Synergistical Lethal Effects Of Arsenic Trioxide Combined With Aclacinomycin On Human Acute Myelogenous Leukemia KG-1a Cell Line And The Study Of Xenotransplant Leukemia Model Using By Zebrafish

Introduction:Arsenic trioxide(As2O3, ATO) is an effective ingredient of arsenic compounds. As a single agent, it has been showed substantial efficiency in treating patients suffering from acute promyelocytic leukemia(APL), a subtype of acute myeloid leukemia(AML). Recent studies report that ATO not only showed effective for hematological malignancies but also for other tumors such as breast cancer, multiple myeloma, neuroblastoma, etc. but the its mechanisms were not full know until today. Some studies revealed that the anti-tumor effect of ATO may be through inhibition of tumor cell proliferation and angiogenesis. Other reports show that the anti-tumor effect of ATO is due to the combiantion with certain drugs which can induce differentiation of tumor cells. In addition to the above point of view, the majority view is that ATO induced apoptosis may be regulated by a variety of intracellular signals, and meanwhile, multiple genes were presented in the apoptosis process. As we know, survivin, P53, Bcl-2, etc. is closely associated with apoptosis. Studies have shown that ATO can down-regulate Bcl-2gene level, and then leads to apoptosis in tumor cells. We know that apoptosis is a type of an orderly or programmed cell death that is a proactive cell death process which is regulated by gene. Due to its importance in healthy process of multi-cellular organisms, causing extensive research on its way to the researchers and become one of the hotspots of life science research. Now cell apoptosis was associated with the following factors:(1) the extracellular apoptosis pathways, also called death receptor(DR) pathway. DRs are members of the tumor necrosis factor(TNF) receptor fuperfamily, and include functional receptors and decoy receptors(DcR). Both types of receptors have an extracellular cysteine rech domain (CRD), but only functional receptors have an intracellular death domain(DD). The receptor superfamily includes TNR-R1, Fsa-/APO1, DR3, TNF-related apoptosis-inducing ligand receptors-1(TRAIL-R1,DR4),-2(TRAIL-R2,DR5), and DR6. Others including TNF-a receptor, FasL/APO1/CD95receptor, and TRAIL/APO2L receptor regulate other biological functions including cell metabolism, proliferation and cytokine production. Pro-apoptotic ligands are receptor-specific and include AP02L/TRAIL (DR1and DR5), FasL (Fas/APO1/CD95), and TNF (TNF-R1). Ligand binding leads to trimerization and subsequent recruitment of several factors that form clustering of receptors-this clustering is thought to amplify the apoptotic response. The adaptor protein Fas-associated death domain protein (FADD) and initiator caspase8and10are then recruited to the intracytoplasmic tail of the receptor, to form a desth-inducing signal complex (DISC). The initiator caspases are so called as their activation is necessary for the process of apoptosis to begin. The proximity of the nintiator caspases to each other within the DISC serves to faciliate their autoatalytic activation allowing them to activate effector caspases3,6and/or7and converging into the intrinsic paytway of apoptosis.(2) The intrinsic pathway. The intrinsic or mitochondrial pathway is so called as it is initiated within the cell, and in particular, within the mitochondria. It is activated in response to cellular stress signals arising from, e.g., DNA damage, hypoxia, a defective cell cycle and loss of cell survival factors. The pathway is tightly regulated by a balance of pro-apoptotic and anti-apoptotic members of the Bcl-2superfamily of proteins. The intrinsic pathway, when activated by cellular stress signals, leads to up regulation of pro-apoptotic BH-3only proteins such as BAD (BCL-2antagonist of the cell death), BID (BH3interacting domain death agonist), BIM (Bcl-2interacting mediator of the cell death) BMF (Bcl-2modifying factor), PUMA (P53up regulated modulatro of apoptosis), and Noxa. These proteins in turn bind to the anti-apoptotic members of the family and inhibit their actions. One subgroup of BH-3only proteins includes direct activators (BID and BIM) which are able to bind and inhibit anti-apoptotic proteins but also can activate the effector proteins, BAK and BAX. The other group called sensitizers includes BAD, Hrk(Karakiri), Noxa and PUMA. These proteins bind the hydrophobic groove of anti-apoptotic Bcl-2proteins, therefore preventing any future interactions between anti-apoptotic and pro-apoptotic proteins. Once the pathway is activated, BAK and BAX homo-oligomerize and form pores in the OMM, leading to outer membrance permeabilization. This results in release of pro-apoptotic proteins like cytochrome c and Smac/DIABLO (second mitochondria-drived activator of caspases/direct inhibitors of apoptosis proteins-binding protein with low ph) into the cytosol. Cytochrome c forms a complex with APAF-1and pro-caspase9, whereas Smac/DIABLO binds to inhibitors of apoptosis proteins(IAPs). These steps lead to activation of caspase9, and subsequent activation of effector caspases3and pro-apoptotic phenotype.(3) other pathways such as the endoplasmic reticulum apoptosis pathway. Endoplasmic reticulum is the main site of protein synthesis, posttranslational modification, and folding, also is a major part of the calcium reserves and calcium signal transduction. When cells were stimulated by internal and external factors such as infection, ischemia and tissu hypoxia and so may lead to changes in the endoplasmic reticulum calcium channels, intracellular calcium imbalance. Ca2+is an important signal transduction factor in eukaryotic cells, its homeostasis plays an important role in normal physiological cells. Numerous studies show that intracellular calcium imbalance is one of the important mechanisms of apoptosis induction occurred. They found that high levels of cytosolic Ca2+lead to activation of the calcium dependent protease, and also can effect mitochondria then change its permeability, thereby promoting apoptosis.Currently, the treatment effect of ATO for AML has been affirmed. The complete remission rate(CR) of this disease has significantly improved by combined with other chomotherapy drugs. However, there are still a considerable number of patients did not achieve CR, or suffer the pain of relapse in a short term after CR. Therefore, develop an chemotherapy which can increase the effect of ATO while reducing the side effects of treatment has become one of the current research focus. Our findings suggest that low-dose of ACM combined with ATO can significantly inhibit the proliferation of human acute myeloid leukemia cell line KG-la cells and induce apoptosis. ACM is an effective anthracycline anti-tumor drugs. Studies have reported that ACM is mainly due to it as topoisomerase I and II inhibitors and thus exert its anti-tumor effect. Currently, clinical use of ACM combined with cytarabine have achieved a certain effect in the treatment of AML. However, the molecular mechanism of apoptosis in AML cells induced by ACM was less. In addition, because high-dose of ACM can lead to serious side effects, thus limiting its clinical application. Researchers in china found that ACM combined with homoharringtonine (HHT) can synergistically kill AML cells. To explore the synergistic killing effect of the combination of drugs for AML, we use ATO combined with ACM as the research object, its low toxicity and efficient was used to evaluate the cytotixic effect and its molecular mechanism after the combination treatment, and therefore provide scientific experimental basis for clinical treatment of AML.As we all know, cancer is one of the most common fatal disease, about14million people suffer from a variety of tumor diseases. All vertebrate species have the potential to develop cancer. By using other vertebrates as model organisms, we have greatly enhanced our understanding of the mechiansms of human carcinogenesis and cancer progression. Furthermore, studies in invertebrate organisms such as flies and worms have been used to decipher the genetic mechanisms relevant to cancer, such as those regulating tissue specification and growth, cell migration, and apoptosis. Zebrafish, a vertebrate model system, combines the advantages of invertebrate organisms with those of mammalian models; large clutch sizes and relative transparency aid in the identification of molecular genetic pathways involved in organ development and homeostasis. Recently, zebrafish have emerged as a model to study cancer susceptibility and carcinogenesis. Since zebrafish has the distinct advantages as a tumor study model. We aimed to use zebrafish embryos to investigate the model of Xenotransplantation. In this study, we use human acute myeloid leukemia cell line KG-la transplanted into zebrafish embryos to investigate the proliferation, migration, and tissue invasion. And therefore may provide a theoretical basis on leukemia study. Objectve:Human acute myeloid leukemia cell line KG-la cells were used as the main subjects, the aim of this section was to investigate the synergistical leath effect and its mechanism in vitro induced by ATO combined with ATO, and then may provide a new idea for the treatment of AML.Methods:Cell Culture.KG-la cell line were kindly presented by Professor Zengxuan Song (Chinese Academy of Medical Sciences and Peking Union of Medical College, China) and cultured in RPMI-1640supplemented with10%inactivated fetal bovine serum, plus1%penicillin and streptomycin at37℃under5%CO2.Cell viability assays in vitro.Inhibition of the proliferation rate of KG-la cells was assessed using cell count kit-8(CCK-8) followed by manufacturer’s instructions. Briefly,1.0×104cells were incubated in triplicate in a96-well plate with or without0-32μM AS2O3treatment or0-1280nM ACM treatment for24,48,72and96h, respectively. The test samples in a final volume of100μl RPMI-1640with10%FBS. Thereafter,10μl CCK-8solution was then added to each well for various lengths of time and incubated for4h at37℃. After4hours, the plates were shaked for5min and the optical density at450nm was detected through a universal microplate reader (ELx800; Bio-TEK, VT, USA). Percent cell viability(%) was calculated as experimental group/the control group×100%. Each experiment was performed at least three dependent measurement.Colony-forming assay. KG-la cells in logarithmic phase were suspended in RPMI1640medium with (0.4,1.5,3.0μM) AS2O3and (75nM) ACM, respectively or the combination treatment, then500cells per well were added to24well-plate supplemented with0.9%methylcellulose and20%FBS at37℃in5%CO2. The colonies were counted at10×10magnification to detect colony size(containing50or more cells) and colony numbers by light microscopy after14days. All cultures were performed in triplicate. Three independent experiments were performed.Wright-Giemsa staining.KG-la cells were treated with0-3μM AS2O3and75nM ACM, respectively or the combination treatment for48h. Morphological signs of apoptosis were determined by Wright-Giemsa staining. The treated and untreated cells were washed with PBS and then stained with Wright-Giemsa solution for10min at room temperature, rinsed with distilled water and air dried. Cell morphology was studied by light microscopy at10x40magnification.Measurement of apoptosis by flow cytometry.KG-la cells were treated for48h with the following concentrations:0.4,1.5and3μM As2O3;75nM ACM; and the combination treatment. Apoptosis assay kit (KeyGEN,Nanjing,China) was used in this experiment followed by the manufacturer’s instructions. Briefly, treated and untreated cells were harvested and washed twice with cold PBS and then resuspended in1×binding buffer at a concentration of6×105cells/ml. A total of200μl of the mixtures was then transferred to a1.5ml cultrue tube and then5μl of fluorescein-conjugated Annexin V (Annexin V-FITC) and5μl of propidium iodide (PI) were added to the culture tube. The cells were gently vortexed and incubated for10min at room temperature in the dark, then the stained cells were analyzed by flow cytometry to determine the percentages of Annexin V+/PI"(early apoptosis) and Annexin V+/PI+(late apoptosis) cells.Cell cycle assay by flow cytometry.The detection of cell cycle by flow cytometry was performed as described by the cell cycle detection kit (KeyGEN, Nanjing, China). AS2O3-or ACM treated KG-la cells were prepared similarly as described in the apoptosis analysis. Approximately 6×105/ml cells in6-well plates were treated with various concentrations of ACM for48h. After treatment, cells were harvested and washed twice with cold PBS, then resuspended in cold70%ethonal for at least6h at4℃. After washing twice with PBS,100μl Rnase A was added to the resuspended cells mixture at37℃for30min, then400μl PI was added to resuspended cells mixture at4℃for30min. Samples were then checked by FACSCalibur folw cytometer (Becton Dickinson, San Jose, CA, USA).Western blot analysis.To analysis the mechanism of synergistic effect of the combination treatment. We evaluated Low-dose of As2O3(1.5μM) and ACM (75nM) in the expression of apoptosis related proterns. The treated and untreated cells were homogenized with150μl of ice-cold lysis buffer (40mM HEPES, PH7.5,120mM NaCl,1mM EDTA,10mM pyrophosphate,10mM glycerophosphate,50mM NaF,1.5mM Na3VO4,1%Triton X-100, and1×EDTA-free protease inhibitors) on ice for30min. Then these homogenates were boiled for10min at100℃and centrifuged at12,000x g for10min at4℃. The pellet was discarded and the supernatant containing the protein was transferred to a clean tube. The prepared proteins wern then analyzed by immunoblotting.Results:In this study, we for the first time use AS2O3combined with ACM to investigate the synergistic cytotoxic effect on acute myeloid leukemia cell line KG-1α CCK-8assay indicated that AS2O3and ACM respectively inhibited the proliferation of KG-1α cells in dose and time manner. However, there is a more prominent effect of antiproliferation when treated by the combination treatment. In KG-1α cells, after the combination treatment with AS2O3and ACM at a40:1ratio, the combination index(CI) values were<lin all treatment groups. This result indicated that the combination treatment had a synergistic effect. Furthermore, we found that the cytotoxic effect of AS2O3on KG-1a cells is mainly due to the induction of apoptosis and the arrest of cell cycle. When KG-1α cells treat with (0.4μM,1.5μM和3.0μM) AS2O3for48h, the proportion of total apoptosis rate(the early apoptosis rate and the late apoptosis rate) was7.6±0.71%,19.1±3.38%and29.5±2.42%, respectively. Minewhile,(10nM,37.5nM,75nM) ACM treat for48h the proportion of total apoptosis rate was18.7±1.68%,26.7±1.87%and28.6±2.76%respectively. Compare to3.1%in the control group. In the cell cycle analysis, when KG-1a cells treat with (0.4μM,1.5μM和3.0μM) AS2O3for48h, the proportion of cells in G0/G1phase was64.32±0.55%,53.04±4.28%and47.13±6.19%, respectively. The S phase proportion was29.07±5.66%,40.13±1.83%and43.13±2.06%, respectively. The G2/M phase proportion was6.61±0.7%,6.83±0.98%and9.74±1.8%, respectively. These results revealed that AS2O3significantly decrease the G0/G1phase and increase the S phase. Similarly to AS2O3treatment, after treated with (10nM,37.5nM,75nM)ACM for48h, the proportion of cells in G0/G1phase was60.15±1.89%,55.19%±1.63and53.08±1.69%, respectively.34.98±1.47%,34.78±0.45%,34.37±4.15%respectively in S phase and4.86±0.1%,10.03±1.7%,12.54±2.41%, respectively in G2/M phase. This result showed that ACM is mainly arrest cells in G2/M phase. However, when cells were treated with the combined concentrations, the results obtained showed that there is a significant arrest in S phase.In order to investigate the possible mechanisms of the synergistic cytotoxic effect of the combination treatment. Protein microarray technology was used to analysis the expression of apoptosis related protein after the combination treatment. Minewhile, the clone formation experiment, Wright-Giemsa staining and western blot were also used to analysis the mechanisms of antiproliferation and the promotion of apoptosis by the combination treatment. The protein microarray assay showed that a variety of pro-apoptsis protein was up-regulated and anti-apoptosis protein down-regulated after the combination treatment. Minewhile, clone formation experiment indicated that the combination treatment had a more prominent cytotoxic effect than the single drug. In addition, Wright-Giemsa staining showed that morhpological changes such as cell shrinkage and nuclear condensation was significantly after the combination treatment with AS2O3and ACM in KG-la cells. Further more, western blot analysis was performed to evaluate the expression of apoptosis related proteins in KG-la cells after treated for48h. The expression level of anti-apoptotic Bcl-2protein showed no significant changes in a low-dose of AS2O3(1.5μM) treatment compare to control group. Meanwhile, caspase-3as an important member of the caspase proteins family which is a critical factor of apoptosis. In our experiment, the results showed that the production of pro-apoptotic caspase-3protein had a modest enhancement after treated with low-dose of As2O3(1.5μM). interestingly, we didn’t obversed obvious changes of anti-apoptotic Bcl-2and pro-apoptotic caspase-3proteins after ACM(75nM) treatment compared to control. However, the combination treatment significantly reduced the production of anti-apoptotic Bcl-2protein and activated the expression of pro-apoptotic caspase-3protein.Conclusions:AS2O3and ACM in certain concentrations can inhibit the proliferation of KG-1a cells, and the effect was time-and dose-dependent increase.Compared to the single drug treatment, more significant cell cycle arrest and apoptosis induction was obversed in the combination treatment group, and we found that the combination index (CI) for all of the combined treatment group are significantly smaller than1, indicating that the combination treatment does have a synergistic leath effect in KG-la cells.ATO combined with ACM may through coactive the caspase pathway and inhibit the proliferation of KG-1a cells, and therefore induce apoptosis. Objectve:The aim of this section was to investigate the feasibility of xenotransplant leukemia model using by zebrafish, thereby providing a more direct in vitro model for the study of leukemia and the development of drug for AML treatment.Methods:(1) The red fluorescent dye-MitoRed was labeled into acute myeloid leukemia cell line KG la in vitro, whether different concentrations of MitoRed have toxic effects on KG1a cells was analysized by MTT assay.(2) The labeled KG1a cells was microinjected into the yolk sac of48hours zebrafish embryos by microinjection transplantion, after injection, we directly observed the survival、proliferate, migrate and metastasize of KG1a cells in the zebrafish embryos using a fluorescence microscope on the1、3、5、7day,respectively.(3) On1day post injection(dpi) and4dpi, we digested the whole zebrafish embryos and counted the number of fluorescent cells in vitro to verify implanted cells survive and proliferate.Results:The results in second section was about the study of xenopransplantation model by using zebrafish embryos. The results showed:(1) High concentrations of MitoRed had toxicity on KG1a cells,while low concentration of MitoRed (200nM-500nM) had no effect on the proliferation of KGla (P>0.05),500nmol/L Mito-Red for30min was optimal for labeling KGla cells in vitro, which labeled KGla cell result in the maximum fluorescence intensity, and allowed detection of KGla as long as7days;(2) After KGla cells were injected into the yolk sac, we observed the KGla cells migration、metastasis and gradually spread to the entire abdominal cavity of the zebrafish in1-7days, the results of cell count in vitro also proved the proliferation of KG la cells,suggesting that implanted cells,survival,prolifer-ation and spread in the abdominal of zebrafish.(3) the survival rate of KG-la injection group was lower than the control and PBS groups;(4) Histological assay showed that the xenotranspantive cells could invade into the tissue of zebrafish.Conclusions:Human acute myeloid leukemia cells KG1a could survive, proliferate, migrate and metastasize in zebrafish, suggesting xenotransplant leukemia model using by zebrafish is feasible.