Ethyl Acetate Extract Of Caesalpinia Sappan L.inhibited Acute Myeloid Leukemia Via ROS-mediated Apoptosis And Differentiation

Background:Acute myeloid leukemia(AML)is a devastating hematological malignancy characterized by unscheduled proliferation and differentiation arrest of immature cells of the myeloid lineage.For several decades,few substantial therapeutic advances have been made for patients with AML as chemotherapy,still remains one of the main treatments.However,many side effects and the multidrug resistance problems caused by chemotherapy still need to be resolved.In recent years,research into the underlying pathogenic mechanisms of AML have led to remarkable advances in our understanding of the disease.Accumulating evidence suggests that therapeutic approaches in AML may be achieved by controlling disordered proliferation and inducing differentiation of abnormal hematopoietic cells.The identification of novel substances capable of restoring sensitivity to apoptosis or promoting cell differentiation are therefore of significant therapeutic importance.Caesalpinia sappan L.,a plant that belongs to the Caesalpiniaceae family,is a medicinal plant widely distributed in Southeast Asia.The dried heartwood of this plant is traditionally used as a herbal medicine in China to increase blood circulation,and treat various illnesses such as pain,tetanus,and blood clotting of ladies.Emerging evidence demonstrates that Caesalpinia sappan L.has induced apoptosis and growth inhibition in a variety of cancer cells such as U266 cells,oral cancer cells,head and neck cancer cells.However,the active fractions and the underlying mechanisms of Caesalpinia sappan L.related to anti-AML activity have not been reported until now.In the present study,a bioactivity guided fractionation was first applied to identify the ethyl acetate extract of Caesalpinia sappan L.(C-A-E)as the bioactive fraction.Then,the effect and underlying mechanisms of C-A-E on the apoptosis,cell cycle and differentiation of AML cells were elucidated.Finally,the anti-AML activity of C-A-E was verified in the NOD/SCID mice model.Objective:This study aims to investigate the effect of the ethyl acetate extract of the dried heartwood of Caesalpinia sappan L.(C-A-E)on the induction of apoptosis and the promotion of differentiation in vitro and anti-AML activity in vivo.Methods:(1)Screening of active components of Caesalpinia sappan L.against acute myeloid leukemiaAfter the water extracted,the Caesalpinia sappan L.is extracted by system solvent extraction to obtain petroleum ether,ethyl acetate,water-saturated n-butanol and water.Then,the different extractions were assayed by HPLC.The SRB method is used to detect the water extract of Caesalpinia sappan L.and its different extraction sites.(2)Study on the inhibition of the growth of acute myeloid leukemia cells by ethyl acetate extract of Caesalpinia sappan L.The SRB assay was used to detect the inhibition rate of human normal cells(293T、HUVEC)and AML cells(Kasumi-1,U937,K562,OCI-AML3,MV-4-11).The trypan blue exclusion method was used to detect the proliferation of AML cells.The cloning ability of AML cells was detected by cloning and formation experiment of methyl cellulose.(3)Study on the ethyl acetate extract of Caesalpinia sappan L.induced mitochondrial apoptosis of AML cellsThe apoptosis level was analyzed combined with Annexin V-FITC/PI double staining by flow cytometry.Mitochondrial membrane potential(MMP)was detected with the JC-1 probe.The activity of caspase-3 and caspase-9 were determined using caspase-3 and caspase-9 activity kits.C-A-E promotes mitochondria release of cytochrome c were determined by immunofluorescence staining.The apoptosis level of AML cells was detected by Western blotting.(4)Study on the ethyl acetate extract of Caesalpinia sappan L.induced mitochondrial fission of AML cellsThe mitochondrial fission protein of AML was detected by Western blotting.The apoptosis level of AML cells was analyzed by flow cytometry combined with Annexin V-FITC/PI double staining.C-A-E promotes mitochondria import of Drp1 were determined by immunofluorescence staining.(5)Study on the cell cycle arrest and differentiation of acute myeloid leukemia induced by ethyl acetate extract of Caesalpinia sappan L.The flow cytometry PI single staining was used to detect the cell cycle distribution of AML cells.Wright’s Giemsa staining assay was used to detect the differentiation of AML cells.The nitrotetrazolium blue(NBT)-reduction activity assay was used to detect the positive cell rate in HL-60 cells by NBT.The expression of CD11b and CD 14 were detected by flow cytometry to evaluate the differentiation of AML cells.The effect of ethyl acetate of C-A-E on the cell cycle related proteins of HL-60 was detected by Western blotting.(6)Effect of ROS on C-A-E-induced cell apoptosis and differentiation of AML cells ROS levels in HL-60 cells were detected using flow cytometry.Then,C-A-E was combined with ROS scavenger(NAC),cell apoptosis was determined by flow cytometry.CD11b and CD14 expression were measured by flow cytometry.The effect of ethyl acetate of C-A-E on the cell cycleere detected by flow cytometry.(7)Effect of ethyl acetate extract of Caesalpinia sappan L.on AML-NOD/SCID mouse model.NOD/SCID mice were irradiated with HL-60 cells in the tail vein to construct a mouse model of AML-NOD/SCID.Automatic animal blood analyzer was used to detect WBC(white blood cells)physiological indicators of mice.HE staining was used to detect liver and spleen infiltration in mice.immunohistochemistry was used to detect the expression of CD45 in liver,spleen and bone marrow of mice.Results:(1)The ethyl acetate extract had significant inhibitory effects on acute myeloid leukemia cellsTo characterize the anti-AML effect of the dried heartwood of Caesalpinia sappan L.in vitro,we first determined the inhibitory effect of C-A on the cell viability of HL-60 cells during our preliminary experiments.C-A showed potent inhibition on HL-60 cells with an IC50 of 0.26 mg/ml.The bio-assay results of the fractions generated by the extraction with different solvent systems clearly demonstrated that C-A-E exhibited the highest inhibitory effect on the cell viability of HL-60 cells(IC50=0.19 mg/mL)when compared to other three fractions.Different extract of the dried wood ofCaesalpiniasappanL.was analyzed on an analytical HPLC.(2)C-A-E inhibited the growth of AML cellsThe inhibitory effect of C-A-E on other AML cells was also evaluated.Similarly,C-A-E potently inhibited Kasumi-1 cells in a concentration-dependent manner(IC50=0.15mg/mL),while relatively weak activities were observed in U937,K562,OCI-AML3 and MV-4-11 cells.Meanwhile,no cytotoxicity for C-A-E was observed in normal 293T and HUVEC cells.Moreover,C-A-E suppressed the proliferation of both HL-60 and Kasumi-1 cells in a concentration-dependent manner.Similar activities were also observed in U937,K562,OCI-AML3 and MV-4-11 cells.Additionally,the clonogenic assay performed with a sustained treatment of HL-60 and Kasumi-1 cells for two weeks also provided an indication that C-A-E significantly reduced the size and number of colonies compared with those of the untreated control,respectively.(3)C-A-E induced mitochondrial apoptosis of AML cellsTo reveal the underlying mechanisms responsible for C-A-E-mediated growth inhibition of AML cells,cell apoptosis was first characterized by flow cytometry with Annexin V/PI staining.C-A-E concentration-dependently induced apoptosis in both HL-60 and Kasumi-1 cells.Meanwhile,C-A-E-induced cell viability inhibition and apoptosis were significantly reversed by Z-VAD-FMK,a pan-caspase inhibitor.C-A-E significantly reduced the MMP level.In addition,C-A-E obviously promoted the release of from the mitochondria.Furthermore,western blotting results demonstrate that C-A-E remarkably induced the Bax expression and reduced the Bcl-2 expression.Moreover,C-A-E clearly decreased the levels of pro-caspase 9 and 3 and increased the activity and the levels of cleaved-caspase 9 and 3.(4)C-A-E induced mitochondrial fission of AML cellsTo verify whether mitochondrial fission was involved in C-A-E-induced mitochondrial apoptosis,a gain-and loss-of-function assay for mitochondrial fission was conducted via the administration of Mdivi-1(mitochondrial fission blocker)and FCCP(mitochondrial fission activator),respectively.Pretreatment with Mdivi-1 significantly attenuated the inhibition of C-A-E on the cell viability of HL-60 cells(p<0.05),whereas no influence was observed for FCCP(p>0.05).Meanwhile,pretreatment with Mdivi-1 remarkably reduced the apoptotic death caused by C-A-E.It is well known that mitochondrial fission is governed by the recruitment of dynamin-related protein 1(DRP1)by adaptor proteins such as mitochondrial fission factor(Mff)and fission 1(Fis1).Our western blotting results further revealed that C-A-E obviously induced the expression of Mff and Fisl in a concentration dependent manner.The immunocytochemical staining results demonstrated that the level of Drp1 clearly increased in mitochondria.(5)C-A-E promoted the differentiation of AML cellsTo examine the effect of C-A-E on the myeloid differentiation of AML cells,Wright-Giemsa staining was first performed to detect the morphologic changes of C-A-E-treated HL-60 and Kasumi-1 cells.There were typical morphological featured of myeloid differentiation,such as:a lower nucleocytoplasmic ratio and chromatin condensation,observed in both C-A-E-treated HL-60 and Kasumi-1 cells.Then,an NBT reduction assay was performed to examine the generation of oxidative bursts during the differentiation.NBT positive cells concentration-dependently increased by C-A-E treatment in both HL-60 and Kasumi-1 cells.Furthermore,C-A-E concentration-dependently enhanced the expression of both CD11b and CD14,two markers of the myeloid differentiation,in HL-60 cells.(6)Involvement of ROS in C-A-E-induced mitochondrial apoptosis and differentiation in AML cellsTo investigate whether ROS was involved in C-A-E-induced cell apoptosis,the potential effect of C-A-E on the cellular ROS production was first examined.Notably,C-A-E significantly enhanced the intracellular ROS level in HL-60 cells with 2.54-foldincrease after 0.2mg/mL C-A-E treatment compared with the untreated control.Pretreatment with NAC,a ROS scavenger,significantly mitigated the inhibition of C-A-E on the cell viability of HL-60 cells(p<0.05).More importantly,pretreatment with NAC dramatically attenuated C-A-E-induced apoptosis of HL-60 cells(p<0.001).Moreover,the enhancement of the levels of CD14 and CDllb by C-A-E was also obviously reduced by NAC pretreatment.(7)Anti-AML activity of C-A-E in NOD/SCID miceThe anti-AML activity of C-A-E was further investigated in vivo using NOD/SCID immunodeficient mice with the injection of HL-60 cells into the tail vein.Fig.6A revealed the dynamic deaths in each group.There were always more deaths in the vehicle group than those in the C-A-E 50 mg/kg and 100 mg/kg groups from the 34th day.The death number of the C-A-E 100 mg/kg group was less than that of the C-A-E 50 mg/kg from the 31st to 35th days.It is noteworthy that the mice in the C-A-E 50 mg/kg and 100 mg/kg groups stopped dying after 35th day,while those in the vehicle group continued dying.The determination of blood physiological parameters showed that WBC obviously decreased in both 50 and 100 mg/kg C-A-E groups compared with those in the vehicle group(p<0.0 01).The peripheral blood and bone marrow smear stained with Wright-Giemsa showed that the cells in C-A-E groups(100 mg/kg in particular)exhibited differentiation-related morphological features compared with those in the vehicle group.Immunohistochemical staining results showed that the expression of CD45,a pan-leukocyte marker,in the liver,spleen and bone marrow was reduced by C-A-E treatment compared to the vehicle group.Moreover,C-A-E significantly reduced hepatosplenomegaly(p<0.05)compared with that of the vehicle group alone.The histological changes of the liver,spleen and bone marrow in femurs were evaluated by HE staining.Notably,C-A-E treatment significantly decreased hepatic edema,indicating the benefit of enhanced liver tissue repair.The spleen of vehicle mice showed extensive atrophy of splenic bodies.In comparison,the spleen from the C-A-E-treated mice showed less atrophy.Histological analysis of femurs showed that C-A-E remarkably increased bone marrow cellularity compared to the vehicle group.Conclusion:C-A-E exhibited an inhibitory effect on AML cells by inducing mitochondrial apoptosis,cell cycle arrest and promoting differentiation,each of which were highly correlated to the activation of ROS.