ABSTRACT
Although the triterpene CDDO and its potent derivatives, CDDO-Im and CDDO-Me, are now in phase I/II studies in the treatment of some pathological conditions, their effects on normal hematopoiesis are not known. In the present study we provide evidence that CDDO-Im exerts in vitro a potent inhibitory effect on erythroid cell proliferation and survival and a stimulatory action on megakaryocytic differentiation. The effect of CDDO-Im on erythroid and megakaryocytic differentiation was evaluated both on normal hemopoietic progenitor cells (HPCs) induced to selective erythroid (E) or megakaryocytic (Mk) differentiation and on erythroleukemic cell lines HEL and TF1. The inhibitory effect of CDDO-Im on erythroid cell survival and proliferation is mainly related to a reduced GATA-1 expression. This conclusion is supported by the observation that GATA-1 overexpressing TF1 cells are partially protected from the inhibitory effect of CDDO-Im on cell proliferation and survival. The stimulatory effect of CDDO-Im on normal megakaryopoiesis is seemingly related to upmodulation of GATA2 expression and induction of mitogen-activated protein kinases ERK1/2.
Subject(s)
Cell Differentiation/drug effects , Imidazoles/pharmacology , Megakaryocytes/drug effects , Oleanolic Acid/analogs & derivatives , Antigens, CD34/metabolism , Apoptosis/drug effects , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Erythroid Cells/cytology , Erythroid Cells/drug effects , Erythroid Cells/metabolism , Flow Cytometry , GATA1 Transcription Factor/metabolism , GATA2 Transcription Factor/metabolism , Granulocytes/cytology , Granulocytes/drug effects , Granulocytes/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Humans , Integrin beta3/metabolism , Leukemia, Erythroblastic, Acute/metabolism , Leukemia, Erythroblastic, Acute/pathology , Megakaryocytes/cytology , Megakaryocytes/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Monocytes/cytology , Monocytes/drug effects , Monocytes/metabolism , Oleanolic Acid/pharmacology , Platelet Membrane Glycoprotein IIb/metabolism , Time FactorsABSTRACT
The effects of endurance or maximal exercise on mobilization of bone marrow-derived hemopoietic and angiogenetic progenitors in healthy subjects are poorly defined. In 10 healthy amateur runners, we collected venous blood before, at the end of, and the day after a marathon race (n = 9), and before and at the end of a 1.5-km field test (n = 8), and measured hemopoietic and angiogenetic progenitors by flow cytometry and culture assays, as well as plasma or serum concentrations of several cytokines/growth factors. After the marathon, CD34(+) cells were unchanged, whereas clonogenetic assays showed decreased number of colonies for both erythropoietic (BFU-E) and granulocyte-monocyte (CFU-GM) series, returning to baseline the morning post-race. Conversely, CD34(+) cells, BFU-E, and CFU-GM increased after the field test. Angiogenetic progenitors, assessed as CD34(+)KDR(+) and CD133(+)VE-cadherin(+) cells or as adherent cells in culture expressing endothelial markers, increased after both endurance and maximal exercise but showed a different pattern between protocols. Interleukin-6 increased more after the marathon than after the field test, whereas hepatocyte growth factor and stem cell factor increased similarly in both protocols. Plasma levels of angiopoietin (Ang) 1 and 2 increased after both types of exercise, whereas the Ang-1-to-Ang-2 ratio or vascular endothelial growth factor-A were little affected. These data suggest that circulating hemopoietic progenitors may be utilized in peripheral tissues during prolonged endurance exercise. Endothelial progenitor mobilization after exercise in healthy trained subjects appears modulated by the type of exercise. Exercise-induced increase in growth factors suggests a physiological trophic effect of exercise on the bone marrow.
Subject(s)
Athletes , Endothelial Cells/physiology , Erythroid Precursor Cells/physiology , Hematopoietic Stem Cells/physiology , Neovascularization, Physiologic , Physical Endurance/physiology , AC133 Antigen , Adult , Angiogenesis Inducing Agents/blood , Antigens, CD/blood , Antigens, CD34/blood , Cadherins/blood , Cytokines/blood , Glycoproteins/blood , Granulocytes/physiology , Hematopoietic Cell Growth Factors/blood , Humans , Male , Middle Aged , Peptides/blood , Running/physiologyABSTRACT
BACKGROUND: The human hemoglobin switch (HbF-->HbA) takes place in the peri/post-natal period. In adult life, however, the residual HbF (<1%) may be partially reactivated by chemical inducers and/or cytokines such as the kit ligand (KL). MicroRNAs (miRs) play a pivotal role in normal hematopoiesis: downmodulation of miR-221/222 stimulates human erythropoietic proliferation through upmodulation of the kit receptor. DESIGN AND METHODS: We have explored the possible role of kit/KL in perinatal Hb switching by evaluating: i) the expression levels of both kit and kit ligand on CD34(+) cells and in plasma isolated from pre-, mid- and full-term cord blood samples; ii) the reactivation of HbF synthesis in KL-treated unilineage erythroid cell cultures; iii) the functional role of miR-221/222 in HbF production. RESULTS: In perinatal life, kit expression showed a gradual decline directly correlated to the decrease of HbF (from 80-90% to <30%). Moreover, in full-term cord blood erythroid cultures, kit ligand induced a marked increase of HbF (up to 80%) specifically abrogated by addition of the kit inhibitor imatinib, thus reversing the Hb switch. MiR-221/222 expression exhibited rising levels during peri/post-natal development. In functional studies, overexpression of these miRs in cord blood progenitors caused a remarkable decrease in kit expression, erythroblast proliferation and HbF content, whereas their suppression induced opposite effects. CONCLUSIONS: Our studies indicate that human perinatal Hb switching is under control of the kit receptor/miR 221-222 complex. We do not exclude, however, that other mechanisms (i.e. glucocorticoids and the HbF inhibitor BCL11A) may also contribute to the peri/post-natal Hb switch.
Subject(s)
Fetal Hemoglobin/metabolism , Hemoglobin A/metabolism , MicroRNAs/physiology , Stem Cell Factor/physiology , Adult , Antigens, CD34/blood , Benzamides , Cell Cycle , Cells, Cultured , Erythroid Cells/cytology , Erythroid Cells/drug effects , Erythroid Cells/metabolism , Erythropoiesis/drug effects , Erythropoiesis/genetics , Fetal Blood/cytology , Fetal Blood/metabolism , Flow Cytometry , Gene Expression , Humans , Imatinib Mesylate , Infant, Newborn , MicroRNAs/genetics , Piperazines/pharmacology , Proto-Oncogene Proteins c-kit/blood , Proto-Oncogene Proteins c-kit/genetics , Pyrimidines/pharmacology , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Stem Cell Factor/blood , Stem Cell Factor/genetics , Time FactorsABSTRACT
Resistance of tumors to cell death signals poses a complex clinical problem. In the present study, we have explored the capacity of proteasome inhibitors to induce cell death of ovarian cancer cells. We explored the sensitivity of primary ovarian cancer cells to a combination of bortezomib (also known as PS-341), a proteasome inhibitor and TRAIL, a death ligand, or mapatumumab or lexatumumab, TRAIL-R1 or TRAIL-R2 targeting agonist monoclonal antibodies, respectively. The results of our study showed that the large majority of primary ovarian cancers are clearly sensitive to the pro-apoptotic action of bortezomib, whose effects are potentiated by the concomitant addition of TRAIL or mapatumumab or lexatumumab. Interestingly, both cisplatin and paclitaxel-chemosensitive and chemoresistant ovarian tumors are equally sensitive to the cytotoxic effect of bortezomib. Bortezomib, combined with TRAIL or TRAIL-R1 or TRAIL-R2 agonist monoclonal antibodies may be a useful treatment for refractory ovarian cancer.
Subject(s)
Apoptosis , Boronic Acids/pharmacology , Ovarian Neoplasms/drug therapy , Proteasome Inhibitors , Pyrazines/pharmacology , Adolescent , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bortezomib , Cell Line, Tumor , Drug Resistance, Neoplasm , Enzyme Inhibitors/pharmacology , Female , Humans , Middle Aged , Ovarian Neoplasms/pathology , TNF-Related Apoptosis-Inducing Ligand/metabolismABSTRACT
The pathophysiology of coronary artery disease (CAD) progression is not well understood. Endothelial progenitor cells (EPCs) may have an important role. In the present observational cohort study we assessed the number of circulating EPCs in 136 patients undergoing elective percutaneous coronary intervention and who had at least one major epicardial vessel with a nonsignificant stenosis [<50% diameter stenosis (DS)], and the relationship between plasma EPC levels and the 24-mo progression of the nonsignificant coronary artery lesion. The following cell populations were analyzed: CD34(+), CD133(+), CD34(+)/KDR(+), CD34(+)/VE cadherin(+), and endothelial cell colony-forming units (CFU-ECs). Progression was defined as a >15% DS increase of the objective vessel at follow-up. At 24 mo, 57 patients (42%) experienced significant progression. Independent predictors of disease progression were LDL cholesterol > 100 mg/dl (OR=1.03; 95% CI 1.01-1.04; P=0.001), low plasma levels of CFU-ECs (OR=3.99; 95% CI 1.54-10.37; P=0.005), and male sex (OR=3.42; 95% CI 1.15-10.22; P=0.027). Circulating levels of EPCs are significantly lower in patients with angiographic CAD progression.
Subject(s)
Coronary Artery Disease/etiology , Coronary Artery Disease/pathology , Endothelium, Vascular/cytology , Stem Cells/metabolism , Cells, Cultured , Cohort Studies , Colony-Forming Units Assay , Coronary Artery Disease/metabolism , Disease Progression , Endothelium, Vascular/metabolism , Female , Flow Cytometry , Humans , Male , Middle Aged , Prognosis , ROC Curve , Risk Factors , Survival Rate , Vascular Endothelial Growth Factor A/metabolismABSTRACT
In the present study we have explored the sensitivity of ovarian cancer cells to the synthetic triterpenoid CDDO-Imidazolide (CDDO-Im). For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/ADR and A2780/CISP, OVCAR3, SKOV3 and HEY cancer cell lines and primary ovarian cancer cells, providing evidence that: (i) the majority of these cell lines are highly sensitive to the pro-apoptotic effects induced by CDDO-Im; (ii) TRAIL, added alone exerted only a weak proapoptotic, but clearly potentiated the cytotoxic effect elicited by CDDO-Im; (iii) the apoptotic effect induced by CDDO-Im involves GSH depletion, c-FLIP downmodulation and caspase-8 activation; (iv) CDDO-Im inhibits STAT3 activation and CDDO-Im sensitivity is inversely related to the level of constitutive STAT3 activation. Importantly, studies on primary ovarian cancer cells have shown that these cells are sensitive to the pro-apoptotic effects of CDDO-Im. These observations support the experimental use of synthetic triterpenoids in the treatment of ovarian cancer.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Imidazoles/pharmacology , Oleanolic Acid/analogs & derivatives , Ovarian Neoplasms/drug therapy , Apoptosis/drug effects , Caspase 8/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm , Enzyme Activation/drug effects , Female , Glutathione/metabolism , Humans , Mitochondria/drug effects , Oleanolic Acid/pharmacology , Ovarian Neoplasms/pathology , Receptors, TNF-Related Apoptosis-Inducing Ligand/analysis , Receptors, Tumor Necrosis Factor/analysis , STAT3 Transcription Factor/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , bcl-2-Associated X Protein/metabolismABSTRACT
Previous studies suggested an important role for vascular endothelial growth factor (VEGF) and its receptors in postnatal haemopoiesis. However, it is unclear how VEGF receptor (VEGFR) signalling could interact with that issued from the activation of haematopoietic growth factor receptors. To elucidate this point we explored VEGF-R2 and granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) membrane localization and cell signalling in TF1-KDR cells (TF1 leukaemic cells that overexpress VEGF-R2/KDR). Activation of either GM-CSFR or VEGF-R2 was shown to determine the migration of both receptor elements (VEGF-R2 and the common beta-chain of the GM-CSFR) to lipid rafts. The study of receptor phosphorylation showed that GM-CSF induced the phosphorylation of its own receptor and the transphosphorylation of VEGF-R2; on the other hand, VEGF triggered the phosphorylation of its receptor and transphosphorylated the beta-chain of the GM-CSFR. Co-stimulation of TF1-KDR cells with both GM-CSF and VEGF-A resulted in massive migration of both the common GM-CSFR beta-chain and VEGF-R2 to lipid rafts and sustained p38 mitogen-activated protein kinase activation. Disruption of lipid rafts inhibited the capacity of both GM-CSF and VEGF-A to activate p38. Experiments with specific p38 inhibitors showed that p38 activation was required to sustain the VEGF- and GM-CSF-dependent proliferation of TF1-KDR and the survival of primary acute myeloid leukaemia blasts.
Subject(s)
Cytokine Receptor Common beta Subunit/metabolism , Interleukin-3/metabolism , Leukemia, Myeloid, Acute/metabolism , Membrane Microdomains/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Apoptosis , Biological Transport , Blotting, Western/methods , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival , Enzyme Activation , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Imidazoles/pharmacology , Immunophenotyping , Immunoprecipitation , Leukemia, Myeloid, Acute/pathology , Phosphorylation , Pyridines/pharmacology , Vascular Endothelial Growth Factor A/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
In a recent study we have explored TfR2 expression in a panel of cancer cell lines and we observed that about 40% of these cell lines clearly express TfR2. Taking advantage of this observation and considering the frequent overexpression of c-Myc in cancer cells we have explored the existence of a possible relationship between c-Myc and TfR2 in these cell lines. Our results provided evidence that TfR2(+) cell lines express low c-Myc levels and low TfR1 levels, while TfR2(-) cell lines express high c-Myc and TfR1 levels. Using the erythroleukemic K562 TfR2(+) cells as a model, we observed that agents that enhance c-Myc expression, such as iron, determine a decrease of TfR2 expression, while molecules that induce a decreased c-Myc expression, such as the iron chelator desferoxamine or the kinase inhibitor ST 1571, induce an enhanced TfR2 expression. On the other hand, we have evaluated a possible effect of hypoxia and nitric oxide on TfR2 expression in erythroleukemia K526 and hepatoma HepG2 cells, providing evidence that: (i) agents inducing cellular hypoxia, such as CoCl(2), elicited a marked upmodulation of TfR1, but a downmodulation of TfR2 expression; (ii) NO(+) donors, such as sodium nitroprusside (SNP), induced a moderate decrease of TfR1, associated with a marked decline of TfR2 expression; (iii) NO donors, such as S-Nitroso-N-Acetylpenicillamine (SNAP), induced a clear increase of TfR1, associated with a moderate upmodulation of TfR2 expression. The ensemble of these observations suggests that in cancer cell lines TfR2 expression can be modulated through stimuli similar to those known to act on TfR1 and these findings may have important implications for our understanding of the role of TfR2 in the regulation of iron homeostasis.
Subject(s)
Antigens, CD/biosynthesis , Proto-Oncogene Proteins c-myc/metabolism , Receptors, Transferrin/biosynthesis , Antigens, CD/drug effects , Antimutagenic Agents/pharmacology , Apoferritins/biosynthesis , Benzamides , Cell Hypoxia/drug effects , Cell Hypoxia/physiology , Cell Line, Tumor , Cobalt/pharmacology , Deferoxamine/pharmacology , Humans , Imatinib Mesylate , Iron/pharmacology , Iron Regulatory Protein 2/biosynthesis , Nitric Oxide/metabolism , Nitric Oxide Donors/pharmacology , Nitroprusside/pharmacology , Penicillamine/analogs & derivatives , Penicillamine/pharmacology , Piperazines , Proto-Oncogene Proteins c-myc/drug effects , Pyrimidines/pharmacology , Receptors, Transferrin/drug effects , Siderophores/pharmacologyABSTRACT
The common beta chain subunit (beta(c)), also known as CDw131, shared by the interleukin-3 (IL-3), granulocytic macrophage colony-stimulating factor (GM-CSF) and IL-5 receptors, is required for high-affinity ligand binding and signal transduction. The present study explored the expression of CDw131 in 105 de novo cases of acute myeloid leukaemia (AML). The levels of CDw131 expression were used to identify two AML subgroups characterized by low (75/105) and high (30/105) expression of this receptor chain. It was observed that (i) the level of CDw131 expression strictly correlated with the level of CD116 (GM-CSFalpha receptor chain) and CD123 (IL-3Ralpha chain); (ii) AMLs with high CDw131 expression were characterized by low CD34 expression and usually high CD11b, CD14 expression; (iii) AMLs with high CDw131 expression frequently co-expressed receptors for angiogenic growth factors (vascular endothelial growth factor R2, Tie-2); (iv) AMLs with high CDw131 expression were more cycling than those with low CDw131 expression; (v) AMLs with high CDw131 frequently displayed Feline Murine Sarcoma (FMS-related) tyrosine kinase 3 (FLT3) internal tandem duplication and constitutively activated Signal Transducer and Activator of Transcription-5 (STAT5). In conclusion, the analysis of the level of CDw131 expression enabled the identification of a subset of AMLs characterized by a high cycling status, the expression of myelo-monocytic markers, mutated FLT3 and the co-expression of receptors for angiogenic growth factors. These findings are of value for the development of new therapeutic strategies for the treatment of these AMLs.
Subject(s)
Cytokine Receptor Common beta Subunit/analysis , Gene Expression Regulation, Leukemic , Leukemia, Myeloid, Acute/immunology , Mutation , fms-Like Tyrosine Kinase 3/genetics , Biomarkers/analysis , Blotting, Western/methods , Cytokine Receptor Common beta Subunit/metabolism , Flow Cytometry , Humans , Immunophenotyping , Interleukin-3 Receptor alpha Subunit/analysis , Interleukin-5 Receptor alpha Subunit/metabolism , Leukocyte Count , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Translocation, Genetic , Tumor Cells, Cultured , Vascular Endothelial Growth Factor Receptor-2/analysisABSTRACT
The synthetic triterpenoid CDDO-Im-induced apoptosis of patient-derived AML blasts: 11/25 AMLs were highly sensitive, while the remaining were moderately sensitive to CDDO-Im. The addition of TRAIL significantly potentiated the cytotoxic effect of CDDO-Im, through mechanisms involving the induction of TRAIL-R1/TRAIL-R2 and downmodulation of TRAIL-R3/TRAIL-R4. Biochemical studies showed that CDDO-Im: induced a rapid and marked GSH depletion and antioxidants (GSH or NAC) completely inhibited its pro-apoptotic effect; sequentially activated caspase-8, -9 and -3; caspase inhibitors partially protected AML blasts from CDDO-Im-induced apoptosis; resistance of AML blasts to CDDO-Im-induced apoptosis correlated with low caspase-8/FADD and high Bcl-X(L) expression in leukemic blasts.
Subject(s)
Caspase 8/metabolism , Fas-Associated Death Domain Protein/metabolism , Imidazoles/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Oleanolic Acid/analogs & derivatives , Apoptosis/drug effects , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Enzyme Activation , Humans , Oleanolic Acid/pharmacology , TNF-Related Apoptosis-Inducing Ligand/analysis , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Tumor Cells, CulturedABSTRACT
The present study explored the sensitivity of leukaemic blasts derived from 30 acute myeloid leukaemia (AML) patients to Bortezomib. Bortezomib induced apoptosis of primary AML blasts: 18/30 AMLs were clearly sensitive to the proapoptotic effects of Bortezomib, while the remaining cases were moderately sensitive to this molecule. The addition of tumour necrosis factor-related-apoptosis-inducing ligand, when used alone, did not induce apoptosis of AML blasts and further potentiated the cytotoxic effects of Bortezomib. The majority of AMLs sensitive to Bortezomib showed immunophenotypic features of the M4 and M5 French-American-British classification subtypes and displayed myelomonocytic features. All AMLs with mutated FLT3 were in the Bortezomib-sensitive group. Biochemical studies showed that: (i) Bortezomib activated caspase-8 and caspase-3 and decreased cellular FLICE [Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme]-inhibitory protein (c-FLIP) levels in AML blasts; (ii) high c-FLIP levels in AML blasts were associated with low Bortezomib sensitivity. Finally, analysis of the effects of Bortezomib on leukaemic cells displaying high aldehyde dehydrogenase activity suggested that this drug induced in vitro killing of leukaemic stem cells. The findings of the present study, further support the development of Bortezomib as an anti-leukaemic drug and provide simple tools to predict the sensitivity of AML cells to this drug.
Subject(s)
Boronic Acids/therapeutic use , Leukemia, Monocytic, Acute/drug therapy , Leukemia, Myelomonocytic, Acute/drug therapy , Protease Inhibitors/therapeutic use , Pyrazines/therapeutic use , Aldehyde Dehydrogenase/metabolism , Apoptosis , Bortezomib , CASP8 and FADD-Like Apoptosis Regulating Protein/analysis , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Caspase 3/metabolism , Caspase 8/metabolism , Cells, Cultured , Fas-Associated Death Domain Protein/analysis , Fas-Associated Death Domain Protein/metabolism , Flow Cytometry , Humans , Immunophenotyping , Leukemia, Monocytic, Acute/pathology , Leukemia, Myelomonocytic, Acute/pathology , Receptors, TNF-Related Apoptosis-Inducing Ligand/analysis , Stem Cells/drug effects , TNF-Related Apoptosis-Inducing Ligand/analysis , X-Linked Inhibitor of Apoptosis Protein/analysis , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
We investigated the expression of Tie-2 in primary blasts from 111 patients with acute myeloid leukemia (AML) to evaluate a possible linkage between the expression of this receptor and the immunophenotypic and biologic properties of leukemic blasts. Tie-2 was expressed at moderate and high levels in 39 and 23 of 111 AMLs, respectively. The analysis of the immunophenotype clearly showed that Tie-2 expression in AML was associated with monocytic features. Interestingly, Tie-2 expression on AML blasts was associated with concomitant expression of other receptors for endothelial growth factors, such as vascular endothelial growth factor receptor 1 (VEGF-R1), -R2, and -R3. Tie-2(+) AMLs were characterized by high blast cell counts at diagnosis, a high frequency of Flt3 mutations, and increased Flt3 expression. The survival of Tie-2(+) AMLs is sustained through an autocrine pattern involving Angiopoietin-1 and Tie-2, as suggested by experiments showing induction of apoptosis in Tie-2(+) AMLs by agents preventing the binding of angiopoietins to Tie-2. Finally, the in vitro growth of Tie-2(+) AMLs in endothelial culture medium supplemented with VEGF and angiopoietins resulted in their partial endothelial differentiation. These observations suggest that Tie-2(+) AMLs pertain to a mixed monocytic/endothelial lineage, derived from the malignant transformation of the normal counterpart represented by monocytic cells expressing endothelial markers. The autocrine angiopoietin/Tie-2 axis may represent a promising therapeutic target to improve the outcome of patients with monocytic AML. Disclosure of potential conflicts of interest is found at the end of this article.
Subject(s)
Endothelial Growth Factors/metabolism , Granulocyte Precursor Cells/metabolism , Leukemia, Myeloid/metabolism , Monocytes/metabolism , Receptor, TIE-2/metabolism , Acute Disease , Angiopoietin-1/metabolism , Angiopoietin-1/pharmacology , Cell Differentiation/drug effects , Cell Survival , Endothelial Cells/cytology , Granulocyte Precursor Cells/cytology , Granulocyte Precursor Cells/drug effects , Granulocyte Precursor Cells/pathology , Humans , Immunophenotyping , Leukemia, Myeloid/diagnosis , Leukemia, Myeloid/pathology , Leukocyte Count , Mutation , Receptors, Vascular Endothelial Growth Factor/metabolism , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A/pharmacology , fms-Like Tyrosine Kinase 3/genetics , fms-Like Tyrosine Kinase 3/metabolismABSTRACT
Different proteins ensure the fine control of iron metabolism at the level of various tissues. Among these proteins, it was discovered a second transferrin receptor (TfR2), that seems to play a key role in the regulation of iron homeostasis. Its mutations are responsible for type 3 hemochromatosis (Type 3 HH). Although TfR2 expression in normal tissues was restricted at the level of liver and intestine, we observed that TfR2 was frequently expressed in tumor cell lines. Particularly frequent was its expression in ovarian cancer, colon cancer and glioblastoma cell lines; less frequent was its expression in leukemic and melanoma cell lines. Interestingly, in these tumor cell lines, TfR2 expression was inversely related to that of receptor 1 for transferrin (TfR1). Experiments of in vitro iron loading or iron deprivation provided evidence that TfR2 is modulated in cancer cell lines according to cellular iron levels following two different mechanisms: (i) in some cells, iron loading caused a downmodulation of total TfR2 levels; (ii) in other cell types, iron loading caused a downmodulation of membrane-bound TfR2, without affecting the levels of total cellular TfR2 content. Iron deprivation caused in both conditions an opposite effect compared to iron loading. These observations suggest that TfR2 expression may be altered in human cancers and warrant further studies in primary tumors. Furthermore, our studies indicate that, at least in tumor cells, TfR2 expression is modulated by iron through different biochemical mechanisms, whose molecular basis remains to be determined.
Subject(s)
Gene Expression Regulation, Neoplastic , Iron/metabolism , Leukemia/metabolism , Melanoma/metabolism , Receptors, Transferrin/biosynthesis , Antigens, CD/biosynthesis , Antigens, CD/genetics , Gene Expression Regulation, Neoplastic/genetics , HL-60 Cells , Hemochromatosis/genetics , Hemochromatosis/metabolism , Homeostasis/genetics , Humans , Intestinal Mucosa/metabolism , K562 Cells , Leukemia/genetics , Liver/metabolism , Melanoma/genetics , Mutation , Receptors, Transferrin/genetics , U937 CellsABSTRACT
OBJECTIVES: Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (Smac) has been described to sensitize for apoptosis. We have explored the proapoptotic activity of a small molecule mimic of Smac/DIABLO on ovarian cancer cell lines (A2780 cells and its chemoresistant derivatives A2780/ADR and A2780/DDP), cancer cell lines and in primary ovarian cancer cells. METHODS: The effects of a small molecule mimic of Smac/DIABLO on ovarian cancer cell lines and primary ovarian cancer cells were determined by cell proliferation, apoptosis and biochemical assays. RESULTS: This compound added alone elicited only a weak proapoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or agonistic TRAILR2 antibody (Lexatumumab) in inducing apoptosis of ovarian cancer cells. CONCLUSIONS: These observations suggest that small molecule mimic of Smac/DIABLO could be useful for the development of experimental strategies aiming to treat ovarian cancer. Interestingly, in addition to its well known proapoptotic effects, Smac/DIABLO elicited a significant increase of pro-caspase-3 levels.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Diynes/pharmacology , Ovarian Neoplasms/drug therapy , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Tetrazoles/pharmacology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Biomimetic Materials/administration & dosage , Biomimetic Materials/pharmacology , Caspase 3/metabolism , Caspase 9/metabolism , Cell Growth Processes/drug effects , Cell Line, Tumor , Diynes/administration & dosage , Drug Synergism , Enzyme Activation/drug effects , Female , Humans , Intracellular Signaling Peptides and Proteins , Mitochondrial Proteins , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Peptide Fragments/administration & dosage , Peptide Fragments/pharmacology , Receptors, TNF-Related Apoptosis-Inducing Ligand/agonists , Receptors, Tumor Necrosis Factor/agonists , TNF-Related Apoptosis-Inducing Ligand/administration & dosage , Tetrazoles/administration & dosage , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
In the present study we have explored the sensitivity of ovarian cancer cells to TRAIL and proteasome inhibitors. Particularly, we have explored the capacity of proteasome inhibitors to bypass TRAIL resistance of ovarian cancer cells. For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/DDP and A2780/ADR, providing evidence that: (i) the three cell lines are either scarcely sensitive (A2780 and A2780/ADR) or moderately sensitive (A2780/DDP) to the cytotoxic effects of TRAIL; (ii) the elevated c-FLIP expression observed in ovarian cancer cells is a major determinant of TRAIL resistance of these cells; (iii) proteasome inhibitors (PS-341 or MG132) are able to exert a significant pro-apoptotic effect and to greatly enhance the sensitivity of both chemosensitive and chemoresistant A2780 cells to TRAIL; (iv) proteasome inhibitors damage mitochondria through stabilization of BH3-only proteins, Bax and caspase activation and significantly enhance TRAIL-R2 expression; (v) TRAIL-R2, but not TRAIL-R1, mediates the apoptotic effects of TRAIL on ovarian cancer cells. Importantly, studies on primary ovarian cancer cells have shown that these cells are completely resistant to TRAIL and proteasome inhibitors markedly enhance the sensitivity of these cells to TRAIL. Given the high susceptibility of ovarian cancer cells to proteasome inhibitors, our results further support the experimental use of these compounds in the treatment of ovarian cancer.
Subject(s)
Apoptosis/physiology , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Protease Inhibitors , Proteasome Endopeptidase Complex , TNF-Related Apoptosis-Inducing Ligand/metabolism , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Boronic Acids/pharmacology , Boronic Acids/therapeutic use , Bortezomib , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Caspases/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Enzyme Activation , Female , Humans , Leupeptins/pharmacology , Leupeptins/therapeutic use , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mitochondria/metabolism , Ovarian Neoplasms/pathology , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Protease Inhibitors/therapeutic use , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Pyrazines/pharmacology , Pyrazines/therapeutic use , RNA, Small Interfering/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolismABSTRACT
Although the arsenic compounds are now widely utilized in clinics in the treatment of various tumors, their effects on normal hematopoiesis do not seem to have been explored. In the present study, we provide evidence that arsenic trioxide (As(2)O(3)) exerts in vitro a potent inhibitory effect on normal erythropoiesis and a stimulatory action on megakaryocytic differentiation. The effect of As(2)O(3) on erythroid and megakaryocytic differentiation was evaluated on both erythroleukemic cell lines K562 and HEL and on normal hemopoietic progenitor cells (HPCs) induced to selective erythroid or megakaryocytic differentiation. The inhibitory effect of As(2)O(3) on erythropoiesis is related to: (a) the inhibition of Stat5 activation with consequent reduced expression of the target genes Bcl-X(L) and glycophorin-A; (b) the activation of an apoptotic mechanism that leads to the cleavage of the erythroid transcription factors Tal-1 and GATA-1, whose integrity is required for erythroid cell survival and differentiation; (c) the reduced expression of heat shock protein 70, required for GATA-1 integrity. The stimulatory effect of As(2)O(3) on normal megakaryocytopoiesis is seemingly related to upmodulation of GATA-2 expression and to stimulation of MAPK activity. These observations may have implications for the patients undergoing anti-leukemic treatment with this compound.
Subject(s)
Antineoplastic Agents/pharmacology , Arsenicals/pharmacology , Cell Differentiation/drug effects , Erythropoiesis/drug effects , Hematopoietic Stem Cells/metabolism , Megakaryocytes/metabolism , Oxides/pharmacology , Apoptosis/drug effects , Arsenic Trioxide , Cell Survival/drug effects , Gene Expression Regulation/drug effects , Glycophorins/biosynthesis , Hematopoietic Stem Cells/cytology , Humans , K562 Cells , MAP Kinase Signaling System/drug effects , Megakaryocytes/cytology , Mitogen-Activated Protein Kinase Kinases/metabolism , Transcription Factors/metabolism , bcl-X Protein/biosynthesisABSTRACT
BACKGROUND AND OBJECTIVES: The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a potential anticancer agent. However, many cancer cells are resistant to apoptosis induction by TRAIL. The present study was designed to evaluate the sensitivity to TRAIL-induced apoptosis in acute myeloblastic leukemias (AML). DESIGN AND METHODS: TRAIL/TRAIL receptor (TRAIL-R) expression and sensitivity to TRAIL-mediated apoptosis were explored in 79 AML patients, including 17 patients with acute promyelocytic leukemia (APL). RESULTS: In non-APL AML we observed frequent expression of TRAIL decoy receptors (TRAIL-R3 and TRAIL-R4), while TRAIL-R1 and TRAIL-R2 expression was restricted to AML exhibiting monocytic features. Total leukemic blasts, as well as AML colony-forming units (AML-CFU), were invariably resistant to TRAIL-mediated apoptosis. APL express membrane-bound TRAIL on their surface and exhibit a pattern of TRAIL-R expression similar to that observed in the other types of AML. Before, during and after retinoic acid treatment APL cells are TRAIL-resistant. The induction of granulocytic maturation of APL cells by retinoic acid was associated with a marked decline of TRAIL expression. INTERPRETATION AND CONCLUSIONS: The analysis of experimental APL models (i.e., U937 cells engineered to express PML/RAR-Eo and NB4 cells) provided evidence that PML/RAR-Eo expression was associated with downmodulation of TRAIL-R1 and with resistance to TRAIL-mediated apoptosis. We suggest that AML blasts, including APL blasts, are resistant to TRAIL-mediated apoptosis, a phenomenon seemingly related to the expression of TRAIL decoy receptors on these cells. Finally, APL blasts express membrane-bound TRAIL that could confer an immunologic privilege to these cells.
Subject(s)
Apoptosis/drug effects , Drug Resistance, Neoplasm/genetics , Leukemia, Myeloid/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/physiology , Receptors, Tumor Necrosis Factor/physiology , Tumor Necrosis Factor Decoy Receptors/physiology , Acute Disease , Adolescent , Adult , Antineoplastic Agents/pharmacology , Caspase 3/analysis , Caspase 8/analysis , Cell Differentiation/drug effects , Cell Membrane/metabolism , Cytarabine/pharmacology , Etoposide/pharmacology , Female , GPI-Linked Proteins , Granulocytes/drug effects , HL-60 Cells/pathology , Humans , Hydroxyurea/pharmacology , Leukemia, Myeloid/drug therapy , Leukemia, Myeloid/pathology , Leukemia, Promyelocytic, Acute/metabolism , Leukemia, Promyelocytic, Acute/pathology , Male , Middle Aged , Monocytes/drug effects , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/physiology , Receptors, Tumor Necrosis Factor, Member 10c , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/physiology , Tretinoin/pharmacology , Tumor Cells, Cultured/drug effects , Tumor Stem Cell Assay , U937 Cells/drug effectsABSTRACT
Mechanisms underlying fetal hemoglobin (HbF) reactivation in stress erythropoiesis have not been fully elucidated. We suggested that a key role is played by kit ligand (KL). Because glucocorticoids (GCs) mediate stress erythropoiesis, we explored their capacity to potentiate the stimulatory effect of KL on HbF reactivation, as evaluated in unilineage erythropoietic culture of purified adult progenitors (erythroid burst-forming units [BFU-Es]). The GC derivative dexamethasone (Dex) was tested in minibulk cultures at graded dosages within the therapeutical range (10(-6) to 10(-9) M). Dex did not exert significant effects alone, but synergistically it potentiated the action of KL in a dose-dependent fashion. Specifically, Dex induced delayed erythroid maturation coupled with a 2-log increased number of generated erythroblasts and enhanced HbF synthesis up to 85% F cells and 55% gamma-globin content at terminal maturation (ie, in more than 80%-90% mature erythroblasts). Equivalent results were obtained in unicellular erythroid cultures of sibling BFU-Es treated with KL alone or combined with graded amounts of Dex. These results indicate that the stimulatory effect of KL + Dex is related to the modulation of gamma-globin expression rather than to recruitment of BFU-Es with elevated HbF synthetic potential. At the molecular level, Id2 expression is totally suppressed in control erythroid culture but is sustained in KL + Dex culture. Hypothetically, Id2 may mediate the expansion of early erythroid cells, which correlates with HbF reactivation. These studies indicate that GCs play an important role in HbF reactivation. Because Dex acts at dosages used in immunologic disease therapy, KL + Dex administration may be considered to develop preclinical models for beta-hemoglobinopathy treatment.
Subject(s)
Dexamethasone/pharmacology , Erythroid Precursor Cells/metabolism , Fetal Hemoglobin/biosynthesis , Stem Cell Factor/pharmacology , Cell Culture Techniques , Cell Division/drug effects , Dose-Response Relationship, Drug , Drug Synergism , Erythroid Precursor Cells/cytology , Erythroid Precursor Cells/drug effects , Erythropoiesis/drug effects , Fetal Hemoglobin/drug effects , Globins/analysis , Humans , Hypoxia , Kinetics , Male , Transcription Factors/analysisABSTRACT
We have investigated the expression of interleukin-3 receptor alpha (IL-3Ralpha) chain in primary blasts from 79 patients with acute myeloid leukemia (AML), 25 patients with B-acute lymphoid leukemia (B-ALL), and 7 patients with T-acute lymphoid leukemia (T-ALL) to evaluate a linkage between the expression of this receptor chain, blast proliferative status, and disease prognosis. Although IL-3Ralpha chain was scarcely expressed in most patients with T-ALL, it was overexpressed in 40% and 45% of patients with B-ALL and AML, respectively, compared with the levels observed in normal CD34(+) progenitors. The biological and clinical significance of this overexpression pattern was investigated in AML. At the biological level, elevated IL-3Ralpha expression was associated with peculiar properties of leukemic blasts, specifically in 3 areas. First, in all patients the blasts expressing elevated IL-3Ralpha levels exhibited higher cycling activity and increased resistance to apoptosis triggered by growth factor deprivation. Second, spontaneous signal transducer and activator of transcription 5 (Stat5) phosphorylation was observed in 13% of AML patients, all pertaining to the group of patients exhibiting high IL-3Ralpha expression. Third, following IL-3 treatment, Stat5 was activated at higher levels in blasts with elevated IL-3Ralpha expression. At the clinical level, a significant correlation was observed between the level of IL-3Ralpha expression and the number of leukemic blasts at diagnosis, and patients exhibiting elevated IL-3Ralpha levels had a lower complete remission rate and survival duration than those showing normal IL-3Ralpha levels. These findings suggest that in AML, deregulated expression of IL-3Ralpha may contribute to the proliferative advantage of the leukemic blasts and, hence, to a poor prognosis.
Subject(s)
Leukemia, Myeloid, Acute/genetics , Receptors, Interleukin-3/genetics , Adult , Aged , Antigens, Surface/analysis , Apoptosis , Blast Crisis , Cell Cycle , Gene Expression Regulation, Neoplastic , Humans , Interleukin-3 Receptor alpha Subunit , Leukemia, Erythroblastic, Acute/genetics , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/mortality , Leukemia, Myeloid, Acute/pathology , Middle Aged , Predictive Value of Tests , Prognosis , Recurrence , Reference Values , Reverse Transcriptase Polymerase Chain Reaction , Survival Rate , Tumor Cells, CulturedABSTRACT
The morphologic, immunophenotypic, genotypic, genomic, and functional features of an undifferentiated acute leukemia with stem cell features are reported. At light and electron microscopy, the leukemic population was represented by primitive progenitor cells with no evidence of differentiation. The blasts were CD34(+), AC133(+), CD71(-), HLA-DR(-), CD38(-/dim+), CD90(+), CD117(dim+), flt3(+); did not express B, T, or myeloid-associated antigens; and showed a germline configuration of the immunoglobulin and T-cell receptor. Genomic profiling documented the expression of early stem cell and myeloid-associated genes. Receptors for early-acting hemopoietic growth factors (HGFs) were detected, while receptors for unilineage HGF were not expressed. Incubation with the flt3 or Kit ligand induced the expression of unilineage HGF receptors, allowing these cells to respond to their respective ligands. Growth without differentiation was sustained only in the presence of early-acting HGF, namely flt3 ligand, while early and unilineage HGF gave rise to all types of hemopoietic colonies.