double staining was used to detect cell apoptosis,flow cytometry to detect CD33 expression in leukemia cells,Western blotting to observe the effect of A on PML-RARα,and to explore the arsenic concentration when the components were combined.The levels of PML-RARα,PARP1,caspase3,Bcl-2,PI3K,mTOR,p-mTOR,AKT,p-AKT,p62 and LC3B in the control group,A group,ITS group and AITS group were observed by Western blotting.Combined with PI3K inhibitor LY294002 and autophagy inhibitor bafilomycin A1(Baf A1)on the basis of composition combination to observe the changes of PML-RARα,mTOR,caspase3,Bcl-2,p62 levels after blocking PI3K or autophagy pathway,and also,the changes of PML-RARα levels were observed by serum starvation-induced autophagy and to observe autophagy by transmission electron microscope.3.Study on the mechanism of anti-HL60-PMLA216V-RARα tumor-bearing nude mice in combination of different RIF components to reverse arsenic resistanceSubcutaneous injection of HL60-PMLA216V-RARα cells to establish arsenic-resistant APL nude mice xenograft model.They were randomly divided into model group,A group,ITS group and AITS group,and another healthy control group was set up.A group was given intraperitoneal injection of A 50 mg/kg daily,ITS group was given daily intraperitoneal injection of I,T,S 20 mg/kg each,AITS group was given A 50 mg/kg,I,T,S 20 mg/kg each daily by intraperitoneal injection,the model group and the healthy control group were intraperitoneally injected with the same volume of vehicle every day for 14 days.During the period,the general state changes of tumor-bearing nude mice were observed,and the volume of the transplanted tumor was measured every other day.After the administration,the mice in each group were sacrificed by de-neck,and the tumors were stripped and weighed to calculate the tumor inhibition rate.The levels of caspase-3 and Bcl-2 in the tumors were detected by Western blotting and the changes of ki67 and caspase3 were detected by immunohistochemistry.The heart,liver,spleen,lung and kidney were isolated and stained with HE to observe the morphological changes.Results1.Establish of the drug-resistant APL cell line HL60-PMLA216V-RARαThe successful insertion of PMLA216V-RARα into HL-60 cells was verified by colony PCR and sequencing,and the successfully transformed strains were verified for seed preservation.Puromycin was used for screening,and more than 90%of the cells were observed under a fluorescence microscope to emit specific green fluorescence.The transfection efficiency was high,and the stable expression cell line HL60-PMLA216V-RARα was successfully established.The cell viability test of HL60-PMLA216V-RARα and HL-60 cells showed that the IC50 values of HL60-PMLA216V-RARα and HL-60 against A were 264.9000 μM vs.0.9386 μM,and the drug resistance fold increased by 282 times.2.Study on the mechanism of anti-HL60-PMLA216V-RARα in the combination of different components of RIF to reverse arsenic resistanceThe cell viability of HL60-PMLA216V-RARα cells in control group,A group,ITS group and AITS group was detected at 0,24,48,72 and 96 h.All groups inhibited the growth of HL60-PMLA216V-RARα cells.The IC50 value of AITS group was significantly lower than the other groups,and the inhibitory effect on HL60-PMLA216V-RARα was AITS group>ITS group>A group>control group.After Hoechst staining,the cell morphology was observed under a fluorescence microscope.The cells in the control group had a complete morphology,a small amount of apoptosis in the A group,and a large number of apoptosis and fragmentation in the AITS group.The mitochondrial membrane potential was observed by JC-1 staining.The AITS group had the most obvious decrease in mitochondrial membrane potential,indicating the early apoptosis accrued.The mitochondrial membrane potential showed a trend of AITS group<ITS group<A group<control group.Annxin V/PI detection of cell apoptosis showed that the cell apoptosis rate was 10.04%in the control group,15.19%in the A group,12.46%in the ITS group,and 28.05%in the AITS group,with significant differences among the groups(P<0.0001).The expression of CD33 by flow cytometry showed that the proportion of CD33 was 79.72%in the control group,79.19%in the ITS group,28.60%in the A group,and 24.46%in the AITS group,with significant differences between the groups(P<0.001 or P<0.0001).When the concentration of A was 150 μM and 300 μM,the expression levels of caspase3 and Bcl-2 decreased,and the difference between the groups was statistically significant(all P<0.0001),and the apoptosis-promoting effect was concentration-dependent.When the concentration of A was 300 μM,the degradation of PML-RARα was statistically significant compared with the control group(P<0.0001).Compared with the control group,the ITS and AITS groups reduced the levels of PML-RARα protein and caspase3(P<0.05 or P<0.0001),and the AITS group was better than the A and ITS groups(both P<0.0001).In the ITS and AITS groups,the levels of Bcl-2,PI3K,mTOR,P-mTOR,AKT,p-AKT and p62 were decreased,and the levels of cleaved-PARP-1 and LC3B were increased(P<0.001 or P<0.0001).After combined with PI3K inhibitor LY294002 or autophagy inhibition of Bafilomycin A1(Bafilomycin A1,Baf A1),compared with the control group,AITS reduced the protein expression of PML-RARα,the levels of caspse3 and Bcl-2,and the levels of mTOR and autophagy-related factor p62(all P<0.0001).When AITS combined with PI3K inhibitor LY294002,compared with AITS group,the levels of PML-RARα,caspse3,Bcl-2 and p62 were increased,and the level of mTOR was decreased(all P<0.0001).When AITS combined with autophagy inhibitor Baf A1,compared with AITS group,the levels of PML-RARα,caspse3,Bcl-2 and p62 were also increased(all P<0.0001),and the mTOR level was increased(P<0.001).3.Study on the mechanism of anti-HL60-PMLA216V-RARα tumor-bearing nude mice in combination of different RIF components to reverse arsenic resistanceThe general condition of nude mice in each group was normal,and no death occurred during the treatment period.The body weight was first low and then high.On the 4th day of administration,the body weight of each group decreased compared with the healthy group;from the 4th day to the 8th day,the differences of body weight of nude mice in the model group,A group,ITS group and AITS group were not statistically significant(all P>0.05).From the 10th day to the end of the study(the 14th day of administration),the body weight increased,and the differences in body weight between the groups were statistically significant(all P<0.0001),showed the trend of healthy control group>AITS group>ITS group>A group>model group.On the 4th day,the tumor volume of the ITS and AITS groups was significantly reduced compared with the model group(all P<0.05);on the 6th day,the difference was statistically significant between the A group and the model group(P<0.05).On the 6th to 12th day,the tumor volume was in the order of AITS group<ITS group<A group<model group(all P<0.05);on day 14(the end of the study),the tumor volume was AITS group<ITS group≈A group<model group,except for A and ITS group,there were statistically significant differences between groups(all P<0.0001).At the end of the study,the tumor weight was(1.42 ± 0.09)g in the model group,(0.96 ± 0.08)g in the A group,(0.93 ± 0.11)g in the ITS group,and(0.39±0.06)g in the AITS group.The differences were statistically significant(all P<0.05),and the tumor weight in AITS group was lighter than that in the A group(P<0.05).In A,ITS,and AITS groups,the tumor volume inhibition rates were 41.06%,46.36%,and 67.55%,respectively,and the tumor weight tumor inhibition rates were 32.29%,34.51%,and 72.53%,respectively.There were significant differences between the groups(all P<0.05).Compared with the model group,the levels of caspase3 and Bcl-2 in each administration group decreased(all P<0.0001),and the AITS group had the lowest levels of caspase3 and Bcl-2(all P<0.05).The positive rates of ki67 and caspase3 were model group>A group>ITS group>AITS group.No obvious morphological changes were found in HE staining of the heart,liver,spleen,lung and kidney of nude mice in each group.Conclusion1.The arsenic-resistant cell line HL60-PMLA216V-RARα transfected with lentivirus was less sensitive to tetraarsenic tetrasulfide,and the drug resistance was 282 times that of the sensitive strain HL-60.2.Tanshinone Ⅱa,indirubin and total saponins of Radix Pseudostellariae enhance the effect of tetraarsenic tetrasulfide in down-regulating PMLA216V-RARα,the mechanism of which is related to the inhibition of mTOR and activation of autophagy.3.Tetraarsenic tetrasulfide alone,indirubin+tanshinone Ⅱa+total saponins of Radix Pseudostellariae and the combination of the four components can inhibit the growth of arsenicresistant APL tumor-bearing nude mice,and the combination of the four components of RIF has the strongest effect,RIF component combination played a synergistic reversal of APL arsenic resistance in vivo.