scholarly journals Anticancer Activity of Neosetophomone B, An Aquatic Fungal Secondary Metabolite, Against Hematological Malignancie S

2021 ◽  
Author(s):  
Shilpa Kuttikrishnan ◽  
Kirti S. Prabhu ◽  
Tamam Elimat ◽  
Ashraf Khalil ◽  
Nicholas H. Oberlies ◽  
...  
Keyword(s):  
Anticancer Activity ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
U937 Cell ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Leukemic Cell Lines ◽  
Caspase Cascade ◽  
Apoptotic Proteins ◽  
Dose Dependent

Cancer is one of the most life threatening diseases, causing nearly 13% death in the worldwide. Leukemia, cancer of the hematopoetic cells is the main cause of cancer death in adults and children. Therapeutic agents used in treatment of cancer are known to have narrow therapeutic window and tendency to develop resistance against some cancer cell lines thus, proposing a need to discover some novel agents to treat cancer. In the present study we investigated the anticancer activity of Neosetophomone B(NSP-B), an aquatic fungal metabolite isolated from Neosetophoma sp against leukemic cells (K562 and U937). MTT results demonstrated a dose dependent inhibition of cell proliferation in K562 and U937 cell lines. Annexin staining using flow cytometry indicated that NSP-B treatment cause a dose dependent apoptosis in leukemic cells.Western blot analysis showed that NSP-B mediated apoptosis involves sequential activation of caspase 9, 3 and poly (ADP-ribose) polymerase (PARP) cleavage. Furthermore NSP-B treatment of leukemic cells resulted in upregulation of pro-apoptotic proteins (Bax) with downregulation of anti-apoptotic proteins ( Bcl-2 ).Thus, present study focuses on exploring the mechanism of anticancer activity of NSP-B on leukemic cells, raising the possibility of its use as a novel therapeutic agent for hematological malignancies. Results: We sought to determine whether NSP-B suppresses the growth of leukemic cell lines. We tested a panel of leukemic cell lines with different doses of NSP-B. Cell viability decreased in a concentration-dependent manner in K562 and U937 cell lines. NSP-B induced apoptosis in K562 and U937 cell lines via downregulation of anti-apoptotic proteins and enhancement of pro-apoptotic proteins. NSP-B induced the activation of caspase cascade signaling pathway. Altogether our results suggest that the NSP-B plays an important role in apoptosis in leukemic cell lines .Conclusions: Our data provides insight on anticancer activities of NSP-B in leukemic cell lines (K562 and U937). NSP-B inhibit cell viability via inducing apoptosis. The NSP-B mediated apoptosis occurs via downregulation of anti-apoptotic proteins and enhancement of pro-apototic proteins, thereby activating the caspase-cascade signaling. Further studies are required to elicit role of NSP-B in regulating molecular pathway involved in the progression of cancer. Taken together, above results suggest that NSP-B may have a future therapeutic role in leukemia and possibly other hematological malignancies.

Chemoprevention Against Hepatocellular Carcinoma of Cornus officinalis in vitro

2004 ◽  
Vol 32 (05) ◽  
pp. 717-725 ◽  
Author(s):  
Jung-San Chang ◽  
Lien-Chai Chiang ◽  
Fen-Fang Hsu ◽  
Chun-Ching Lin
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Xtt Assay ◽  
Leukemic Cell Lines ◽  
Cornus Officinalis ◽  
Anti Oxidant ◽  
Dose Dependent

The water extracts of Cornus officinalis Sieb. et Zuce against hepatocellular carcinoma (HCC) was studied for its chemopreventive potential. Three HCC cell lines (HepG2, SK-Hep1 and PLC/PRF/5) and three leukemic cell lines (U937, K562 and Raji) were tested with XTT assay. Extracts of C. officinalis inhibited all these HCC cells and leukemic cells at a concentration of 100 μg/ml (P<0.05) and was dose-dependent (P<0.0001). P53 (P<0.0001) and Ras (P=0.001) significantly affected its activity against HCC. Extracts of C. officinalis also possessed the anti-oxidant activity through free radicals scavenging activity at a concentration of 50 μg/ml (P<0.05). In summary, our experiment implied that C. officinalis might be a candidate for chemopreventive agent against HCC through the antioxidant and anti-neoplastic effects.

Differential expression of a novel proline-rich homeobox gene (Prh) in human hematolymphopoietic cells

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1237-1245 ◽  
Author(s):  
G Manfioletti ◽  
V Gattei ◽  
E Buratti ◽  
A Rustighi ◽  
A De Iuliis ◽  
...  
Keyword(s):  
Cell Lines ◽  
Constitutive Expression ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Maturation Stage ◽  
Lymphoid Tissues ◽  
Specific Cell ◽  
B Cell Differentiation ◽  
Leukemic Cell Lines

Proline-rich homeobox (Prh) is a novel human homeobox-containing gene recently isolated from the CD34+ cell line KG-1A, and whose expression appears mainly restricted to hematopoietic tissues. To define the pattern of Prh expression within the human hematopoietic system, we have analyzed its constitutive expression in purified cells obtained from normal hematopoietic tissues, its levels of transcription in a number of leukemia/lymphoma cell lines representing different lineages and stages of hematolymphopoietic differentiation, and its regulation during in vitro maturation of human leukemic cell lines. Prh transcripts were not detected in leukemic cells of T-lymphoid lineage, irrespective of their maturation stage, and in resting or activated normal T cells from peripheral blood and lymphoid tissues. In contrast, high levels of Prh expression were shown in cells representing early stages of B lymphoid maturation, being maintained up to the level of circulating and tissue mature B cells. Terminal B-cell differentiation appeared to be conversely associated with the deactivation of the gene, since preplasmacytic and plasmocytoma cell lines were found not to express Prh mRNA. Prh transcripts were also shown in human cell lines of early myelomonocytic, erythromegakaryocytic, and preosteoclast phenotypes. Prh expression was lost upon in vitro differentiation of leukemic cell lines into mature monocyte-macrophages and megakaryocytes, whereas it was maintained or upregulated after induction of maturation to granulocytes and osteoclasts. Accordingly, circulating normal monocytes did not display Prh mRNA, which was conversely detected at high levels in purified normal granulocytes. Our data, which show that the acquisition of the differentiated phenotype is associated to Prh downregulation in certain hematopoietic cells but not in others, also suggest that a dysregulated expression of this gene might contribute to the process of leukemogenesis within specific cell lineages.

scholarly journals Signal transduction and glycophosphatidylinositol-linked proteins (lyn, lck, CD4, CD45, G proteins, and CD55) selectively localize in Triton- insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3783-3794 ◽  
Author(s):  
I Parolini ◽  
M Sargiacomo ◽  
MP Lisanti ◽  
C Peschle
Keyword(s):  
Signal Transduction ◽  
G Proteins ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Leukemic Cell ◽  
Receptor Activation ◽  
Leukemic Cells ◽  
Human Leukemic Cell ◽  
Leukemic Cell Lines ◽  
B Lymphoid

Src-family nonreceptor protein tyrosine kinases (NRPTK) are associated with cell surface receptors in large detergent-resistant complexes: in epithelial cells, yes is selectively located in vesicle structures containing caveolin (“caveolae”). These formations are typically also endowed with glycophosphatidylinositol (GPI)-anchored proteins. In the present study, we observed lck, lyn, src, hck, CD4, CD45, G proteins, and CD55 (decay-accelerating factor) expression in the buoyant low- density Triton-insoluble (LDTI) fraction of selected leukemic cell lines and granulocytes. We provide a detailed analysis of the two most highly expressed NRPTK, p53/p56lyn and p56lck, which are involved in the transduction of signals for proliferation and differentiation of monocytes/B lymphocytes and T lymphocytes, respectively. We show that lyn is selectively recovered in LDTI complexes isolated from human leukemic cell lines (promyelocytic [HL-60], erythroid [K562] and B- lymphoid [697]) and from normal human granulocytes, and that lck is recovered from LDTI fractions of leukemic T- and B-lymphoid cell lines (CEM, 697). In LDTI fractions of leukemic cells, lck and lyn are enriched 100-fold as compared with the total cell lysates. Analysis of these fractions by electron microscopy shows the presence of 70- to 200- nm vesicles: lyn and lck are homogenously distributed in the vesicles, as revealed by an immunogold labeling procedure. These novel results propose a role for these vesicles in signal transduction mechanisms of normal and neoplastic hematopoietic cells. In support of this hypothesis, we further observed that molecules participating in B- and T-cell receptor activation cofractionate in the LDTI fractions, CD45/lyn (B cells) and CD45/lck/CD4 (T cells).

scholarly journals Loss of suppression of normal bone marrow colony formation by leukemic cell lines after differentiation is induced by chemical agents

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 100-106 ◽  
Author(s):  
HN Steinberg ◽  
AS Tsiftsoglou ◽  
SH Robinson
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Colony Formation ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Cell Phenotype ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Leukemic Cell Lines

Abstract The human leukemic cell lines K562 and HL-60 were cocultured with normal bone marrow (BM) cells. Coculture with 10(4) K562 or HL-60 cells results in 50% inhibition of normal CFU-E and BFU-E colony formation. However, when the same number of K562 and HL-60 cells is first treated for two to five days with agents that induce their differentiation, a gradual loss in their capacity to inhibit CFU-E and BFU-E colony formation is observed. The inhibitory material in K562 cells is soluble and present in conditioned medium from cultures of these cells. The degree to which leukemic cell suppression of CFU-E and BFU-E growth is reversed is correlated with the time of exposure to the inducing agent. Suppression is no longer evident after five days of prior treatment with inducers. In fact, up to a 90% stimulation of CFU-E growth is observed in cocultures with K562 cells that have been pretreated with 30 to 70 mumol/L hemin for five days. K562 cells treated with concentrations of hemin as low as 30 mumol/L demonstrate increased hemoglobin synthesis and grow normally, but no longer have an inhibitory effect on CFU-E growth. Hence, reversal of normal BM growth inhibition must be caused by the more differentiated state of the K562 cells and not by a decrease in the number of these cells with treatment. Thus, induction of differentiation in cultured leukemic cells not only alters the malignant cell phenotype but also permits improved growth of accompanying normal marrow progenitor cells. Both are desired effects of chemotherapy.

Pioglitazone Inhibits the Growth of Human Leukemic Cell Lines and Primary Leukemic Cells in Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4493-4493 ◽  
Author(s):  
Yoshihiro Hatta ◽  
Minoru Saiki ◽  
Yuko Enomoto ◽  
Shin Aizawa ◽  
Umihiko Sawada ◽  
...  
Keyword(s):  
Cell Lines ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Hematopoietic Cells ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Peroxisome Proliferator ◽  
Leukemic Cell Lines ◽  
Ppar Γ

Abstract Troglitazone and pioglitazone are one of thiazolidinediones that are high affinity ligand for the nuclear receptor called peroxisome proliferator-activated receptor gamma (PPAR-γ). Troglitazone is a potent inhibitor of clonogenic growth of acute myeloid leukemia cells when combined with a retinoid. However, the effect of pioglitazone to neoplastic cells and normal hematopoietic cells has not been studied yet. Adult T-cell leukemia (ATL), prevalent in western Japan, is a highly aggressive malignancy of mature T lymphocyte. Therefore, we studied antitumor effect of pioglitazone against leukemic cells including ATL as well as normal hematopoietic cells. With 300 μM of pioglitazone, colony formation of ATL cell lines (MT1, MT2, F6T, OKM3T, and Su9T01) was completely inhibited. Colony formation of HUT102, another ATL cell line, was 12 % compared to untreated control. Clonogenic cells of other leukemic cell lines (K562, HL60, U937, HEL, CEM, and NALM1) was also inhibited to 0–30% of control. Colony formation of primary leukemic cells from 5 AML patients was decreased to 15 %. However, normal hematopoietic cells were weakly inhibited with 300 μM pioglitazone; 77 % of CFU-GM, 70 % of CFU-E, and 33 % of BFU-E survived. Cell cycle analysis showed that pioglitazone decreased the ratio of G2/M phase in HL60 cells, suggesting the inhibition of cell division. By Western blotting, PPAR-γ protein level was similar in all leukemic cells and normal bone marrow mononuclear cells. Taken together, pioglitazone effectively eliminate leukemic cells and could be used as an antitumor agent in vivo.

Expression of Vascular Endothelial-Cadherin on Leukemic Cells Mediates Their Interaction with Vascular Endothelium.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2761-2761
Author(s):  
Leslie R. Ellis ◽  
Loic Vincent ◽  
Sergey Shmelkov ◽  
Andrea Hooper ◽  
Scott Avecilla ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Minimal Residual Disease ◽  
Cell Lines ◽  
Residual Disease ◽  
Leukemic Cell ◽  
Angiogenic Factors ◽  
Leukemic Cells ◽  
Vascular Endothelial ◽  
Leukemic Cell Lines ◽  
Minimal Residual

Abstract It has already been established that subsets of leukemic cells express receptors for pro-angiogenic factors, such as vascular endothelial growth factor receptor-2 (VEGFR-2). Further studies have shown that these same leukemic cells also produce the ligand for these VEGF receptors, VEGF-A. This autocrine loop supports the invasion and proliferation of these particular leukemic cells. The VEGFR-2 signaling pathway is further dependent upon the co-activation of other pro-angiogenic factors, such as vascular endothelial (VE)-cadherin. VE-cadherin is an endothelial cell-specific transmembrane cellular adhesion protein that when bound results in the dephosphorylation of VEGFR-2 and contributes to neo-vessel formation. Recent studies have suggested that VE-cadherin may be expressed by a unique subset of hematopoietic cells, raising the possibility that leukemic cells may express VE-cadherin as well. We therefore sought to identify the expression of VE-cadherin on leukemic cell lines and primary samples, and further determine its role in the interaction with VE-cadherin-positive endothelial cells. Leukemic cell lines and primary leukemias were screened for the presence of VE-cadherin by both RT-PCR and Western blot analysis. Primary leukemic samples, as well as established cell lines for human erythroblastic leukemia (HEL) and acute myelogenous leukemia (KG-1a) were found to express VE-cadherin. VE-cadherin expression was further confirmed by flow cytometry and immunocytochemistry, which demonstrated that approximately 15% of the total population was VE-cadherin-positive. Proliferation and migration assays utilizing neutralizing monoclonal antibodies to VE-cadherin were performed with no obvious effects. However, immunofluorescent staining of a co-culture performed with the above leukemic cells grown on a layer of human umbilical vein endothelial cells (HUVECs) demonstrated that the leukemic cells and HUVECs interact via VE-cadherin. Furthermore, when these leukemic cells were injected into NOD-SCID mice subcutaneously, the VE-cadherin-positive cells localized around the vessels present within the leukemic chloroma. These data set forth the concept that a subset of leukemic cells expresses the protein VE-cadherin and that VE-cadherin is involved in the cell-to-cell interaction between a subset of leukemic cells and vascular endothelial cells. Heterotypic VE-cadherin interaction between leukemic cells and neo-vessels may increase the survival of leukemic cells and contribute to the generation of minimal residual disease. Therefore, inhibition of VEGFR-2 in conjunction with VE-cadherin may provide a novel strategy to eradicate minimal residual disease.

Expression and Function of the CXCR7 Chemokine Receptor in Leukemias

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2423-2423
Author(s):  
Sergej Konoplev ◽  
Hongbo Lu ◽  
Michael A Fiegl ◽  
Zhihong Zeng ◽  
Wenjing Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
And Function

Abstract Background: Bone marrow produced stromal-derived factor-1a (SDF-1a) is a key chemokine involved in chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. While the majority of well-defined functions of SDF-1a are mediated via its receptor CXCR4, recent studies have characterized CXCR7 as an alternative receptor capable of binding SDF-1a. Although the functions of CXCR7 are still incompletely understood, the receptor was reported to promote migration and adhesion in certain cell types and function as a pro-survival factor in breast cancer cells. CXCR7 expression and function in human leukemia cells has not been characterized. In this study, we examined CXCR7 expression in leukemia cell lines and primary samples from patients with acute lymphoblastic leukemia (ALL) and utilized a small molecule inhibitor of CXCR7 to probe CXCR7’s function. Materials and methods: CXCR4 and CXCR7 expression was determined by flow cytometry, real-time PCR (RT-PCR) and immunocytochemistry (ICC) in leukemic cell lines including AML (OCI-AML2, OCI-AML3, HL60, U937 NB4, Molm13), ALL (REH, Raji, RS4; 11, Nalm6, Molt4) and CML (KBM5, K562) cells. In primary ALL patient samples, CD34+CD19+ gating was applied to detect CXCR7 expression on pre-B leukemic cells by flow cytometry. The migration of leukemic cells towards SDF-1a was studied using a transwell system. CXCR4 inhibitor AMD3100 was purchased from Sigma, and CXCR7 inhibitor CCX-733 was provided by ChemoCentryx Inc., Mountain View, CA. Results: CXCR4 was found to be ubiquitously expressed on the cell surface of all leukemic cell lines tested. CXCR7 mRNA and protein expression was detectable only in Burkitt lymphoma Raji cells, as analyzed by flow cytometry (clone 11G8, R&D systems), RT-PCR and ICC. Curiously, CXCR7 expression was significantly induced in MOLM13 cells under hypoxic (6% O2) conditions (p=0.01). Low levels of surface CXCR7 were found in 8 of the 9 primary ALL samples by flow cytometry. To determine the respective roles of CXCR4 and CXCR7 in migration of leukemic cells, we utilized CXCR4 inhibitor AMD3100 and CXCR7 inhibitor CCR733 in Raji (CXCR7 positive) and RS4;11 (CXCR7 negative) cells. AMD3100 at 25μM significantly inhibited SDF-1a induced migration (from 38.5% to 12%); CCR733 at 10μM also inhibited SDF-1a induced migration (from 38.5% to 24%) and the combination of AMD3100 and CCR733 resulted in 81% inhibition of migration (from 38.5% to 7.2%). AMD3100 blocked SDF-1a induced migration of CXCR4+CXCR7− RS4;11 cells (from 36.5% to 15.8%), while CCR733 had no effect (36.5% and 39.2%). In conclusion, these studies demonstrate functional expression of the SDF-1 receptor CXCR-7 on Raji and primary ALL cells and suggest that CXCR7 plays an active role in the migration of leukemic cells. CXCR-7 may serve as an alternative receptor to CXCR4. Studies addressing the role of CXCR7 in adhesion, SDF-1a-mediated signaling and survival of leukemic cells are in progress.

scholarly journals Co-Treatments of Edible Curcumin from Turmeric Rhizomes and Chemotherapeutic Drugs on Cytotoxicity and FLT3 Protein Expression in Leukemic Stem Cells

Molecules ◽  
2021 ◽  
Vol 26 (19) ◽  
pp. 5785
Author(s):  
Fah Chueahongthong ◽  
Singkome Tima ◽  
Sawitree Chiampanichayakul ◽  
Cory Berkland ◽  
Songyot Anuchapreeda
Keyword(s):  
Stem Cells ◽  
Protein Expression ◽  
Cell Lines ◽  
Synergistic Effects ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Chemotherapeutic Drugs ◽  
Protein Levels ◽  
Leukemic Cell Lines

This study aims to enhance efficacy and reduce toxicity of the combination treatment of a drug and curcumin (Cur) on leukemic stem cell and leukemic cell lines, including KG-1a and KG-1 (FLT3+ LSCs), EoL-1 (FLT3+ LCs), and U937 (FLT3− LCs). The cytotoxicity of co-treatments of doxorubicin (Dox) or idarubicin (Ida) at concentrations of the IC10–IC80 values and each concentration of Cur at the IC20, IC30, IC40, and IC50 values (conditions 1, 2, 3, and 4) was determined by MTT assays. Dox–Cur increased cytotoxicity in leukemic cells. Dox–Cur co-treatment showed additive and synergistic effects in several conditions. The effect of this co-treatment on FLT3 expression in KG-1a, KG-1, and EoL-1 cells was examined by Western blotting. Dox–Cur decreased FLT3 protein levels and total cell numbers in all the cell lines in a dose-dependent manner. In summary, this study exhibits a novel report of Dox–Cur co-treatment in both enhancing cytotoxicity of Dox and inhibiting cell proliferation via FLT3 protein expression in leukemia stem cells and leukemic cells. This is the option of leukemia treatment with reducing side effects of chemotherapeutic drugs to leukemia patients.

Glycogen Synthase Kinase-3β (GSK-3β) Inhibition Induces Apoptosis In Leukemic Cells through Mitochondria-Dependent Pathway

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2889-2889
Author(s):  
Mohammad Reza Mirlashari ◽  
Ingrid Randen ◽  
Jens Kjeldsen-Kragh
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Cell Types ◽  
Leukemic Cells ◽  
Caspase 8 ◽  
Apoptotic Pathway ◽  
Leukemic Cell Lines ◽  
Cell Death Pathway ◽  
Gsk 3Β

Abstract Abstract 2889 GSK-3β is a multifunctional kinase that plays a role in several signaling pathways. Due to the contradictory roles of GSK-3β as a mediator of both cell survival and apoptosis, we have examined the role of GSK-3β for proliferation and apoptosis in leukemic cell lines KG1a, K562 and CMK. GSK-3β was selectively inhibited by the small-molecule SB-415286. Treatment of leukemia cells with SB-415286 (40 μM) for 72 hr approximately halved cell growth in all three cell lines. SB-415286 also showed a concentration-dependent stabilization of intracellular β-catenin: In KG1a cells the mean fluorescence intensity (MFI) [± 95% CI] was 3.1 [± 1.7] in untreated cells vs. 423 [± 24] in treated cell. The figures for the K562 and CMK cell lines were: 2.8 [± 1.6] vs. 353.2 [± 11.1], and 6.8 [± 4.0] vs. 320.2 [± 23.7], respectively. Cell cycle analysis was carried out to examine if the growth inhibition was caused by arrest in cell cycle and/or induction of apoptosis. We found that SB-415286 caused cell cycle arrest in the G2/M phase and accumulation of events corresponding to the subG1 phase, indicative of DNA fragmentation. The subG1 population was 45%, 34% and 17% in KG1a, K562 and CMK cells, respectively. To confirm that the increase of the subG1 fraction represented an apoptotic effect of the GSK-3β inhibition, we analyzed phosphatidylserine (PS) externalization and plasma membrane integrity. We found that SB-415286 caused a considerable increase of the proportion of early apoptotic cells, i.e. cells that were annexin V-positive and 7-AAD-negative: Mean [± 95% CI] in KG1a cells increased from 6.2% [± 1.2%] in untreated cells to 38% [± 3.1%] in treated cells. The figures for the K562 and CMK cell lines were: 3.0% [± 1.2%] vs. 29% [± 3.3%], and 3.9% [± 1.0%] vs. 16.0% [± 1.1%], respectively. Apoptosis signaling can be initiated by extracellular (death receptor) and/or intracellular (mitochondrial) signals. Flow cytometric analysis of cells stained by a dual-fluorescent mitochondrial dye JC-1 showed that 5–11% of untreated leukemic cells had low mitochondrial membrane potential. After 72 hr exposure to SB-415286 the mean [±95% CI] loss of the mitochondrial potential was found in 23% [± 2.0%], 33% [± 3.5%] and 42% [± 3.8%], in CMK, K562 and KG1a cells, respectively. Since drug treatment in some cell types may result in activation of both the intrinsic or extrinsic cell-death pathway in a parallel manner, we investigated if the external pathway is involved in SB-415286-induced apoptosis. For this purpose we assessed caspase-8 activation by flow cytometry. After 72 hr of treatment of CMK, K562 and KG1a cells the caspase-8 activities compared, to untreated cells, had increased 3.7-fold, 3.9-fold, and 4.4-fold, respectively. In some cell types, the extrinsic cell-death pathway leads to the cleavage of Bid (pro-apoptotic member of the Bcl-2 family) by caspase-8, generating a truncated version of the protein (tBid) which in turn activates the mitochondrial apoptotic pathway. Therefore, we determined whether depolarization of the mitochondrial membrane in the leukemic cell lines was an effect of activated caspase-8 or a direct effect of SB-415286. For this purpose Z-IETD-FMK (25 μM), a specific inhibitor of caspase-8, was applied to the cells for 2 hr. We found that inhibition of caspase-8 did not prevent SB-415286-induced apoptosis assessed by PS externalization. This indicates that activation of caspase-8 is part of the intrinsic apoptotic pathway and occurs downstream of mitochondria membrane potential depolarization mediated by other caspases. Taken together, our observations suggest that inhibition of GSK-3β induces apoptosis of leukemic cells by depolarizing the mitochondria membrane. Thus, inhibition of GSK-3β could be an attractive target for treatment of leukemia. Disclosures: No relevant conflicts of interest to declare.

Metabolic Capability to Induce the Pasteur Effect Mediates Sensitivity of Human Leukemic Cells to Metformin

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2601-2601
Author(s):  
Sarah Scotland ◽  
Estelle Saland ◽  
Lindsay Peyriga ◽  
Rémi Peyraud ◽  
Elizabeth Micklow ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Pasteur Effect ◽  
Cytotoxic Response ◽  
Leukemic Cell Lines

Abstract Abstract 2601 An emerging hallmark of cancer cells is the reprogramming of intermediary and energy metabolism these cells undergo. Several epidemiological studies have shown that metformin, widely used to treat patients with type 2 diabetes, may reduce their risk of cancer. Despite several reports of anti-neoplastic activity of metformin, the mechanisms responsible for this activity have not been fully elucidated in cancer or leukemic cells. We hypothesized that metformin elicits a metabolic reprogramming driven by alterations in mitochondrial function and signaling, which induces apoptosis in leukemic cells, and that metabolic flexibility determines the variation(s) of the cytotoxic response to metformin among different leukemic cell lines. We first demonstrated that metformin markedly decreased oxygen consumption of six leukemic cell lines in a concentration-dependent manner. We also observed that the cytotoxic effect of metformin varies between cell lines reflecting their energetic capacity to compensate for the mitochondrial inhibition induced by metformin (eg. to induce the Pasteur effect). Importantly, metformin-insensitive leukemic cells did not exhibit a Pasteur effect in response to metformin. All leukemic cells exhibited high basal conversion of glucose to lactate (eg. aerobic glycolysis) and specific expression of key metabolic genes as compared to normal mononuclear cells. Despite dependence on glucose catabolism, metformin sensitivity was associated with relative resistance to glucose starvation. Metformin effects in drug-resistant cells were potentiated by the addition of a glycolytic inhibitor, but not by inhibitors of the pentose phosphate pathway or glutaminolysis. Leukemic cells with broad metabolic capacities to utilize other energetic substrates in response to diverse nutrient starvation showed insensitivity to metformin. Metformin induced a significant decrease in metabolites of the upper segment of glycolysis and the oxidative branch of the pentose phosphate pathway as well as a clear increase of PRPP and IMP biosynthesis. Energy charge, the nucleotide phosphate pool and lactate/glucose ratio remained stable after metformin treatment. Furthermore, our results showed that basal glucose uptake/consumption and the activity of the lower segment of the glycolytic pathway are key determinants of a cytotoxic response to metformin. In addition, high glutathione, malate, IMP and orotate content were observed in metformin-insensitive leukemic cells. Moreover, the cytotoxic effect of metformin was independent of AMPK/LKB1 status of the leukemic cells while p53 expression abrogated this effect. The presence of wild-type p53 appears to partially protect tumor cells from glucose starvation and metformin cytotoxicity and prevents the induction of the Pasteur effect. Finally, we demonstrated that metformin increased the cytotoxicity of chemotherapy agent, cytarabine, on all leukemic cell lines in vitro and significantly reduced leukemic colony-forming units (CFU-L) from six primary AML patient samples in a concentration-dependent manner. Additional experiments on metabolic and signaling pathways as well as in vivo studies are in progress to better understand the cytotoxic response of metformin in both AML cell lines and primary AML patient specimens that impact the therapeutic potential of metformin in vivo. Disclosures: Carroll: Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.

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