Study On The Effect Mechanism Of Croton And Glycyrrhiza In The Treatment Of Acute Myeloid Leukemia

Aim:To study pharmacodynamics and mechanism of component and monomer compatibility of croton and glycyrrhiza on human leukemia cell line MV4-11 the in vivo and pharmacodynamics and mechanism of component compatibility of croton and glycyrrhiza on human leukemia cell line MV4-11 in vitro.Methods:In vitro experiments of combination components1.The in vitro inhibitory activity of croton alkaloids(main pharmacodynamic components of croton),glycyrrhiza flavonoids(main pharmacological components of licorice),and their different compatibility ratios on human leukemia cell line MV4-11was determined by MTT assay,respectively.The half-maximal inhibitory concentration(IC₅₀)was calculated,and the in vitro inhibitory activity of croton alkaloids and glycyrrhiza flavonoids on Molm-13,MCF-7,Hela,231,H460,Hepg-2cell lines was determined,respectively;2.MTT assay was used to determine the in vitro inhibitory activity of croton alkaloids and glycyrrhiza flavonoids on Ba/F3-FLT3-ITD cell line with or without interleukin 3(IL-3);3.Detected the respectively inhibitory and post-inhibition effects of croton alkaloids and glycyrrhiza flavonoids on the growth of MV4-11 cell line by cell counting assays.Their combined effects were also detected by the same method;4.MDC fluorescent staining method was used to detect the autophagy effects of croton alkaloids and glycyrrhiza flavonoids respectively,and their combined effects in inducing autophagy of MV4-11;5.The Hoechst 33258 staining method was used to detect the sole effects and the combined effects of croton alkaloids and glycyrrhiza flavonoids on the morphology and quantity of MV4-11 cells'nucleus;6.The solo effects and the combined effects of croton alkaloids and glycyrrhiza flavonoids on cell cycle and apoptosis of MV4-11 were detected using flow cytometry;7.Western Blot was used to detect the effects of croton alkaloids,glycyrrhiza flavonoids,and their combination on related signal proteins(FLT3,P-FLT3,Stat5,P-Stat5,Erk1/2,P-Erk1/2,AKT,P-AKT,m TOR,P-m TOR,LC3-?,LC3-?,Bax,Bcl-2,C-MYC)in MV4-11 cells;In vivo experiments of combination components1.MV4-11 xenografted immunodeficient mice(SCID mice)models were applied for evaluating the in vivo inhibitory effects of croton alkaloids,glycyrrhiza flavonoids,and their combination on leukemia cells and evaluate the toxicity,respectively;2.The effect of Croton alkaloids and glycyrrhiza flavonoids on the expression of Ki67 protein in tumor tissue of SCID mice was detected by immunohistochemistry,and the apoptosis of tumor tissue was detected by TUNEL staining;In vitro experiments of combination monomers1.Screened several monomer components extracted from croton and licorice.MTT assay was used to evaluate their in vitro inhibitory activity on MV4-11 cell line and Molm-13,MCF-7,Hela,231,H460,Hepg-2 cell lines.Conducted a combination drug evaluation as well;2.Western blot was used to detect the effects of crotonoside,isoliquiritin,and their combination on the expression of FLT3 signaling-related proteins(FLT3,P-FLT3,Erk1/2,P-Erk1/2,STAT5,P-STAT5).Result:In vitro experiments of combination components1.Croton alkaloids and glycyrrhiza flavonoids had in vitro inhibitory activity on MV4-11 cells,with IC₅₀ of 1.35±0.12?g/m L,6.43±0.46?g/m L,respectively,and they have no activity or weak activity to other tumor type cell lines and have certain selectivity.The combination of croton alkaloids and glycyrrhiza flavonoids had a synergism on the inhibition of MV4-11 cells in vitro.The IC₅₀ of the combination was0.65±0.10?0.66±0.08?0.69±0.11?0.95±0.24?1.01±0.23?1.68±0.14?1.67±0.25?2.28±0.15?1.96±0.09?1.99±0.05?1.93±0.13?1.91±0.10?1.90±0.12?g/m L within the compatibility ratio of 20:1?10:1?5:1?4:1?3:1?2:1?1:1?1:2?1:3?1:4?1:5?1:10?1:20.The CI of the combination index was less than 1;2.Croton alkaloids and glycyrrhiza flavonoids had in vitro inhibitory activity on Ba/F3-FLT3-ITD cell line,with IC₅₀ of 3.96±0.33?g/m L and 9.26±1.97?g/m L respectively.After adding 100ng/m L of IL-3,the proliferation and transplantation of the Ba/F3-FLT3-ITD cell line in the croton alkaloid and glycyrrhiza flavonoids could be partially or completely restored;3.Croton alkaloids and glycyrrhiza flavonoids had a post-inhibitory effect on the growth of MV4-11 cells,which could delay the peak of the growth curve.The efficacy of the combination had the best effect on delaying the peak of the growth curve;4.The results of Hoechst 33258 staining showed that croton alkaloids,glycyrrhiza flavonoids,and their combination made the nucleus shrink in MV4-11 and increased the number of apoptotic bodies respectively;5.Croton alkaloids,glycyrrhiza flavonoids,and their combination could induce autophagy in MV4-11,while the number of autophagosomes was significantly increased;6.Croton alkaloids and glycyrrhiza flavonoids could induce cell cycle arrest in the G1 phase of MV4-11.The proportion of cells in the G1 and S phases of the croton alkaloid group was 70.50±1.31%and 23.27±1.36%.The proportion of cells in the G1and S phases of the glycyrrhiza flavonoids group was 67.40±0.82%?27.33±1.11%.The proportion of cells in the G1 and S phases of the combination group was 78.67±0.57%?15.53±0.21%.All results of administration groups had significant differences compared with the control group(**P<0.01).Differences between the two separate medication groups and the combined medication group also had significant differences(**P<0.01);Croton alkaloids and glycyrrhiza flavonoids could induce apoptosis of MV4-11cells.The percentage of apoptotic cells of control group,croton alkaloid group,glycyrrhiza flavonoids group and combination group was 4.72%±2.82%,54.07±7.60%,32.33±2.91%,64.90±4.61%respectively.All results of administration groups had significant differences compared with the control group(**P<0.01).Differences between the two separate medication groups and the combined medication group also had significant differences(**P<0.01);7.Croton alkaloids and glycyrrhiza flavonoids could affect related proteins of the FLT3 pathway,inhibiting the phosphorylation of FLT3,Erk1/2,STAT5,AKT,and m TOR.Except for the reduction of the total proteins of STAT5,the total proteins of the other pathways were not affected.Croton alkaloids,glycyrrhiza flavonoids,and their combination showed inhibitory effects on the protein expression of Bcl-2 and c-Myc while no significant effect showed on total protein.Croton alkaloids showed an inhibitory effect on the expression of Bax protein,while the glycyrrhiza flavonoids performed the opposite.The expression of Bax in the combination group was significantly higher than that in the control group;In vivo experiments of combination components1.80mg/kg of croton alkaloids and 200mg/kg of glycyrrhiza flavonoids were administered to SCID mice alone or in combination.The drug was stopped for 1 day per 6 days for a total of 14 days.The tumor inhibition rates were 46.79%,24.61%,and62.17%,respectively.The tumor sizes of the croton alkaloids group,glycyrrhiza flavonoids group,and the combination group were smaller than that of the control group.In terms of toxicity,Croton alkaloids can cause significant weight loss,increase of alt,decrease of ALB,Glu and CK,while glycyrrhiza flavonoids can significantly alleviate its toxicity(P<0.05).These results showed that the combination of croton alkaloids and glycyrrhiza flavonoids had synergistic and attenuation effects.2.Ki67 and TUNEL staining showed the positivity of Ki67 in tumor tissues of SCID mice treated with croton alkaloids,glycyrrhiza flavonoids,and their combination,while the TUNEL positivity was significantly increased.The effect of croton alkaloids was better than that of glycyrrhiza flavonoids.The combination group was significantly better than the two-drug single-use group,indicating that the croton alkaloids,glycyrrhiza flavonoids,and their combination could inhibit the proliferation of tumor cells in mice and induced tumor cell apoptosis in vivo.The medication efficacy of the combination group was better than that in the other two groups;In vitro experiments of combination components1.crotonoside,isoliquiritigenin,licochalcone A and Luteolin from croton and glycyrrhiza had good inhibitory or activity on mv4-11 cells,IC₅₀ were 3.30±0.57,0.88±0.36,3.77±0.83 and 3.64±0.95?g/m L,respectively.By analyzing the inhibitory activity of MCF-7,Hep G-2 and other non AML cells,it was found that crotonoside and isoliquiritigenin had certain selectivity.In addition,the combination of the crotonoside and isoliquiritin,which mixed in a ratio of 4:1,had a synergism on the inhibition of the MV4-11 cell line in vitro.The IC₅₀ of the combination was 1.23±0.21?g/m L,and the CI was 0.74±0.11;2.The crotonoside and isoliquiritin combination which mixed in a ratio of 4:1could significantly inhibit the phosphorylation of FLT3,Erk1/2,and STAT5,but have no significant effect on their total protein.Conclusion:Croton alkaloids and glycyrrhiza flavonoids could inhibit the proliferation of MV4-11 cells in vivo and in vitro,inducing autophagy and apoptosis of tumor cells,and the combina tion efficacy was better than that of the individual drug.The mechanisms might be related to the croton alkaloids,glycyrrhiza flavonoids,and their combination,which involved in regulating the phosphorylation of FLT3 signaling-related proteins FLT3,Erk1/2,STAT5,regulating the phosphorylation of PI3K/Akt/m TOR signaling-related proteins Akt,m TOR,inhibiting the expression of c-Myc,inducing autophagy up-regulated by the expression of LC3-?,and regulating the expression of apoptosis-related proteins Bax and Bcl-2 to induce apoptosis.At the same time,the glycyrrhiza flavonoids has the effect of reducing the toxicity of Croton alkaloids in vivo.Crotonoside,isoliquiritin,and their combination could significantly inhibit the proliferation of MV4-11 cells in vitro,and the mechanism was related to the inhibition of the phosphorylated FLT3 signaling-related proteins FLT3,Erk1/2,and STAT5.