The Effect And Mechanism Of Novel Mitochondrial Uncoupling Agent BAM15 On Acute Myeloid Leukemia

BackgroundAML is a type of neoplastic diseases derived from the hematopoietic system.The"7+3" regimen of cytarabine combined with anthracycline chemotherapy agents is the standard induction chemotherapy regimen for other types of AML except acute promyelocytic leukemia,and high-dose cytarabine is the preferred treatment after remission of AML.Although these chemotherapy regiments can obtain a high response rate,they still have problems of high recurrence rate and low survival rate.The reason is that some leukemia cells are insensitive to traditional chemotherapy drugs,or chemotherapy drugs are toxic,resulting in delayed or interrupted treatment.Therefore,it is necessary to develop drugs with good anti-leukemia effect and low toxicity to normal cells.In recent years,abnormal cell metabolism has been found to be related to the occurrence and development of tumors.Mitochondria,as the center of cell metabolism,participate in the energy metabolism and biosynthesis of almost all cells.Reactive oxygen species(ROS)are important substances produced by mitochondrial metabolism.When mitochondrial metabolism is disturbed,the ROS content in cells increases,resulting in a series of changes related to mitochondrial injury such as oxidative stress and imbalance of redox homeostasis.Tumor cells were found to have abnormally elevated ROS and increased sensitivity to ROS.Abnormally elevated ROS exert anti-tumor effects by oxidizing biomacromolecules in tumor cells,causing microvascular damage in tumor tissues or organs,and increasing drug-induced apoptosis of tumor cells.It was found that acute myeloid leukemia cells and other malignant tumor cells have unique mitochondrial characteristics,such as decreased ATP storage and increased dependence on oxidative phosphorylation.Therefore,exploring AML specific changes in mitochondrial metabolism and exploring compounds that inhibit mitochondrial abnormal oxidative phosphorylation has become a new strategy for AML intervention.Screening of compound library is an important method for the development of new drugs and the exploration of new indications.The compound library is composed of small molecular compounds with specific structures or good biological safety,making it an effective way to explore new therapeutic drugs for AML.Ara-C,a cytosine analogue antitumor drug,has been widely used in AML induction therapy,consolidation therapy,central nervous system leukemia prevention and maintenance therapy.However,cytarabine,especially in large doses,has toxic and side effects such as bone marrow suppression and hepatotoxicity.Therefore,increasing sensitivity of cytarabine,reducing dosage and increasing its safety have become clinical concerns in the treatment of acute myeloid leukemia.Objective1.Through a large scale compound library,a novel mitochondrial uncoupling agent,BAM 15,was screened out and its biological effects and clinical application value were verified.2.Through the combined application of cytarabine and BAM15 in vitro and in vivo,the synergistic effect of the two drugs was verified,and the sensitization effect of BAM 15 on traditional chemotherapy drugs was analyzed.Methods and Results1.The mitochondrial uncoupling agent BAM15 was screened from the library of small molecular compounds.(1)4704 compounds from the library were screened:three AML cell lines(U937,Kasumil and THP1)were incubated with 10 μM small molecule compounds for 72 h,respectively,and the proliferation of AML cells was detected by CCK-8 cell proliferation assay.Finally,the inhibitory rate of mitochondrial uncoupling agent BAM15 on the proliferation of the three AML cell lines was more than 90%.According to literature review,BAM 15 has not been studied in leukemia.2.BAM15,a novel mitochondrial uncoupling agent,inhibits the proliferation and promotes the apoptosis of leukemia cells by inducing ROS production.(1)BAM 15 significantly inhibited the proliferation of AML cell lines:The effect of BAM 15 on the proliferation of AML cell lines was investigated by CCK8,EdU and clonogenesis cell proliferation experiments,and the proliferation ability of BAM15 and traditional mitochondrial uncoupling agents CCCP and FCCP on AML cell lines and CD34+ hematopoietic progenitor cells(CD34+HPCs)was compared.The results showed that BAM 15 can significantly inhibit the proliferation of AML cell lines,and has a stronger anti-tumor activity than traditional mitochondrial uncoupling agents,and is less toxic to normal hematopoietic progenitor cells,so it is safer.(2)Screening the causes of cell death caused by BAM15:Death pathway inhibitors,including iron death inhibitors Fer-1,Lipro,Necrosis pathway inhibitors Necro and apoptosis pathway inhibitors ZVAD,were used to screen the anti-AML effects of BAM15,and it was found that ZVAD could effectively recover the anti-AML effects of BAM 15.These results indicated that apoptosis was the main cause of AML cell death induced by BAM 15.The apoptosis of AML cells induced by BAM 15 and the expression of apoptotic molecules Bcl-2,Bax,PUMA and NOXA were tested by FCM and Western blotting.The results showed that BAM 15 significantly increased the apoptosis rate of AML cells.And the expression of Bax increased,the expression of Bcl-2 decreased.(3)Effects of BAM 15 on mitochondrial function:Flow cytometry was used to detect the ROS production and membrane potential changes of leukemia cell lines treated with BAM15,and the changes of ATP production after BAM 15 treatment.It was found that BAM 15 significantly increased ROS content in AML cell lines,decreased cell membrane potential level,and reduced ATP production.(4)BAM 15 interferes with ROS to produce balance and affect cell activity:NAC and BAM 15 were combined to act on AML cell lines.Flow cytometry,CCK8,EdU and Western blotting were used to verify the effect of BAM 15 on AML cell lines after ROS level changes.The results showed that NAC combined with BAM 15 reduced ROS production,increased cell membrane potential,decreased apoptosis level and increased proliferation activity in AML cells.These results suggest that ROS equilibrium changes are responsible for the biological changes of AML induced by BAM15.(5)Anti-proliferation activity of BAM 15 in vivo:1×106 C1498 leukemia cells were injected into the caudal vein of C57 mice to establish C1498 leukemia model.PBS or BAM 15 were intraperitoneally injected into the control group and experimental mice,respectively.The anti-leukemia effect of BAM 15 in vivo was verified by leukocyte count,bone marrow cytology,liver and spleen weighing,HE staining,Kaplan-Meier survival analysis and other methods.The results showed that BAM15 could significantly inhibit the number of leukocytes and primitive cells in AML model group,reduce tumor infiltration in liver and spleen,and prolong the survival time of leukemia mice.(6)BAM 15 inhibited the activity of primary cells from newly diagnosed AML patients:Bone marrow from newly diagnosed AML patients and umbilical cord blood from healthy volunteers were collected.And primary cells or CD34+HPCs were extracted respectively to verify the changes of cell apoptosis and activity under BAM15.The results showed that BAM 15 could significantly inhibit the activity of primary AML cells and increase the apoptosis rate,resulting in increased expression of apoptosis protein Bax and decreased expression of apoptosis inhibitory protein Bcl-2.But BAM15 had little effect on the activity of CD34+HPCs.3.BAM15 has synergistic effect with Ara-C in the treatment of acute myeloid leukemia(1)Verify the synergistic effect of BAM15 and Ara-C:BAM15 and Ara-C were used alone or in combination in U937 and Kasumil cells,and the synergistic effects of BAM 15 and Ara-C were analyzed using Compusyn software according to The Chou-Talalay formula.The results showed that BAM 15 combined with cytarabine could synergistically inhibit AML cells.(2)Combination of BAM 15 and Ara-C inhibits proliferation and induces apoptosis of AML cells:The experiment was divided into Vehicle group,BAM 15 group,Ara-C group and BAM15+Ara-C group.Cell proliferation was detected by clonogenesis and EdU methods,cell apoptosis level was tested by FCM and apoptosis-related molecules were tested by WB.The results presented that Ara-c combined with BAM 15 depressed the activity of AML cells and synergistically promoted the apoptosis of AML cells compared with the treatment group alone.Ara-C achieve synergistic treatment of acute myeloid leukemia through DNA damage:After BAM 15 and Ara-c were applied to AML cell lines alone or in combination,the expression levels of DNA damage-related molecules were detected.It was found that BAM 15 and Ara-C could significantly promote the expression levels of DNA damage-related molecules such as r-H2AX,p-ATM and p-ATR,and the combination of BAM 15 and Ara-C could induce stronger DNA damage.(4)Synergistic effect of BAM 15 and Ara-c in vivo:C1498 mouse leukemia model was established by tail vein injection of C1498 cells,and the differences of leukocytes,bone marrow primal cells,liver and spleen size,weight and survival time of mice treated with AML model(Vehicle)group,BAM 15 group,Ara-C group and BAM 15 combined with Ara-C group were compared.The results showed that the combined treatment group could significantly inhibit the number of white blood cells and bone marrow primitive cells,reduce the tumor infiltration of liver and spleen,and prolong the survival time of mice.(5)Synergistic effect of BAM 15 and Ara-C in primary cells:After BAM 15 and Ara-C alone or in combination were applied to bone marrow primary cells of newly diagnosed AML patients,the changes of cell proliferation,apoptosis,Bax and Bcl-2 were detected respectively.Compared with the single treatment group,the results showed that the combined treatment group could significantly promote the apoptosis of primary cells and inhibit cell activity.Conclusion(1)The mitochondrial uncoupling agent BAM 15 was found to inhibit the proliferation of AML cells through the screening of small molecular compound library.Compared with the traditional mitochondrial decoupling agents CCCP and FCCP,BAM15 has the advantages of strong inhibition of AML cell proliferation and good safety.Through cellular and molecular level experiments,it was found that BAM 15 acted on leukemia cells by inducing the generation of mitochondrial ROS,interfering with oxidative balance,leading to the decrease of mitochondrial cell membrane potential,thus promoting cell apoptosis and inhibiting cell proliferation.In mice,BAM 15 was found to delay the growth of tumor cells in AML model mice and prolong the survival time of mice.BAM 15 is a promising drug candidate for the treatment of AML.(2)BAM 15 combined with Ara-c has stronger anti-proliferation and pro-apoptotic effects than both AML cell lines and primary leukemia cells treated with the drug alone.In vivo studies in mice also demonstrated that BAM 15 combined with Ara-C can inhibit tumor proliferation better than that treated with the drug alone.The mechanism is related to the fact that combined application can significantly promote the expression of molecules p-ATM、p-ATR and r-H2AX,and synergistically increase DNA damage.