ABSTRACT
Alcoholic extract of Piper betle (Piper betle L.) leaves was recently found to induce apoptosis of CML cells expressing wild type and mutated Bcr-Abl with imatinib resistance phenotype. Hydroxy-chavicol (HCH), a constituent of the alcoholic extract of Piper betle leaves, was evaluated for anti-CML activity. Here, we report that HCH and its analogues induce killing of primary cells in CML patients and leukemic cell lines expressing wild type and mutated Bcr-Abl, including the T315I mutation, with minimal toxicity to normal human peripheral blood mononuclear cells. HCH causes early but transient increase of mitochondria-derived reactive oxygen species. Reactive oxygen species-dependent persistent activation of JNK leads to an increase in endothelial nitric oxide synthase-mediated nitric oxide generation. This causes loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, cleavage of caspase 9, 3 and poly-adenosine diphosphate-ribose polymerase leading to apoptosis. One HCH analogue was also effective in vivo in SCID mice against grafts expressing the T315I mutation, although to a lesser extent than grafts expressing wild type Bcr-Abl, without showing significant bodyweight loss. Our data describe the role of JNK-dependent endothelial nitric oxide synthase-mediated nitric oxide for anti-CML activity of HCH and this molecule merits further testing in pre-clinical and clinical settings.
Subject(s)
Apoptosis/drug effects , Drug Resistance, Neoplasm/drug effects , Eugenol/analogs & derivatives , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , MAP Kinase Kinase 4/metabolism , Mitochondria/drug effects , Nitric Oxide Synthase Type III/metabolism , Piper betle/chemistry , Reactive Oxygen Species/metabolism , Animals , Antineoplastic Agents/pharmacology , Benzamides , Blotting, Western , Eugenol/chemistry , Eugenol/pharmacology , Flow Cytometry , Fusion Proteins, bcr-abl/genetics , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Male , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Nude , Mice, SCID , Mitochondria/metabolism , Nitric Oxide/metabolism , Phosphorylation/drug effects , Piperazines/pharmacology , Plant Extracts/pharmacology , Plant Leaves/chemistry , Pyrimidines/pharmacology , Tumor Cells, CulturedABSTRACT
Chlorogenic acid (Chl) has been reported to possess a wide range of biological and pharmacological properties including induction of apoptosis of Bcr-Abl(+) chronic myeloid leukemia (CML) cell lines and clinical leukemia samples via inhibition of Bcr-Abl phosphorylation. Here we studied the mechanisms of action of Chl in greater detail. Chl treatment induced an early accumulation of intracellular reactive oxygen species (ROS) in Bcr-Abl(+) cells leading to downregulation of Bcr-Abl phosphorylation and apoptosis. Chl treatment upregulated death receptor DR5 and induced loss of mitochondrial membrane potential accompanied by release of cytochrome c from the mitochondria to the cytosol. Pharmacological inhibition of caspase-8 partially inhibited apoptosis, whereas caspase-9 and pan-caspase inhibitor almost completely blocked the killing. Knocking down DR5 using siRNA completely attenuated Chl-induced caspase-8 cleavage but partially inhibited apoptosis. Antioxidant NAC attenuated Chl-induced oxidative stress-mediated inhibition of Bcr-Abl phosphorylation, DR5 upregulation, caspase activation and CML cell death. Our data suggested the involvement of parallel death pathways that converged in mitochondria. The role of ROS in Chl-induced death was confirmed with primary leukemia cells from CML patients in vitro as well as in vivo in nude mice bearing K562 xenografts. Collectively, our results establish the role of ROS for Chl-mediated preferential killing of Bcr-Abl(+) cells.
Subject(s)
Apoptosis/physiology , Chlorogenic Acid/pharmacology , Fusion Proteins, bcr-abl/biosynthesis , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Reactive Oxygen Species/metabolism , Animals , Apoptosis/drug effects , Chlorogenic Acid/isolation & purification , Fusion Proteins, bcr-abl/physiology , Gene Knockdown Techniques/methods , Humans , K562 Cells , Mice , Mice, Nude , Piper betle , Plant Leaves , Tumor Cells, Cultured , U937 Cells , Xenograft Model Antitumor Assays/methodsABSTRACT
INTRODUCTION: Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H(2)O(2), which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. MATERIALS AND METHODS: Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl(+) cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl(+) CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl(+) cells. CONCLUSION: NAC enhances imatinib-induced apoptosis of Bcr-Abl(+) cells by endothelial nitric oxide synthase-mediated production of nitric oxide.
Subject(s)
Acetylcysteine/pharmacology , Apoptosis/physiology , Free Radical Scavengers/pharmacology , Nitric Oxide Synthase Type III/metabolism , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Annexin A5/pharmacology , Apoptosis/drug effects , Benzamides , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Fusion Proteins, bcr-abl/metabolism , Hematologic Neoplasms/metabolism , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Membrane Potential, Mitochondrial/physiology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/drug effects , Reactive Oxygen Species/analysis , Up-Regulation/drug effects , Up-Regulation/physiologyABSTRACT
Granulocyte-macrophage colony-stimulating factor (GM-CSF) has long been found to have growth-promoting effects on multipotent haematopoietic lineages, specifically granulocytes and macrophages. GM-CSF combined with interleukin-4 (IL-4) drives monocytes to become myeloid dendritic cells (mDCs) in vitro. We report that culturing human monocytes with GM-CSF alone generates myeloid cells (GM-Mono) that have lower expression of CD14 than monocytes and that fail to express DC-SIGN. GM-Monos, however, express CD83 and the transcription factor PU.1, although at a lower level than the conventional mDCs generated in the presence of GM-CSF and IL-4. On stimulation with tumour necrosis factor-alpha, interferon-gamma and anti-CD40 monoclonal antibody, the GM-Monos predominantly produced IL-10 but were less efficient in IL-12 production. In a primary allogeneic mixed lymphocyte reaction, GM-Monos induced hyporesponsiveness and IL-10-biased cytokine production in CD4(+) T cells. In fresh mixed lymphocyte reaction, GM-Monos inhibited conventional mDC-induced allogeneic CD4(+) T-cell proliferation. GM-Mono-induced inhibition of allogeneic CD4(+) T-cell proliferation was partially attributed to IL-10. Interestingly, GM-Monos neither induced hyporesponsiveness in allogeneic CD8(+) T cells nor inhibited conventional mDC-induced allogeneic CD8(+) T-cell proliferation. Taken together, we characterize monocyte-derived CD14(low) CD83(+) cells generated by GM-CSF that can induce tolerance or stimulation of T cells depending on T-cell subsets.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Monocytes/immunology , T-Lymphocyte Subsets/immunology , Antigens, CD/analysis , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Adhesion Molecules/analysis , Cell Differentiation/immunology , Cell Proliferation , Cells, Cultured , Flow Cytometry , Humans , Immune Tolerance/immunology , Immunoglobulins/analysis , Interleukin-10/biosynthesis , Lectins, C-Type/analysis , Lipopolysaccharide Receptors/analysis , Lymphocyte Culture Test, Mixed , Membrane Glycoproteins/analysis , Phagocytosis/immunology , Proto-Oncogene Proteins/metabolism , Receptors, Cell Surface/analysis , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism , Trans-Activators/metabolism , CD83 AntigenABSTRACT
The role played by dendritic cells (DCs) in Leishmania donovani infection is poorly understood. Here, we report that L. donovani amastigotes efficiently infect human peripheral-blood monocyte-derived DCs. Opsonization with normal human serum enhanced the infectivity of amastigotes and promastigotes only marginally. Surface attachment versus internalization was distinguished by incubation of DCs with live, fluorescein isothiocyanate-labeled parasites, followed by quenching with crystal violet. Infection with amastigotes was accompanied by DC maturation, as was evident from the up-regulation of maturation-associated cell-surface markers, the nuclear translocation of RelB, and the release of cytokines. Amastigote-primed DCs produced inflammatory cytokines in response to subsequent treatment with interferon- gamma or anti-CD40 monoclonal antibody. When cocultured, amastigote-infected DCs induced T helper cell type 1 (Th1) responses both in naive allogeneic CD4(+) T cells and in autologous CD4(+) T cells from patients with kala-azar and up-regulated the expression of T-bet. Our data reveal that infection with L. donovani amastigotes induces a Th1 cytokine milieu in both DCs and T cells.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Dendritic Cells/parasitology , Leishmania donovani/immunology , Leishmaniasis, Visceral/immunology , Th1 Cells/immunology , Animals , Cell Differentiation/immunology , Cricetinae , GATA3 Transcription Factor/biosynthesis , GATA3 Transcription Factor/immunology , Humans , Leishmania donovani/growth & development , Leishmania donovani/pathogenicity , Life Cycle Stages , Lymphocyte Activation , Mesocricetus , Myeloid Cells/immunology , Myeloid Cells/parasitology , T-Box Domain Proteins/biosynthesis , T-Box Domain Proteins/immunologyABSTRACT
Leishmaniasis is the second-most dreaded parasitic disease in the modern world, behind malaria. The lack of effective vaccines demand improved chemotherapy along with the development of lead compounds and newer targets. We report here that the pentacyclic triterpenoid, dihydrobetulinic acid (DHBA), is a novel lead compound for antileishmanial therapy. It acts by targeting DNA topoisomerases. DNA topoisomerase I and II activity was studied using relaxation and decatenation assays. Mechanistic studies were based on the decreased mobility of enzyme-bound DNA compared with free DNA and the differential mobility of nicked and supercoiled monomers in 1% agarose gel. Pulsed field gradient gel electrophoresis, confocal microscopy, and transmission electron microscopy were performed to assess cytotoxicity of the compound and ultrastructural damage of the parasite. Apoptosis was studied by the isolation of DNA from DHBA-treated parasites and subsequent electrophoresis in 1% agarose gel. DHBA inhibits growth of Leishmania donovani promastigotes and amastigotes with an IC50 of 2.6 and 4.1 microM respectively. The compound is a dual inhibitor of DNA topoisomerases that fails to induce DNA cleavage and acts by preventing the formation of enzyme-DNA binary complex, ultimately inducing apoptosis. Treatment of infected golden hamsters with the compound markedly reduces (> 92%) parasitic burden, both in spleen and liver. Interestingly, the 17-decarboxylated analogue, dihydrolupeol, does not inhibit DNA topoisomerase I and II, has no effect on parasitic growth, and also fails to induce apoptosis. DHBA is a potent antileishmanial agent that induces apoptosis by primarily targeting DNA topoisomerases. Therefore it is a strong candidate for use in designing new antileishmanial drugs.
Subject(s)
Antiprotozoal Agents/pharmacology , Apoptosis/drug effects , Leishmania donovani/enzymology , Leishmaniasis, Visceral/drug therapy , Topoisomerase I Inhibitors , Topoisomerase II Inhibitors , Triterpenes/pharmacology , Animals , Cricetinae , DNA Damage/drug effects , DNA Topoisomerases, Type I/metabolism , DNA Topoisomerases, Type II/metabolism , DNA, Kinetoplast/metabolism , Enzyme Inhibitors/pharmacology , Mesocricetus , Microscopy, ConfocalABSTRACT
Dendritic cells (DCs) have been proposed to play a critical role as adjuvants in vaccination and immunotherapy. In this study we evaluated the combined effect of soluble Leishmania donovani Ag (SLDA)-pulsed syngeneic bone marrow-derived DC-based immunotherapy and antimony-based chemotherapy for the treatment of established murine visceral leishmaniasis. Three weekly injections of SLDA-pulsed DCs into L. donovani-infected mice reduced liver and splenic parasite burden significantly, but could not clear parasite load from these organs completely. Strikingly, the conventional antileishmanial chemotherapy (sodium antimony gluconate) along with injections of SLDA-pulsed DCs resulted in complete clearance of parasites from both these organs. Repetitive in vitro stimulation of splenocytes from uninfected or L. donovani-infected mice with SLDA-pulsed DCs led to the emergence of CD4(+) T cells with characteristics of Th1 cells. Our data indicate that DC-based immunotherapy enhances the in vivo antileishmanial potential of antimony or vice versa.