Heme Oxygenase-1 (HO-1): A Novel KIT D816V-Dependent Target in Neoplastic Human Mast Cells (HMC-1).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3521-3521 ◽  
Author(s):  
Rudin Kondo ◽  
Matthias Mayerhofer ◽  
Karoline Gleixner ◽  
Anja Vales ◽  
Maria-Theresa Krauth ◽  
...  
Keyword(s):  
Mast Cells ◽  
Heme Oxygenase ◽  
Kinase Inhibitor ◽  
Northern Blotting ◽  
Heme Oxygenase 1 ◽  
Zinc Protoporphyrin ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Survival Factor ◽  
Kit D816v

Abstract Systemic mastoyctosis is a myeloid neoplasm characterized by abnormal growth and accumulation of mast cells (MC). Most patients express the D816V-mutated variant of c-KIT, which mediates resistance against most available tyrosine kinase inhibitors. Therefore, current research is focusing on novel targets in MC. We analyzed expression and function of the survival factor heme oxygenase-1 (HO-1) in neoplastic MC. As assessed by Northern blotting and RT-PCR, the human MC line HMC-1 that exhibits KIT D816V, was found to express HO-1 mRNA. Expression of the HO-1 protein was demonstrable by immunocytochemistry and Western blotting. To examine the role of mutated KIT in expression of HO-1, Ba/F3 cells with doxycycline-inducible expression of c-KIT D816V were employed. In these experiments, KIT D816V was found to induce HO-1 promoter activity and expression of HO-1 mRNA and HO-1 protein in these cells. Moreover, the KIT-D816V-targeting compound PKC412 was found to downregulate expression of HO-1 in HMC-1 cells. The KIT D816V-induced upregulation of HO-1 in Ba/F3 cells was completely blocked by a combination of LY294002 (PI3-kinase inhibitor) and PD89059 (MEK inhibitor), but not by single agents, suggesting involvement of multiple signaling pathways. We next examined whether targeting of HO-1 is associated with decreased survival. As assessed by 3H-thymidine uptake, the pegylated HO-1 inhibitor zinc-protoporphyrin (PEG-ZnPP) reduced proliferation of HMC-1 cells in a dose-dependent manner (IC50: 5 μM). The PEG-ZnPP-induced inhibition of growth was found to be associated with induction of apoptosis (control: 1±0.6 vs PEG-ZnPP, 5 μM: 55±5% apoptotic cells, p<0.05). The HO-1 inductor hemin (10 μM) increased the expression of HO-1 in HMC-1 cells and partly rescued these cells from PKC412-induced growth-inhibition (PKC412, 1 μM: 39±8% vs PKC412 + hemin: 23±11%). Finally, PKC412 and PEG-ZnPP were found to produce cooperative (additive) growth-inhibitory effects on HMC-1 cells. In summary, our data show that HO-1 is a novel survival factor and interesting target in neoplastic human MC exhibiting the D816V-mutated variant of KIT.

Identification of heat shock protein 32 (Hsp32) as a novel survival factor and therapeutic target in neoplastic mast cells

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 661-669 ◽  
Author(s):  
Rudin Kondo ◽  
Karoline V. Gleixner ◽  
Matthias Mayerhofer ◽  
Anja Vales ◽  
Alexander Gruze ◽  
...  
Keyword(s):  
Mast Cells ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Therapeutic Target ◽  
Systemic Mastocytosis ◽  
Heme Oxygenase 1 ◽  
Zinc Protoporphyrin ◽  
Survival Factor ◽  
Myeloid Neoplasm ◽  
Kit D816v

AbstractSystemic mastocytosis (SM) is a myeloid neoplasm characterized by increased survival and accumulation of neoplastic mast cells (MCs). In most patients, the D816V-mutated variant of KIT is detectable. We report here that heat shock protein 32 (Hsp32), also known as heme oxygenase-1 (HO-1), is a novel KIT-inducible survival factor in neoplastic MCs. As assessed by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and Western blotting, the KIT D816V+ MC line HMC-1.2 as well as highly enriched primary neoplastic MCs were found to express Hsp32 mRNA and the Hsp32 protein. Moreover, KIT D816V and stem cell factor (SCF)–activated wild-type KIT were found to induce Hsp32 promoter activity, expression of Hsp32 mRNA, and expression of the Hsp32 protein in Ba/F3 cells. Correspondingly, the KIT D816V-targeting drug PKC412 decreased the expression of Hsp32 as well as proliferation/survival in neoplastic MCs. The inhibitory effects of PKC412 on the survival of HMC-1.2 cells were counteracted by the HO-1 inductor hemin or lentiviral-transduced HO-1. Moreover, 2 Hsp32-targeting drugs, pegylated zinc protoporphyrin (PEG-ZnPP) and styrene maleic acid copolymer micelle-encapsulated ZnPP (SMA-ZnPP), were found to inhibit proliferation and to induce apoptosis in neoplastic MCs. Furthermore, both drugs were found to cooperate with PKC412 in producing growth inhibition. Together, these data show that Hsp32 is an important survival factor and interesting new therapeutic target in neoplastic MCs.

The Heme Oxygenase-1-Targeting Compound PEG-ZnPP Inhibits Growth of Imatinib-Resistant BCR/ABL-Transformed Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1986-1986 ◽  
Author(s):  
Matthias Mayerhofer ◽  
Karl J. Aichberger ◽  
Stefan Florian ◽  
Maria-Theresa Krauth ◽  
Sophia Derdak ◽  
...  
Keyword(s):  
Heme Oxygenase ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Pi3 Kinase ◽  
Leukemic Cells ◽  
Heme Oxygenase 1 ◽  
Zinc Protoporphyrin ◽  
Inhibitory Effects ◽  
Growth Inhibitory ◽  
Imatinib Resistant

Abstract Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the BCR/ABL oncogene and an increased survival of leukemic cells. The BCR-ABL tyrosine kinase inhibitor imatinib has successfully been introduced as a treatment of CML. However, resistance after an intitial response is common in patients with advanced disease, and it is not yet clear if responses in early disease phases will be durable. Therefore, current studies focus on novel potential drug-targets in CML cells. We have recently identified heme oxygenase-1 (HO-1) as a novel BCR/ABL-dependent survival-molecule in primary CML cells. In this study, we analyzed signal transduction pathways underlying BCR/ABL-induced expression of HO-1 and evaluated the role of HO-1 as a potential new target of drug therapy. We found that the PI3-kinase inhibitor LY294002 and MEK inhibitor PD98059 downregulate expression of HO-1 in CML cells. In addition, constitutively active Ras- and Akt -mutants were found to promote expression of HO-1 in Ba/F3 cells, further supporting the involvement of the PI3-kinase/Akt as well as the MAPK pathway in regulating HO-1 expression. To establish a role for HO-1 in survival of CML cells, expression of HO-1 was silenced by siRNAs which resulted in apoptosis of K562 cells. Next, HO-1 was targeted in CML cells by pegylated zinc protoporphyrin (PEG-ZnPP), a competitive inhibitor of HO-1. Exposure to PEG-ZnPP resulted in growth inhibition and induction of apoptosis in primary CML cells as well as in the CML-derived cell lines K562 and KU812 with IC50 values ranging between 1–10 μM. The growth-inhibitory effects of PEG-ZnPP were not only observed in CML cells responsive to imatinib, but also in imatinib-resistant K562 cells and Ba/F3 cells expressing various imatinib-resistant mutants of BCR/ABL (T315I, E255K, M351T, Y253F, Q252H, H396P). Moreover, imatinib and PEG-ZnPP were found to exert synergistic growth inhibitory effects on imatinib-resistant leukemic cells. Together, these data suggest that HO-1 represents a novel drug target in cells expressing BCR/ABL, including those with resistance to imatinib.

Effects of the Mcl-1/Bcl-2 Inhibitor GX015-070 (Obatoclax®) on Growth and Viability of Canine and Human Neoplastic Mast Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 861-861
Author(s):  
Barbara Peter ◽  
Karl J. Aichberger ◽  
Karoline V. Gleixner ◽  
Veronika Ferenc ◽  
Alexander Gruze ◽  
...  
Keyword(s):  
Mast Cells ◽  
Cell Line ◽  
Systemic Mastocytosis ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Growth And Survival ◽  
Kit D816v

Abstract Mcl-1 is a Bcl-2 family-member that has been described to act anti-apoptotic in various myeloid neoplasms. We and others have recently shown that neoplastic mast cells (MC) in patients with systemic mastocytosis (SM) display Mcl-1, Bcl-2, and Bcl-xL. In the present study, we examined the effects of the Mcl-1/Bcl-2-targeting drug GX015-070 (obatoclax®; GeminX, Montréal, Quebéc, Canada) on growth and viability of primary neoplastic MC obtained from patients with SM (n=3), the human MC leukemia cell line HMC-1, and the canine mastocytoma cell line C2. Two HMC-1 subclones, one lacking KIT D816V (HMC- 1.1) and one expressing KIT D816V (HMC-1.2) were examined. As assessed by RT-PCR and immunostaining, primary neoplastic MC as well as HMC-1 cells (both subclones) were found to express Mcl-1 mRNA and the Mcl-1 protein in a constitutive manner, but did not express significant amounts of proapoptotic Bim. Transfection of HMC-1 cells with Mcl-1-specific siRNA resulted in reduced proliferation and increased apoptosis compared to cells transfected with a control siRNA. GX015-070 was found to inhibit 3H-thymidine uptake and thus proliferation in HMC-1 cells in a dose-dependent manner, with higher IC50 values obtained in HMC-1.2 cells (0.5 μM) compared to HMC-1.1 cells (0.05 μM). GX015-070 also inhibited the growth and survival in the canine mastocytoma cell line C2 (IC50: 0.5-1 μM). Moreover, GX015-070 was found to inhibit the proliferation of primary human neoplastic MC in all SM patients tested (IC50: 0.05-0.1 μM). We next attempted to combine obatoclax with a modulator of Mcl-1/Bim expression in MC, in order to enhance drug effects. Since Bim is degraded via the proteasome, we applied the proteasome inhibitor bortezomib. Whereas GX015-07 did not modulate the production/expression of Mcl-1 or Bim in HMC-1 cells, bortezomib was found to promote the expression of Bim in our Western blot experiments. In addition, bortezomib was found to suppress 3H-thymidine uptake in both HMC-1 subclones. Finally, bortezomib was found to cooperate with GX015-070 in producing apoptosis in HMC-1.1 cells, HMC-1.2 cells, and C2 cells. Together, our data show that the Mcl-1/Bcl-2-targeting drug GX015-070 is a potent inhibitor of in vitro growth and survival of canine and human neoplastic MC. Targeting of Mcl-1 in neoplastic MC alone or in combination with a Bim-regulator may be an interesting pharmacologic approach in advanced SM.

The Plk-1 Inhibitor BI 2536 Counteracts the Growth of Neoplastic Mast Cells and Synergizes with the KIT D816V-Targeting Drug Midostaurin (PKC412) in Producing Growth-Inhibition.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3554-3554
Author(s):  
Veronika Ferenc ◽  
Karoline V. Gleixner ◽  
Alexander Gruze ◽  
Michael Kneidinger ◽  
Christian Baumgartner ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mast Cells ◽  
Growth Inhibition ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Ic50 Values ◽  
Bi 2536 ◽  
Kit D816v

Abstract Systemic mastocytosis (SM) is a myeloid neoplasm characterized by abnormal growth and accumulation of mast cells (MC) in various internal organs. In most patients, the D816V-mutated variant of c-KIT, which mediates resistance against several tyrosine kinase (TK) inhibitors like imatinib, is found. In advanced SM, the response of neoplastic MC to conventional drugs is poor and the prognosis is grave. Therefore current research is attempting to identify novel targets in neoplastic MC. Polo-like kinase 1 (Plk-1) is a serine/threonine kinase that plays an essential role in mitosis and has recently been introduced as a new target in myeloid leukemias. In the present study, we analyzed expression and function of Plk-1 in neoplastic human MC, and asked whether Plk-1 can serve as a target of therapy in SM. As determined by immunohistochemistry, primary neoplastic MC were found to display activated/phosphorylated Plk-1 in all patients examined (n=5). The human MC leukemia cell line HMC-1 was also found to exhibit activated Plk-1. In addition, we found that primary neoplastic MC as well as HMC-1 cells express Plk-1 mRNA in RT-PCR experiments. As assessed by 3H-thymidine-uptake experiments, the Plk-1-targeting drug BI 2536 (Boehringer Ingelheim GmbH, Germany) was found to inhibit the proliferation of HMC-1 cells in a dose-dependent manner (IC50 5–15 nM). The effect of BI 2536 was seen in both subclones of HMC-1, i.e. in HMC-1.1 cells displaying KIT G560V (but not KIT D816V), and HMC-1.2 cells exhibiting both KIT G560V and KIT D816V, with comparable IC50 values. Moreover, BI 2536 was found to inhibit the proliferation of primary neoplastic cells, with IC50 values ranging between 5 and 50 nM. The growth-inhibitory effects of BI 2536 on HMC-1 cells were found to be associated with mitotic arrest and G2-M cell cycle arrest as well as consecutive apoptosis. In normal bone marrow or peripheral blood mononuclear cells, neither mitotic cell arrest nor apoptosis were observed after treatment with BI 2536. In a consecutive phase of the study, we asked whether combined targeting of KIT D816V and Plk-1 would lead to synergistic drug-interactions. For this purpose, HMC-1 cells and primary neoplastic MC were coincubated with BI 2536 and midostaurin (PKC412), a multitargeted kinase inhibitor that blocks KIT D816V TK activity. In these experiments, BI 2536 was found to synergize with midostaurin in counteracting the proliferation of HMC-1 cells and primary neoplastic MC. In conclusion, our data show that activated Plk-1 is detectable in MC neoplasms and plays a role in cell cycle progression and viability of neoplastic MC. Targeting of Plk-1 with BI 2536 leads to growth inhibition and apoptosis in neoplastic MC. Furthermore, BI 2536 synergizes with midostaurin in counteracting growth of neoplastic MC. Targeting of Plk-1 may be an attractive new pharmacologic concept in advanced SM.

The Hsp32/HO-1-targeted drug SMA-ZnPP counteracts the proliferation and viability of neoplastic cells in solid tumors and hematologic neoplasms

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 14122-14122 ◽  
Author(s):  
K. V. Gleixner ◽  
M. Mayerhofer ◽  
A. Vales ◽  
A. Gruze ◽  
W. F. Pickl ◽  
...  
Keyword(s):  
Cell Growth ◽  
Solid Tumors ◽  
Cell Lines ◽  
Malignant Cell ◽  
Northern Blotting ◽  
Zinc Protoporphyrin ◽  
Thymidine Uptake ◽  
Neoplastic Cells ◽  
Induction Of Apoptosis ◽  
Kit D816v

14122 Background: Heat shock protein 32 (Hsp32) is a stress-related survival factor that is overexpressed in various neoplastic cells. Recently, specific Hsp32- targeting drugs such as styrene maleic acid encapsulated zinc protoporphyrin (SMA-ZnPP) have been developed. Methods: We examined the effects of SMA-ZnPP on proliferation and survival of various tumor cell-lines, including U97MG (glioblastoma), A549 (lung cancer), MDA-MB-231 (breast cancer), BxPC-3 (pancreatic), HepG2 (hepatocellular), Colo201, Colo320DM, DLD-1 (colon), OvCar3 (ovarian carcinoma), KG1, U937, HL60, K562 (myeloid leukemias), RAJI, NALM-6 (lymphatic leukemias), RPMI 8226, U266 (multiple myeloma) as well as on primary neoplastic cells. Moreover, Ba/F3 cells with doxycycline-inducible expression of oncoproteins (RAS-G12V, BCR/ABL, KIT-D816V) were analyzed. Expression of Hsp32 mRNA was examined by RT-PCR and Northern blotting, and expression of the Hsp32 protein by Western blotting. To silence Hsp32 in neoplastic cells, we used specific siRNA as well as SMA-ZnPP. Proliferation was analyzed by 3H-thymidine uptake and apoptosis by light microscopy. Results: All neoplastic cells tested were found to express Hsp32 mRNA and the Hsp32 protein in a constitutive manner. In Ba/F3 cells, induction of RAS-G12V, BCR/ABL, or KIT D816V enhanced the expression of Hsp32. The Hsp32 siRNA was found to lead to a reduced viability and induction of apoptosis. Treatment of malignant cells with SMA-ZnPP resulted in a significant decrease in proliferation and induction of apoptosis. The effects of SMA- ZnPP on primary neoplastic cells and cell lines were dose-dependent and occurred at pharmacologic concentrations (IC50 1–30 μM). Moreover, SMA-ZnPP was found to synergize with various anti-neoplastic drugs (cisplatin, cytarabine, tyrosine kinase inhibitors, bortezomib) in producing growth-inhibition in neoplastic cells. Conclusions: The Hsp32-targeting drug SMA-ZnPP counteracts malignant cell growth and sensitizes neoplastic cells against various other targeted or conventional antineoplastic drugs. Hsp32-targeting drugs may represent an interesting new aproach to inhibit malignant cell growth in solid tumors and leukemias. No significant financial relationships to disclose.

scholarly journals 15-Deoxy-Δ12,14-prostaglandin J2Induces Heme Oxygenase-1 Gene Expression in a Reactive Oxygen Species-dependent Manner in Human Lymphocytes

2004 ◽  
Vol 279 (21) ◽  
pp. 21929-21937 ◽  
Author(s):  
Moisés Álvarez-Maqueda ◽  
Rajaa El Bekay ◽  
Gonzalo Alba ◽  
Javier Monteseirín ◽  
Pedro Chacón ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reactive Oxygen Species ◽  
Heme Oxygenase ◽  
Human Lymphocytes ◽  
Reactive Oxygen ◽  
Heme Oxygenase 1 ◽  
Dependent Manner ◽  
Oxygen Species

scholarly journals 6-Shogaol protects against ischemic acute kidney injury by modulating NF-κB and heme oxygenase-1 pathways

2019 ◽  
Vol 317 (3) ◽  
pp. F743-F756 ◽  
Author(s):  
Sang Jun Han ◽  
Mihwa Kim ◽  
Vivette D. D’Agati ◽  
H. Thomas Lee
Keyword(s):  
Acute Kidney Injury ◽  
Proximal Tubule ◽  
Heme Oxygenase ◽  
Kidney Injury ◽  
Heme Oxygenase 1 ◽  
Zinc Protoporphyrin ◽  
Mrna Synthesis ◽  
Proximal Tubule Cells ◽  
Tnf Α ◽  
Tubule Cells

Acute kidney injury (AKI) due to renal ischemia-reperfusion (I/R) is a major clinical problem without effective therapy. Ginger is one of the most widely consumed spices in the world, and 6-shogaol, a major ginger metabolite, has anti-inflammatory effects in neuronal and epithelial cells. Here, we demonstrate our novel findings that 6-shogaol treatment protected against renal I/R injury with decreased plasma creatinine, blood urea nitrogen, and kidney neutrophil gelatinase-associated lipocalin mRNA synthesis compared with vehicle-treated mice subjected to renal I/R. Additionally, 6-shogaol treatment reduced kidney inflammation (decreased proinflammatory cytokine and chemokine synthesis as well as neutrophil infiltration) and apoptosis (decreased TUNEL-positive renal tubular cells) compared with vehicle-treated mice subjected to renal I/R. In cultured human and mouse kidney proximal tubule cells, 6-shogaol significantly attenuated TNF-α-induced inflammatory cytokine and chemokine mRNA synthesis. Mechanistically, 6-shogaol significantly attenuated TNF-α-induced NF-κB activation in human renal proximal tubule cells by reducing IKKαβ/IκBα phosphorylation. Furthermore, 6-shogaol induced a cytoprotective chaperone heme oxygenase (HO)-1 via p38 MAPK activation in vitro and in vivo. Consistent with these findings, pretreatment with the HO-1 inhibitor zinc protoporphyrin IX completely prevented 6-shogaol-mediated protection against ischemic AKI in mice. Taken together, our study showed that 6-shogaol protects against ischemic AKI by attenuating NF-κB activation and inducing HO-1 expression. 6-Shogaol may provide a potential therapy for ischemic AKI during the perioperative period.

Intestinal preconditioning prevents inflammatory response by modulating heme oxygenase-1 expression in endotoxic shock model

2007 ◽  
Vol 293 (6) ◽  
pp. G1308-G1314 ◽  
Author(s):  
Fabienne Tamion ◽  
Vincent Richard ◽  
Sylvanie Renet ◽  
Christian Thuillez
Keyword(s):  
Inflammatory Response ◽  
Multiple Organ Failure ◽  
Organ Failure ◽  
Heme Oxygenase ◽  
Ischemia Reperfusion ◽  
Multiple Organ ◽  
Intestinal Injury ◽  
Heme Oxygenase 1 ◽  
Zinc Protoporphyrin ◽  
Lps Challenge

Gut mucosal injury observed during ischemia-reperfusion is believed to trigger a systemic inflammatory response leading to multiple organ failure. It should be interesting to demonstrate this relationship between gut and multiple organ failure in a sepsis model. Intestinal preconditioning (PC) can be used as a tool to assess the effect of intestinal ischemia in inflammatory response after LPS challenge. The aim of this study was to investigate the protective effect of PC against LPS-induced systemic inflammatory and intestinal heme oxygenase-1 (HO-1) expression. ES was performed with LPS (10 mg/kg iv) with or without PC, which was done before LPS. Rats were first subjected to sham surgery or PC with four cycles of 1 min ischemia and 4 min of reperfusion 24 h before LPS challenge or saline administration. PC significantly reduced fluid requirements, lung edema, intestinal lactate production, and intestinal injury. Inflammatory mRNA expressions for intestine and lung ICAM and TNF were significantly reduced after PC, and these effects were significantly abolished by zinc-protoporphyrin (a specific HO-1 activity inhibitor) and mimicked by bilirubin administration. Intestinal PC selectively increased HO-1 mRNA expression in intestine, but we have observed no expression in lungs. These findings demonstrate that intestinal injury is a important event for inflammatory response and multiple organ injury after LPS challenge. Intestinal HO-1 expression attenuates LPS-induced multiple organ failure by modulating intestine injury and its consequences on inflammatory response. Identification of the exact mechanisms responsible for intestine HO-1 induction may lead to the development of new pharmacological interventions.

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