A Novel Peptide Derived from Ginger Induces Apoptosis through the Modulation of p53, BAX, and BCL2 Expression in Leukemic Cell Lines

Planta Medica ◽  
2021 ◽  
Author(s):  
Chawalit Chatupheeraphat ◽  
Sittiruk Roytrakul ◽  
Narumon Phaonakrop ◽  
Kamolchanok Deesrisak ◽  
Sucheewin Krobthong ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Raji Cell ◽  
Leukemic Cell ◽  
B Cell Lymphoma ◽  
Leukemic Cells ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Peptide Fraction

AbstractDespite the efficacy of chemotherapy, the adverse effects of chemotherapeutic drugs are considered a limitation of leukemia treatment. Therefore, a chemotherapy drug with minimal side effects is currently needed. One interesting molecule for this purpose is a bioactive peptide isolated from plants since it has less toxicity to normal cells. In this study, we extracted protein from the Zingiber officinale rhizome and performed purification to acquire the peptide fraction with the highest cytotoxicity using ultrafiltration, reverse-phase chromatography, and off-gel fractionation to get the peptide fraction that contained the highest cytotoxicity. Finally, a novel antileukemic peptide, P2 (sequence: RALGWSCL), was identified from the highest cytotoxicity fraction. The P2 peptide reduced the cell viability of NB4, MOLT4, and Raji cell lines without an effect on the normal peripheral blood mononuclear cells. The combination of P2 and daunorubicin significantly decreased leukemic cell viability when compared to treatment with either P2 or daunorubicin alone. In addition, leukemic cells treated with P2 demonstrated increased apoptosis and upregulation of caspase 3, 8, and 9 gene expression. Moreover, we also examined the effects of P2 on p53, which is the key regulator of apoptosis. Our results showed that treatment of leukemic cells with P2 led to the upregulation of p53 and Bcl-2-associated X protein, and the downregulation of B-cell lymphoma 2, indicating that p53 is involved in apoptosis induction by P2. The results of this study are anticipated to be useful for the development of P2 as an alternative drug for the treatment of leukemia.

scholarly journals Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
M. R. Ricciardi ◽  
R. Licchetta ◽  
S. Mirabilii ◽  
M. Scarpari ◽  
A. Parroni ◽  
...  
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Leukemic Cell ◽  
Bioactive Compound ◽  
Primary Cells ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Leukemic Cell Lines

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor, on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity.

Aberrant DNA methylation ofp57KIP2 gene in the promoter region in lymphoid malignancies of B-cell phenotype

Blood ◽  
2002 ◽  
Vol 100 (7) ◽  
pp. 2572-2577 ◽  
Author(s):  
Yinghua Li ◽  
Hirokazu Nagai ◽  
Toshihito Ohno ◽  
Masaaki Yuge ◽  
Sonoko Hatano ◽  
...  
Keyword(s):  
Dna Methylation ◽  
B Cell ◽  
Cell Lines ◽  
Promoter Region ◽  
Mononuclear Cells ◽  
B Cell Lymphoma ◽  
Cell Phenotype ◽  
Sequencing Analysis ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood

The cyclin-dependent kinase inhibitorp57KIP2 is thought to be a potential tumor suppressor gene (TSG). The present study examines this possibility. We found that the expression ofp57KIP2 gene is absent in various hematological cell lines. Exposing cell lines to the DNA demethylating agent 5-aza-2′-deoxycytidine restoredp57KIP2 gene expression. Bisulfite sequencing analysis of its promoter region showed thatp57KIP2 DNA was completely methylated in cell lines that did not express thep57KIP2 gene. Thus, DNA methylation of its promoter might lead to inactivation of thep57KIP2 gene. DNA methylation of this region is thought to be an aberrant alteration, since DNA was not methylated in normal peripheral blood mononuclear cells or in reactive lymphadenitis. Methylation-specific polymerase chain reaction analysis found frequent DNA methylation of thep57KIP2 gene in primary diffuse large B-cell lymphoma (54.9%) and in follicular lymphoma (44.0%), but methylation was infrequent in myelodysplastic syndrome and adult T-cell leukemia (3.0% and 2.0%, respectively). These findings directly indicate that the profile of the p57KIP2gene corresponds to that of a TSG.

Monoglycerides induce apoptosis in human leukemic cells while sparing normal peripheral blood mononuclear cells

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 292-294 ◽  
Author(s):  
Fabianne Philippoussis ◽  
Chantal Arguin ◽  
Véronique Mateo ◽  
Ann-Muriel Steff ◽  
Patrice Hugo
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Major Drawback ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Blood Mononuclear Cells

Abstract A major drawback of the current antineoplastic treatments is their lack of specificity toward cancer cells, because they are most often cytotoxic to normal cells, thus creating related side effects. Hence, the identification of new apoptosis-inducing agents, specifically targeting malignant cells while sparing their normal counterparts, is of crucial interest. We show here that monoglycerides, a family of lipids consisting of a single fatty acid attached to a glycerol backbone, induce cell death in several human leukemic cell lines. Importantly, treatment of primary leukemic cells, obtained from B-cell chronic lymphocytic leukemia patients, resulted in rapid apoptosis. In striking contrast, resting or activated human peripheral blood mononuclear cells from healthy individuals were resistant to the same treatment. Therefore, these compounds could represent potential antileukemic drugs or could allow for the design of novel therapeutic agents applied to leukemia.

scholarly journals Mechanistic Study of Triazole Based Aminodiol Derivatives in Leukemic Cells—Crosstalk between Mitochondrial Stress-Involved Apoptosis and Autophagy

2020 ◽  
Vol 21 (7) ◽  
pp. 2470
Author(s):  
She-Hung Chan ◽  
Wohn-Jenn Leu ◽  
Sharada Prasanna Swain ◽  
Jui-Ling Hsu ◽  
Duen-Ren Hou ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Cholesterol Biosynthesis ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Mechanistic Study ◽  
Nuclear Staining ◽  
Peripheral Blood Mononuclear ◽  
Mitochondrial Stress ◽  
Stat3 Phosphorylation

Various derivatives that mimic ceramide structures by introducing a triazole to connect the aminodiol moiety and long alkyl chain have been synthesized and screened for their anti-leukemia activity. SPS8 stood out among the derivatives, showing cytotoxic selectivity between leukemic cell lines and human peripheral blood mononuclear cells (about ten times). DAPI nuclear staining and H&E staining revealed DNA fragmentation under the action of SPS8. SPS8 induced an increase in intracellular Ca2+ levels and mitochondrial stress in HL-60 cells identified by the loss of mitochondrial membrane potential, transmission electron microscopy (TEM) examination, and altered expressions of Bcl-2 family proteins. SPS8 also induced autophagy through the detection of Atg5, beclin-1, and LC3 II protein expression, as well as TEM examination. Chloroquine, an autophagy inhibitor, promoted SPS8-induced apoptosis, suggesting the cytoprotective role of autophagy in hindering SPS8 from apoptosis. Furthermore, SPS8 was shown to alter the expressions of a variety of genes using a microarray analysis and volcano plot filtering. A further cellular signaling pathways analysis suggested that SPS8 induced several cellular processes in HL-60, including the sterol biosynthesis process and cholesterol biosynthesis process, and inhibited some cellular pathways, in which STAT3 was the most critical nuclear factor. Further identification revealed that SPS8 inhibited the phosphorylation of STAT3, representing the loss of cytoprotective activity. In conclusion, the data suggest that SPS8 induces both apoptosis and autophagy in leukemic cells, in which autophagy plays a cytoprotective role in impeding apoptosis. Moreover, the inhibition of STAT3 phosphorylation may support SPS8-induced anti-leukemic activity.

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.

Honokiol Overcomes Conventional Drug Resistance in Human Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1488-1488
Author(s):  
Kenji Ishitsuka ◽  
Teru Hideshima ◽  
Makoto Hamasaki ◽  
Raje Noopur ◽  
Kumar Shaji ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Flow Cytometric Analysis ◽  
Caspase 8 ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Human Multiple Myeloma ◽  
Induced Apoptosis

Abstract Honokiol is an active component isolated and purified from Magnolia, a plant used in traditional Chinese medicine. It is an anti-oxidant, and inhibits both xanthine oxidase and angiogenesis. In this study, we first examined the direct toxicity of honokiol against human multiple myeloma (MM) cell lines in vitro. Honokiol significantly inhibited growth of MM cell lines (RPMI8226, U266 and MM.1S) via induction of G1 growth arrest, followed by apoptosis, with IC50 values at 48h of 5 to 10 μg/ml. Moreover, honokiol similarly inhibited growth of doxorubicin (Dox)-resistant (RPMI-Dox40), melphalan resistant (RPMI-LR5), and dexamethasone (Dex)-resistant (MM.1R) cell lines. Furthermore, flow cytometric analysis demonstrated that honokiol (6–10 μg/ml, 48h) induced death of CD38+CD138+ tumor cells isolated from 5 patients with relapsed refractory MM. In contrast, no toxicity was observed in normal peripheral blood mononuclear cells or long term-cultured bone marrow stromal cells (BMSCs) treated with honokiol (≤20 mg/ml). Neither culture of MM cells with BMSCs nor interleukin-6 (IL-6) and insulin like growth factor-1 (IGF-1) protected against honokiol-induced cytotoxicity in MM.1S cells. We next delineated the mechanism of honokiol-triggered cytotoxicity. Honokiol triggered increased expression of Bax and Bad; down regulated Mcl-1 protein expression, followed by caspase-8/9/3 cleavage. Importantly, the pan-caspase inhibitor z-VAD-fmk only partially inhibited honokiol-induced apoptosis in MM.1S cells. Furthermore, honokiol induced apoptosis even in SU-DHL4 cells, which express low level of caspase-8 and -3 and are resistant to both conventional (doxorubicin, melphalan, dexamethason) and novel (bortezomib, revimid) drugs. These results suggest that honokiol may induce apoptosis via both caspase-dependent and -independent pathways. Finally, honokiol inhibited IL-6-induced phosphorylation of ERK1/2, STAT3, and Akt, known to mediate growth, survival, and drug resistance, respectively. Taken together, our results suggest that providing the rationale for clinical evaluation of honokiol to improve patient outcome in MM.

SL-401, A Novel Targeted Therapy Directed To The Interleukin-3 Receptor (IL-3R), Inhibits Plasmacytoid Dendritic Cell (pDC)-Induced Myeloma Cell Growth and Overcomes Drug Resistance

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3170-3170 ◽  
Author(s):  
Arghya Ray ◽  
Deepika Sharma Das ◽  
Ze Tian ◽  
Vincent Macri ◽  
Christopher L. Brooks ◽  
...  
Keyword(s):  
New York ◽  
Drug Resistance ◽  
Targeted Therapy ◽  
Cell Growth ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Plasmacytoid Dendritic Cell ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Low Concentrations

Abstract Introduction Multiple Myeloma (MM) remains incurable despite the advent of novel drugs, highlighting the need for further identification of factors mediating disease progression and resistance. The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in MM cells. Our earlier study using both in vitro and in vivo MM xenograft models showed increased numbers of plasmacytoid dendritic cells (pDCs) in the MM BM, which promote MM cell growth and survival (Chauhan et al., Cancer Cell 2009, 16:309-323). We found increased IL-3 levels upon pDC-MM interaction, which in turn, trigger MM cell growth and pDC survival. Interestingly, the IL-3 receptor (IL-3R) is highly expressed on pDCs. In this study, we utilized SL-401, a novel targeted therapy directed to IL-3R, to examine whether blockade of the IL-3-IL3R signaling axis inhibits pDC-induced MM cell growth. Methods To study the anti-MM activity of SL-401, we utilized MM cell lines, patient MM cells, and pDCs from normal healthy donors or MM patients. The pDCs and MM cells were cultured alone or together in the presence or absence of SL-401, and cell growth or viability was analyzed using WST/MTT assays. Results MM cells or pDCs were freshly isolated and treated with various concentrations of SL-401. SL-401 significantly decreased the viability of pDCs even at low concentrations (IC50: 0.83 ng/ml; 14.6 pM) (mean ± SD; n=4, P < 0.005). SL-401 decreased the viability of MM cells at clinically achievable doses, without significantly affecting the viability of normal peripheral blood mononuclear cells. Co-culture of pDCs with MM cells triggered growth of various MM cell lines, which was blocked in the presence of low concentrations of SL-401 (0.8 ng/ml). MM patient-derived pDCs also induced proliferation of MM cell lines and primary MM cells; and importantly, SL-401 inhibited pDC-triggered MM cell growth (P < 0.005). Moreover, 3 of 5 samples were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, SL-401 blocked pDC-induced growth of dexamethasone-, doxorubicin- or melphalan-resistant MM cell lines (MM.1R, Dox-40 and LR5 cell lines, respectively). Finally, combinations of SL-401 with bortezomib, melphalan, or lenalidomide showed synergistic anti-MM activity. Conclusions Our preclinical study provides the basis for directly targeting pDCs and inhibiting the pDC-MM interaction, as well as targeting MM, in novel therapeutic strategies using SL-401 to enhance MM cytotoxicity, overcome drug-resistance, and improve patient outcome. Disclosures: Macri: Stemline Therapeutics, Inc., New York, NY USA : Employment. Brooks:Stemline Therapeutics, Inc., New York, NY USA : Employment. Rowinsky:Stemline Therapeutics, Inc., New York, NY USA : Employment.

The BH3-only mimetic ABT-737 synergizes the antineoplastic activity of proteasome inhibitors in lymphoid malignancies

Blood ◽  
2008 ◽  
Vol 112 (7) ◽  
pp. 2906-2916 ◽  
Author(s):  
Luca Paoluzzi ◽  
Mithat Gonen ◽  
Govind Bhagat ◽  
Richard R. Furman ◽  
Jeffrey R. Gardner ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Cell Lymphoma ◽  
Proteasome Inhibitors ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Lymphoma Cell ◽  
Peripheral Blood Mononuclear ◽  
Mitochondrial Membrane Depolarization

Abstract Overexpression of antiapoptotic members of the Bcl-2 family is observed in approximately 80% of B-cell lymphomas, contributing to intrinsic and acquired drug resistance. Nullifying the antiapoptotic influence of these proteins can potentially overcome this resistance, and may complement conventional chemotherapy. ABT-737 is a BH3-only mimetic and potent inhibitor of the antiapoptotic Bcl-2 family members Bcl-2, Bcl-XL, and Bcl-w. In vitro, ABT-737 exhibited concentration-dependent cytotoxicity against a broad panel of lymphoma cell lines including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). ABT-737 showed synergism when combined with the proteasome inhibitors bortezomib or carfilzomib in select lymphoma cell lines and induced potent mitochondrial membrane depolarization and apoptosis when combined with either. ABT-737 plus bortezomib also induced significant apoptosis in primary samples of MCL, DLBCL, and chronic lymphocytic leukemia (CLL) but no significant cytotoxic effect was observed in peripheral blood mononuclear cells from healthy donors. In severe combined immunodeficient beige mouse models of MCL, the addition of ABT-737 to bortezomib enhanced efficacy compared with either drug alone and with the control. Collectively, these data suggest that ABT-737 alone or in combination with a proteasome inhibitor represents a novel and potentially important platform for the treatment of B-cell malignancies.

IFNα-stimulated neutrophils and monocytes release a soluble form of TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) displaying apoptotic activity on leukemic cells

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3837-3844 ◽  
Author(s):  
Cristina Tecchio ◽  
Veronica Huber ◽  
Patrizia Scapini ◽  
Federica Calzetti ◽  
Daniela Margotto ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Soluble Form ◽  
Human Neutrophils ◽  
Interferon Α ◽  
Cytotoxic Activities ◽  
Peripheral Blood Mononuclear ◽  
Trail Receptors

Abstract Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily exerting cytotoxic activities toward tumor cells. Herein, we demonstrate that therapeutic concentrations of interferon α (IFNα) stimulate the expression of high levels of TRAIL mRNA and the release of elevated amounts of a soluble bioactive form of TRAIL (sTRAIL) in both human neutrophils and monocytes. Supernatants harvested from IFNα-treated neutrophils/monocytes elicited, on TRAIL-sensitive leukemic cell lines, proapoptotic activities that were significantly reduced by either a combination of TRAIL-R1/Fc and TRAIL-R2/Fc chimeras or neutralizing anti-TRAIL, anti–TRAIL-R1, and anti–TRAIL-R2 antibodies, suggesting that they were mediated by released sTRAIL acting on both TRAIL receptors. Since diseases such as chronic myeloid leukemia (CML) and melanoma are effectively treated with IFNα,we also demonstrate that CML neutrophils and peripheral blood mononuclear cells (PBMCs) cultured with IFNα at therapeutic concentrations retain the capacity of releasing sTRAIL, suggesting that CML leukocytes, in vivo, might represent an important source of sTRAIL. In this regard, we show that sTRAIL serum levels as well as leukocyte-associated TRAIL significantly increase in melanoma patients following IFNα administration. Collectively, these findings indicate that sTRAIL released by IFNα-activated neutrophils and monocytes contributes not only to the immunoregulatory actions but also to the therapeutic activities of IFNα.

scholarly journals SKIP Is Underexpressed in AML Leading to Sphingosine Kinase Hypofunction

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5324-5324
Author(s):  
Essam Ghazaly ◽  
Paul Smith ◽  
Chathunissa Gnanaranjan ◽  
Bryan D Young ◽  
Simon Joel ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Leukemic Cell ◽  
Sphingosine Kinase ◽  
Fold Increase ◽  
Peripheral Blood Mononuclear ◽  
Leukemic Cell Lines ◽  
Intracellular Concentrations

Abstract Background: Sphingosine kinase interacting protein (SKIP) has been shown to be mostly silenced by hypermethylation in AML [1]. SKIP interacts with and regulates the function of sphingosine kinase (SK) enzyme. SK activity results in phosphorylation of sphingosine (SPH) to form sphingosine 1 phosphate (S1P), which promotes cell survival and resistance to apoptosis. On the other hand, S1P precursors ceramide (CER) and SPH mediate antiproliferative and apoptotic responses. SKIP has been reported to negatively regulate SK1 activity in fibroblasts. Therefore, we investigated the consequences of SKIP silencing in primary AML cells. In addition, we studied the effects of SKIP re-expression in leukemic cell lines. Methods: CTS and K562 cells were transfected with SKIP gene using standard techniques. SKIP is normally silenced in both cell lines. Intracellular and extracellular S1P, SPH and CER were measured by UPLC-MS/MS. In addition, intracellular SK activity was determined based on C17 S1P production from C17 SPH substrate. Chemosensitivity to doxorubicin, Imatinib and Ara-C in transfected cells was also studied. Results: In Primary AML cells, intracellular S1P and CER concentrations were reduced compared to G-CSF-mobilized peripheral blood mononuclear cells. In addition, SK activity was found to be downregulated in AML primary cells. When we transfected leukemic cell lines with SKIP gene, S1P and CER showed at least 2 fold increase in intracellular and extracellular basal levels compared to vector alone control (Figure 1). Further studies confirmed a significant increase in intracellular SK activity in SKIP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p< 0.05). This increase in S1P and CER was associated with increasing apoptotic signals as evidenced by Annexin V staining and cleaved PARP expression. Moreover, chemosensitivity to Imatinib and AraC was significantly increased in SKIP transfected cell lines. These experiments confirm the regulation of SK1 function by SKIP. Conclusion: These data indicate that SKIP is downregulated in AML leading to reduced SK activity, which ultimately inhibits the apoptosis response. Figure 1. Effect of SKIP transfection on intracellular concentrations of S1P. Figure 1. Effect of SKIP transfection on intracellular concentrations of S1P. 1. Saied, M.H., et al., Genome wide analysis of acute myeloid leukemia reveal leukemia specific methylome and subtype specific hypomethylation of repeats. PLoS One, 2012. 7(3): p. e33213. EAG and PS contributed equally JG and DT contributed equally Disclosures No relevant conflicts of interest to declare.

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