Investigation Of The Cytotoxic Activity And Mechanism Of Novel Mitochondria-Targeted Small Molecule IR-26 On Acute Myeloid Leukemia Cells

Background:Acute myeloid leukemia(AML)is a deadly hematological malignancy,comprised with genetically and clinically heterogeneous groups of aggressive malignant cells.Although the development of advanced mutation-targeted agents,such as FLT3 and IDH1inhibitors has improved treatment for AML in individual patients,the current therapies fail to eliminate all the malignant cells which eventually lead to disease relapse.This is due to the genetic abnormalities heterogeneity,genomic complexity and the close proximation of leukemia cells to the normal hematopoietic cells,which makes it difficult to target leukemia cells accurately and then induces severe side-effects.Thus,new therapeutic strategies towards targeted therapy based on the general vulnerabilities of leukemia cells may help to design personalized treatments and improve the therapeutic efficiency.Mitochondria are involved in bioenergetic,biosynthetic and signaling activities in cells,and conferred vita l roles in regulating tumorigenesis and progression in tumor.The cancer-specific alterations in mitochondria have already been recognized as a critical target for cancer cells targeting and treatment.In AML,the malignant cells are demonstrated dependent more on mitochondrial oxidative phosphorylation and have increased mitochondrial biogenesis,decreased spare reserve capacity.The mitochondrial complex of the electron transport chain(ETC)is mainly used by cells to generate energy.Therefore,increasing efforts have been made to target mitochondrial ETC proteins and mitochondrial metabolism in AML treatment strategies.In the previous study,we have synthesized and identified a near infrared(NIR)fluorescent small-molecule,IR-26 to specifically target AML cells.And IR-26 have been demonstrated to accumulate in mitochondria.Especially,IR-26 can be used to imaging of AML cells in a real-time and noninvasive way using the in vivo flow cytometry system(IVFC),which holds significances in monitoring disease progression and treatment response in AML.As is recognized that mitochondria play a vital role in AML,we intend to investigate whether IR-26 is capable of eradicating AML cells and improve the efficacy of AML treatment.Thus,our study may provide a rationally new strategy to integrate leukemia cells imaging,targeted treatment and monitoring of therapeutic outcomes.Methods and Results:1.Explore the cytotoxic effect of IR-26 on AML cells in vitro.IR-26 effectively killed HL-60 and THP-1 cells in a concentration dependent manner,the half maximal inhibitory concentration(IC50)were 2.629μM and 2.351μM respectively.Annexin V and PI staining by flow cytometry detection also indicated IR-26 obviously induced AML cells death in the both cell lines.Western-blot analysis indicated IR-26induced HL-60 and THP-1 cells apoptosis by inducing of PARP,caspase-3 and caspase-9cleavages.The cytotoxicity of IR-26 on AML cell and normal cells were compared,results showed IR-26 obviously induced HL-60 cells death with no obvious cytotoxicity in the PBMCs.Moreover,IR-26 was also demonstrated to inhibit cell colony formation in HL-60cells,which suggested the inhibiting effects on AML progenitor cells.2.Explore the cytotoxic effect of IR-26 on AML cells in vivo.To further assess the AML treatment effects of IR-26 in vivo,two AML mouse models were established and used.First,the leukemia xenografts mouse models were established by subcutaneously injection of HL-60 cells in the nude mice.After tumors became palpable,the mice were randomly divided into 3 groups and received intraperitoneally injection of PBS,cytarabine(50 mg/kg)and IR-26(5.0 mg/kg)respectively.It turns out that a distinct inhibition of tumor growth was observed after IR-26 treatment,while the treatment effect of cytarabine was limited.The tumor weight which was measured after mice sacrifice also indicated the dramatically antitumor effect of IR-26 in vivo.Compared with control group,IR-26 treatment did not cause body weight change and obvious pathologic changes in vital organs,indicating its satisfactory security for in vivo treatment.Moreover,nonob ese diabetic/severe combined immunodeficient(NOD/SCID)mice were engrafted with GFP-labeled human AML cells HL-60 through tail vein.One week post primary transplant,mice were treated with cytarabine or IR-26 with intraperitoneally injection.At day 25,the Kodak In-Vivo FX professional Imaging System showed that dissected organs from IR-26group displayed no obvious GFP fluorescence while organs from other groups showed strong GFP fluorescence.The detection of GFP labeled AML cells in peripheral blood o f mice using IVFC also indicated IR-26 treatment significantly decreased AML cells level.IR-26 remarkably reduced tumor burden,manifested with a prominent decrease in percentage of hCD45~+cells in bone marrows.H&E staining revealed significantly reduced infiltration of leukemic cells and increased preservation of normal tissue structures in the organs of the mice treated with IR-26 when compared with control group mice.Besides,IR-26 dramatically prolonged the median survival of mice in Kaplan-Meier analysis when compared with the control group.3.Reveal the mechanism of IR-26 on AML cells.As the effective therapeutic benefits of IR-26 in AML cells,further investigated the underlying mechanisms of IR-26 induced cell death is needed.A subcellular location assay was performed and indicated that IR-26 specifically accumulated in the mitochondria of AML cells by co-localization with the mitochondrial specific probe Mitotracker-Green.Moreover,transmission electron microscope showed IR-26 treatment induced mitochondria swelling and vacuolated in AML cells.JC-1 cytometry analysis showed that IR-26 also decreased mitochondrial membrane potential in AML cells,suggesting mitochondria might be an important therapeutic target for IR-26.Further MitoSOX cytometry analysis showed that IR-26 treatment increased mitochondrial ROS production in AML cells,and the mitochondrial ROS specific scavenger MitoTEMPO significantly reversed the IR-26inducing of ROS productions and cell death.Real-time analysis of mitochondrial respiration profile using a Seahorse XFe analyzer indicated that IR-26 reduced the basal mitochondrial oxygen consumption rates(OCR)and the maximal respiration of mitochondria in HL-60 cells.And,the ATP production was significantly decreased after cells treated with IR-26 detected by ATP assay kit.NAD~+/NADH Assay Kit with WST-8showed the NADH/NAD~+ratio in HL-60 cells were increased after treatment with IR-26.As the mitochondrial complex in the ETC is a major source of ROS production in mitochondria.Our study indicated IR-26 inhibited the activity of mitochondria complexⅡand complexⅤdramatically.Conclusion:1.IR-26 effectively induces apoptosis in AML cells and inhibits AML progenitor cells colony formation,while at the concentrations that appear pharmacologically achievable does not have similar negative effect on normal cells.IR-26 inhibits AML cell growth and prolong median survival in two AML mouse models.2.IR-26 reduced mitochondrial OXPHOS of AML cells and disturbed electron transporting in the ETC for the ROS production,which is a key factor for IR-26 inducing of cell apoptosis in AML.