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

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.

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

Planta Medica ◽

10.1055/a-1408-5629 ◽

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.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21072470 ◽

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.

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OX40 Expressed on Fresh Leukemic Cells From Adult T-Cell Leukemia Patients Mediates Cell Adhesion to Vascular Endothelial Cells: Implication for the Possible Involvement of OX40 in Leukemic Cell Infiltration

Blood ◽

10.1182/blood.v89.8.2951 ◽

1997 ◽

Vol 89 (8) ◽

pp. 2951-2958 ◽

Cited By ~ 55

Author(s):

Akihiro Imura ◽

Toshiyuki Hori ◽

Kazunori Imada ◽

Shin Kawamata ◽

Yuetsu Tanaka ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

T Cell ◽

Mononuclear Cells ◽

Vascular Endothelial Cells ◽

Leukemic Cell ◽

Leukemic Cells ◽

T Cell Leukemia ◽

Cell Leukemia

Abstract We demonstrated previously that OX40 and its ligand, gp34, directly mediate adhesion of activated normal CD4+ T cells, as well as human T-cell leukemia virus type I (HTLV-I)–transformed T cells to vascular endothelial cells. In the present study, we examined expression of OX40 on fresh leukemic cells from patients with adult T-cell leukemia (ATL) and its possible involvement in cell adhesion. Flow cytometric analysis showed that peripheral blood mononuclear cells (PBMC) or lymph node tumor cells from 15 of 17 cases expressed significant levels of OX40 without stimulation. On the other hand, gp34 was not expressed on these cells, although its expression is also known to be associated with HTLV-I-infection. In Western blot analysis, a 50-kD protein band was detected by anti-OX40 monoclonal antibody (MoAb) in two ATL cases examined, as well as phytohemagglutinin (PHA) blasts and Hut102, an HTLV-I–infected T-cell line, but not in resting PBMC or Jurkat. Expression of OX40 mRNA was shown by reverse transcriptase-polymerase chain reaction in all ATL cases tested, PHA-blasts, and Hut102, but not in resting PBMC or Jurkat. We could not detect expression of HTLV-I viral mRNA in any of the cases tested. Cell adhesion assay was performed and in at least three cases, fresh ATL cells exhibited adhesion to human umbilical vein endothelial cells that could be considerably inhibited by either anti-OX40 MoAb or anti-gp34 MoAb. Immunohistochemical staining of skin biopsy specimens indicated that infiltrating mononuclear cells express OX40 in vivo. Taken together, these data indicate that leukemic cells from most, but not all, ATL patients constitutively express OX40, which may play a role in leukemic cell infiltration in addition to cell adhesion in vivo.

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Identification of the soluble granulocyte-macrophage colony stimulating factor receptor protein in vivo

Blood ◽

10.1182/blood.v95.2.461 ◽

2000 ◽

Vol 95 (2) ◽

pp. 461-469 ◽

Cited By ~ 19

Author(s):

Farzana Sayani ◽

Felix A. Montero-Julian ◽

Valerie Ranchin ◽

Jay M. Prevost ◽

Sophie Flavetta ◽

...

Keyword(s):

Cell Lines ◽

State Level ◽

Leukemic Cell ◽

Protein Product ◽

Receptor Protein ◽

Soluble Form ◽

Human Neutrophils ◽

Gm Csf ◽

Leukemic Cell Lines

On the basis of the finding of alternatively spliced mRNAs, the -subunit of the receptor for GM-CSF is thought to exist in both a membrane spanning (tmGMR) and a soluble form (solGMR). However, only limited data has been available to support that the solGMR protein product exists in vivo. We hypothesized that hematopoietic cells bearing tmGMR would have the potential to also produce solGMR. To test this hypothesis we examined media conditioned by candidate cells using functional, biochemical, and immunologic means. Three human leukemic cell lines that express tmGMR (HL60, U937, THP1) were shown to secrete GM-CSF binding activity and a solGMR-specific band by Western blot, whereas a tmGMR-negative cell line (K562) did not. By the same analyses, leukapheresis products collected for autologous and allogeneic stem cell transplants and media conditioned by freshly isolated human neutrophils also contained solGMR. The solGMR protein in vivo displayed the same dissociation constant (Kd = 2-5 nmol) as that of recombinant solGMR. A human solGMR ELISA was developed that confirmed the presence of solGMR in supernatant conditioned by the tmGMR-positive leukemic cell lines, hematopoietic progenitor cells, and neutrophils. Furthermore, the ELISA demonstrated a steady state level of solGMR in normal human plasma (36 ± 17 pmol) and provided data suggesting that plasma solGMR levels can be elevated in acute myeloid leukemias.

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Monocyte-Derived Dendritic Cells from HLA-Matched Allogeneic Donors Showed Superiority To Induce Leukemic Cell-Specific T Cells in Comparison to Leukemic Cell-Derived Dendritic Cells or Monocyte-Derived Dendritic Cells from AML Patients.

Blood ◽

10.1182/blood.v104.11.1798.1798 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1798-1798

Author(s):

Je-Jung Lee ◽

Bo-Hwa Choi ◽

Myong-Suk Park ◽

Deok-Hwan Yang ◽

Yeo-Kyeoung Kim ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Mononuclear Cells ◽

Leukemic Cell ◽

Leukemic Cells ◽

Cytotoxic Activities ◽

Cell Lysates ◽

Gm Csf ◽

Tnf Α

Abstract Leukemic-dendritic cells (leukemic-DCs) have certain limitations, which include difficult generation in 30–40% of patients, and low levels of expression of several key molecules. To overcome these limitations, autologous mononocyte-derived DCs (autologous-DCs) from the patients can use as an alternative approach, but the DCs may have some defective functions. In this study, we investigated the feasibility of immunotherapy for AML using leukemic-cell specific cytotoxic T lymphocytes that were stimulated by allogeneic monocyte-derived DCs (allogeneic-DCs) pulsed with leukemic cell lysates in vitro. To generate autologous- or allogenic-DCs, CD14+cells isolated from mononuclear cells (MNCs) of the patients or of HLA-matched donors were cultured in the medium with GM-CSF and IL-4 for 6 days. Their maturation was induced by adding TNF-α, IL-1β, IL-6, and PGE2 for 2 days, and then, leukemic cell lysates were pulsed. To generate leukemic-DCs, MNCs obtained from AML patients were cultured in the presence of GM-CSF, IL-4, and TNF-α for 8–12 days. The expressions of several key molecules on allogeneic-DCs were significantly higher than that on leukemic-DCs or autologous-DCs. Allogeneic-DCs exhibited a higher capacity to stimulate allogeneic CD3+ T cells compared to leukemic-DCs or autologous-DCs in allogeneic mixed lymphocyte reaction assay. Autologous CD3+ T cells stimulated by HLA-matched allogeneic-DCs pulsing with leukemic cell lysates showed more potent cytotoxic activities against autologous leukemic cells than those stimulated by leukemic-DCs or autologous-DCs. In addition, autologous-DC showed an intermediate level between leukemic-DCs and allogeneic-DCs in phenotypic expressions, allogeneic T-cell stimulatory capacities, or cytotoxic activities. In conclusion, these results suggest that monocyte derived-DCs from HLA-matched allogeneic donors can alternatively use to generate leukemic cell-specific cytotoxic T cells and to overcome the limitation of leukemic-DCs or autologous-DCs.

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Autocrine/paracrine cytokine stimulation of leukemic cell proliferation in smoldering and chronic adult T-cell leukemia

Blood ◽

10.1182/blood-2010-04-277418 ◽

2010 ◽

Vol 116 (26) ◽

pp. 5948-5956 ◽

Cited By ~ 28

Author(s):

Jing Chen ◽

Mike Petrus ◽

Bonita R. Bryant ◽

Vinh Phuc Nguyen ◽

Carolyn K. Goldman ◽

...

Keyword(s):

Mononuclear Cells ◽

Ex Vivo ◽

Leukemic Cell ◽

Leukemic Cells ◽

Cell Leukemia ◽

Peripheral Blood Mononuclear ◽

Adult T Cell Leukemia ◽

Blood Mononuclear Cells ◽

Cytokine Stimulation ◽

Stimulation Of

AbstractAdult T-cell leukemia (ATL), a heterogeneous disease, can be divided into smoldering, chronic, lymphoma, and acute types clinically. In addition to different clinical manifestations, different stages of ATL have different molecular signatures. Here, we demonstrated that smoldering/chronic ATL peripheral blood mononuclear cells spontaneously proliferated ex vivo in a cytokine (interleukin -12 [IL-12]/IL-9/IL-15)–dependent manner, while acute-type ATL peripheral blood mononuclear cells did not proliferate or proliferated independent of cytokines. Smoldering/chronic ATL cells produced IL-2 and IL-9 in 6-day ex vivo cultures. Interestingly, the addition of an anti–IL-2R-α monoclonal antibody profoundly inhibited IL-9 expression, suggesting optimal expression of IL-9 was dependent on IL-2 signaling in these patients. To determine whether there would be autonomous proliferation of ATL leukemic cells, we purified leukemic cells from patients with smoldering/chronic ATL. Purified leukemic cells cultured alone produced IL-2/IL-9, and the downstream Janus kinase/signal transducer and activator of transcription pathway was activated. However, the leukemic cells did not proliferate independently, but required coculture with autologous monocytes to induce proliferation. Moreover, interaction between leukemic cells and monocytes was contact dependent, and major histocompatibility complex class II expression may have contributed to this interaction. In conclusion, our data provide evidence that there is autocrine/paracrine cytokine stimulation of leukemic cell proliferation in patients with smoldering/chronic ATL that could be targeted for treatment.

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171 Preclinical studies support therapeutic application of the leukemic cell-based cancer relapse vaccine DCP-001 in ovarian cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0171 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A185-A185

Author(s):

Maho Nagasawa ◽

Remco Bos ◽

Haoxiao Zuo ◽

Kiave Yune Ho Wang Yin ◽

Marie-José van Lierop ◽

...

Keyword(s):

Ovarian Cancer ◽

T Cells ◽

Cd8 T Cells ◽

Mononuclear Cells ◽

Leukemic Cell ◽

Peripheral Blood Mononuclear ◽

Cancer Relapse ◽

Blood Mononuclear Cells

BackgroundOvarian cancer (OC) is the gynecological malignancy with the highest mortality due to the late diagnosis of disease and a high rate of relapse following initial therapy. Immunotherapy in combination with standard treatment modalities has emerged as an encouraging treatment approach to surmount this unmet medical need. DCP-001 is a cancer relapse vaccine derived from the DCOne human leukemic cell line and is currently progressing through clinical trials in hematological malignancies. During manufacturing, DCOne cells are shifted towards a mature dendritic cell phenotype, rendering the cells highly immunogenic and providing the basis for DCP-001, which is administered as an intradermal vaccine. DCOne cells express multiple common tumor associated antigens (TAA) such as WT-1, RHAMM, PRAME and MUC-1, which have been documented as potential target antigens in ovarian cancer. This observation suggests that DCP-001 vaccination may also have an anti-tumor effect in OC. To support this hypothesis, we assessed the capacity of DCP-001 to induce immune responses against OC in human peripheral blood mononuclear cells (PBMCs) and a humanized mouse model for OC.MethodsThe effect of DCP-001 on T cells from OC patients or healthy donors was evaluated after a 3 week culture of peripheral blood mononuclear cells (PBMCs) with or without DCP-001. Cytotoxic activity was analysed by specific IFNg production and CD107a expression when these cells were subsequently cultured with the OC cell line SKOV3. The effect of DCP-001 vaccination in vivo was evaluated in humanised NCG mouse subcutaneously engrafted with SKOV3 OC cells. Mice received intra-peritoneal (i.p.) vaccination with DCP-001 either after or prior to SKOV3 engraftment and tumor size was measured to evaluate the efficacy of DCP-001.ResultsIn vitro, DCP-001 was shown to activate both CD4+ as well as CD8+ T cells and to induce formation of memory T cells. Importantly, DCP-001-stimulated CD8+ T cells from OC patients were shown to exert a HLA class I dependent, cytotoxic immune response to OC cells. In vivo, in an ovarian tumor mouse model, significant reduction of tumor growth rate and partial or even complete tumor regressions were observed in mice vaccinated with DCP-001, particularly when administered as relapse vaccine (prior to tumor engraftment), as compared to PBS treated mice.ConclusionsThese pre-clinical in vitro and in vivo results support the potential use of DCP-001 as a cancer relapse vaccine in ovarian cancer, with the aim to reduce disease recurrence following initial standard of care therapy.

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Prevention of leptin binding to its receptor suppresses rat leukemic cell growth by inhibiting angiogenesis

Blood ◽

10.1182/blood-2001-11-0134 ◽

2002 ◽

Vol 100 (12) ◽

pp. 4123-4128 ◽

Cited By ~ 30

Author(s):

Per Ole Iversen ◽

Christian Andre Drevon ◽

Janne Elin Reseland

Keyword(s):

Bone Marrow ◽

Cell Growth ◽

Leptin Receptor ◽

Mononuclear Cells ◽

Leukemic Cell ◽

Leukemic Cells ◽

Acute Myelocytic Leukemia ◽

In Vitro Proliferation

Leptin promotes the growth and viability of hematopoietic cells, and it also stimulates microvessel formation, indicating a role for leptin in angiogenesis. Acute myelocytic leukemia (AML) remains a disease with poor prognosis. Similar to solid tumors, it probably requires angiogenesis to ensure adequate supplies of nutrients. We studied rats with transplanted AML to test if a neutralizing anti–leptin receptor monoclonal antibody (mAb) (anti–OB-R) could inhibit leukemogenesis. At 4 weeks after transplantation, the bone marrow contained about 80% leukemic cells as assayed with a specific mAb and flow cytometry. Microscopic examination of bone marrow sections stained with an anti–von Willebrand mAb revealed a marked increase in microvessel density in the leukemic rats compared with controls. Treatment with anti–OB-R for 3 weeks more than halved the content of bone marrow leukemic cells with a concomitant, substantial decrease in angiogenesis. A parallel experiment using an irrelevant anticasein mAb showed no effect on either leukemic cell growth or angiogenesis. We could not detect surface expression of the leptin receptor on the leukemic cells, but on mononuclear cells from healthy rats. The anti–OB-R did not affect in vitro proliferation of leukemic cells whereas proliferation of the mononuclear cells was markedly impaired. The anti–OB-R had no effect on either leukemic cell growth or angiogenesis in leukemic fa/fa rats with a mutated leptin receptor. We conclude that leptin stimulates leukemic cell growth in vivo by promoting angiogenesis. Inhibition of binding of leptin to its receptor might be a new adjunct therapy in AML.

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Pharmacologic Modulation of STAT1 and Wnt Signaling in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.4150.4150 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4150-4150

Author(s):

Christina Wu ◽

Fitzgerald S Lao ◽

Michael Y. Choi ◽

Dennis A. Carson

Keyword(s):

Wnt Signaling ◽

Mononuclear Cells ◽

Leukemia Cell ◽

Xenograft Model ◽

Advisory Board ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Peripheral Blood Mononuclear ◽

Normal Peripheral Blood

Abstract BACKGROUND: The STAT and Wnt signaling pathways play critical roles in early lymphocyte development and function, with considerable cross-talk between them. STAT1 has been reported to be constitutively phosphorylated at SER727, and often at TYR701, in CLL patients. STAT1 activation can regulate the function of the IRF8 transcription factor. Polymorphisms of the IRF8 gene have been associated with both familial and sporadic CLL, and can influence Wnt signaling. Hence, both STAT1 and Wnt are potential pharmacologic targets for CLL therapy, particularly in patients that are resistant to, or intolerant of BTK and PI3K inhibitors. We previously reported that the electrophilic drug dimethylfumarate (DMF, Tecfidera, Fumaderm) could inhibit Wnt signaling in CLL. Here we have investigated its effects on constitutive and inducible STAT1 signaling, and on IRF8 expression, in primary CLL cells. METHODS: Primary leukemia cells from patients with CLL were cultured with DMF at pharmacologically relevant doses (3 uM to 30 uM) for 4 hours prior to analysis of STAT1 phosphorylation and IRF8 expression. We assessed the effect of DMF with and without pre-stimulation by lipopolysaccharide (LPS), Wnt3a, or a TLR7 agonist. To further evaluate the effect on CLL cells in a microenvironment that stimulates these pathways, we utilized a murine CLL xenograft model. We implanted primary leukemic cells from patients with CLL into the peritoneum of Rag2 deficient immune-compromised mice. Groups of mice were then treated with DMF at a dose of 10 mg/kg by oral gavage. CLL cells were retrieved 4 to 5 hours later by peritoneal lavage and analyzed. RESULTS: We confirmed that both SER727 and TYR701 are phosphorylated in the majority of primary CLL cells from both VH mutated (high risk) and unmutated (low risk) clones, but not in normal peripheral blood mononuclear cells (PBMC). A 4 hours exposure of CLL cells to DMF inhibited TYR701 phosphorylation, but had no effect on SER727 phosphorylation nor on total STAT1 levels. We also confirmed that IRF8 levels are 6-8 fold elevated in CLL cells, compared to normal PBMC. IRF8 expression was inhibited by DMF treatment in 7 out of 11 CLL patients. Similar effects were observed in vivo. Wnt or LPS stimulation increased IRF8 levels in normal PBMC, but did not alter the already elevated IRF8 levels in CLL cells. CONCLUSIONS: Activation of the STAT1/IRF8 pathway, like the Wnt pathway, is highly characteristic of CLL. DMF treatment of CLL cells can interrupt these signaling cascades, interfering with leukemia cell survival. Disclosures Choi: AbbVie: Consultancy, Other: Advisory Board, Research Funding; Gilead: Consultancy, Other: Advisory Board, Speakers Bureau.

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