scholarly journals Antileukemic Effects of Anti-miR-146a, Anti-miR-155, Anti-miR-181a, and Prednisolone on Childhood Acute Lymphoblastic Leukemia

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Burak Durmaz ◽  
Bakiye Goker Bagca ◽  
Ozgur Cogulu ◽  
Sunde Yilmaz Susluer ◽  
Araz Alpay ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Trypan Blue ◽  
B Lymphocyte ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Protein Coding ◽  
Epigenetic Regulators

Prednisolone has been used frequently in the treatment of acute lymphoblastic leukemia. However, to overcome the challenges of the treatment, the development of additional therapies is of great importance. Small, non-protein-coding RNAs, namely, microRNAs (miRNAs), are critical epigenetic regulators with physiological and pathological importance. This study is aimed at determining the effects of miR-146a, miR-155, and miR-181a inhibition with their corresponding anti-miRs on both leukemic and healthy cells, individually and with prednisolone. Leukemic (SUP-B15) and healthy B-lymphocyte (NCI-BL 2171) cell lines were used in this study. A total of 12 experimental groups included individual and combinational silenced ALL-associated miRNAs (hsa-miR-155, hsa-miR-146a, and hsa-miR-181a) and their combination with prednisolone. Cytotoxicity, proliferation, cell cycle, and apoptosis analyses were performed by using WST-1, trypan blue, APC-BrdU, Annexin V, and JC-1 methods in each study group, respectively. To control the effectiveness of anti-miR transfection and prednisolone application, miRNA expression analysis was performed from all groups. Anti-miR application was effective on the viability, proliferation, cell cycle, and apoptosis of leukemia cells, and this effect was increased with prednisolone administration. In addition, this activity was found to be very low on healthy cells. In conclusion, anti-miR applications may have the potential for clinical use of adjuvant to or as an alternative to conventional therapies for childhood acute lymphoblastic leukemia.

scholarly journals Autophagy Inhibition Sensitizes Acute Lymphoblastic Leukemia Cells to L-Asparaginase

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3772-3772 ◽  
Author(s):  
Hiroyoshi Takahashi ◽  
Jun Inoue ◽  
Kimiyoshi Sakaguchi ◽  
Masatoshi Takagi ◽  
Shuki Mizutani ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Combination Treatment ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Leukemia Cells ◽  
G1 Phase ◽  
Autophagy Inhibition

Abstract Autophagy is an intracellular protein and organelle degradation system, and is upregulated in response to cellular stress such as amino acid starvation. On the other hand, L-asparaginase (L-asp) plays an essential role in acute lymphoblastic leukemia (ALL) therapy by reducing intracellular asparagine and glutamine in ALL cells. However, the relationship of L-asp and autophagy is largely unknown. Here we show that L-asp induced cytoprotective autophagy. Three ALL cell lines of varied genetic background were used for in vitro experiments (REH, ETV6-RUNX1+ B-cell precursor (BCP) ALL; 697, E2A-PBX1+ BCP-ALL; TS-2, MEF2D-DAZAP1+ BCP-ALL). The cells were exposed to chroloquine (CQ) or bafilomycin A1 as autophagy inhibitors for 3 hours. LC3B-II, autophagy flux marker, was significantly increased under L-asp treatment with CQ as compared to only CQ condition, which was confirmed in independent experiments at immunofluorescence staining. Transmission electron microscopy showed that both the number and the area of autophagic vesicles per cell were markedly increased in L-asp with CQ condition. Thus, autophagy was induced by L-asp increasing turnover and clearance of autophagosomes in ALL cells. The toxic effect of 4 groups (control, CQ, L-asp, and L-asp plus CQ) by flow cytometry using Annexin-V staining indicated that combination treatment with L-asp and CQ for 48 hours induced significant cell death in the three ALL cell lines. Furthermore, inhibition of autophagy by CQ comparably sensitize REH cells to L-asp as ATG7 silencing by short interfering RNA. Cell growth assays for 6-9 days showed that L-asp monotreatment suppressed cell growth but did not increase the percentage of dead cells. In contrast, combination treatment with L-asp and CQ decreased the number of living cells and significantly increased the percentage of dead cells in time-dependent manner. Cell cycle analysis showed that cell cycle arrest at G1 phase was induced and the percentage of cells in sub-G1 phase remained a small increase by L-asp monotreatment, indicating leukemia cells endured amino acid deficiency by G1 arrest. In contrast, addition of CQ to L-asp significantly increased the sub-G1 population instead of decreasing G1 population. The apoptosis-related protein expressions using western blot analysis showed that combination treatment with L-asp and CQ induced cleavage of caspase 3 and PERP. In addition, a pan-caspase inhibitor benyloxycarbonyl Val-Ala-Asp (O-methyl)-fluoro-methylketone (z-VAD) significantly reduced the percentage of Annexin-V positive cells in the combination treatment with L-asp and CQ, which suggested that the autophagy inhibition upon L-asp treatment induced apoptotic cell death. We next transduced REH cell line with a luciferase-expressing viral vector. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were transplanted with these cells via tail vein and 6,000 U/kg L-asp and/or 50 mg/kg CQ were injected intraperitoneally once per day for survival analysis. The combination treatment with L-asp and CQ clearly reduced the leukemia burden as detected by luciferase intensity and improved outcome (L-asp plus CQ vs L-asp at day 28 after administration: 82% vs 0%, respectively. p <0.001). Of note, LC3B dots detected by immunofluorescence staining were apparently increased by the combination treatment with L-asp and CQ in the ALL cells derived from peripheral blood, bone marrow, and central nervous system at day 14 after transplantation. Taken together, these data suggest that autophagy plays cytoprotective role in L-asp-treated ALL cells both in vitro and in vivo, and autophagy inhibition upon L-asp treatment may be a useful approach for ALL. Disclosures No relevant conflicts of interest to declare.

Zebularine induces chemosensitization to methotrexate and efficiently decreases AhR gene methylation in childhood acute lymphoblastic leukemia cells

2014 ◽  
Vol 25 (1) ◽  
pp. 72-81 ◽  
Author(s):  
Augusto F. Andrade ◽  
Kleiton S. Borges ◽  
Angel M. Castro-Gamero ◽  
Vanessa S. Silveira ◽  
Veridiana K. Suazo ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Gene Methylation ◽  
Ahr Gene

scholarly journals BAG3 Protein Regulates Cell Survival in Childhood Acute Lymphoblastic Leukemia Cells

2003 ◽  
Vol 2 (5) ◽  
pp. 508-510 ◽  
Author(s):  
Maria Fiammetta Romano ◽  
Michelina Festa ◽  
Antonello Petrella ◽  
Maria Pascale ◽  
Rita Bisogni ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Survival ◽  
Lymphoblastic Leukemia ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Leukemia Cells

Abstract B62: Cell cycle alterations in paired diagnosis-relapse childhood acute lymphoblastic leukemia.

2013 ◽  
Author(s):  
Roberta Bortolozzi ◽  
Giampietro Viola ◽  
Maddalena Paganin ◽  
Silvia Bresolin ◽  
Silvia Disarò ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Cell Cycle Alterations

Bafilomycin A1 Targets Both Autophagy and Apoptosis Pathways in Pediatric t(1;19) Pre-B Acute Lymphoblastic Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3699-3699
Author(s):  
Na Yuan ◽  
Lin Song ◽  
Suping Zhang ◽  
Weiwei Lin ◽  
Yan Cao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Western Blotting ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Primary Cells ◽  
Bafilomycin A1 ◽  
Low Concentration

Abstract The t (1; 19) subtype leukemia accounts for a quarter of pre-B acute lymphoblastic leukemia (ALL) and up to 5% of all ALL patients. Despite plausible remission rate, current treatment regimen on the pediatric pre-B ALL is associated with side effects and CNS relapse, which poses the need for more effective and safer drugs. Bafilomycin A1 (Baf-A1) is known as an inhibitor of late phase of autophagy by inhibiting fusion between autophagosomes and lysosomes as well as by inhibiting lysosomal degradation. Here we show that Baf-A1 of low concentration (1 nM) effectively and specifically inhibits and kills the pre-B ALL cells. E2A/Pbx1 fusion gene positivepre-B ALL 697 cells were used for In vitro experiments. The results of flow cytometry analysis and western blotting showed that Baf-A1 induced cell cycle arrest and proliferation inhibition of ALL cells by upregualting cell cycle negative regulators and downregulating cell cycle positive regulators. In contrast, AML and CML cell lines were insensitive to Baf-A1 treatment. Western blotting and confocal observation on protein LC3 also showed that Baf-A1 at 1 nM blocked basal autophagic flux. Baf-A1 treatment activated mTOR signaling and induced the formation of Becn1–Bcl-2 complex to inhibit the induction of autophagy. Furthermore, apoptosis was induced in ALL cells treated with Baf-A1 for 72 h. However, procaspase-3 and poly-(ADP-ribose) polymerase (PARP) were not cleaved in these cells. We observed that AIF relocalized to the nucleus after 72h Baf-A1 treatment by confocal and immunoblotting. Knockdown of AIF significantly attenuated apoptosis induced by Baf-A1. These data suggest that Baf-A1 targets mitochondria membrane to trigger apoptosis via AIF pathway. In the in vivo experiment, Baf-A1 treatment extended survival and improved pathology of 697 xenograft mice, and significantly reduced the E2A/PBX1 positive leukemia cells in the bone marrow of mice. In vivomouse toxicity assay confirms Baf-A1 as a safe compound. The bone marrow cells of pre-B ALL leukemia patients were sorted against CD133+CD19+ markers, and treatment with Baf-A1 induced a clear inhibition on the CD133+CD19+ primary cells with a significant increased cell death in the sorted B-ALL patient samples. Conversely, Baf-A1 had no inhibitory effect on the bone marrow cells isolated from acute myeloid leukemia patients and healthy people. In summary, Baf-A1 treatment at low concentration effectively and specifically inhibited autophagy by activating mTOR and inducing beclin1-Bcl-2 interaction and induced AIF-dependent apoptosis in t (1; 19) pre-B ALL 697 cells. In the pre-B ALL xenograft mouse model, Baf-A1 specifically targets the leukemia cells while sparing normal cells. More importantly, Baf-A1 potently inhibits and kills the primary cells from pediatric pre-B ALL patients both at initial diagnosis and relapse without compromising normal human hematopoietic cells, all proposing Baf-A1 as a promising drug candidate for this pre-B ALL. Disclosures No relevant conflicts of interest to declare.

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