FTY720 Has Potent Anti-Leukemic Effects on Acute Lymphoblastic Leukemia Cells and Results In Caspase Independent Cell Death

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3260-3260
Author(s):  
Craig T. Wallington-Beddoe ◽  
John Hewson ◽  
Kenneth Francis Bradstock ◽  
Linda J. Bendall
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cell Lines ◽  
Western Blotting ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Parp Cleavage ◽  
Oxygen Species ◽  
Ic50 Values

Abstract Abstract 3260 Introduction: Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer, which usually responds to chemotherapy. Long-term survival in adults is poor with most developing disease relapse, whilst Ph+ ALL has a particularly poor prognosis. FTY720 is an immunosuppressive drug that has recently demonstrated efficacy in phase 3 trials of relapsing/remitting multiple sclerosis. FTY720 also appears promising in a number of malignancies with the proposed mechanism being the reactivation of PP2A, a protein serine/threonine phosphatase whose activity may be reduced in malignant cells. Here we report findings of in vitro testing of FTY720 on Ph+ and negative ALL cell lines and primary patient samples, describing mechanisms of cell death. Methods: ALL cell lines and primary patient samples were treated with 1 nM - 100 μM FTY720 for 24 hours. Viability was measured by flow cytometry using propidium iodide and annexin V staining. Cellular proliferation was measured by 3H-thymidine incorporation. Flow cytometry and western blotting were used to measure caspase 3 activation whilst western blotting was used to assess caspase 3, PARP cleavage and LC3II formation. Electron microscopy permitted a detailed examination of cell ultra-structure and confocal microscopy with lysosensor blue staining enabled visualisation of acidic vacuoles. Reactive oxygen species generation was assessed by flow cytometry using the cell permeable dye carboxy-H2DCFDA. Results: FTY720 produced a profound reduction in proliferation and viability of Ph+ (ALL1 cells) and Ph− (REH, NALM6 and LK63 cells) cell lines and patient samples (n=7) in the low micromolar range. IC50 values for loss of viability at 24 hours ranged from 5.3 μM for ALL1 to 7.9 μM for LK63. The IC50 values for proliferation at 24 hours were 1.4 μM for ALL1 and 3.5 μM for REH. Caspase 3 activation was observed only at very low levels by flow cytometry whilst both caspase 3 and PARP cleavage were not detected by western blotting. Inhibition of caspases by ZVAD-FMK failed to rescue ALL cells from FTY720 induced cell death, demonstrating a caspase independent cell death mechanism. Light microscopy revealed prominent cytoplasmic vacuolation, and electron microscopy showed features consistent with autophagy and necrosis. Western blotting demonstrated strong LC3II bands and confocal microscopy, using lysosensor blue, revealed prominent acidic vacuolation, all confirming the induction of autophagy. Reactive oxygen species were generated in response to FTY720 treatment and partial reversal of this by N-acetyl-cysteine produced a concomitant increase in cell viability. PP2A inhibition with okadaic acid failed to rescue cells from FTY720-induced cell death. Conclusion: FTY720 is a highly active drug in vitro in ALL cell lines and patient samples. Evidence supports a caspase independent mechanism of cell death with the occurrence of autophagy and necrosis. PP2A activation is not solely responsible for leukemic cell death. Data on the in vivo effects of FTY720 on ALL cells in NOD-SCID mice will be presented. Disclosures: Bendall: Genzyme: Honoraria.

Sphingosine Kinase Inhibition Has Pre-Clinical Activity in Acute Lymphoblastic Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3573-3573
Author(s):  
Craig T. Wallington-Beddoe ◽  
David Ho ◽  
Kenneth Francis Bradstock ◽  
Linda J. Bendall
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Term Survival ◽  
Intracellular Location ◽  
Ic50 Values

Abstract Abstract 3573 Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer, which usually responds to chemotherapy. Long-term survival in adults is poor with most developing disease relapse, whilst Ph+ ALL has a particularly poor prognosis. Sphingosine 1-phosphate (S1P) is a lipid mediator of diverse cellular functions, most notably of lymphocyte trafficking, angiogenesis, cell proliferation and survival. S1P is produced intracellularly by the sphingosine kinases (SK) of which there are two isoforms, SK1 and SK2. SK1 is over expressed in a number of malignancies and evidence points overwhelmingly to a pro-survival role. The role of SK2 is much less well defined and appears to be dependent on its intracellular location with some reports of opposite effects to those of SK1. Inhibition of either SK1 or SK2 is currently under investigation as a novel anti-cancer strategy and potent anti-leukemic effects are likely. Application of the combined SK1/SK2 inhibitor SKI II and the selective SK2 inhibitor ABC294640 to ALL cells produced a reduction in cellular proliferation as measured by 3H-thymidine incorporation in all cell lines (REH, NALM6, LK63, ALL1, 2070 and TOM1) tested with IC50 values of 1μM – 7μM for SKI II and <40μM for ABC294640. Viability, measured by flow cytometry using annexin V and propidium iodide (PI) staining, was also reduced in all cell lines except the Ph+ ALL1 and 2070 cells treated with SKI II with IC50 values ranging from 2μM to >10μM for SKI II and 50–60μM for ABC294640. SKI II resulted in caspase-dependent cell death, as determined by flow cytometric assessment of intracellular caspase-3 cleavage and apoptotic morphology on light microscopy, with cell death prevented by pre-incubation with 100μM of the pan-caspase inhibitor Z-VAD-FMK. However, ABC294640 induced caspase-3 cleavage at lower than expected levels and cell death was not prevented by Z-VAD-FMK. Both agents significantly reduced intracellular S1P concentrations by 24 hours as determined by ELISA, thereby confirming the ability of these compounds to inhibit SK1 or SK2 activity. A search for agents that synergized with the SK inhibitors revealed that when Ph+ ALL cells were treated with the combination of imatinib and either ABC294640 or SKI II, a further reduction in cell death occurred than with either agent alone, thereby enhancing the therapeutic effect of ABC294640 and overcoming resistance seen with SKI II alone. Furthermore, the combination of mildly cytotoxic concentrations of ABC294640 and the novel pan histone deacetylase inhibitor AR-42 were found to significantly increase leukemic cell death at 24 and 48 hours in Ph+ and negative ALL cells. In vivo assessment of the SK inhibitors was determined by injecting NOD/SCID IL2gc−/− mice with 2–5 million human B-ALL cells and treating with 100 mg/kg/day ABC294640 or vehicle by intraperitoneal injection for 21 days after which all animals were sacrificed. Assessment of leukemia in blood, bone marrow and spleen was determined by flow cytometry using antibodies to human CD19 and murine CD45. Significant reductions in the levels of leukemia in all examined tissues were found in ABC294640-treated animals using three different human ALL xenografts, including the Ph+ positive xenograft 2070. Average absolute levels of leukemia in the bone marrow of ABC294640-treated mice for xenografts 2070, 1345 and 0398 were reduced by 40% (p = 0.00007), 55% (p = 0.004) and 72% (p = 0.000001) respectively. No overt toxicity was noted. SK inhibition, resulting in reduced intracellular S1P, is an exciting novel anti-leukemic strategy potentially adding to the repertoire of non-chemotherapeutic agents for the treatment of ALL. Combinations of SK inhibitors with newer targeted agents show promise of greater leukemia reduction. Disclosures: No relevant conflicts of interest to declare.

Obinutuzumab (GA101) Significantly Enhances Cell Death and ADCC Compared to Rituximab Against CD20+ sensitive and Rituximab Resistant B-Cell Non-Hodgkin Lymphoma (NHL) and Lymphoblastic Leukemia (BLL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4865-4865 ◽  
Author(s):  
Aradhana Awasthi Tiwari ◽  
Janet Ayello ◽  
Carmella van de Ven ◽  
Danielle Glassman ◽  
Anthony Sabulski ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Dismal Prognosis

Abstract Abstract 4865 Background: Patients who relapse with CD20+ B-NHL and B cell lymphoblastic leukemia (B-LL) have a dismal prognosis, often associated with chemotherapy resistance (Cairo et al. JCO, 2012,Mils/Cairo et al. BJH,2012) and often require alternative therapeutic strategies. Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe, well-tolerated treatment that is associated with > 90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Cairo M.S. et al. ASCO, 2010). Resistance to RTX, however, may predispose patients with CD20+ NHL to an increase risk of relapse and or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, mediates enhanced cell death vs RTX and has a glycoengineered Fc region that induces significantly enhanced ADCC (Mössner et al. Bld, 2010; Niederfellner G. et al. Bld, 2011; Bologna L et al. JI, 2012). Objective: To evaluate the in-vitro efficacy of GA101 compared to RTX against RTX sensitive and resistant CD20+ B-NHL and B-LL cell lines. Methods: Raji (CD20+,ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany), Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) and Raji-2R and Raji-4RH (generously supplied by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS and incubated with GA101 and/or RTX at 100 μg/ml for 24 hrs (n=6), 48 and 72 hrs (n=5). Cell death was evaluated by staining with AnnexinV/7AAD and flow-cytometry. Loucy cells (CD20−) were used as the negative control. The caspase 3/7 activity was measured by FAM caspase 3/7 assay kit by FLICA™ methodology. RSCL, RRCL, U698-M and Loucy were incubated with GA101 and RTX treatment for 24, 48 and 72 hrs, and caspase3/7 activity was detected by FACS using 488 nm excitation and emission filter (n=3). ADCC were performed with K562-IL-15–41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) as well as IL-2 expanded NK cells, at 20:1 effector: target ratio (E: T, n=3) using europium release assay (Perkin-Elmer). Results: GA101 induced significantly more cell death compared to RTX in B-NHL and BLL cell lines. (Table-1) GA101 vs RTX shows a significantly increase in caspase 3/7 activity in Raji 16.92±0.84% vs 11.76±0.08% compared to Raji2R 6.7±0.62% vs 2.8±0.7%, Raji4RH 5.8±0.35% vs 2.0±0.3% and U698-M 12.54±0.44% vs 9.6±0.95% compared to Loucy 3.22±0.45% vs 2.59±0.05%, respectively, at 24 hrs of treatment (p<0.0001). GA101 vs RTX also elicited a significant increase a ADCC with K562-IL15–41BBL expanded NK cells, in Raji 73.8±8.1% vs 56.81±4.6% compared to Raji-2R 38.0±2.0% vs 21.6±1.2%, Raji-4RH 40.0±1.6% vs 0.5±1.1% and U698-M 70.0±1.6% vs 45.56±0.1%, compared to Loucy 21.67±0.48% vs 15.92±0.52%, respectively (p<0.001) at day 7.The IL-2 alone expanded Hu-NK cells demonstrated a reduction of 10–20% cytotoxicity compared to K562-IL15–41BBL Hu-NK cells at day 7 against BLL, RSCL and RRCL, in-vitro. Conclusion: Obinutuzumab compared to RTX significantly enhanced cell death, caspase3/7 activity and NK mediated ADCC in sensitive and RTX resistant B-NHL and B-LL. Obinutuzumab represents a promising candidate for treating RTX sensitive and resistant CD20+ B-Cell Lymphomas and lymphoblastic leukemia. Further studies will investigate the combination of activated NK cells or chemotherapy that may enhance or synergize with the efficacy of GA101 (Obinutuzumab) both in -vitro and in-vivo in xenografted NOD/SCID mice. Disclosures: No relevant conflicts of interest to declare.

Evaluation Of Sphingosine Kinase 1 As a Therapeutic Target In B-Lineage Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1426-1426
Author(s):  
Craig T Wallington-Beddoe ◽  
Stuart M Pitson ◽  
Jason A Powell ◽  
Kenneth F Bradstock ◽  
Linda J Bendall
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Chemotherapeutic Agents ◽  
Parp Cleavage ◽  
Signalling Pathways ◽  
Wide Range ◽  
Ic50 Values

Abstract Sphingosine 1-phosphate (S1P) is a bioactive lipid with roles in cell proliferation and survival. S1P is produced by the sphingosine kinases, SphK1 and SphK2. SphK1 is over-expressed in a number of malignancies and evidence points overwhelmingly to a pro-survival role. Furthermore, SphK1 has been shown to correlate with the clinical outcome of certain tumors. Here we focus on SphK1 as an important oncogenic target in acute lymphoblastic leukemia (ALL). We have previously shown SphK1 protein to be over-expressed and activated (Ser225 phosphorylation) in ALL cell lines and primary patient samples compared to normal CD34+CD19+ B-cell progenitors. Furthermore, we have reported the importance of SphK1 in the development of ALL by transducing B-cell progenitors isolated from WT or SphK1-/- mice with the ALL associated p185 form of the oncogenic fusion gene BCR/ABL and injecting transduced cells into sub-lethally irradiated WT mice. The absence of SphK1 significantly reduced the incidence of ALL in recipient mice (ASH 2012). Inhibition of SphK1 by the selective inhibitor SK1-I significantly reduced intra-cellular S1P concentrations (p=0.017 and p=0.003 at 24 and 48 hours respectively) in 3 cell lines examined, indicating that the drug targets this enzyme. SK1-I killed ALL cells as determined by annexin V/PI flow cytometric analysis with IC50 values ranging from 12 µM to 18 µM at 72 hours. Furthermore, SK1-I induced cell death in primary patient ALL cells by 16 hours. This agent resulted in virtually no caspase-3 cleavage and cell death was not prevented by the pan-caspase inhibitor Z-VAD-FMK (p=0.45, n=4). Marked cytoplasmic vacuolation was detected by light microscopy, with LC3 processing present by Western blot, consistent with the development of autophagy. However, the autophagy inhibitor 3MA failed to prevent SK1-I-mediated cell death. These results suggest that the cell death associated with inhibition of SphK1 in ALL cells is caspase-independent and cannot be attributed to autophagy. Surprisingly, conventional chemotherapeutic agents such as doxorubicin and vincristine failed to synergize with SK1-I, however, synergistic killing was observed when SK1-I was combined with 500 nM imatinib over 72 hours in Philadelphia-positive (BCR/ABL+) ALL cells. We have developed a novel SphK1 inhibitor, MP8, that targets the enzyme via a different mechanism to SK1-I, since it blocks ATP binding. MP8 reduced intra-cellular S1P in Jurkat cells by 43% compared to untreated controls, and killed Jurkat and SUP-B15 cells over 24 to 48 hours with IC50 values of 8 µM. Additionally, MP8 induced cell death in primary patient ALL cells by 24 hours. This agent resulted in classic apoptotic cell death, which was rescued by Bcl-2 over-expression, resulting in near complete reversal of PARP cleavage. SphK1 has indisputable tumor-promoting properties and lies downstream of a number of signalling pathways known to be dysregulated in ALL. Here we show that SphK1 is over-expressed and activated in ALL cells and targeting SphK1 has potent cytotoxic effects in a wide range of cell lines and patient samples. Furthermore, genetic deletion of Sphk1 significantly reduced the incidence of murine BCR/ABL-driven ALL. These findings suggest further examination of the role SphK1 plays in ALL will uncover novel interactions with oncogenic signalling pathways and paves the way for the inclusion of SphK1 inhibitors in future pre-clinical trials. Disclosures: No relevant conflicts of interest to declare.

Reactive oxygen species and caspase activation mediate silica-induced apoptosis in alveolar macrophages

2001 ◽  
Vol 280 (1) ◽  
pp. L10-L17 ◽  
Author(s):  
Han-Ming Shen ◽  
Zhuo Zhang ◽  
Qi-Feng Zhang ◽  
Choon-Nam Ong
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
Alveolar Macrophages ◽  
Caspase 3 ◽  
Reactive Oxygen ◽  
Parp Cleavage ◽  
Oxygen Species ◽  
Caspase Activation ◽  
Caspase 9 ◽  
Induced Apoptosis

Alveolar macrophages (AMs) are the principal target cells of silica and occupy a key position in the pathogenesis of silica-related diseases. Silica has been found to induce apoptosis in AMs, whereas its underlying mechanisms involving the initiation and execution of apoptosis are largely unknown. The main objective of the present study was to examine the form of cell death caused by silica and the mechanisms involved. Silica-induced apoptosis in AMs was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and cell cycle/DNA content analysis. The elevated level of reactive oxygen species (ROS), caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage in silica-treated AMs were also determined. The results showed that there was a temporal pattern of apoptotic events in silica-treated AMs, starting with ROS formation and followed by caspase-9 and caspase-3 activation, PARP cleavage, and DNA fragmentation. Silica-induced apoptosis was significantly attenuated by a caspase-3 inhibitor, N-acetyl-Asp-Glu-Val-Asp aldehyde, and ebselen, a potent antioxidant. These findings suggest that apoptosis is an important form of cell death caused by silica exposure in which the elevated ROS level that results from silica exposure may act as an initiator, leading to caspase activation and PARP cleavage to execute the apoptotic process.

scholarly journals 883 An anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human acute myeloid leukemia (AML) cell lines in vitro

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A925-A925
Author(s):  
Alessandra Romano ◽  
Nunziatina Parrinello ◽  
Sara Marino ◽  
Enrico La Spina ◽  
Massimo Fantini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Functional Analysis ◽  
Epithelial Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Phase 1 ◽  
Adcc Activity ◽  
Phenotypic Analysis

BackgroundNEO-201 is an IgG1 mAb targeting variants of CEACAM5/6 and has demonstrated tumor sensitivity and specificity in epithelial cells. Functional analysis has revealed that NEO-201 can engage innate immune effector mechanisms including ADCC and CDC to directly kill tumor cells expressing its target. A recent Phase 1 clinical trial at the NCI has determined both safety and recommended Phase 2 dosing. We have also seen the expression of the NEO-201 target on hematologic cells, specifically Tregs and neutrophils. Due to epitope being expressed both on malignant epithelial cells as well as several hematologic cells, we designed this study to explore the reactivity of NEO-201 against hematological neoplastic cells in vitro.MethodsPhenotypic analysis was conducted by flow cytometry. Cell lines used were six AML (HL60, U937, MOLM13, AML2, IMS-M2 and OCL-AML3), two multiple myelomas (MM) (OPM2, MM1.S), two acute lymphoblastic leukemia (ALL) (SUP-B15, RPMI8402) and four mantle cell lymphoma (MCL) (Jeko-1, Z138, JVM2 and JVM13). Markers used for flow cytometry analysis were CD15, CD45, CD38, CD138, CD14, CD19 and NEO-201. Functional analysis was performed by evaluating the ability of NEO-201 to mediate ADCC activity against AML cell lines using human NK cells as effector cells.Results5 of 6 AML cell lines tested bind to NEO-201 and the% of positive cells were 47%, 99.5%,100%,100% and 97.8% for HL60, U937, MOLM13, AML3 and IMS-M2, respectively. The% of positive cells in the two MM cell line were 99% and 18% for OPM2 and MM1.S, respectively. NEO-201 binding was not detected in the two ALL and the four MCL cell lines tested. Functional analysis has demonstrated that NEO-201 can mediate ADCC activity against the AML cell line (HL60) tested.ConclusionsThis study demonstrates that NEO-201 mAb’s target is expressed in most of the AML cell lines tested in vitro. In addition, we have shown it can mediate ADCC activity against HL60 cells (AML). Together, these findings provide a rationale for further investigation of the role of NEO-201 in AML as well as MM, further exploring patient PBMCs and bone marrow samples.

Molecular Docking and In Vitro Anticancer Screening of Synthesized Arylthiazole linked 2H-indol-2-one Derivatives as VEGFR-2 Kinase Inhibitors

2021 ◽  
Vol 21 ◽  
Author(s):  
Nishtha Shalmali ◽  
Sandhya Bawa ◽  
Md Rahmat Ali ◽  
Sourav Kalra ◽  
Raj Kumar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Anticancer Activity ◽  
Cell Lines ◽  
Cancer Cell ◽  
Active Sites ◽  
Colorectal Adenocarcinoma ◽  
Kinase Inhibitors ◽  
Adenocarcinoma Cells ◽  
Ic50 Values

Background: Indoline-2,3-dione comprises a leading course group of heterocycles endowed with appealing biological actions, including anticancer activity. There are significant justifications for exploring the anticancer activity of Schiff base derivatives of isatin as a vast number of reports have documented remarkable antiproliferative action of isatin nucleus against various cancer cell lines. Aims and Objectives: A series of arylthiazole linked 2H-indol-2-one derivatives (5a-t) was designed and synthesized as potential VEGFR-2 kinase inhibitors keeping the essential pharmacophoric features of standard drugs, like sunitinib, sorafenib, nintedanib, etc. They were evaluated for their in vitro anticancer activity. The aim of this study was to investigate and assess the anticancer potential of isatin-containing compounds along with their kinase inhibition activity. Methods: The title compounds were synthesized by reacting substituted isatins with para-substituted arylthiazoles using appropriate reaction conditions. Selected synthesized derivatives went under preliminary screening against a panel of 60 cancer cell lines at NCI, the USA, for single-dose and five dose assays. Molecular docking was performed to explore the binding and interactions with the active sites of the VEGFR-2 receptor (PDB Id: 3VHE). Derivatives 5a, 5b, 5c, 5d, 5g, 5h, and 5m were assessed for in vitro inhibition potency against Human VEGFR-2 using ELISA (Enzyme-Linked Immunosorbent Assay) kit. All the target compounds were determined against human colon cancer cell line SW480 (colorectal adenocarcinoma cells). Cellular apoptosis/necrosis was determined by flow cytometry using annexin V-FITC. DNA content of the cells was analyzed by flow cytometry and the cycle distribution was quantified. Results: Compounds 5a and 5g exhibited noteworthy inhibition during a five-dose assay against a panel of 60 cell lines with MID GI50 values of 1.69 and 1.54 µM, respectively. Also, both the lead compounds 5a and 5g demonstrated promising VEGFR-2 inhibitory activity with IC50 values of 5.43±0.95 and 9.63±1.32 µM, respectively. The aforesaid potent compounds were found effective against SW480 (colorectal adenocarcinoma cells) with IC50 values of 31.44 µM and 106.91 µM, respectively. Compound 5a was found to arrest the cell cycle at the G2/M phase, increasing apoptotic cell death. The docking study also supported VEGFR-2 inhibitory activity as both compounds 5a and 5g displayed promising binding and interactions with the active sites of VEGFR-2 receptor (PDB: 3VHE) with docking scores -9.355 and -7.758, respectively. All the compounds obeyed Lipinski’s rule of five. Conclusion: Indoline-2,3-dione and thiazole have huge potential to be considered a steer combination approach for developing promising kinase inhibitors as cancer therapeutics.

scholarly journals Oxymatrine Ameliorates Memory Impairment in Diabetic Rats by Regulating Oxidative Stress and Apoptosis: Involvement of NOX2/NOX4

2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Yongpan Huang ◽  
Xinliang Li ◽  
Xi Zhang ◽  
Jiayu Tang
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Brain Injury ◽  
Western Blotting ◽  
Caspase 3 ◽  
Memory Function ◽  
Diabetic Rats ◽  
Oxygen Species ◽  
Expression Levels

Oxymatrine (OMT) is the major quinolizidine alkaloid extracted from the root of Sophora flavescens Ait and has been shown to exhibit a diverse range of pharmacological properties. The aim of the present study was to investigate the role of OMT in diabetic brain injury in vivo and in vitro. Diabetic rats were induced by intraperitoneal injection of a single dose of 65 mg/kg streptozotocin (STZ) and fed a high-fat and high-cholesterol diet. Memory function was assessed using a Morris water maze test. A SH-SY5Y cell injury model was induced by incubation with glucose (30 mM/l) to simulate damage in vitro. The serum fasting blood glucose, insulin, serum S100B, malondialdehyde (MDA), and superoxide dismutase (SOD) levels were analyzed using commercial kits. Morphological changes were observed using Nissl staining and electron microscopy. Cell apoptosis was assessed using Hoechst staining and TUNEL staining. NADPH oxidase (NOX) and caspase-3 activities were determined. The effects of NOX2 and NOX4 knockdown were assessed using small interfering RNA. The expression levels of NOX1, NOX2, and NOX4 were detected using reverse transcription-quantitative PCR and western blotting, and the levels of caspase-3 were detected using western blotting. The diabetic rats exhibited significantly increased plasma glucose, insulin, reactive oxygen species (ROS), S-100B, and MDA levels and decreased SOD levels. Memory function was determined by assessing the percentage of time spent in the target quadrant, the number of times the platform was crossed, escape latency, and mean path length and was found to be significantly reduced in the diabetic rats. Hyperglycemia resulted in notable brain injury, including histological changes and apoptosis in the cortex and hippocampus. The expression levels of NOX2 and NOX4 were significantly upregulated at the protein and mRNA levels, and NOX1 expression was not altered in the diabetic rats. NOX and caspase-3 activities were increased, and caspase-3 expression was upregulated in the brain tissue of diabetic rats. OMT treatment dose-dependently reversed behavioral, biochemical, and molecular changes in the diabetic rats. In vitro, high glucose resulted in increases in reactive oxygen species (ROS), MDA levels, apoptosis, and the expressions of NOX2, NOX4, and caspase-3. siRNA-mediated knockdown of NOX2 and NOX4 decreased NOX2 and NOX4 expression levels, respectively, and reduced ROS levels and apoptosis. The results of the present study suggest that OMT alleviates diabetes-associated cognitive decline, oxidative stress, and apoptosis via NOX2 and NOX4 inhibition.

scholarly journals P06.01 Delta24-ACT oncolytic adenovirus as a therapeutic approach for DIPG

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii36-iii36
Author(s):  
V Laspidea ◽  
M Puigdelloses ◽  
M García-Moure ◽  
I Iñigo-Marco ◽  
J Gallego ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cell Lines ◽  
In Vivo Studies ◽  
Immunogenic Cell Death ◽  
Murine Models ◽  
Antitumoral Effect ◽  
Infected Cells

Abstract BACKGROUND Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor, being the leading cause of pediatric death caused by cancer. We previously showed that administration of the oncolytic virus Delta-24-RGD to DIPG murine models was safe and led to an increase in the median survival of these animals. However, not all the animals responded, underscoring the need to improve this therapy. In order to increase the antitumoral effect of the virus, we have engineered Delta-24-RGD with the costimulatory ligand 4-1BBL (Delta24-ACT). 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. In this project, we evaluated the oncolytic effect of Delta24-ACT and the antitumor immune response in DIPG murine models. MATERIALS AND METHODS We use the NP53 and XFM murine DIPG cell lines. Flow cytometry was used to assess cell infectivity and ligand expression. We analyzed viral replication using a method based in hexon detection, and viral cytotoxic effect using the MTS assay. For immunogenic cell death analysis, we measured ATP secretion by a luminometric assay and calreticulin location by flow cytometry and immunofluorescence. For in vivo studies, cells and virus were injected in the pons of the mice, using the screw-guided system. RESULTS In vitro, Delta24-ACT was able to infect and induce cell death in a dose-dependent manner in murine DIPG cell lines. In addition, Delta24-ACT was able to replicate in these tumor cells and to express viral proteins. Moreover, infected cells expressed 41BBL in their membranes. Delta24-ACT could induce immunogenic cell death due to an increased secretion of ATP and calreticulin translocation to the membrane of infected cells (in no-infected cells it located in the ER), DAMPs that can trigger the immune response activation. In vivo, Delta24-ACT demonstrated to be safe in all the tested doses and was able to induce a significant increase in the median survival of the treated animals. Moreover, long-term survivors display immunological memory. CONCLUSIONS Delta24-ACT treatment led to antitumoral effect in DIPG murine cell lines in vitro. Of significance, we have demonstrated that in vivo administration of Delta24-ACT is safe and results in an enhanced antitumor effect. Future in vivo studies will explore the underlying immune mechanism of the virus.

Glucocorticoid Induced Cell Death Is Mediated by Its Effect on Cellular Metabolism and Is Independent of Caspase Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3475-3475
Author(s):  
Sandeep Gurbuxani
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Lactate Concentration ◽  
Cellular Metabolism ◽  
Mechanisms Of Resistance ◽  
Metabolic State ◽  
Childhood All ◽  
Mitochondrial Mass

Glucocorticoids (GCs) such as prednisone and dexamethasone are a crucial component of acute lymphoblastic leukemia (ALL) therapy protocols. Multiple studies in childhood ALL have demonstrated that resistance to GC mediated cell death in vitro and in vivo is the single most important predictor of treatment outcome in childhood ALL. However, the mechanisms of GC mediated cell death as well as the mechanisms of resistance are poorly understood. The present study was undertaken to better understand the mechanism of GC induced cell death and to delineate possible mechanisms of resistance. In the initial experiments performed, multiple ALL cell lines, when treated with dexamethasone, underwent a reduction in the amount of reactive oxygen species (ROS) followed by cell cycle arrest and finally cell death which was unaffected by the presence of a pan-caspase inhibitor z-VAD-fmk. Since the amount of ROS present in a cell is an indicator of the metabolic state of the cell, specifically the amount of oxidative phosphorylation going on in the mitochondria, additional experiments were performed to directly estimate the mitochondrial mass as well as the metabolic state of the cells treated with GCs. While the mitochondrial mass measured by Mitotracker green labeling of mitochondria in the viable cells remained unchanged in cell lines susceptible to low concentrations (nano or micromolar) of dexamethasone, there was a prominent reduction in mitochondrial mass 36 hours after dexamethasone exposure in MOLT-4 cell line that requires several fold higher (millimolar) concentration of dexamethasone to induce cell death. The reduction in ROS was not accompanied by an increase in glycolysis as determined by the measurement of lactate concentration in the culture supernatants either in the susceptible or the resistant cells. Since one possible mechanism of reduction in ROS is increased scavenging by molecules that are dependent on the presence of NADPH generated during glucose metabolism via the pentose phosphate pathway (PPP), additional experiments were performed to determine if chemical inhibition of this pathway could augment dexamethasone induced cell death in ALL cell lines. Indeed, addition of transandosterone, an inhibitor of G6PD, the rate limiting enzyme of the PPP, resulted in significantly increased dexamethasone toxicity. Based on these experiments it can be concluded that GC induced cell death is mediated by its effect on cellular metabolism. Furthermore, this cell death is caspase independent and likely proceeds via a pathway mechanistically distinct from classical apoptosis. Finally, cells resistant to GC induced cell death have evolved mechanisms to adapt to GC induced changes in cellular metabolism and may maintain energy production via alternative pathways such as the PPP shunt that are independent of mitochondria.

Intracellular NAD+ Depletion Enhances Bortezomib-Induced Myeloma Cytotoxicity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 330-330
Author(s):  
Antonia Cagnetta ◽  
Michele Cea ◽  
Chirag Acharya ◽  
Teresa Calimeri ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Cell Death ◽  
Synergistic Effect ◽  
Cell Lines ◽  
Caspase 3 ◽  
Statistical Significance ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Nicotinamide Phosphoribosyltransferase

Abstract Abstract 330 Background: Our previous study demonstrated that inhibition of nicotinamide phosphoribosyltransferase (Nampt) acts by severely depleting intracellular NAD+ content and thus eliciting mitochondrial dysfunction and autophagic MM cell death. The proteasome inhibitor Bortezomib induces anti-MM activity by affecting a variety of signaling pathways. However, as with other agents, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we demonstrate that combining Nampt inhibitor and bortezomb induces synergistic anti-MM cell death both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. Material and Methods: We utilized MM.1S, MM.1R, RPMI-8226, and U266 human MM cell lines, as well as purified tumor cells from patients relapsing after prior therapies. Cell viability and apoptosis assays were performed using Annexin V/PI staining. Intracellular NAD+ level and proteasome activity were quantified after 12, 24, and 48h exposure to single/combination drugs by specific assays. In vitro angiogenesis was assessed by Matrigel capillary-like tube structure formation assay. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, and tubulin. CB-17 SCID male mice (n = 28; 7 mice/EA group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum free RPMI-1640 medium. When tumors were measurable (3 weeks after MM cell injection), mice were treated for three weeks with vehicle alone, FK866 (30mg/kg 4 days weekly), Bortezomib (0.5 mg/kg twice weekly), or FK866 (30 mg/kg) plus Bortezomib (0.5 mg/kg). Statistical significance of differences observed in FK866, Bortezomib or combination-treated mice was determined using a Student t test. Isobologram analysis was performed using “CalcuSyn” software program. A combination index < 1.0 indicates synergism. Results/Discussion: Combining FK866 and Bortezomib induces synergistic anti-MM activity in vitro against MM cell lines (P<0.005, CI < 1) or patient CD138-positive MM cells (P< 0.004). FK866 plus Bortezomib-induced synergistic effect is associated with: 1)activation of caspase-8, caspase-9, caspase-3, and PARP; 2) improved intracellular NAD+ dissipation; 3) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteolytic activities; 4) inhibition of NF-kappa B signaling; and 5) inhibition of angiogenesis. Importantly, the ectopic overexpression of Nampt rescues this observed synergistic effect; conversely, Nampt knockdown by RNAi significantly enhances the anti-MM effect of bortezomib. In the murine xenograft MM model, low dose combination FK866 (30 mg/kg) and Bortezomib (0.5 mg/kg) is well tolerated, significantly inhibits tumor growth (P < 0.001), and prolongs host survival (2–2.5 months in mice receiving combined drugs, P = 0.001). These findings demonstrate that intracellular NAD+ levels represent a major determinant in the ability of bortezomib to induce apoptosis of MM cells, providing the rationale for clinical protocols evaluating FK866 together with Bortezomib to improve patient outcome in MM. Disclosures: Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

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