scholarly journals Changes in cell death of peripheral blood lymphocytes isolated from children with acute lymphoblastic leukemia upon stimulation with 7 Hz, 30 mT pulsed electromagnetic field

2015 ◽  
Vol 20 (1) ◽  
Author(s):  
Jolanta Kaszuba-Zwoińska ◽  
Magdalena Ćwiklińska ◽  
Walentyna Balwierz ◽  
Paulina Chorobik ◽  
Bernadeta Nowak ◽  
...  
Keyword(s):  
Cell Death ◽  
Electromagnetic Field ◽  
Acute Lymphoblastic Leukemia ◽  
Propidium Iodide ◽  
Peripheral Blood ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Pulsed Electromagnetic Field ◽  
Pulsed Electromagnetic ◽  
In Cells

AbstractPulsed electromagnetic field (PEMF) influenced the viability of proliferating in vitro peripheral blood mononuclear cells (PBMCs) isolated from Crohn’s disease patients as well as acute myeloblastic leukemia (AML) patients by induction of cell death, but did not cause any vital changes in cells from healthy donors. Experiments with lymphoid U937 and monocytic MonoMac6 cell lines have shown a protective effect of PEMF on the death process in cells treated with death inducers.The aim of the current study was to investigate the influence of PEMF on native proliferating leukocytes originating from newly diagnosed acute lymphoblastic leukemia (ALL) patients.The effects of exposure to PEMF were studied in PBMCs from 20 children with ALL. PBMCs were stimulated with three doses of PEMF (7 Hz, 30 mT) for 4 h each with 24 h intervals. After the last stimulation, the cells were double stained with annexin V and propidium iodide dye to estimate viability by flow cytometric analysis.The results indicated an increase of annexin V positive as well as double stained annexin V and propidium iodide positive cells after exposure to threefold PEMF stimulation.A low-frequency pulsed electromagnetic field induces cell death in native proliferating cells isolated from ALL patients. The increased vulnerability of proliferating PBMCs to PEMF-induced interactions may be potentially applied in the therapy of ALL.The analysis of expression of apoptosis-related genes revealed changes in mRNA of some genes engaged in the intrinsic apoptotic pathway belonging to the Bcl-2 family and the pathway with apoptosis-inducing factor (AIF) abundance upon PEMF stimulation of PBMCs.

Pulsed electromagnetic field and relief of hypoxia‐induced neuronal cell death: The signaling pathway

2019 ◽  
Vol 234 (9) ◽  
pp. 15089-15097 ◽  
Author(s):  
Stefania Gessi ◽  
Stefania Merighi ◽  
Serena Bencivenni ◽  
Enrica Battistello ◽  
Fabrizio Vincenzi ◽  
...  
Keyword(s):  
Cell Death ◽  
Electromagnetic Field ◽  
Signaling Pathway ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Pulsed Electromagnetic Field ◽  
Pulsed Electromagnetic

Obatoclax (GX15-070) Overcomes Glucocorticoid-Resistance In Acute Lymphoblastic Leukemia through Induction of Apoptosis and Autophagy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1809-1809
Author(s):  
Hisashi Harada ◽  
Nastaran Heidari ◽  
Mark Hicks
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Small Molecule ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Single Agent ◽  
Annexin V ◽  
Glucocorticoid Resistance ◽  
Down Regulation

Abstract Abstract 1809 Glucocorticoids (GC) are common components in many chemotherapeutic protocols for lymphoid/myeloid malignancies, including acute lymphoblastic leukemia (ALL). However, patients often develop resistance to GC on relapse. Resistance to GC in ALL can be associated with defects in apoptosis machinery, but not in the GC receptor. Thus, targeting downstream molecules may lead to the development of new therapeutic strategies. GC-induced apoptosis is through the intrinsic mitochondria-dependent pathway. The BCL-2 family proteins are central regulatory proteins in this pathway. We hypothesized that targeting anti-apoptotic MCL-1 might be effective among the BCL-2 family proteins, since (1) we recognized that treatment with dexamethasone (Dex) in CCRF-CEM or Molt-4 T-ALL cells slightly induce MCL-1 and the expression level of MCL-1 is higher in Dex-resistant ALL cells compared with that in Dex-sensitive cells; (2) recent studies have demonstrated that increased expression of MCL-1 associates with GC resistance. In support of our hypothesis, down-regulation of MCL-1 by shRNA enhances Dex-induced cell death. We then pharmacologically inactivate MCL-1 function by GX15-070 (obatoclax), a BH3 mimetic small molecule that targets anti-apoptotic BCL-2 family proteins including BCL-2, BCL-XL, and MCL-1. Treatment with GX15-070 in both Dex-sensitive and -resistant ALL cells shows effective growth inhibition and cell death. GX15-070 induces caspase-3 cleavage and increases Annexin V-positive population, indicative of apoptosis. Before the onset of apoptosis, GX15-070 induces LC3 conversion as well as p62 degradation, both of which are autophagic cell death markers. A pro-apoptotic molecule BAK is released from BAK/MCL-1 complex following GX15-070 treatment. Consistently, down-regulation of BAK reduces caspase-3 cleavage and cell death, but does not alter LC3 conversion. In contrast, down-regulation of ATG5, an autophagy regulator, decreases LC3 conversion and cell death, but does not alter caspase-3 cleavage, suggesting that apoptosis and autophagy induced by GX15-070 are independently regulated. Down-regulation of Beclin-1, which is capable of crosstalk between apoptosis and autophagy, affects GX15-070-induced cell death through apoptosis but not autophagy. Taken together, GX15-070 treatment in ALL could be an alternative regimen to overcome glucocorticoid resistance by inducing BAK-dependent apoptosis and ATG5-dependent autophagy. Enhanced anti-apoptotic BCL-2 family protein expression has been observed in several types of tumors. Targeting these proteins is therefore an attractive strategy for restoring the apoptosis process in tumor cells. Among the small molecule BCL-2 inhibitors, ABT-737 and its analog ABT-263 are the leading compounds currently in clinical development. However, these molecules have an affinity only with BCL-2 and BCL-XL, but not with MCL-1. Thus, ABT-737 can not be effective as a single agent therapeutic for ALL when MCL-1 is overexpressed. In contrast, GX15-070 can overcome the resistance conferred by high level of MCL-1. Our results suggest that GX15-070 could be useful as a single agent therapeutic against ALL and that the activity/expression of anti-apoptotic proteins could be a biomarker to determine the treatment strategy to ALL patients. (Supported by NIH R01CA134473 and the William Lawrence and Blanche Hughes Foundation) Disclosures: No relevant conflicts of interest to declare.

scholarly journals Preclinical Investigation of the p53-MDM2 Antagonist Idasanutlin (RG7388) Demonstrates Significant Activity in High Risk Adult Acute Lymphoblastic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 38-38
Author(s):  
Hayden L Bell ◽  
Mankaran Singh ◽  
Helen J Blair ◽  
Frederik W. van Delft ◽  
Anthony V. Moorman ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Target Gene ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Tp53 Mutations ◽  
Mdm2 Antagonist ◽  
P53 Signaling ◽  
Transcriptional Target

Outcomes for adult patients with acute lymphoblastic leukemia (ALL) are dismal and have not kept pace with their pediatric counterparts, with five-year survival rates of less than 45%. TP53 mutations are infrequent in ALL, but activity of the oncoprotein MDM2 may otherwise phenotypically disrupt and circumvent normal p53 function, positing the p53-MDM2 signaling axis as a potential therapeutic target for the engagement of intrinsic cell death programs. Given the clinical safety and responses to p53-MDM2 antagonist idasanutlin (RG7388) in other hematological cancers and solid tumors, we aimed to evaluate the therapeutic potential of idasanutlin in ALL. Single-agent activity of idasanutlin was investigated in 17 high-risk ALL patient and patient-derived xenograft samples (aged 4 to 51 years), both B-ALL (n=15) and T-ALL (n=2), including KMT2A-rearranged, TCF3-rearranged, and Philadelphia-positive ALL. An ex vivo coculture of ALL blasts and hTERT-immortalized mesenchymal stem cells (MSC) was employed to support growth of the ALL blasts during short-term culture, complemented by a fluorescent image-based microscopy platform which identifies and discriminates the two cellular compartments using random forest machine learning algorithms based on cellular nuclear staining. Idasanutlin demonstrated sub-micromolar, dose-dependent anti-leukemic activity against 15 of 17 samples tested, with half maximal effective concentrations (EC50) in the range of 10 to 220 nM (mean EC50 = 45.1nM); the two exceptions were later determined to harbor homozygous inactivating TP53 mutations; p.Y220C and p.S241P within the p53 DNA binding domain. The idasanutlin EC50 concentrations determined are clinically achievable, well below the peak plasma concentrations reported in patients for other disease indications. Furthermore, idasanutlin concentrations below 10µM had no impact upon MSC survival. Conforming to p53-MDM2 auto-regulatory feedback mechanisms, we demonstrated that idasanutlin efficiently stabilized and activated p53 at the protein level within 6 hours when treated with their respective idasanutlin EC50, to a level greater than 4-fold increased relative to their respective vehicle-only controls (p=0.001, n=7). Further, p53-regulated transcriptional target gene products, MDM2 and p21, were increased by 5-fold and 2-fold respectively, validating engagement of the p53 pathway by idasanutlin (p=0.036 and 0.125, respectively). By contrast, idasanutlin did not elicit increased expression of either p53-regulated transcriptional target gene product in the identified TP53-mutant patient samples (n=2). On-target specificity of idasanutlin was further confirmed in a NALM6 isogenic cell line model, whereby the TP53 wildtype line was sensitive to idasanutlin (EC50 = 74nM) and effectively activated p53 signaling whereas the TP53 homozygous null line was highly resistant (EC50 = ~10µM). To determine whether the decreased cell numbers and engagement of p53 signaling observed were accompanied by cell death, the capacity of idasanutlin to induce apoptosis in the ALL samples was next investigated. Patient-derived ALL samples (n=6) were treated with vehicle or idasanutlin at their respective EC50s for 24 and 48 hours, and then analyzed by flow cytometry. There was an increase in annexin-V positive cells within 24 hours compared to the vehicle-only treated cells (mean±SD 14.3±6.6% vs 27.0±21.2% respectively (p=0.125). By 48 hours significant apoptosis was attained, with a mean±SD of 59.0±23.8% annexin-V positive cells compared to the mean of vehicle-treated cells at 29.1±11.6% (p=0.004). Cleaved poly(ADP-ribose) polymerase (PARP) levels were also increased greater than 3-fold compared to vehicle-only control cells as assessed by immunoblotting (p=0.045, n=3), corroborating these findings. These data emphasize the potential of pharmacologically targeting the p53-MDM2 axis in ALL, demonstrating potent, on-target, cytotoxic activity in a range of high-risk ALL cytogenetic subgroups. Taken together, these findings support further preclinical investigations into idasanutlin and other p53-MDM2 antagonists and potential combinations to improve the treatment of adult ALL. Disclosures Irving: F. Hoffmann-La Roche: Research Funding.

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.

Targeting Bone Marrow Lymphoid Niches In Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1398-1398
Author(s):  
Geoffrey L. Uy ◽  
Wendy Stock ◽  
Yen-Michael S. Hsu ◽  
Jane E. Churpek ◽  
Peter Westervelt ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Peripheral Blood ◽  
Single Case ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Trophic Factors ◽  
Maximal Effect ◽  
Similar Data ◽  
Proliferation And Apoptosis

Abstract Similar to normal lymphoid progenitors, acute lymphoblastic leukemia (ALL) blasts receive key growth and survival signals from the bone marrow microenvironment which contributes to chemotherapy resistance and disease relapse. We previously reported that treatment with G-CSF in mice results in a marked a marked suppression of lymphopoiesis by targeting lymphoid niches in the bone marrow (Day et al., ASH abstract #1246, 2012). Specifically, G-CSF suppresses the expression of lymphoid trophic factors from osteoblasts and CXCL12-abundant reticular (CAR) cells, including CXCL12, stem cell factor, and interleukin-7, and interleukin-6. We hypothesized that disruption of lymphoid niches in the bone marrow by G-CSF mediated suppression of lymphoid trophic factors will render ALL cells more sensitive to chemotherapy. We conducted a pilot study of G-CSF priming in patients with relapsed or refractory ALL (NCT01331590). Our preclinical data indicates that the timing of G-CSF in relation to chemotherapy is critical and that G-CSF must be given for at least 4 days prior to chemotherapy to achieve its maximal effect on the BM microenvironment. In this study, G-CSF (10 µg/kg/d) was administered continuously from day 1 until neutrophil recovery followed by ifosfamide 3330 mg/m2/d CIVI on days 4-6, etoposide 150 mg/m2 IV BID, and dexamethasone 5 mg/m2 BID on Days 4-10. Peripheral blood and BM samples are obtained at baseline and after 4 days of G-CSF (prior to chemotherapy). Where sufficient material was available, the baseline and day 4 G-CSF blood and BM samples were analyzed to assess: 1) changes in the BM microenvironment; 2) ALL cell mobilization into the blood; and 3) ALL cell proliferation and apoptosis. To assess expression of B trophic factors by BM stromal cells, we analyzed RNA from BM core biopsies at baseline and at day 4. Ten patients with with relapsed or refractory ALL with a median age of 47 years (range 21-69) have been enrolled. One subject developed leukocytosis to 67K/mm3 from baseline of 20K/mm3 requiring temporary cessation of G-CSF and earlier initiation of chemotherapy. One treatment related death occured on study. Three of 10 patients have achieved a CR/CRi (30%). Importantly, there were no cases of delayed hematologic recovery. Compared to healthy volunteers, the baseline expression of CXCL12, IL-7, and osteocalcin mRNA in the bone marrow was markedly variable, with some samples displaying very low expression. Nonetheless, G-CSF resulted in a 2-fold or greater decrease in CXCL12 expression in 6 of 7 cases tested. These data suggest that, although the BM microenvironment is disrupted at baseline in many patients with relapsed/refractory ALL, G-CSF can suppress lymphoid trophic factors even further. In 4 of 5 cases, mobilization of ALL blasts after G-CSF to the peripheral blood was observed (average fold-increase: 3.1 ± 0.95; range 1.3 to 6.2-fold). Interestingly, in the single case with minimal mobilization, little change in bone marrow expression of CXCL12 with G-CSF treatment was observed. ALL blast proliferation and apoptosis in the bone marrow was measured by flow cytometry before and after G-CSF priming. In all 3 samples that were analyzed, a significant increase in apoptosis, as measured by Annexin V surface expression, was observed [% Annexin V+ ALL blasts: 2.9 ± .81% (baseline) versus 13.2 ± 4.0% (G-CSF); p = 0.01]. Similar data were observed measuring apoptosis by activated caspase 3 expression. No change in ALL cell blast proliferation (as measured by Ki67 and DAPI staining) was observed. This study demonstrates the safety and feasibility of G-CSF priming in combination with chemotherapy in patients with relapsed or refractory ALL. Furthermore, G-CSF priming mobilizes ALL blasts into the blood and induces apoptosis these cells in the BM. Collectively, these data provide the proof of principle framework for ongoing studies targeting marrow lymphoid niches in ALL. Disclosures: Off Label Use: sorafenib for AML.

scholarly journals Pulsed electromagnetic field stimulation may improve fusion rates in cervical arthrodesis in high-risk populations

2018 ◽  
Vol 7 (2) ◽  
pp. 124-130 ◽  
Author(s):  
D. Coric ◽  
D. E. Bullard ◽  
V. V. Patel ◽  
J. T. Ryaby ◽  
B. L. Atkinson ◽  
...  
Keyword(s):  
Electromagnetic Field ◽  
Risk Factor ◽  
High Risk ◽  
Clinical Risk Factor ◽  
Field Stimulation ◽  
Pulsed Electromagnetic Field ◽  
Exact Test ◽  
High Risk Populations ◽  
Pulsed Electromagnetic ◽  
Pemf Treatment

Objectives Pulsed electromagnetic field (PEMF) stimulation was evaluated after anterior cervical discectomy and fusion (ACDF) procedures in a randomized, controlled clinical study performed for United States Food and Drug Administration (FDA) approval. PEMF significantly increased fusion rates at six months, but 12-month fusion outcomes for subjects at elevated risk for pseudoarthrosis were not thoroughly reported. The objective of the current study was to evaluate the effect of PEMF treatment on subjects at increased risk for pseudoarthrosis after ACDF procedures. Methods Two evaluations were performed that compared fusion rates between PEMF stimulation and a historical control (160 subjects) from the FDA investigational device exemption (IDE) study: a post hoc (PH) analysis of high-risk subjects from the FDA study (PH PEMF); and a multicentre, open-label (OL) study consisting of 274 subjects treated with PEMF (OL PEMF). Fisher’s exact test and multivariate logistic regression was used to compare fusion rates between PEMF-treated subjects and historical controls. Results In separate comparisons of PH PEMF and OL PEMF groups to the historical control group, PEMF treatment significantly (p < 0.05, Fisher’s exact test) increased the fusion rate at six and 12 months for certain high-risk subjects who had at least one clinical risk factor of being elderly, a nicotine user, osteoporotic, or diabetic; and for those with at least one clinical risk factor and who received at least a two- or three-level arthrodesis. Conclusion Adjunctive PEMF treatment can be recommended for patients who are at high risk for pseudoarthrosis. Cite this article: D. Coric, D. E. Bullard, V. V. Patel, J. T. Ryaby, B. L. Atkinson, D. He, R. D. Guyer. Pulsed electromagnetic field stimulation may improve fusion rates in cervical arthrodesis in high-risk populations. Bone Joint Res 2018;7:124–130. DOI: 10.1302/2046-3758.72.BJR-2017-0221.R1.

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