cyclin-d1 and Leukemia--Promyelocytic--Acute

Acute promyelocytic leukaemia (APL) is a malignant hematological tumour characterized by the presence of promyelocytic leukaemia-retinoic acid receptor A (PML-RARA) fusion protein. Cinobufagin (CBG) is one of the main effective components of toad venom with antitumor properties. However, only a few reports regarding the CBG treatment of APL are available.. We explored the effect and mechanism of action of CBG on NB4 and NB4-R1 cells.. We evaluated the viability of NB4 and NB4-R1 cells treated with 0, 20, 40, and 60 nM CBG for 12, 24, and 48 h. After treatment with CBG for 24 h, Bcl-2 associated X (Bax), B-cell lymphoma 2 (Bcl-2), β-catenin, cyclin D1, and c-myc expression was detected using western blotting and real-time polymerase chain reaction. Caspase-3 and PML-RARA expression levels were detected using western blotting.. CBG inhibited the viability of NB4 and NB4-R1 cells. The IC. CBG induced NB4 and NB4-R1 cell apoptosis and PML-RARA degradation in a caspase-dependent manner by inhibiting the β-catenin signalling pathway. This study proposes a novel treatment strategy for patients with APL, particularly those with ATRA-resistant APL.

Topics: Amphibian Venoms; Apoptosis; bcl-2-Associated X Protein; beta Catenin; Bufanolides; Caspase 3; Caspases; Cyclin D1; Humans; Leukemia, Promyelocytic, Acute; Oncogene Proteins, Fusion; Receptors, Retinoic Acid

In acute promyelocytic leukemia (APL), all-trans retinoic acid (ATRA) treatment induces granulocytic differentiation and maturation. MicroRNAs play pivotal roles in formation of the leukemic phenotype. Previously, microRNA-382-5p (miR-382-5p) was upregulated in acute myeloid leukemia (AML) with t(15;17). In the present study, we found that miR-382-5p expression was elevated with ATRA-induced differentiation of APL. To investigate the potential functional role of miR-382-5p in APL differentiation, an APL cell line was transfected with miR-382-5p mimics, inhibitors, or negative control (NC). The results showed in APL cell line NB4 that miR-382-5p downregulation upon ATRA treatment was a key event in the drug response. Mechanistic investigations revealed that miR-382-5p targeted the ATRA-regulated tumor suppressor gene PTEN through direct binding to its 3' UTR. Enforced expression of miR-382-5p or specific PTEN inhibitors inhibited ATRA-induced granulocytic differentiation via regulation of the cell cycle regulator cyclinD1. Conversely, PTEN overexpression promoted differentiation and enhanced sensitivity of NB4 cell line to physiological levels of ATRA. Finally, we found that PTEN overexpression restored PML nuclear bodies (NBs). Taken together, these results demonstrated that up-regulated miR-382-5p in NB4 cell line inhibited granulocytic differentiation through the miR-382-5p/PTEN axis, uncovering PTEN as a critical element in the granulocytic differentiation program induced by ATRA in APL.

Topics: Antineoplastic Agents; Cell Differentiation; Cyclin D1; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; MicroRNAs; PTEN Phosphohydrolase; THP-1 Cells; Tretinoin

Within the genus Scutellaria various species are used in different folk medicines throughout Asia. Traditional Chinese Medicine (TCM) uses S. baicalensis (Labiatae) to treat various inflammatory conditions. The root shows strong anticancer properties in vitro and was suggested for clinical trials against multiple myeloma. Further, S. barbata was successfully tested against metastatic breast cancer in a phase I/II trial. Therefore, we investigated the anti-cancer properties of S. orientalis L. ssp. carica Edmondson, an endemic subspecies from the traditional medicinal plant S. orientalis L. in Turkey, which is used to promote wound healing and to stop haemorrhage.. Freeze-dried plant material was extracted with petroleum ether, dichloromethane, ethyl acetate, and methanol and the bioactivity of these extracts was analysed by proliferation assay, cell death determination, and by investigating protein expression profiles specific for cell cycle arrest and apoptosis.. The strongest anti-leukemic activity was shown by the methanol extract, which contained apigenin, baicalein, chrysin, luteolin and wogonin, with an IpC50 of 43 microg/ml (corresponding to 1.3mg/ml of dried plant material) which correlated with cyclin D1- and Cdc25A suppression and p21 induction. At 132 microg/ml (=4 mg/ml of the drug) this extract caused genotoxic stress indicated by substantial phosphorylation of the core histone H2AX (gamma-H2AX) followed by activation of caspase 3 and signature-type cleavage of PARP resulting in a 55% apoptosis rate after 48 hours of treatment.. Here, we report for the first time that S. orientalis L. ssp. carica Edmondson exhibited potent anti-leukaemic properties likely through the anti-proliferative effect of baicalein and the genotoxic property of wogonin.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; cdc25 Phosphatases; Chromatography, High Pressure Liquid; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Histones; HL-60 Cells; Humans; Inhibitory Concentration 50; Leukemia, Promyelocytic, Acute; Phosphorylation; Phytotherapy; Plant Extracts; Poly(ADP-ribose) Polymerases; Scutellaria; Turkey

More than 60% of conventional drugs are derived from natural compounds, some of the most effective pharmaceuticals (e.g. aspirin, quinine and various antibiotics) originate from plants or microbes, and large numbers of potentially valuable natural substances remain to be discovered. Plants with considerable medicinal potential include members of the genus Acalypha. Notably, extracts of A. platyphilla, A. fruticosa, A. siamensis, A. guatemalensis and A. wilkesiana have been recently shown to have antioxidant, antimicrobial and cytotoxic effects. In the study presented here we investigated the anti-inflammatory, anti-proliferative and pro-apoptotic activities of A. alopecuroidea, which is endemic in parts of Central America and is traditionally used by the Mopan- and Itza-Maya in the form of decoctions to treat skin conditions, and as a tea to treat stomach and urinary complaints. We demonstrate here that extracts of A. alopecuroidea can inhibit TNFalpha-induced E-selectin production, providing a mechanistic validation of its traditional use against inflammatory diseases. Furthermore, a fraction of A. alopecuroidea root extracts purified by solid phase extraction and separated by HPLC displayed strong cell cycle inhibitory activity by down-regulating and inactivating two proto-oncogenes (cyclin D1 and Cdc25A), and simultaneously inducing cyclin A, thereby disturbing orchestrated cell cycle arrest, and thus (presumably) triggering caspase 3-dependent apoptosis. The results of this study indicate that there are high prospects for purifying an active principle from A. alopecuroidea for further in vivo and preclinical studies.

Topics: Anti-Inflammatory Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Caspase 3; cdc25 Phosphatases; Cell Cycle; Cell Proliferation; Cell Survival; Checkpoint Kinase 2; Chromatin Assembly and Disassembly; Cyclin A; Cyclin D1; Dose-Response Relationship, Drug; E-Selectin; Endothelial Cells; Euphorbiaceae; Female; HL-60 Cells; Humans; Inflorescence; Leukemia, Promyelocytic, Acute; Mutation; Plant Leaves; Plant Shoots; Protein Serine-Threonine Kinases; Time Factors; Transfection; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

With the aim of determining the differential expression of WNT and FZD genes, before and after induction of apoptosis in BCR-ABL positive cells, we treated the myeloid cell line K562 and control cell line HL60 with imatinib mesylate and etoposide, and analyzed relative mRNA expression levels of WNT, FZD and sFRP genes under normal and apoptotic conditions by real-time RT-PCR. We observed marked increase in mRNA levels of FZD4, FZD5, FZD7 and WNT5b, correlating with apoptotic activity and independent of the agent or cell line used. Our results suggest the involvement of non-canonical Wnt signaling in executing programmed cell death in myeloid cell lines.

Topics: Antineoplastic Agents; Benzamides; Caspase 3; Cell Line, Tumor; Cyclin D1; Etoposide; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Piperazines; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Wnt Proteins

The marine prostanoid clavulones were shown to exert cytotoxicity against several cancer cells. In the present study, we illustrate the pathways utilized by clavulone II to trigger apoptotic signaling in human acute promyelocytic leukemia HL-60 cells. Exposure of cells to clavulone II resulted in early induction of phosphatidylserine externalization, mitochondrial dysfunction, and alteration of the cell cycle. Down-regulated expression of cyclin D1 explained the effect of clavulone II on G1 phase arrest of the cell cycle. Clavulone II induced the disruption of mitochondrial membrane potential and activation of caspase-8, -9 and -3 in a time- and concentration-dependent manner. Furthermore, the effect of 3 microM clavulone II was accompanied by the up-regulation of Bax, down-regulation of Mcl-1, and cleavage of Bid. Taken together, it is suggested that low concentrations of clavulone II induce the antiproliferative effect through the down-regulation of cyclin D1 expression and G1 arrest of the cell cycle, while that of high concentration induce the apoptotic cell death via the modulation of members of caspases and Bcl-2 family proteins in HL-60 cells.

Topics: Animals; Anthozoa; Antineoplastic Agents; Apoptosis; Blotting, Western; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Cycle; Cell Proliferation; Cell Separation; Cyclin D1; DNA; Dose-Response Relationship, Drug; Down-Regulation; Flow Cytometry; G1 Phase; HL-60 Cells; Humans; Inhibitory Concentration 50; Intracellular Membranes; Leukemia, Promyelocytic, Acute; Membrane Potentials; Models, Chemical; Oxidation-Reduction; Oxidative Stress; Prostaglandins; Prostaglandins A; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Time Factors

All-trans retinoic acid (ATRA) induces clinical remission in patients with t(15;17) acute promyelocytic leukemia (APL) carrying leukemogenic promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) fusion protein by overcoming PML-RARalpha transcriptional repression and inducing myeloid differentiation. To identify more potent chemical differentiation inducers, a screening assay was developed utilizing an ATRA-insensitive NB4 cell line (NB4-c) in which differentiation could be measured after 48 hours when primed with ATRA followed by other potential inducers. Over 300 cytostatic agents selected from the National Cancer Institute library were screened using this established method. Three compounds, NSC656243, NSC625748, and NSC144168, were identified to amplify ATRA-induced differentiation with acceptable cytotoxicity in NB4-c cells. In the absence of ATRA, these compounds also induced HL-60 and murine erythroleukemia cells to undergo partial differentiation. NSC656243, a benzodithiophene compound, was selected for further studies to examine the underlying mechanism of action. The differentiation effect of NSC656243 was associated with enhanced ATRA-mediated up-regulation of cell cycle regulatory proteins p21waf1 and p27kip1, retinoblastoma dephosphorylation, expression of RIG-E and RIG-G, and myelomonocytic differentiation-specific down-regulation of the myeloperoxidase (MPO) gene. Moreover, at 2- to 3-fold higher concentrations than those used to synergize with ATRA, NSC656243 induced apoptosis in NB4-c cells by reactive oxygen species-mediated pathways. The dual effects of benzodithiophenes (i.e., differentiation and apoptosis induction) support further development of these compounds as therapeutic agents for leukemia.

Topics: Animals; Antioxidants; Apoptosis; Cell Differentiation; Cyclin D1; Dose-Response Relationship, Drug; HL-60 Cells; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Leukemia, Promyelocytic, Acute; Mice; Structure-Activity Relationship; Thiophenes; Tretinoin

We studied the cytotoxic effect of an organic arsenical compound, phenylarsine oxide (PAO) on an acute promyelocytic leukemia (APL) cell line (NB4) and an As2O3-resistant NB4 subline (NB4/As). Cell growth was inhibited by 50% (IC50) upon 2-day treatment with As2O3 or PAO at 0.54 and 0.06 microM, respectively in NB4 cells (P = 0.025), and 2.80 and 0.08 microM, respectively in NB4/As (P = 0.030). 0.1 microM PAO increased the proportion of hypodiploid cells (50.3%) by a greater degree than the same dose of As2O3 (3.8%) in NB4 cells. In NB4 cells, 0.1 microM PAO reduced the mitochondrial transmembrane potential (20.5% in a PI(negative)-Rhodamine123(low) fraction) by a greater degree than 1 microM As2O3 (7.1%). Western blotting showed that 0.1 microM PAO downregulated the expression of both Bcl-2 and Bcl-X(L) proteins, whereas I microM As2O3 downregulated only Bcl-2 expression. These results suggest that the cytotoxic effect of PAO on an APL cell line and As2O3-resistant subline is significantly higher than that of As2O3. PAO-induced apoptosis seems to be related to the activation of the mitochondrial pathway and downregulation of both Bcl-2 and Bcl-X(L). PAO is a considerable agent for relapsed/refractory APL and for purging APL cells following stem cell transplantation.

Topics: Apoptosis; Arsenic Trioxide; Arsenicals; bcl-X Protein; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance, Neoplasm; Humans; Leukemia, Promyelocytic, Acute; Membrane Potentials; Mitochondria; Oxides; Proto-Oncogene Proteins c-bcl-2

The majority of the promyelocytic leukemia (PML) protein is present in nuclear bodies which are altered in several pathogenic conditions including acute promyelocytic leukemia. PML nuclear bodies are found in nearly all cells yet their function remains unknown. Here, we demonstrate that PML and the eukaryotic initiation factor 4E (elF-4E) co-localize and co-immunopurify. eIF-4E is involved in nucleocytoplasmic transport of specific mRNAs including cyclin D1. eIF-4E overexpression leads to increased cyclin D1 protein levels; whereas, overexpression of PML leads to decreased cyclin D1 levels. Neither PML nor eIF-4E cause significant changes in cyclin D1 mRNA levels. The association with eIF-4E led us to investigate if PML could alter mRNA distribution as a possible post-transcriptional mechanism for suppressing cyclin D1 production. We show that overexpression of PML results in nuclear retention of cyclin D1 mRNA and that intact PML nuclear bodies are required. Addition of eIF-4E overcomes PML induced retention and alters the morphology of PML bodies suggesting a mechanism by which eIF-4E can modulate PML function. These results raise the possibility that PML nuclear bodies may participate in the regulation of nucleocytoplasmic transport of specific mRNAs.

Topics: 3T3 Cells; Animals; Biological Transport; Cell Line; Cyclin D1; Cytoplasm; Eukaryotic Initiation Factor-4E; Fibroblasts; Humans; Leukemia, Promyelocytic, Acute; Macromolecular Substances; Mice; Neoplasm Proteins; Nuclear Proteins; Organelles; Peptide Initiation Factors; Promyelocytic Leukemia Protein; Protein Structure, Tertiary; Recombinant Fusion Proteins; RNA, Messenger; RNA, Neoplasm; Subcellular Fractions; Transcription Factors; Transcription, Genetic; Transfection; Tumor Suppressor Proteins

It has been demonstrated that protein expression of p16, the inhibitor of cyclin-dependent kinase 4 and 6, increases 4 fold at the G1/S transition when serum-arrested cells are restimulated to logarithmic growth. We examined the cell cycle regulation of this cyclin-dependent kinase inhibitor in cells separated according to their cell cycle phases by centrifugal elutriation. Neither p16 mRNA nor its protein expression are regulated during the cell cycle of normal phytohemagglutinin-stimulated lymphocytes, retinoblastoma protein-negative cells, papilloma virus-transformed cells, and acute promyelocytic leukemia cells. p16 mRNA is constitutively expressed in cells in which we detected the normal E2F-dependent S-phase specific expression of thymidine kinase mRNA. We further observed a G1-phase specific expression of cyclin D1 mRNA in the same cells separated by centrifugal elutriation.

Topics: Blotting, Western; Carrier Proteins; Cell Cycle; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclins; Enzyme Inhibitors; Eye Neoplasms; Gene Expression; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Leukemia, Promyelocytic, Acute; Oncogene Proteins; Protein Kinase Inhibitors; Retinoblastoma; Retinoblastoma Protein; RNA, Messenger; Thymidine Kinase; Tumor Cells, Cultured