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.

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.

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.

scholarly journals BCL-2, a Therapeutic Target for High Risk Hypodiploid B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 280-280 ◽  
Author(s):  
Ernesto Diaz-Flores ◽  
Evan Q. Comeaux ◽  
Kailyn Kim ◽  
Kyle Beckman ◽  
Kara L. Davis ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Advisory Committees ◽  
Chromosomal Number ◽  
Patient Derived Xenograft

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood. Specific genetic subsets, including hypodiploid ALL, are associated with particularly high rates of relapse. Despite the poor outcomes of hypodiploid B-ALL with traditional therapeutic approaches, there have been no known effective alternative therapies or novel candidates tested to improve outcome. We hypothesized that new therapeutic targets could by identified by integrated biochemical and genomic profiling, combined with functional drug assays in order to determine which pathways play an essential role in transformation. For biochemical profiling, we analyzed multiple pathways commonly deregulated in leukemias using phosphoflowcytometry (including receptor tyrosine kinases, JAK/STAT, MAPK, PI3K, PTEN, Bcl-2 survival and pro-apoptotic family members and p53). We subjected hypodiploid cell lines (NALM-16, MHH-CALL2) and patient derived xenograft samples in vitro to inhibitors against each of these pathways (PP2:Src family;Ruxolitinib: JAK/STAT; PD235901/CI1040: MAPK; GDC-0941, PI-90, PI-103, p110 (a, b, g, d): PI3K isoform specific; PP-242:mTOR; ABT-263/ABT-737: Bcl-2/Bcl-xl, and ABT-199: Bcl-2 specific). We found that the Bcl-2 inhibitors (ABT-263, ABT-737 and ABT-199) and to a lesser extent PI3K pathway inhibitors GDC-0941 and PP-242, but not the MAPK or RTK inhibitors, efficiently reduced proliferation of hypodiploid cells. However, only ABT-263/ABT-199 induced high levels of apoptosis at nanomolar concentrations. Based on the consistent efficacy observed with ABT-199 against hypodiploid patient-derived cells and cell lines in culture, we selected eight cryopreserved, previously xenografted (F3 generation) hypodiploid patient samples (4 low hypodiploid, chromosomal number between 32 and 39; and 4 Near Haploid, chromosomal number between 24 and 31) and three non-hypodiploid patient samples (Ph-positive,Ph-Like and Erg+) for a preclinical trial in immunodeficient mice. Each patient sample was engrafted into six mice, which were randomized to receive vehicle or ABT-199 daily over 60 days (Figure 1). Treatment started when the peripheral blood (PB) human CD45 count reached 15%. A rapid decrease in PB blasts was noted at 7 days (Figure 1). Eighty-five percent of the hypodiploid xenografts survived 60 days with either undetectable or low levels of leukemia in the PB. In contrastPh+ andPh-Like xenografts died within 10-20 days regardless of treatment. Importantly, hypodiploid leukemic blasts gradually emerged after discontinuing ABT-199 after 60 days. Additionally, despite low or undetectable levels of leukemic blasts in PB and reduced levels in bone marrow and spleen, all mice had high percentages of leukemic cells in the liver (Figure 2). In conclusion we have identified the survival protein Bcl-2 as a promising molecular target in hypodiploid B-ALL. ABT-199 for dramatically reduced leukemia cells in vitro and in vivo in patient-derived xenograft models of hypodiploid B-ALL. However, the liver represented a protective niche for these leukemias. In addition, our biochemical characterization of the organ infiltrating blasts collected from mice on trial indicate that the sensitivity of hypodiploid ALL to ABT-199 relies not only on high levels of Bcl-2 and deficiency for other survival proteins such as Bcl-xl but also on high levels of proapoptotic proteins, providing two different signatures that correlate with response to ABT-199. Using genome editing (CRISPR/Cas9) we interrogated the necessity for individual proapoptotic genes, including PUMA, NOXA, and BAD, for ABT-199-induced cell death. This study provides encouraging preclinical data that Bcl-2 may be a promising target for the treatment of hypodiploid B-ALL. Our studies identify signature biomarkers that correlate with drug response and identify essential proteins mediating ABT-199-induced cell death. Importantly, this report also identifies the limitations of using ABT-199 as single drug, and provides the rationale for using combinatorial therapies in order to improve the efficacy of the drug. Disclosures Mullighan: Loxo Oncology: Research Funding; Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees. Loh:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

Leukemia Driven By a NUP98-Phd Domain Fusion Is Highly Sensitive To Disruption Of H3K4me3-Phd Domain Binding By a Small Molecule Inhibitor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3759-3759
Author(s):  
Sheryl M Gough ◽  
Fan Lee ◽  
Robert L Walker ◽  
Fan Yang ◽  
Yuelin Zhu ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Small Molecule ◽  
Wide Spectrum ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Signature ◽  
Leukemic Cells ◽  
Common Theme ◽  
Phd Domain

Abstract NUP98 gene fusions, created by non-random chromosomal translocations, are associated with a wide spectrum of high risk hematologic malignancies, and typically lead to overexpression of abdominal-b HOXA genes, a common theme shared by ∼50% of AML patients. We have generated a transgenic mouse model of the NUP98-PHF23 (NP23) gene fusion, initially identified in patients with AML, which develop AML, erythroleukemia, pre-T lymphoblastic leukemia (pre-T LBL), and a novel pre-B1 B cell acute lymphoblastic leukemia (pre-B1 ALL). A common theme in the leukemias and the premalignant hematopoietic tissues, is the overexpression of a Hoxa/b +Meis1 stem cell-like gene expression signature. GSEA analysis reveals this signature to be enriched in both human AML and ALL malignancies, and in human HSPC profiles. In addition, we found Bahcc1, a gene not previously associated with malignancy, to consistently segregate with the Hoxa/b+Meis1 signature in the NP23 leukemias and the premalignant tissues, independent of hematopoietic cell lineage. Furthermore, data-mining revealed BAHCC1 to be markedly overexpressed in AML patients with HOXA9/MEIS1 overexpression, and in a subset of MLL rearranged pre-B-ALL patients, suggesting BAHCC1 may be a previously unsuspected marker of leukemic transformation. NUP98-PHF23 belongs to a subset of fusion oncoproteins (including some MLL- and NUP98-fusions) that are potently tumorigenic and act by abrogating the normal reading, writing and erasure of histone methylation. Wild type PHF23 binds H3K4me3 residues via a PHD domain, therefore we used ChIP-seq to characterize global chromatin H3K4me3 and NP23 enrichment in NP23 leukemia derived cell lines. The vast majority (88%) of NP23 binding sites were enriched for H3K4me3 binding. Conversely, the NP23 protein co-localized at only 1.6% of all H3K4me3 enriched sites (including Hoxa, Hoxb and Meis1 loci) identifying these sites as direct targets of the NP23 fusion protein. Given that the NP23 fusion appears to function, at least in part, via binding to H3K4me3 sites at specific loci, we hypothesized that NP23 cells would be sensitive to disruption of the H3K4me4 binding by the NP23 PHD domain. Treatment of leukemic NP23 cells with Tetraethylthiuram disulfide (Disulfiram), a small molecule shown to inhibit PHD domain binding of H3K4me3 marks in vitro, rapidly and selectively killed NP23 myeloblasts but not control myeloblast cell lines (188G3, 189E6 and 32D) at 2 µM. Cell death was rapid, being 100% complete within 24 hours. Cell death was preceded by decreased levels of NP23 protein bound at target loci and decreased expression of these loci (e.g., Hoxa7/9/10, Hoxb5 and Meis1). We conclude that inhibitors of H3K4me3 PHD domain readers are promising therapeutic compounds that can kill leukemic cells driven by proteins that aberrantly read or write the histone code. The NP23 model provides a robust platform on which to identify and improve such compounds. Disclosures: Denu: Sirtris-GSK: Consultancy.

Vitamin K3 Selectively Induces Apoptosis in Acute Lymphoblastic Leukemia Cells with Constitutive Activation of IKKα/NF-kB Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 859-859
Author(s):  
Ningxi Zhu ◽  
Lubing Gu ◽  
Harry W. Findley ◽  
Kuang-Yueh Chiang ◽  
Muxiang Zhou
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Cytotoxic Effect ◽  
Human Cancer ◽  
Lymphoblastic Leukemia ◽  
Vitamin K3 ◽  
Neoplastic Cells

Abstract Although the cytotoxic effect of vitamin K3 (VK3) on human cancer cells has been repeatedly reported, no clear conclusions from either in vitro or in vivo tests have so far been made for VK3 as an anticancer agent due to marked inter-tumor variability of efficacy in response to VK3 treatment. Here, we report that sensitivity of neoplastic cells to VK3-induced killing depends on IKKα expression/NF-kB activation in the cells. We tested the sensitivity to VK3 of 14 leukemic cell lines established from children with acute lymphoblastic leukemia (ALL). The 14 lines were classified into three groups: IKKα +/NF-kB+, IKKα +/NF-kB−, IKKα−/NF-kB−. IKKα +/NFkB+ cell lines that are generally resistant to doxorubicin are more sensitive to VK3 induced cell death than are the IKKα +/NFkB− lines that are usually sensitive to doxorubicin. The median of IC 50 values of VK3 and doxorubicin as tested by WST analysis for IKKα +/NFkB+ cells were 3.92 mM and 1.58 mM, respectively, compared to IKKα +/NFkB− cells (7.3 mM of VK3 and 0.71 mM of doxorubicin, p&lt;0.01, t-test). Assays by testing activation of caspase and cleavage of death substrate PARP as well as flow cytometry showed that apoptosis was induced in a line with high levels of IKKα/NF-kB activation at 2 h after VK3 treatment. In contrast, apoptosis was not induced by VK3 even at 48 h post-treatment in two lines that lack IKKa expression and NF-kB activation. To test if IKKα/NF-kB is a molecular target of VK3 inducing apoptosis in ALL, we examined the expression and activation of IKKα/NF-kB in VK3-treated cells. VK3 specifically reduced IKKα expression and inhibited NF-kB activation, resulting in downregulation of NF-kB-mediated gene expression and apoptosis. These results suggest that inhibition of IKKα/NF-kB signaling pathway is essential for VK3 to induce cell death, and that VK3, a dietary factor with no cytotoxic effect on normal cells, would be a useful adjuvant in the treatment of ALL and other cancer patients whose neoplastic cells express constitutive NF-kB and are resistant to chemotherapy.

Stew in its Own Juice: Protein Homeostasis Machinery Inhibition Reduces Cell Viability in Multiple Myeloma Cell Lines

2019 ◽  
Vol 19 (2) ◽  
pp. 112-119 ◽  
Author(s):  
Mariana B. de Oliveira ◽  
Luiz F.G. Sanson ◽  
Angela I.P. Eugenio ◽  
Rebecca S.S. Barbosa-Dantas ◽  
Gisele W.B. Colleoni
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Viability ◽  
Cell Lines ◽  
Treatment Options ◽  
Compensatory Mechanism ◽  
Protein Homeostasis ◽  
Protein Response ◽  
Rpmi 8226

Introduction:Multiple myeloma (MM) cells accumulate in the bone marrow and produce enormous quantities of immunoglobulins, causing endoplasmatic reticulum stress and activation of protein handling machinery, such as heat shock protein response, autophagy and unfolded protein response (UPR).Methods:We evaluated cell lines viability after treatment with bortezomib (B) in combination with HSP70 (VER-15508) and autophagy (SBI-0206965) or UPR (STF- 083010) inhibitors.Results:For RPMI-8226, after 72 hours of treatment with B+VER+STF or B+VER+SBI, we observed 15% of viable cells, but treatment with B alone was better (90% of cell death). For U266, treatment with B+VER+STF or with B+VER+SBI for 72 hours resulted in 20% of cell viability and both treatments were better than treatment with B alone (40% of cell death). After both triplet combinations, RPMI-8226 and U266 presented the overexpression of XBP-1 UPR protein, suggesting that it is acting as a compensatory mechanism, in an attempt of the cell to handle the otherwise lethal large amount of immunoglobulin overload.Conclusion:Our in vitro results provide additional evidence that combinations of protein homeostasis inhibitors might be explored as treatment options for MM.

Simvastatin Induces Death of Multiple Myeloma Cell Lines

2004 ◽  
Vol 52 (5) ◽  
pp. 335-344 ◽  
Author(s):  
Naomi Gronich ◽  
Liat Drucker ◽  
Hava Shapiro ◽  
Judith Radnay ◽  
Shai Yarkoni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cytoplasmic Calcium ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
Rpmi 8226

BackgroundAccumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy.MethodsU266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity.ResultsExposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected.ConclusionsSimvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

scholarly journals Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase.

1994 ◽  
Vol 180 (4) ◽  
pp. 1547-1552 ◽  
Author(s):  
M G Cifone ◽  
R De Maria ◽  
P Roncaioli ◽  
M R Rippo ◽  
M Azuma ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Apoptotic Cell ◽  
Human Tumor ◽  
Apoptotic Cell Death ◽  
Cell Surface Receptor ◽  
Cross Linking ◽  
Cell Extracts

Intracellular pathways leading from membrane receptor engagement to apoptotic cell death are still poorly characterized. We investigated the intracellular signaling generated after cross-linking of CD95 (Fas/Apo-1 antigen), a broadly expressed cell surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 monoclonal antibody was produced by immunizing mice with human CD95-transfected L cells. Crosslinking of CD95 with DX2 resulted in the activation of a sphingomyelinase (SMase) in promyelocytic U937 cells, as well as in other human tumor cell lines and in CD95-transfected murine cells, as demonstrated by induction of in vivo sphingomyelin (SM) hydrolysis and generation of ceramide. Direct in vitro measurement of enzymatic activity within CD95-stimulated U937 cell extracts, using labeled SM vesicles as substrates, showed strong SMase activity, which required pH 5.0 for optimal substrate hydrolysis. Finally, all CD95-sensitive cell lines tested could be induced to undergo apoptosis after exposure to cell-permeant C2-ceramide. These data indicate that CD95 cross-linking induces SM breakdown and ceramide production through an acidic SMase, thus providing the first information regarding early signal generation from CD95, and may be relevant in defining the biochemical nature of intracellular messengers leading to apoptotic cell death.

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.

scholarly journals Therapeutic evaluation of palbociclib and its compatibility with other chemotherapies for primary and recurrent nasopharyngeal carcinoma

2020 ◽  
Author(s):  
zhichao xue ◽  
Vivian Wai Yan Lui ◽  
Yongshu Li ◽  
Jia Lin ◽  
Chanping You ◽  
...  
Keyword(s):  
Cell Death ◽  
Nasopharyngeal Carcinoma ◽  
Cell Lines ◽  
Chemotherapeutic Drugs ◽  
Induce Cell Cycle Arrest ◽  
Ki 67 ◽  
Concurrent Treatment ◽  
Xenograft Models

Abstract Background: Recent genomic analyses revealed that druggable molecule targets were detectable in approximately 6% of patients with nasopharyngeal carcinoma (NPC). However, a dependency on dysregulated CDK4/6–cyclinD1 pathway signaling is an essential event in the pathogenesis of NPC. In this study, we aimed to evaluate the therapeutic efficacy of a specific CDK4/6 inhibitor, palbociclib, and its compatibility with other chemotherapeutic drugs for the treatment of NPC by using newly established xenograft models and cell lines derived from primary, recurrent, and metastatic NPC. Methods: We evaluated the efficacies of palbociclib monotherapy and concurrent treatment with palbociclib and cisplatin or suberanilohydroxamic acid (SAHA) in NPC cell lines and xenograft models. RNA sequencing was then used to profile the drug response–related pathways. Palbociclib-resistant NPC cell lines were established to determine the potential use of cisplatin as a second-line treatment after the development of palbociclib resistance. We further examined the efficacy of palbociclib treatment against cisplatin-resistant NPC cells. Results: In NPC cells, palbociclib monotherapy was confirmed to induce cell cycle arrest in the G1 phase in vitro . Palbociclib monotherapy also had significant inhibitory effects in all six tested NPC tumor models in vivo , as indicated by substantial reductions in the total tumor volumes and in Ki-67 proliferation marker expression. In NPC cells, concurrent palbociclib treatment mitigated the cytotoxic effect of cisplatin in vitro . Notably, concurrent treatment with palbociclib and SAHA synergistically promoted NPC cell death both in vitro and in vivo . This combination also further inhibited tumor growth by inducing autophagy-associated cell death. NPC cell lines with induced palbociclib or cisplatin resistance remained sensitive to treatment with cisplatin or palbociclib, respectively. Conclusions: Our study findings provide essential support for the use of palbociclib as an alternative therapy for NPC and increase awareness of the effective timing of palbociclib administration with other chemotherapeutic drugs. Our results provide a foundation for the design of first-in-human clinical trials of palbociclib regimens in patients with NPC.

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