Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia. Apoptosis (programmed cell death) is the genetically controlled ablation of cells during normal development. Inappropriately regulated apoptosis is implicated in disease states such as Alzheimer disease, stroke and cancer. Nearly 5 million Americans have heart failure today, with an incidence approaching population among persons older than 65 years of age. Heart failure is the reason for at least 20 percent of all hospital admissions. Microglia home page at microglia.net; The Role of Microglia in the Central Nervous System — Clinical Microbiology Reviews October 2004, p. 4; Creeping into your Head - A Brief Introduction to Microglia. Yeast metacaspase (Yca1p) is required for the execution of apoptosis upon a wide range of stimuli. However, the specific degradome of this yeast protease has not been unraveled so far. By combining different. Q: What are the functions of each ingredient? Tribulus terrestris is used to enhance. 1 Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Portland, OR, USA 2 Department of Integrative Biosciences, Oregon Health & Science University, Portland, OR, USA Correspondence: M Azuma, Senju. Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B- cell acute lymphoblastic leukemia. Introduction. Most cases of pediatric acute lymphoblastic leukemia (ALL) are of B- cell origin. One common B- cell acute lymphoblastic leukemia (B- ALL) subtype, first reported by Volgler et al. Patients with the oncogenic E2. A- PBX1 translocation had a dismal prognosis two decades ago,4,5 but their prognosis has been greatly improved by the more aggressive multiagent therapies that are currently the standard of care. Treatment protocols including intensive chemotherapy regimens for various B- ALL have improved cure rates from 1. However, about 2. Currently, major approaches include new formulations of existing chemotherapeutic agents, new antimetabolites and nucleoside analogs, monoclonal antibodies directed against leukemia- associated antigens, and molecularly targeted drugs, such as tyrosine kinase inhibitors (TKI) and FMS- related tyrosine kinase 3 inhibitors. Although the overall outcome of children with t(1; 1. B- ALL are often associated with adverse effects and a higher risk of central nervous system relapse, necessitating more effective and safer agents. Bafilomycin A1, a macrolide antibiotic isolated from the Streptomyces species, is an inhibitor of vacuolar H+ ATPase (V- ATPase). It binds to the V0 sector subunit c of the V- ATPase complex and inhibits H+ translocation, causing an accumulation of H+ in the cytoplasm of treated cells. Bafilomycin inhibits cell growth. These anticancer effects of bafilomycin A1 are considered to be attributable to the intracellular acidosis caused by V- ATPase inhibition. Bafilomycin A1 was also found to inhibit the growth of cancer cells under hypoxic conditions by expressing hypoxia- inducible factor- 1. Apart from their physiological role in the maintenance of cellular homeostasis, apoptosis and autophagy serve as important targets of tumor therapeutics. Whereas apoptosis is implicated in the removal of damaged or unwanted cells, autophagy is a cellular catabolic pathway that is involved in lysosomal degradation and recycling of proteins and organelles, and is therefore considered as an important survival mechanism for both normal cells and cancer cells in response to metabolic stress or chemotherapy. In hematologic malignancies, autophagy can either act as a chemoresistance mechanism or have tumor suppressive functions, depending on the context. In addition, autophagy is involved in other important aspects of blood cancers as it promotes immune competence and anticancer immunity, and may even help to enhance patients’ tolerance to standard treatments. Here, we present data demonstrating that a low concentration of bafilomycin A1 effectively inhibits and kills pediatric B- ALL cells. By using in vitro, ex vivo and in vivo models, we provide compelling evidence that bafilomycin A1 attenuates cytoprotective autophagy, induces apoptosis, and delays the onset of leukemia in a xenograft mouse model and inhibits and kills leukemic primary cells. An in vivo toxicity assay confirmed that bafilomycin A1 is safe. These data validate bafilomycin A1 as a novel, candidate therapeutic drug for pediatric B- ALL. Methods. Major reagent and cell lines. Bafilomycin A1 from Sigma- Aldrich (St. Louis, MO, USA) was used at a concentration of 1 n. M unless indicated with different doses. Leukemia cell lines RS4; 1. NB4, HL- 6. 0, K5. BV1. 73 were purchased from the ATCC (Manassas, VA, USA). Leukemia cell lines 6. Nalm- 6 were from DSMZ, Braunschweig, Germany. The leukemia cells were grown in RPMI 1. Hyclone, USA) with 1. Gibco, USA) at 3. Experimental cultures were initiated by reculturing exponentially growing cells at a density of 0. The viability of the leukemia cells collected from the medium was determined by counting total and trypan blue cells under a microscope. Patients’ samples. Primary samples from leukemia patients either cytogenetically identified as myeloid leukemia cells or sorted against CD1. BALL cells were obtained with informed consent according to institutional guidelines and were cultured in triplicate in flat- bottomed, 2. The 6. 97 B- ALL cells were injected at a dose of 5. Cells were allowed to proliferate in vivo for 6 days and then the transplanted mice were injected intraperitoneally with phosphate- buffered saline or bafilomycin A1 (0. Mice were killed on day 3. Peripheral blood, bone marrow, livers and spleens were analyzed for the presence of leukemic cells by flow cytometry. Engraftment was detected by flow cytometry using antibodies recognizing E2. A/PBX1 (BD, USA). Liver and spleen cells were collected for analysis. All experiments with animals complied with institutional protocols on animal welfare and were approved by the Ethics Committee of Soochow University. Image. Stream analysis. B- ALL cells (5. Samples were visualized and analyzed for the expression of marker proteins with IDEAS 5. Amnis, Seattle, USA). Cells were gated with the area and aspect ratio features for single cells or with the Gradient RMS feature for focused cells. These subsets were plotted for log intensities of channel with LC3. The LC3 spot count was analyzed by spot count wizard. Autophagy levels were calculated by measuring cell percentage with LC3high spot cells. The cell proliferation assay, small interfering RNA, fluorescence microscopy, analysis of mitochondrial membrane potential, analysis of intracellular free Ca. H, and routine blood examinations are described in the Online Supplementary Information. Statistical analysis. Results are shown as the mean . The Student t- test was used for comparisons between groups, with P values less than 0. Results. Bafilomycin A1 preferentially inhibits in vitro growth of pediatric B- cell acute lymphoblastic leukemia cells. Bafilomycin A1, at concentrations between 0. The cells were cultured in the presence of increasing concentrations of bafilomycin A1 (0 n. M, 0. 5 n. M, 1 n. M). Cell proliferation was measured using an MTT assay. The results showed that various concentrations of bafilomycin A1 profoundly inhibited the growth of three pediatric B- ALL cell types in culture (Figure 1. A). A flow cytometric assay also revealed that bafilomycin A1 effectively inhibited cell division of the three pediatric B- ALL cell lines (Figure 1. B). In contrast, acute and chronic myeloid leukemia cell lines were virtually insensitive to the action of this growth inhibitory agent (Figure 1. A,B, Online Supplementary Figure S1. A,B). Figure 1. Bafilomycin- A1 at low concentration preferentially inhibits the growth of pediatric B- ALL cells. Low- dose bafilomycin A1 preferentially inhibits B- ALL cells over 9. A1 treatment with the concentrations indicated. Cell division of various leukemia cell lines was measured by labeling with CFSE. The cells were cultured for 7. A1. The B- ALL cells were treated with bafilomycin A1, and then the cell cycle was analyzed by flow cytometry after PI staining. Bafilomycin A1 increased the percentage of cells in the G0/G1 phase and decreased the percentage of cells in the S and G2/M phases of the cell cycle. The representative histogram data and the percentage of cells in each stage of the cell cycle after 7. Although the remission rate of these types of B- ALL is high, central nervous system relapse is frequent. To understand the mechanisms by which a low concentration of bafilomycin A1 (1 n. M) inhibits the growth of pediatric B- ALL cells, we performed flow cytometric analysis of propidium iodide (PI)- stained samples of the B- ALL cell lines cultured for 7. A1. Bafilomycin A1 increased the percentage of B- ALL 6. Nalm- 6 cells in the G0/G1 phase of the cell cycle, with a concomitant decrease in cells in S and G2/M phases (Figure 1. C, Online Supplementary Figure S1. C). To elucidate how bafilomycin A1 slows down cell cycle progression, we examined the levels of critical protein components of cell cycle regulation in B- ALL cells. The time course of bafilomycin A1 treatment revealed that p. Nalm- 6 cells (the effects became obvious at 2. Figure 1. D, Online Supplementary Figure S1. D). Cyclin D3 in the 6. D1, D3 in Nalm- 6 cells were decreased after 7. A1 treatment (Figure 1. D, Online Supplementary Figure S1. D). Cyclin E2 was decreased in the late stage of bafilomycin A1 treatment in both the 6. Nalm- 6 cells, but the irregular change in cyclin E2 level in the early stage of culture and drug treatment of 6. Figure 1. D, Online Supplementary Figure S1. D). Bafilomycin A1 treatment also decreased both total Rb and phosphorylated Rb in the 6. Nalm- 6 cells (Figure 1. D, Online Supplementary Figure S1. D). Although there is a discrepancy between the dynamic changes of the cell cycle regulators in different B- ALL cell lines, the overall pattern appears to be similar, with low concentration bafilomycin A1 up- regulating the protein levels of certain cell cycle- dependent kinase (CDK) inhibitors, but down- regulating the protein levels of certain cyclin members. Notably, the discrepancy between the percentage of G0/G1 cells and the CFSE flow cytometry data or cell cycle profile in response to bafilomycin A1 treatment suggests that, in addition to inhibiting proliferation, bafilomycin A1 also causes cell death. The growth arrest may also result from fewer cells in the S phase after bafilomycin A1 treatment. Nevertheless, the above data suggest that bafilomycin A1 may induce cell cycle arrest and inhibit proliferation of pediatric B- ALL cells, at least in part, by up- regulating negative regulators and down- regulating positive regulators of the cell cycle. Bafilomycin A1 at low concentration effectively inhibits autophagy of pediatric B- cell acute lymphoblastic leukemia cells via multiple targets. To confirm that bafilomycin A1 targets acidic vesicle lysosomes in pediatric B- ALL cells, we analyzed the long- term effects of bafilomycin A1 on the p. H of intracellular vesicles in B- ALL cell lines. Lyso. Sensor staining showed that 1 n.
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