scholarly journals Biochemical and Cellular Characterization of New Radio-Resistant Cell Lines Reveals a Role of Natural Flavonoids to Bypass Senescence

2021 ◽  
Vol 23 (1) ◽  
pp. 301
Author(s):  
Maria Russo ◽  
Carmela Spagnuolo ◽  
Stefania Moccia ◽  
Idolo Tedesco ◽  
Fabio Lauria ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Adult Population ◽  
Bone Neoplasms ◽  
Resistant Cell ◽  
Clinical Settings ◽  
Γ Irradiation ◽  
New Radio

Cancer is one of the main causes of death worldwide, and, among the most frequent cancer types, osteosarcoma accounts for 56% of bone neoplasms observed in children and colorectal cancer for 10.2% of tumors diagnosed in the adult population. A common and frequent hurdle in cancer treatment is the emergence of resistance to chemo- and radiotherapy whose biological causes are largely unknown. In the present work, human osteosarcoma (SAOS) and colorectal adenocarcinoma (HT29) cell lines were γ-irradiated at doses mimicking the sub-lethal irradiation in clinical settings to obtain two radio-resistant cellular sub-populations named SAOS400 and HT500, respectively. Since “therapy-induced senescence” (TIS) is often associated with tumor response to radiotherapy in cancer cells, we measured specific cellular and biochemical markers of senescence in SAOS400 and HT500 cells. In detail, both cell lines were characterized by a higher level of expression of cyclin-dependent kinase inhibitors p16INK4 and p21CIP1 and increased positivity to SAβ-gal (senescence-associated β-galactosidase) with respect to parental cells. Moreover, the intracellular levels of reactive oxygen species in the resistant cells were significantly lower compared to the parental counterparts. Subsequently, we demonstrated that senolytic agents were able to sensitize SAOS400 and HT500 to cell death induced by γ-irradiation. Employing two natural flavonoids, fisetin and quercetin, and a BH3-mimetic, ABT-263/navitoclax, we observed that their association with γ-irradiation significantly reduced the expression of p16INK4, p21CIP1 and synergistically (combination index < 1) increased cell death compared to radiation mono-alone treatments. The present results reinforce the potential role of senolytics as adjuvant agents in cancer therapy.

Displacement of Bim From Anti-Apoptotic Proteins Is the Primary Factor for Determining ABT-737 Activity in Multiple Myeloma Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2851-2851
Author(s):  
Alejo A Morales ◽  
Metin Kurtoglu ◽  
David Siefker ◽  
Shannon M Matulis ◽  
Delia M Gutman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Cell Types ◽  
Myeloma Cell ◽  
Resistant Cell ◽  
The Other ◽  
Multiple Myeloma Cell ◽  
Other Hand

Abstract Abstract 2851 Poster Board II-827 ABT-737 and its orally active analog ABT-263 are Bcl-2-family inhibitors that are currently in clinical trials for a variety of cancers including hematological malignancies such as multiple myeloma. Previously, we reported that the sensitivity of multiple myeloma cell lines to ABT-737 correlates with the interactions, but not the expression, of Bcl-2 proteins. Analysis of 6 multiple myeloma cell lines revealed that expression of Bcl-2 proteins did not correlate with sensitivity, however the sensitive cells (8226/S, MM.1S and KMS-11) have a substantial amount of their pro-apoptotic Bcl-2 protein, Bak, bound to Bcl-xL. On the other hand, in the insensitive cell lines (U266, KMS-11 and OPM2), Bak was found to be associated with Mcl-1, a family member that does not bind ABT-737 and thereby confers resistance to this drug. Furthermore, we also showed that release of the BH3-only protein Bim by ABT-737 from Bcl-xL and Bcl-2 also contributes to cell death in 8226/S and MM.1S. The purpose of the current study is to further investigate the role of Bim in ABT-737-induced cell death in the multiple myeloma lines. Similar to Bak, a substantial amount of Bim is bound to Bcl-xL and Bcl-2 in the ABT-737-sensitive cell lines, MM.1S and KMS-18, while in the insensitive cell lines, it is highly bound to Mcl-1. Surprisingly, in the ABT-737-sensitive 8226/S cells, Bim appears to bind to Mcl-1. However in these cells, ABT-737 treatment resulted in upregulation of Noxa, which is a BH3-only protein that binds Mcl-1 and can release Bim. Taken together these data suggest that although binding of Bim to Mcl-1 may confer resistance to ABT-737, in certain cell types this treatment could also induce Noxa expression that antagonizes Mcl-1-mediated resistance. Consistent with this hypothesis, Mcl-1 overexpression as well as knockdown of Noxa expression significantly protected 8226/S cells from ABT-737-induced cell death while they had no effect in MM.1S cells. To further demonstrate the role of Bim in ABT-737-induced cell death, ABT-resistant 8226/S, KMS-11, KMS-18 and U266 cell lines were generated. In the resistant cell lines of 8226/S and KMS-18, Bim is exclusively bound to Mcl-1, which was overexpressed as compared to the parental cells. Bak binding was not affected by acquisition of ABT-737 resistance. This result is in agreement with the findings that interaction of Bim and Mcl-1 confers resistance to ABT-737. On the other hand, in ABT-resistant U266 and KMS-11 cell lines, Bim expression was down-regulated while Mcl-1 levels were not changed. Thus, it appears that in cells where Bim is already bound to Mcl-1, further resistance is achieved by down-regulating the expression of this BH3-only protein. Overall, these results suggest that the complex interactions between Bcl-2 proteins need to be investigated in order to understand how multiple myeloma cells may respond to ABT-737 treatment. Disclosures: Boise: University of Chicago: Patents & Royalties.

scholarly journals Kinase Inhibitors of DNA-PK, ATM and ATR in Combination with Ionizing Radiation Can Increase Tumor Cell Death in HNSCC Cells While Sparing Normal Tissue Cells

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 925
Author(s):  
Eva-Maria Faulhaber ◽  
Tina Jost ◽  
Julia Symank ◽  
Julian Scheper ◽  
Felix Bürkel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Side Effects ◽  
Ionizing Radiation ◽  
Cell Lines ◽  
Normal Tissue ◽  
Kinase Inhibitors ◽  
Induced Response ◽  
Tumor Control ◽  
Tumor Cell Death

(1) Kinase inhibitors (KI) targeting components of the DNA damage repair pathway are a promising new type of drug. Combining them with ionizing radiation therapy (IR), which is commonly used for treatment of head and neck tumors, could improve tumor control, but could also increase negative side effects on surrounding normal tissue. (2) The effect of KI of the DDR (ATMi: AZD0156; ATRi: VE-822, dual DNA-PKi/mTORi: CC-115) in combination with IR on HPV-positive and HPV-negative HNSCC and healthy skin cells was analyzed. Cell death and cell cycle arrest were determined using flow cytometry. Additionally, clonogenic survival and migration were analyzed. (3) Studied HNSCC cell lines reacted differently to DDRi. An increase in cell death for all of the malignant cells could be observed when combining IR and KI. Healthy fibroblasts were not affected by simultaneous treatment. Migration was partially impaired. Influence on the cell cycle varied between the cell lines and inhibitors; (4) In conclusion, a combination of DDRi with IR could be feasible for patients with HNSCC. Side effects on healthy cells are expected to be limited to normal radiation-induced response. Formation of metastases could be decreased because cell migration is impaired partially. The treatment outcome for HPV-negative tumors tends to be improved by combined treatment.

scholarly journals Prolonged exposure to FLT3 inhibitors leads to resistance via activation of parallel signaling pathways

Blood ◽  
2006 ◽  
Vol 109 (4) ◽  
pp. 1643-1652 ◽  
Author(s):  
Obdulio Piloto ◽  
Melissa Wright ◽  
Patrick Brown ◽  
Kyu-Tae Kim ◽  
Mark Levis ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Prolonged Exposure ◽  
Cellular Transformation ◽  
Mapk Signaling ◽  
Resistant Cell ◽  
Myelogenous Leukemia ◽  
Continuous Treatment

Abstract Continuous treatment of malignancies with tyrosine kinase inhibitors (TKIs) may select for resistant clones (ie, imatinib mesylate). To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in acute myelogenous leukemia (AML), we developed resistant human cell lines through prolonged coculture with FLT3 TKIs. FLT3 TKI-resistant cell lines and primary samples still exhibit inhibition of FLT3 phosphorylation on FLT3 TKI treatment. However, FLT3 TKI-resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these signaling pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI-resistant cell lines revealed activating N-Ras mutations in 2 cell lines that were not present in the parental FLT3 TKI-sensitive cell line. Taken together, these data indicate that FLT3 TKI-resistant cells most frequently become FLT3 independent because of activation of parallel signaling pathways that provide compensatory survival/proliferation signals when FLT3 is inhibited. Anti-FLT3 mAb treatment was still cytotoxic to FLT3 TKI-resistant clones. An approach combining FLT3 TKIs with anti-FLT3 antibodies and/or inhibitors of important pathways downstream of FLT3 may reduce the chances of developing resistance.

scholarly journals Role of notch pathway and HNF1 in cell death induced by HDACs inhibitors in ovarian cancer cell lines, serous and clear cells

2010 ◽  
Vol 4 (S2) ◽  
Author(s):  
Fernanda Silva ◽  
Jacinta Serpa ◽  
Germana Domingues ◽  
Gabriela Silva ◽  
António Almeida ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Death ◽  
Cell Lines ◽  
Cancer Cell ◽  
Ovarian Cancer Cell ◽  
Notch Pathway ◽  
Cancer Cell Lines ◽  
Clear Cells ◽  
Ovarian Cancer Cell Lines

Cannabis-induced cytotoxicity in leukemic cell lines: the role of the cannabinoid receptors and the MAPK pathway

Blood ◽  
2005 ◽  
Vol 105 (3) ◽  
pp. 1214-1221 ◽  
Author(s):  
Thomas Powles ◽  
Robert te Poele ◽  
Jonathan Shamash ◽  
Tracy Chaplin ◽  
David Propper ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Death ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Cytotoxic Agents ◽  
Complex Signal ◽  
Signal Transduction Pathways ◽  
Potent Inducer ◽  
Leukemic Cell Lines

Abstract Δ9-Tetrahydrocannabinol (THC) is the active metabolite of cannabis. THC causes cell death in vitro through the activation of complex signal transduction pathways. However, the role that the cannabinoid 1 and 2 receptors (CB1-R and CB2-R) play in this process is less clear. We therefore investigated the role of the CB-Rs in mediating apoptosis in 3 leukemic cell lines and performed microarray and immunoblot analyses to establish further the mechanism of cell death. We developed a novel flow cytometric technique of measuring the expression of functional receptors and used combinations of selective CB1-R and CB2-R antagonists and agonists to determine their individual roles in this process. We have shown that THC is a potent inducer of apoptosis, even at 1 × IC50 (inhibitory concentration 50%) concentrations and as early as 6 hours after exposure to the drug. These effects were seen in leukemic cell lines (CEM, HEL-92, and HL60) as well as in peripheral blood mononuclear cells. Additionally, THC did not appear to act synergistically with cytotoxic agents such as cisplatin. One of the most intriguing findings was that THC-induced cell death was preceded by significant changes in the expression of genes involved in the mitogen-activated protein kinase (MAPK) signal transduction pathways. Both apoptosis and gene expression changes were altered independent of p53 and the CB-Rs.

Arsenic Trioxide-Induced Cell Death Occurs Partially Independent of the Pro-Apoptotic Bcl-2-Family Members Bax and Bak.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4585-4585
Author(s):  
Christian Scholz ◽  
Antje Richter ◽  
Anja Richter ◽  
Bernd Dörken ◽  
Peter T. Daniel
Keyword(s):  
Cell Death ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Family Members ◽  
Death Receptor ◽  
Hematologic Malignancies ◽  
Mitochondrial Pathway ◽  
Cell Death Pathways

Abstract Arsenic trioxide (As2O3, arsenite) efficiently kills cells from various hematologic malignancies and has successfully been employed for the treatment of acute promyelocytic leukaemia, myelodysplastic syndrome, and multiple myeloma. Investigating the mechanisms of arsenic trioxide-induced cell death, we recently demonstrated that arsenite-mediated cell demise has a partially necrotic phenotype, occurs independently of the extrinsic death receptor pathway of apoptosis, and is not hampered by the absence of functioning caspases. On the contrary, cell death proceeded entirely via an intrinsic, mitochondrial pathway and was efficiently blocked by the anti-apoptotic Bcl-2 family members Bcl-2 or Bcl-xL. Here, we address the role of the pro-apoptotic multi-domain Bcl-2 family members Bax and Bak. By employing different cell lines deficient for Bax and/or Bak, we demonstrate that Bax- or Bak-deficiency as well as the combined absence only partially blocks arsenite-induced cell death. While the detection of an additive effect of the combined Bax-/Bak-deficiency argues for a non redundant function of Bax and Bak, the persistence of a substantial percentage of arsenite-mediated cell demise in different double deficient cell lines nevertheless suggests a mode of arsenic trioxide-mediated cell death independent from these central inducers of apoptotic cell demise. The presented data add to the notion that arsenic trioxide kills tumor cells independent of the apoptotic machinery, and warrants further investigation on the efficacy of this compound in malignancies with deficiencies of the apoptotic cell death pathways.

Targeting Proteinkinase C Alters ER-Stress and b-Catenin Signaling in Multiple Myeloma: Therapeutic Implications.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 258-258
Author(s):  
Marc S. Raab ◽  
Klaus Podar ◽  
Jing Zhang ◽  
Giovanni Tonon ◽  
Johannes H. Fruehauf ◽  
...  
Keyword(s):  
Cell Death ◽  
Stress Response ◽  
Growth Inhibition ◽  
Er Stress ◽  
Cell Lines ◽  
Wnt Pathway ◽  
Dependent Manner ◽  
P53 Family ◽  
Er Stress Response

Abstract We have previously shown that the novel orally available small molecule inhibitor of PKC enzastaurin (Eli Lilly and Company) inhibits MM cell growth, survival and angiogenesis both in vitro and in vivo. To date, however, the downstream effects contributing to growth inhibition and cell death remain to be determined. Here, we performed global gene expression profiling on enzastaurin treated MM cells and identified 200 Genes to be differentially regulated with a &gt; 2-fold cut off. Strikingly, two major groups of up-regulated probe sets were associated with either of two pathways - endoplasmatic reticulum (ER)-stress response or WNT-signaling. Importantly, MM cells, producing high levels of paraprotein, are highly susceptible to perturbation of ER function and protein folding. Moreover, PKC isoforms have been reported to directly regulate the canonical WNT pathway via phosphorylation of b-catenin (CAT), leading to its ubiquination and proteasomal degradation. Specifically, we fist evaluated the role of enzastaurin in mediating ER-stress in MM cells. The transcriptional up-regulation of genes involved in ER-stress (GADD153/CHOP, GADD34, ATF3), triggered by enzastaurin at 3h, was confirmed by western blot analysis, accompanied by induction of the molecular ER chaperone BiP/grp78, phosphorylation of eIF2a consistent with PERK activation, and up-regulation of p21. These events were preceded by an early (1h) increase of intracellular calcium levels, a hallmark of ER-stress, assessed by FLUO4 staining. These data suggest an important role of ER-stress response in the early growth inhibition of MM cells caused by enzastaurin. Second, we delineated effects of enzastaurin on WNT pathway in MM and other tumor cell lines. Upon enzastaurin treatment, CAT was dephosphorylated at Ser33, 37, 41 in a dose- and time-dependent manner in all cell lines tested (10 MM, 3 colon cancer, HeLa, as well as human embryonic kidney 293 cells). Consequently, accumulation of CAT occurred in both cytosolic and nuclear fractions of treated MM cells, associated with activated TOPflash LUC-reporter system, confirming nuclear transactivating activity. Specific inhibition of CAT by siRNA partially rescued HeLa, HEK 293, and MM cells from cell death induced by enzastaurin. Analysis of downstream target molecules revealed a CAT-dependent up-regulation of c-Jun, but not of c-Myc or Cyclin D1. c-Jun has been reported to stabilize p73, a pro-apoptotic p53-family member; CAT induction by enzastaurin led to p73 (but not p53) activation and was also abrogated by CAT-specific siRNA. In turn, specific knockdown of p73 by siRNA rescued cells from enzastaurin-induced apoptosis. Finally, ectopic overexpression of CAT in HeLa and MM cells induced c-Jun expression and p73 activation, followed by apoptotic cell death. Our studies therefore indicate that ER-stress response contributes to the immediate inhibition of proliferation by enzastaurin, followed by CAT accumulation leading to p73 activation, contributing to enzastaurin-mediated cell death. These findings provide a novel link between CAT and p53-family members. Moreover p73, which is only rarely mutated in human cancers, represents a novel therapeutic target in MM.

The Role of Iron in the Cytotoxicity Induced in Hematopoietic Malignant Cell Lines by an Anti-Transferrin Receptor IgG3-Avidin Fusion Protein and Gambogic Acid.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4204-4204
Author(s):  
Elizabeth Ortiz-Sanchez ◽  
Tracy R. Daniels ◽  
Gustavo Helguera ◽  
Otoniel Martinez-Maza ◽  
Benjamin Bonavida ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Surface ◽  
Cell Lines ◽  
Transferrin Receptor ◽  
Cytotoxic Effect ◽  
Malignant Cell ◽  
Gambogic Acid ◽  
Iron Starvation ◽  
Human Transferrin Receptor

Abstract The human transferrin receptor (hTfR) is a cell surface glycoprotein responsible for iron uptake, which is required for several cellular reactions including respiration and DNA synthesis. In cancer cells, the hTfR is over-expressed due to the high amount of iron required to maintain the uncontrolled cell proliferation. This makes the cancer cells highly sensitive to death induced by iron starvation. In addition to iron uptake, the TfR may also play a role in cell growth signaling. We have developed a mouse/human chimeric anti-human transferrin receptor IgG3-avidin fusion protein (anti-hTfR IgG3-Av) that induces cell death in human malignant B-cells such as IM9 and ARH-77 (B lymphoblastoid cell lines). The TfR is constitutively internalized and recycled back to cell surface. Anti-hTfR IgG3-Av binding to the hTfR disrupts this cycling and redirects the hTfR to lysosomal compartments where hTfR is presumably degraded. This promotes a state of iron starvation that induces cancer cell death by apoptosis, which can be blocked by iron supplementation. However, the cytotoxic effect induced by anti-hTfR IgG3-Av was minor or absent in U266 (multiple myeloma cell line). Gambogic acid (GA), a natural compound from traditional Chinese medicine, was previously shown to bind the TfR, blocking its internalization and inducing apoptosis in an iron independent manner in Jurkat (acute T cell leukemia) and HL-60 (acute promyelocytic leukemia) cell lines. The goal of the present work is to explore the cytotoxic effect of anti-hTfR IgG3-Av and GA alone or in combination in a panel of hematopoietic malignant cell lines and understand the relevance of iron in the cytotoxicity. We demonstrate that GA inhibits the proliferation of Ramos, HS-sultan, Raji (Burkitt’s lymphomas), IM9, U266, Jurkat and HL-60 cell lines. Our results showed that 0.3 μM GA at 24 h decreases cell proliferation by 70–90%, and 95–99.9% at 48 h, in all cell lines tested. We also observed morphological changes in the cells treated with GA consistent with cell death. Importantly, using ferric ammonium citrate as an iron supplement, we confirmed that the cytotoxic effect of GA in the cell lines tested is iron independent. This work also demonstrates, for the first time, that anti-hTfR IgG3-Av has a potent cytotoxic effect in the panel of leukemia and lymphoma cell lines tested above. Additionally, we confirmed that anti-hTfR IgG3-Av induces cell death in an iron dependent manner. Since both anti-hTfR IgG3-Av and GA target the TfR, we tested the effect of the combination of these components. Despite a potential antagonistic effect due to the fact that anti-hTfR IgG3-Av decreases the cell surface levels of the hTfR and GA blocks its internalization, the combination of anti-hTfR IgG3-Av plus GA enhanced the cytotoxicity, including cases of synergy, of either one alone in the cell lines tested. Studies are currently underway to understand the role of iron in the enhancement of toxicity. Our results suggest that anti-hTfR IgG3-Av and GA, alone or in combination, could be an effective treatment option for various hematopoietic malignancies.

Functional Defects In Neutrophils Derived From Ezh2 Null Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1556-1556
Author(s):  
Albert Perez-Ladaga ◽  
Bennett Caughey ◽  
Huafeng Xie ◽  
Stuart H. Orkin ◽  
David B. Sykes ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Progenitor Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Resting Cells ◽  
Surface Markers ◽  
Advisory Committees ◽  
Spontaneous Cell Death

Abstract Introduction We investigate the role of Ezh2 in neutrophil function using murine progenitor cells differentiated into neutrophils lacking the Ezh2 gene. Ezh2 is the catalytic component of the polycomb repressive complex 2, which methylates lysine 27 of histone H3. It is frequently disrupted in myelodysplastic syndromes (MDS) leading to loss of function (Ernst et al., 2010). Mutations in EZH2 are found in 6% of MDS patients and while not strongly linked to cytopenias or blast proportion, they are independently associated with worse overall survival compared to patients with wildtype EZH2 (Bejar R. et al., 2011 and 2012). We hypothesize that Ezh2 mutations may cause qualitative defects in myeloid cells that impact their function and could contribute to the adverse prognosis observed in EZH2 mutant MDS. Methods Bone marrow from Ezh2 null (Ezh2-/-) and littermate control mice (WT) were transduced with HOXB8 fused to the estrogen receptor ligand-binding domain to produce immortalized myeloid progenitor cells. Removal of estrogen from the media allows these cells differentiate into mature neutrophils (Wang G.G., 2006). Differentiated cells were characterized for surface markers by flow cytometry and for gene expression by PCR of mRNA. Spontaneous cell death was measured by annexin/PI staining. Cell cycle patterns were determined by measuring the red emission of PI. Chemotactic function was assessed by counting cells that migrated across a transwell in presence/absence of the attractant zymosan. For phagocytosis experiments, cells were incubated with Fluoresbrite YG carboxylate beads at 37°C or 4°C. Reactive oxygen species (ROS) generation was measured by the oxidation of dihydrorhodamine 123 into fluorescent rhodamine 123. Results Estrogen withdrawal caused differentiation of both WT and Ezh2-/- lines into cells with mature neutrophil morphology after six days (Figure 1a). Both differentiated lines expressed the neutrophil surface markers CD11b and CD62L and the neutrophil-specific genes lactoferrin and Itgb2l. Ezh2 -/- cells had an increased rate of spontaneous cell death compared to WT in undifferentiated (32.81% vs. 20.33%) and mature cells (32.82% vs. 14.23%). Nevertheless, both progenitor cell lines showed similar cell cycle patterns, demonstrating that Ezh2 absence had no other effect on cell cycle progression. Ezh2 -/- neutrophils failed to migrate towards zymosan (Figure 1b). Expression of Tlr2, which binds zymosan, and other Toll-like receptors (Tlr4/5/9) were similar between the differentiated cell lines. Cells incubated with FITC-zymosan at 37°C showed no fluorescence differences between cell lines, indicating similar adherence. Experiments with neutrophils from an MDS patient with homozygous EZH2 mutations demonstrated a similar migration defect. Additional studies in MDS patient samples are ongoing and will be presented. Phagocytosis was reduced in Ezh2-/-cells. Unstimulated, the number of cells ingesting and adhering YG-beads was significantly greater with WT cells than with Ezh2-/-cells. When activated with fMLP, both lines showed increased adherence of YG-beads but the number of phagocytosing Ezh2-/- cells was reduced. The average number of beads ingested by each cell was lower for Ezh2-/- cells compared to WT (5.95 vs 2.94, p < 0.001) in resting cells, and 9.47 vs. 3.73 in fMLP-activated cells, p < 0.01. The fraction of Ezh2-/- neutrophils generating ROS when stimulated with PMA is 2.4-fold higher than for WT cells. ROS production was greatly reduced in the presence of diphenyleneiodonium (DPI), confirming the role of NADPH oxidase in the generation of ROS. Conclusion Our results indicate impaired function of neutrophils derived from Ezh2-/- mice, demonstrating increased spontaneous cell death, impaired migration, decreased phagocytosis, and overproduction of ROS. Qualitative defects observed in neutrophils deficient for EZH2 may help explain the adverse prognosis associated with these mutations in MDS patients. Disclosures: Bejar: Genoptix: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

Alox-5 As a Potent Therapeutic Target on Overcoming TKI-Resistance in Chronic Myeloid Leukemia with T315I Mutation in Bcr-Abl

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4835-4835 ◽  
Author(s):  
Jishi Wang ◽  
Dan Ma ◽  
Ping Wang ◽  
Weibing Wu ◽  
Lu Cao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Kinase Inhibitors ◽  
Mrna Level ◽  
Resistant Cell ◽  
Tki Resistance ◽  
T315i Mutation ◽  
Pkc Β

Abstract Background&Significance: Chronic myeloid leukemia(CML) is a malignant disease of a primitive haematological cell, characterised by inappropriate expansion of myeloid cells. Although the disease is readily controlled by Tyrosine kinase inhibitors, approximately one third of patients will eventually fail treatment. And we believed it corresponds to insensitive leukemia stem cells(LSCs) with unresponsive genes to the kinase inhibitors in CML and mutation of Bcr-Abl. 5-lipoxygenase gene(Alox5) was proved as a novel therapeutic target in cancer stem cells of CML. It encodes a member of the lipoxygenase gene family and plays a role in the synthesis of leukotrienes from arachidonic acid. Without Alox5, Bcr-Abl fails to induce CML in mice due to the impairments of the functions of LSCs. However, recent report suggest that Alox5 regulation may not be important for the development of CML in human. Interestingly, we found specific upregulation of Alox5 in CML patients with strongly positive expression of p210 in mRNA level, including the patients primary diagnosed as CML and who suffered in relapse for TKI resistance. Therefore, we characterized the function and regulation of Alox5 in TKI-resistant CML. Results: Firstly, we verified the upregulation of Alox5 by real-time PCR on sorted human CML progenitor populations with strongly positive expression of Bcr-Abl(p210), but not on CML patients obtained remission after treatment of TKI. To evaluate the function and regulation of Alox5, we silenced Alox5 by siRNA and chemical inhibitior in human CML cell lines K562, its TKI-resistant cell lines K562R, murine CML cell lines BaF3wild, and its TKI-resistant cell line BaF3T315I. As a result, the apoptotic rate induced by Alox5 inhibition alone in K562 and BaF3wild cells was lower than by TKI. Conversely, blockage of Alox5 in BaF3T315I cells caused increasingly apoptotic rate, which was higher than imatinib treatment. To further study if Alox5 could play an important role in impairing leukemia stem cells in CML, we cultured LSCs(Lin-c-Kit+Sca-1+) derived from CML patients with Bcr-Abl-T315I mutation in the presence of Alox5 inhibitor or imatinib alone. For 24h treatment, obvious apoptosis was observed in cells cultured with Zileuton, a kind of Alox5 inhibitor, but not in cells cultured with imatinib. Therefore, apoptosis related genes were detected, significant downregulation of Bcl-2 was found compared to in LSCs without Alox5 knockouted. In addition, downregulation of Alox5 followed Bcr-Abl inhibition in CML, we hypothesised that Alox5 was a downstream of Bcr-Abl, genomic array was used to uncover the signaling pathway connected Alox5 with Bcr-Abl. The results shown us that PKC-β was significantly downregulated when Bcr-Abl was inactivated by TKI. Selectivly inhibited PKC-β could decreased Alox5 expression in mRNA level. As for this mechanism, we found p38/MAPK signaling pathway mediated regulation of Alox5 by PKC-β. Next, we evaluated the in vivo anti-CML effect of Alox5 inhibition with a xenograft mice model. Two weeks after the transplantation of human CML cells with mutant Bcr-Abl-T315I(n=12). NOD/SCID/IL2Rg-KO(KOG) mice were treated with normal saline, 10mg/kg Zileuton or imatinib intraperitoneally on everyday schedule. At first, we confirmed that Zileuton doesn't affect normal hematopoiesis(n=3), but eventually inhibite LSCs growth(n=3). Eight to ten weeks after the transplantation, the frequencies of human CD45+ CML cells and LSCs were significantly reduced by Zileuton treatment in bone marrow((BM) of the recipient mice compared with normal saline-treated control mice and imatinib treated mice, indicating that Alox5 inhibition can inhibit the survival of CML-T315I mutant cells and LSCs in vivo. Conclusion: Together, these results suggest that Alox5 would be a potent therapeutic target on overcoming TKI-resistance involved in Bcr-Abl-T315I mutation. Disclosures No relevant conflicts of interest to declare.

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