Involvement of p38 Activation and Mitochondria in Death of Human Leukemia Cells Induced by an Agonistic Human Monoclonal Antibody Fab Specific to TRAIL Receptor 1

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death with minimal damage to normal cells; however, some cancer cells are resistant to TRAIL. TRAIL resistance may be overcome by agonistic antibodies to TRAIL receptors. In this study, we report the toxic effects of a novel recombinant agonistic human anti–TRAIL receptor 1 (DR4) monoclonal antibody Fab fragment, DR4-4, on various TRAIL-resistant and -sensitive cancer cell lines. The mechanisms of DR4-4 Fab–induced cell death in a human T cell leukemia cell line (Jurkat) were investigated using cell viability testing, immunoblotting, immunoassays, flow cytometry, and morphological observation. DR4-4 Fab–induced caspase-independent necrosis was observed to occur in Jurkat cells in association with p38 mitogen-activated protein kinase activation, cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein degradation, decreased mitochondrial membrane potential, and increased mitochondrial reactive oxygen species production. Increased cytotoxic effects of DR4-4 Fab were observed in combination with TRAIL or γ-irradiation. Our results indicate that the novel DR4-4 Fab might overcome TRAIL-resistance and induce death in leukemia cells via cellular mechanisms different from those activated by TRAIL. DR4-4 Fab may have application as a potential therapeutic antibody fragment in single or combination therapy for cancer.

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Establishment of TRAIL-Resistance Kasumi-1 Cell Line and the Analysis of It different mRNA Expression Profile with the Original Kasumi-1 Cell Line

Blood ◽

10.1182/blood.v124.21.5221.5221 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5221-5221

Author(s):

Xuewei Jiang ◽

Pan Zengkai ◽

Chen Jin ◽

Yu Pengfei ◽

Li-Gen Liu

Keyword(s):

Mrna Expression ◽

Human Genome ◽

Cell Line ◽

Expression Profile ◽

Leukemia Cell Line ◽

Leukemia Cells ◽

Mrna Expression Profile ◽

Affymetrix Human Genome ◽

Trail Receptors ◽

Trail Resistance

Abstract Introduction Tumor necrosis factor related apoptosis inducing ligand (TRAIL) can induce the apoptosis of many human leukemia cells while sparing of normal cells, but its resistance is also universal. Our previous study on apoptosis of t(8;21) positive acute myeloid leukemia cell line Kasumi-1 induced by rhTRAIL showed that the survival rate no longer decreased significantly when rsTRAIL reached a certain concentration which implied Kasumi-1 cells might have a resistant tendency to TRAIL. Then, we established a TRAIL-resistant Kasumi-1 cell line (Kasumi-1 TR) by intermittently escalating rsTRAIL concentration in culture media, and compared the mRNA expression profile with the original Kasumi-1 cell line by using Affymetrix Human Genome U133 Plus 2.0 Array. Methods Kasumi-1 TR cell line was established by intermittently treated Kasumi-1 cells with progressively escalating rsTRAIL concentration. Proliferation of leukemia cells were measured by CCK-8 assay, and rsTRAIL IC50 of cells and resistance index were calculated according to proliferation of cells treated with rsTRAIL at different concentrations. TRAIL and TRAIL receptors 1-4 on cells surface were detected by flow cytometry. Expression profiles of Kasumi-1 cells and Kasumi-1 TR cells were analyzed by Affymetrix Human Genome U133 Plus 2.0 Array to identify differentially expressed genes, and the search of genes possibly related with TRAIL-resistance were using by GO functional analysis and pathway enrichment analysis. Results 1) Kasumi-1 TR cells proliferation was faster than that of Kasumi-1 cells(Fig 1A); 2) IC50 of 24 hours for Kasumi-1 cells was 756.833ng/ml (logIC50 2.879 ± 0.148), IC50 of 24 hours for Kasumi-1 TR cells was 1634646.005ng/ ml (logIC50 6.213 ± 0.637), the RI of 24h was 2159 (Fig 1B); IC50 of 48 hours for Kasumi-1 cells was 345.390ng/ml (logIC50 2.538 ± 0.153), IC50 of 48 hours for Kasumi-1 TR cells was 33642.641ng/ml (logIC50 is 4.257 ± 0.317), the RI for 48h was 97 (Fig 1C); 3) Cell surface expression of TRAIL and its receptors 1-4 had no difference between two cell lines(Fig 1D). 4) There were 1537 genes up regulated by more than 2 times while 487 genes down regulated by more than 2 times in Kasumi-1 TR cells compared with the original Kasumi-1 cells (Fig 1E). Of which BCL-2 family antiapoptotic gene BCL2 is increased by 3.153 times and BCL2A1 increased by 18.23 times, IFNAR1 involved in JAK/STAT pathway increased by 12.841 times and TRAIL death receptor TNFRSF10A down regulated by 3.256 times(Fig 1F). Conclusions: The Kasumi-1 cell line with rsTRAIL resistance (Kasumi-1 TR) is established, and its resistance may be associated with the up expression of BCL2, BCL2A1, IFNAR1 and down regulated expression of DR4. Acknowledgment This work was supported by grants from NSFC (30672415) and STCSM (054119528). Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Transferrin-Bound Doxorubicin Enhances Apoptosis and DNA Damage through the Generation of Pro-Inflammatory Responses in Human Leukemia Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21249390 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9390

Author(s):

Monika Jedrzejczyk ◽

Katarzyna Wisniewska ◽

Katarzyna Dominika Kania ◽

Agnieszka Marczak ◽

Marzena Szwed

Keyword(s):

Dna Damage ◽

Cell Death ◽

Mononuclear Cells ◽

Inflammatory Responses ◽

Leukemia Cell ◽

Leukemia Cells ◽

Morphological Observation ◽

Human Leukemia ◽

Dependent Manner ◽

Normal Peripheral Blood

Doxorubicin (DOX) is an effective antineoplastic drug against many solid tumors and hematological malignancies. However, the clinical use of DOX is limited, because of its unspecific mode of action. Since leukemia cells overexpress transferrin (Tf) receptors on their surface, we proposed doxorubicin–transferrin (DOX–Tf) conjugate as a new vehicle to increase drug concentration directly in cancer cells. The data obtained after experiments performed on K562 and CCRF-CEM human leukemia cell lines clearly indicate severe cytotoxic and genotoxic properties of the conjugate drug. On the other hand, normal peripheral blood mononuclear cells (PBMCs) were more resistant to DOX–Tf than to DOX. In comparison to free drug, we observed that Tf-bound DOX induced apoptosis in a TRAIL-dependent manner and caused DNA damage typical of programmed cell death. These fatal hallmarks of cell death were confirmed upon morphological observation of cells incubated with DOX or DOX–Tf. Studies of expression of TNF-α, IL-4, and IL-6 at the mRNA and protein levels revealed that the pro-inflammatory response plays an important role in the toxicity of the conjugate. Altogether, the results demonstrated here describe a mechanism of the antitumor activity of the DOX–Tf conjugate.

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Vitamin K2 Induces Autophagy and Apoptosis Simultaneously in Leukemia Cells and Bcl-2 Expression Level Determines the Phenotype of Their Cell Death.

Blood ◽

10.1182/blood.v110.11.3478.3478 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3478-3478

Author(s):

Keisuke Miyazawa ◽

Tomohisa Yokoyama ◽

Munekazu Naito ◽

Juri Toyotake ◽

Testuzo Tauchi ◽

...

Keyword(s):

Cell Death ◽

Leukemia Cell ◽

Vitamin K2 ◽

Expression Level ◽

Leukemia Cell Line ◽

Leukemia Cells ◽

Potent Inducer ◽

Acridine Orange Staining ◽

Induced Apoptosis

Abstract Vitamin K2 (menaquinone-2: VK2) is now known to be a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of human bcl-2 gene into HL-60 leukemia cell line, show 5-fold greater expression of Bcl-2 protein compared with that in HL-60neo cells, a control clone transfected with vector alone. Although HL-60neo cells are induced apoptosis in response to VK2, HL-60bcl-2 cells are resistant against apoptosis induction but still show cell growth inhibition along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed autophagosomes and autolysosomes formation in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVO) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunonoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported enhanced autophagy induction in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, autophagosome formation was rather prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells were protected from rapid apoptotic death by higher expression level of Bcl-2.

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Glucose Deprivation Induces ATF4-Mediated Apoptosis through TRAIL Death Receptors

Molecular and Cellular Biology ◽

10.1128/mcb.00479-16 ◽

2017 ◽

Vol 37 (10) ◽

Cited By ~ 40

Author(s):

Raffaella Iurlaro ◽

Franziska Püschel ◽

Clara Lucía León-Annicchiarico ◽

Hazel O'Connor ◽

Seamus J. Martin ◽

...

Keyword(s):

Cell Death ◽

Stress Response ◽

Er Stress ◽

Death Receptor ◽

Metabolic Stress ◽

Glucose Deprivation ◽

Trail Receptor ◽

Normal Tissues ◽

Trail Receptors ◽

The Unfolded Protein Response

ABSTRACT Metabolic stress occurs frequently in tumors and in normal tissues undergoing transient ischemia. Nutrient deprivation triggers, among many potential cell death-inducing pathways, an endoplasmic reticulum (ER) stress response with the induction of the integrated stress response transcription factor ATF4. However, how this results in cell death remains unknown. Here we show that glucose deprivation triggered ER stress and induced the unfolded protein response transcription factors ATF4 and CHOP. This was associated with the nontranscriptional accumulation of TRAIL receptor 1 (TRAIL-R1) (DR4) and with the ATF4-mediated, CHOP-independent induction of TRAIL-R2 (DR5), suggesting that cell death in this context may involve death receptor signaling. Consistent with this, the ablation of TRAIL-R1, TRAIL-R2, FADD, Bid, and caspase-8 attenuated cell death, although the downregulation of TRAIL did not, suggesting ligand-independent activation of TRAIL receptors. These data indicate that stress triggered by glucose deprivation promotes the ATF4-dependent upregulation of TRAIL-R2/DR5 and TRAIL receptor-mediated cell death.

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Chemotherapy and TRAIL-mediated colon cancer cell death: the roles of p53, TRAIL receptors, and c-FLIP

Molecular Cancer Therapeutics ◽

10.1158/1535-7163.mct-05-0262 ◽

2005 ◽

Vol 4 (12) ◽

pp. 2026-2036 ◽

Cited By ~ 108

Author(s):

Leeona Galligan ◽

Daniel B. Longley ◽

Miranda McEwan ◽

Timothy R. Wilson ◽

Kirsty McLaughlin ◽

...

Keyword(s):

Colon Cancer ◽

Cell Death ◽

Cancer Cell ◽

Colon Cancer Cell ◽

Cancer Cell Death ◽

Trail Receptors

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NADPH Oxidase Regulates Cytarabine-Induced Apoptotic Death in Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood.v118.21.4258.4258 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4258-4258

Author(s):

Nazmul H Khan ◽

Kevin J Sexton ◽

Melissa J Grimm ◽

Brahm H Segal ◽

Carlos E Vigil

Keyword(s):

Cell Death ◽

Nadph Oxidase ◽

Myeloid Leukemia ◽

Apoptotic Cell ◽

Leukemia Cell ◽

Apoptotic Cell Death ◽

Leukemia Cell Line ◽

Leukemia Cells ◽

Myelomonocytic Leukemia

Abstract Abstract 4258 Background: Cellular metabolism and oxidative stress are important in the biology and pathophysiology of malignancies. Both increased reactive oxygen species [ROS] levels and induction of anti-oxidative pathways have been described in several malignancies, and may be modulate tumor biology and susceptibility to chemotherapy. Limited studies point to metabolic pathways, including ROS production, influencing pathogenesis and chemo-sensitivity of leukemia. NADPH oxidase is a critical enzyme in antimicrobial host defense and its activation results in ROS generation in myeloid leukemia cells. Our prior studies show that NADPH oxidase can activate Nrf2, a transcriptional factor that induces anti-oxidant and cytoprotective pathways. However the role of NADPH oxidase in chemotherapy-mediated apoptosis induction in leukemic cells is not well-known. Hypothesis: NADPH oxidase-derived ROS will increase sensitivity of AML cells to chemotherapy, whereas Nrf2 will be associated with chemotherapy resistance Methods: We evaluated the role of NADPH oxidase and Nrf2 in regulating cytarabine-induced cell death in wild-type [WT] and engineered PLB-985 cells, a human acute myelomonocytic leukemia cell line derivative. NADPH oxidase-deficient PLB-985 cells were generated by recombination with mutant gp91phox, a necessary component of NADPH oxidase. Nrf2-deficient cells were generated by shRNA (Nrf2shRNA) and depletion (>70%) of Nrf2 mRNA was confirmed by quantitative-PCR. WT and engineered PLB-985 cells were treated with cytarabine (12.5 to 750ng/ml for 24 – 48 hours) and cell death was determined by trypan blue exclusion and Annexin V/7-AAD staining. Results: NADPH oxidase-deficient PLB-985 cells were significantly more resistant to cytarabine compared to WT cells. Cytarabine (500 ng/ml for 48h) induced apoptotic cell death in 25% of NADPH oxidase-deficient vs. 53% of WT PLB-985 cells. Additional dose-response studies confirmed a significant effect of NADPH oxidase in potentiating cytarabine-induced cell death. Nrf2shRNA PLB-985 cells had either similar or modestly increased susceptibility to cytarabine-induced cell death compared to WT PLB-985 cells with empty vectors. NADPH-deficient/Nrf2shRNA PLB-985 cells had similar susceptibility to cytarabine as NADPH-deficient cells with empty vector. Conclusions: Our results show that NADPH oxidase potentiates apoptotic cell death by cytarabine in a myelomonocytic leukemia cell line. However, we did not observe a consistent effect of Nrf2 depletion on apoptotic cell death by cytarabine. These studies suggest that modulation of redox-stress may be a potential therapeutic approach in AML that merits further study. Disclosures: No relevant conflicts of interest to declare.

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Thioridazine Enhances TRAIL Cytotoxicity in Human Myeloid Leukemic Cells By up-Regulating DR5 and Modulating PI3K-AKT-NF-Kb Pathway

Blood ◽

10.1182/blood.v126.23.4938.4938 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4938-4938

Author(s):

Yi Wang ◽

Yangyi Bao ◽

Leiming Xia ◽

Liu Liu ◽

Kunyuan Guo ◽

...

Keyword(s):

Cancer Research ◽

Tumor Necrosis Factor ◽

Tumor Necrosis ◽

Leukemic Cells ◽

Leukemia Cells ◽

Down Regulation ◽

Trail Receptors ◽

Trail Resistance ◽

Induced Apoptosis ◽

Necrosis Factor

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in cancer cells but not in most normal cells, and is identified to be effective in various cancers, include myeloid leukemic cells[1]. Although some leukemia cell lines, K562 and KG-1, are sensitive to TRAIL, many showed certain degrees of resistance to TRAIL-mediated apoptosis[2,3], and the mechanism remains largely unknown, which forced us to find out ways to solve the problem. In this study, we investigated whether thioridazine, a phenothiazine derivative, could overcome the TRAIL resistance in K562 and KG-1 cells. Recently, we showed that Compared to treatment with thioridazine or TRAIL alone, co-treatment with thioridazine and TRAIL-induced apoptosis in K562 and KG-1 cells synergistically. This combination led to activation of caspase-8 and Bid, the cytosolic cumulation of cytochrome c from mitochondria as well as caspase-3 activated downstream. Treatment with thioridazine induced down-regulation of PI3K-AKT-NF-κB pathway. meanwhile, thioridazine dropped the level of NF-κB-dependent Bcl-xL, leading caspase activated and Bid cleaved. the expression of TRAIL-receptors in both K562 and KG-1 cells underwentthe treatment of thioridazine investigated that thioridazine significantly up-regulated DR5 by up to 51.22%, but not other TRAIL-receptors such as DR4, decoy receptor 1, and DcR2. Therefore, our results indicate that the combination of TRAIL with thioridazine overturn TRAIL resistance through Up-regulating the expression of DR5 and down-regulation of AKT protein, and combination treatment with thioridazine and TRAIL may be a novel therapeutic strategy in leukemia. Reference: Srivastava R K. TRAIL/Apo-2L: mechanisms and clinical applications in cancer.[J]. Neoplasia, 2001, 3(6):535-546. Nimmanapalli R, Porosnicu M, Nguyen D, et al. Cotreatment with STI-571 enhances tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL or apo-2L)-induced apoptosis of Bcr-Abl-positive human acute leukemia cells.[J]. Clinical Cancer Research An Official Journal of the American Association for Cancer Research, 2001, 7(2):350-357. Yang T, Lan J, Huang Q, et al. Embelin sensitizes acute myeloid leukemia cells to TRAIL through XIAP inhibition and NF-κB inactivation.[J]. Cell Biochemistry & Biophysics, 2015, 71(1):291-297. Disclosures No relevant conflicts of interest to declare.

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HGS-ETRI, a Fully Human TRAIL-Receptor I Monoclonal Antibody, Induces Cell Death in Multiple Tumour Types In Vitro and In Vivo

Yearbook of Oncology ◽

10.1016/s1040-1741(08)70261-6 ◽

2006 ◽

Vol 2006 ◽

pp. 376-377

Author(s):

M.S. Gordon

Keyword(s):

Monoclonal Antibody ◽

Cell Death ◽

Trail Receptor ◽

Multiple Tumour

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Biomarkers In Cell Death of Imatinib-Resistant Ph-Leukemia Cells During Treatment with mTOR Inhibitor, Everolimus

Blood ◽

10.1182/blood.v116.21.3988.3988 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3988-3988

Author(s):

Miho Minami ◽

Yosuke Minami ◽

Yachiyo Kuwatsuka ◽

Hitoshi Kiyoi ◽

Tomoki Naoe

Keyword(s):

Cell Death ◽

Anticancer Agents ◽

Research Funding ◽

Mtor Inhibitor ◽

Leukemia Cell ◽

Tumor Formation ◽

Leukemia Cell Line ◽

Leukemia Cells ◽

T315i Mutation

Abstract Abstract 3988 Aberrant activation of mammalian target of rapamycin (mTOR) signaling pathway has been reported in hematological malignancies including leukemia initiating cells. Although rapamycin and its analogs have proven effective as anticancer agents, the mechanism of action and the solid biomarkers of response have not been fully elucidated. We investigated detailed biomarkers during the cell death of imatinib (IM)-resistant Ph-positive (Ph+) leukemia cells due to quiescence or mutations at the ABL-kinase domain after treatment with mTOR inhibitor, everolimus (Eve, RAD001). Ph+ leukemic NOD/SCID/IL2rγnull (NOG) mice cells were long co-cultured with S17 stromal cells and treated with IM and Eve. While slow-cycling (Hoechst 33342low/Pyronin Ylow) CD34+ cells were insensitive to IM in spite of BCR-ABL-dephosphorylation, combination treatment with IM and Eve induced substantial cell death including the CD34+ population. In Baf3/p210T315I cells, IM-resistant Ph+ leukemia cell line harboring T315I-mutation, Eve also induced cell death with low IC50 values in PI-exclusion assays. In murine model cutaneously injected with Baf3/p210T315I cells, in vivo-treatment with Eve decreased tumor formation. In these systems during treatment with Eve, we did not observe evident dephosphorylations of BCR-ABL, mTOR itself and 4EBP1, but rapid S6K-dephosphorylation with lower doses and decreased expression of MCL-1. Furthermore, the feedback-loop effects such as reversely increased phosphorylations of AKT (Ser473) and FOXO1/3a were also detected during the cell death. We are now investigating more efficient strategies using inhibitors screening kit and also planning to examine new generation of mTOR inhibitors to overcome the IM-resistance due to quiescence or T315I-mutation. Disclosures: Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Janssen: Research Funding.

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