nitrophenols and Leukemia

Melatonin (N-acetyl-5-methoxytryptamine, MEL), secreted by the pineal gland, plays an important role in regulation of various functions in the human body. There is evidence that MEL exhibits antitumor effect in various types of cancer. We studied the combined effect of MEL and drugs from different pharmacological groups, such as cytarabine (CYT) and navitoclax (ABT-737), on the state of the pool of acute myeloid leukemia (AML) tumor cell using the MV4-11 cell line as model. The combined action of MEL with CYT or ABT-737 contributed to the decrease in proliferative activity of leukemic cells, decrease in the membrane potential of mitochondria, and increase in the production of reactive oxygen species (ROS) and cytosolic Ca2+. We have shown that introduction of MEL together with CYT or ABT-737 increases expression of the C/EBP homologous protein (CHOP) and the autophagy marker LC3A/B and decreases expression of the protein disulfide isomerase (PDI) and binding immunoglobulin protein (BIP), and, therefore, could modulate endoplasmic reticulum (ER) stress and initiate autophagy. The findings support an early suggestion that MEL is able to provide benefits for cancer treatment and be considered as an adjunct to the drugs used in cancer therapy.

Topics: Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Endoplasmic Reticulum Stress; Humans; Leukemia; Melatonin; Nitrophenols

Obatoclax and ABT-737 belong to a new class of anticancer agents known as BH3-mimetics. These agents antagonize the anti-apoptotic members of Bcl-2 family. The Bcl-2 proteins modulate sensitivity of many types of cancer cells to chemotherapy. Therefore, the objective of the present study was to examine and compare the antileukemic activity of obatoclax and ABT-737 applied alone, and in combination with anticancer agent, mafosfamide and daunorubicin. The in vitro cytotoxic effects of the tested agents on human leukemia cells were determined using the spectrophotometric MTT test, Coulter electrical impedance method, flow cytometry annexin V-fluorescein/propidium iodide assay, and light microscopy technique. The combination index analysis was used to quantify the extent of agent interactions. BH3 mimetics significantly decreased the leukemia cell viability and synergistically enhanced the cytotoxic effects induced by mafosfamide and daunorubicin. Obatoclax affected the cell viability to a greater degree than did ABT-737. In addition, various patterns of temporary changes in the cell volume and count, and in the frequency of leukemia cells undergoing apoptosis, were found 24 and 48 h after the tested agent application. ABT-737 combined with anticancer agents induced apoptosis more effectively than obatoclax when given in the same combination regimen. The results of the present study point to the different antileukemic activities of obatoclax and ABT-737, when applied alone, and in combination with anticancer agents. A better understanding of the exact mechanisms of BH3 mimetic action is of key importance for their optional use in cancer therapy.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Cell Survival; Flow Cytometry; Humans; Indoles; Leukemia; Nitrophenols; Piperazines; Pyrroles; Sulfonamides

Two clinical-stage anticancer drugs, the Bcl-2 inhibitor ABT-263, and the MDM2 inhibitor SAR405838 achieve complete tumor regression in animal models of leukemia but also induce acquired resistance. Elucidation of acquired resistance mechanisms and development of strategies to overcome the resistance are critical for their successful clinical development.. We employed RS4;11 and MV4;11 cell lines, two acute leukemia models, to investigate acquired resistance mechanisms for both drugs in vitro and in vivo and evaluated several treatment regimens in xenograft mouse models to improve long-term, complete tumor regression.. Resistance to either SAR405838 or ABT-263 (or its analogue ABT-737) develops in acute leukemia models in vitro and in vivo. RS4;11 and MV4;11 tumors treated with SAR405838 acquire resistance to the drug by mutation of the TP53 gene or compromise of p53 protein function. RS4;11 tumors treated with either ABT-263 or ABT-737 acquire resistance primarily through downregulation of BAX but not BAK. When acute leukemia cells become highly resistant to the MDM2 inhibitor, they retain their sensitivity to the Bcl-2 inhibitors, or vice versa. Certain sequential or combination treatment of SAR405838 and ABT-263 can achieve longer-term tumor regression than treatment with either agent alone.. Our study provides new insights into the mechanisms of acquired resistance of Bcl-2 and MDM2 inhibitors in acute leukemia models and suggests that certain sequential or combination treatment of these two distinct classes of apoptosis-inducing agents should be tested as new treatment strategies for acute leukemia in the clinic.

Topics: Animals; Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Indoles; Leukemia; Mice; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Spiro Compounds; Sulfonamides; Xenograft Model Antitumor Assays

Topics: Animals; Biphenyl Compounds; Drug Resistance, Neoplasm; Humans; Leukemia; Nitrophenols; Proteasome Endopeptidase Complex; Proteolysis; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Chronic lymphocytic leukemia (CLL) is the most frequent form of leukemia in adult population and despite numerous studies, it is considered an incurable disease. Since CLL is characterized by overexpression of pro-survival Bcl-2 family members, treatments with their antagonists, such as ABT-737, represent a promising new therapeutic strategy. ABT-737 is a BH3 mimetic agent which binds Bcl-2, Bcl-XL and Bcl-w with high affinity, while weakly interacts with Mcl-1 and Bfl-1. Previous studies demonstrated that quercetin, a flavonoid naturally present in food and beverages, was able to sensitize B-cells isolated from CLL patients to apoptosis when associated with death ligands or fludarabine, through a mechanism involving Mcl-1 down-regulation. Here, we report that the association between ABT-737 and quercetin synergistically induces apoptosis in B-cells and in five leukemic cell lines (Combination Index <1). Peripheral blood mononuclear cell from healthy donors were not affected by quercetin treatment. The molecular pathways triggered by quercetin have been investigated in HPB-ALL cells, characterized by the highest resistance to both ABT-737 and quercetin when applied as single molecules, but highly sensitivity to the co-treatment. In this cell line, quercetin down-regulated Mcl-1 through the inhibition of PI3K/Akt signaling pathway, leading to Mcl-1 instability. The same mechanism was confirmed in B-cells. These results may open new clinical perspectives based on a translational approach in CLL therapy.

Topics: Antineoplastic Agents; Apoptosis; B-Lymphocytes; Biphenyl Compounds; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Humans; Leukemia; Leukemia, Lymphocytic, Chronic, B-Cell; Mitogen-Activated Protein Kinase Kinases; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Phosphoinositide-3 Kinase Inhibitors; Piperazines; Protein Stability; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Quercetin; Signal Transduction; Sulfonamides

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1-2 μM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.

Topics: Animals; bcl-X Protein; Biphenyl Compounds; Drug Resistance, Neoplasm; HL-60 Cells; Humans; Jurkat Cells; K562 Cells; Leukemia; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proteasome Endopeptidase Complex; Proteolysis; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides

The BH3-mimetic ABT-737 and an orally bioavailable compound of the same class, navitoclax (ABT-263), have shown promising antitumor efficacy in preclinical and early clinical studies. Although both drugs avidly bind Bcl-2, Bcl-x(L), and Bcl-w in vitro, we find that Bcl-2 is the critical target in vivo, suggesting that patients with tumors overexpressing Bcl-2 will probably benefit. In human non-Hodgkin lymphomas, high expression of Bcl-2 but not Bcl-x(L) predicted sensitivity to ABT-263. Moreover, we show that increasing Bcl-2 sensitized normal and transformed lymphoid cells to ABT-737 by elevating proapoptotic Bim. In striking contrast, increasing Bcl-x(L) or Bcl-w conferred robust resistance to ABT-737, despite also increasing Bim. Cell-based protein redistribution assays unexpectedly revealed that ABT-737 disrupts Bcl-2/Bim complexes more readily than Bcl-x(L)/Bim or Bcl-w/Bim complexes. These results have profound implications for how BH3-mimetics induce apoptosis and how the use of these compounds can be optimized for treating lymphoid malignancies.

Topics: Aniline Compounds; Animals; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-X Protein; Biphenyl Compounds; Cell Death; Cytoprotection; Drug Resistance, Neoplasm; Etoposide; Gene Expression Regulation, Neoplastic; Humans; Leukemia; Lymphoma; Membrane Proteins; Mice; Molecular Targeted Therapy; Mutant Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

The traditional view is that cancer cells predominately produce ATP by glycolysis, rather than by oxidation of energy-providing substrates. Mitochondrial uncoupling--the continuing reduction of oxygen without ATP synthesis--has recently been shown in leukemia cells to circumvent the ability of oxygen to inhibit glycolysis, and may promote the metabolic preference for glycolysis by shifting from pyruvate oxidation to fatty acid oxidation (FAO). Here we have demonstrated that pharmacologic inhibition of FAO with etomoxir or ranolazine inhibited proliferation and sensitized human leukemia cells--cultured alone or on bone marrow stromal cells--to apoptosis induction by ABT-737, a molecule that releases proapoptotic Bcl-2 proteins such as Bak from antiapoptotic family members. Likewise, treatment with the fatty acid synthase/lipolysis inhibitor orlistat also sensitized leukemia cells to ABT-737, which supports the notion that fatty acids promote cell survival. Mechanistically, we generated evidence suggesting that FAO regulates the activity of Bak-dependent mitochondrial permeability transition. Importantly, etomoxir decreased the number of quiescent leukemia progenitor cells in approximately 50% of primary human acute myeloid leukemia samples and, when combined with either ABT-737 or cytosine arabinoside, provided substantial therapeutic benefit in a murine model of leukemia. The results support the concept of FAO inhibitors as a therapeutic strategy in hematological malignancies.

Topics: Adenosine Triphosphate; Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Biphenyl Compounds; Cell Proliferation; Cytarabine; Fatty Acid Synthases; Fatty Acids; Humans; Ion Channels; Leukemia; Mice; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Proteins; Nitrophenols; Oxidation-Reduction; Piperazines; Reactive Oxygen Species; Sulfonamides; Uncoupling Protein 2

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Caspase Inhibitors; Caspases; Drug Synergism; Humans; Immunoblotting; Leukemia; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Nucleosides; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Sulfonamides; Tumor Cells, Cultured

The Bcl-2 antagonist ABT-737 kills transformed cells in association with displacement of Bim from Bcl-2. The histone deactetylase (HDAC) inhibitor suberoyl bis-hydroxamic acid (SBHA) was employed to determine whether and by what mechanism ABT-737 might interact with agents that upregulate Bim. Expression profiling of BH3-only proteins indicated that SBHA increased Bim, Puma, and Noxa expression, while SBHA concentrations that upregulated Bim significantly potentiated ABT-737 lethality. Concordance between SBHA-mediated Bim upregulation and interactions with ABT-737 was observed in various human leukemia and myeloma cells. SBHA-induced Bim was largely sequestered by Bcl-2 and Bcl-x(L), rather than Mcl-1; ABT-737 attenuated these interactions, thereby triggering Bak/Bax activation and mitochondrial outer membrane permeabilization. Knockdown of Bim (but not Puma or Noxa) by shRNA or ectopic overexpression of Bcl-2, Bcl-x(L), or Mcl-1 diminished Bax/Bak activation and apoptosis. Notably, ectopic expression of these antiapoptotic proteins disabled death signaling by sequestering different proapoptotic proteins, i.e., Bim by Bcl-2, both Bim and Bak by Bcl-x(L), and Bak by Mcl-1. Together, these findings indicate that HDAC inhibitor-inducible Bim is primarily neutralized by Bcl-2 and Bcl-x(L), thus providing a mechanistic framework by which Bcl-2 antagonists potentiate the lethality of agents, such as HDAC inhibitors, which upregulate Bim.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; bcl-X Protein; Biphenyl Compounds; Cell Line, Tumor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia; Membrane Proteins; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Protein Binding; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Sulfonamides; Up-Regulation

In this study, we investigated the mechanism of apoptosis induction of obatoclax (GX15-070), a novel Bcl-2 homology domain-3 (BH3) mimetic, in acute myeloid leukemia (AML) cell lines and primary AML samples. Obatoclax inhibited cell growth of HL-60, U937, OCI-AML3, and KG-1 cell lines. Apoptosis induction contributed to the observed antiproliferative effects at concentrations of this agent that mirror its affinity for antiapoptotic Bcl-2 proteins. We show that obatoclax can promote the release of cytochrome c from isolated leukemia cell mitochondria and that apoptosis induced by this agent is preceded by the release of Bak from Mcl-1, liberation of Bim from both Bcl-2 and Mcl-1, and the formation of an active Bak/Bax complex. Notably, apoptosis was diminished, but not fully prevented, in the absence of Bak/Bax or Bim, suggesting that obatoclax has additional targets that contribute to its cytotoxicity. At growth inhibitory doses that did not induce apoptosis or decrease viability, obatoclax induced an S-G(2) cell-cycle block. Obatoclax induced apoptosis in AML CD34+ progenitor cells with an average IC(50) of 3.59 +/- 1.23 micromol/L although clonogenicity was inhibited at concentrations of 75 to 100 nmol/L. Obatoclax synergized with the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells and synergistically induced apoptosis in combination with AraC in leukemic cell lines and in primary AML samples. In conclusion, we show that obatoclax potently induces apoptosis and decreases leukemia cell proliferation and may be used in a novel therapeutic strategy for AML alone and in combination with other targeted agents and chemotherapeutics.

Topics: Animals; Antineoplastic Agents; Apoptosis; AraC Transcription Factor; Biomimetics; Biphenyl Compounds; Cells, Cultured; Drug Synergism; HL-60 Cells; Humans; Indoles; Leukemia; Mice; Mice, Knockout; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Nitrophenols; Piperazines; Protein Structure, Tertiary; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides; Tumor Stem Cell Assay; U937 Cells

The Bcl-2 antagonist ABT-737 targets Bcl-2/Bcl-xL but not Mcl-1, which may confer resistance to this novel agent. Here, we show that Mcl-1 down-regulation by the cyclin-dependent kinase (CDK) inhibitor roscovitine or Mcl-1-shRNA dramatically increases ABT-737 lethality in human leukemia cells. ABT-737 induces Bax conformational change but fails to activate Bak or trigger Bax translocation. Coadministration of roscovitine and ABT-737 untethers Bak from Mcl-1 and Bcl-xL, respectively, triggering Bak activation and Bax translocation. Studies employing Bax and/or Bak knockout mouse embryonic fibroblasts (MEFs) confirm that Bax is required for ABT-737+/-roscovitine lethality, whereas Bak is primarily involved in potentiation of ABT-737-induced apoptosis by Mcl-1 down-regulation. Ectopic Mcl-1 expression attenuates Bak activation and apoptosis by ABT-737+roscovitine, whereas cells overexpressing Bcl-2 or Bcl-xL remain fully sensitive. Finally, Mcl-1 knockout MEFs are extremely sensitive to Bak conformational change and apoptosis induced by ABT-737, effects that are not potentiated by roscovitine. Collectively, these findings suggest down-regulation of Mcl-1 by either CDK inhibitors or genetic approaches dramatically potentiate ABT-737 lethality through cooperative interactions at two distinct levels: unleashing of Bak from both Bcl-xL and Mcl-1 and simultaneous induction of Bak activation and Bax translocation. These findings provide a mechanistic basis for simultaneously targeting Mcl-1 and Bcl-2/Bcl-xL in leukemia.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; bcl-X Protein; Biphenyl Compounds; Cyclin-Dependent Kinases; Down-Regulation; Drug Synergism; HL-60 Cells; Humans; Jurkat Cells; Leukemia; Mice; Mice, Knockout; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Nitrophenols; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Purines; RNA Interference; Roscovitine; Sulfonamides; U937 Cells

We show that the antiapoptotic proteins BCL-2, BCL-XL, MCL-1, BFL-1, and BCL-w each bear a unique pattern of interaction with a panel of peptides derived from BH3 domains of BH3-only proteins. Cellular dependence on an antiapoptotic protein for survival can be decoded based on the pattern of mitochondrial sensitivity to this peptide panel, a strategy that we call BH3 profiling. Dependence on antiapoptotic proteins correlates with sequestration of activator BH3-only proteins like BID or BIM by antiapoptotic proteins. Sensitivity to the cell-permeable BCL-2 antagonist ABT-737 is also related to priming of BCL-2 by activator BH3-only molecules. Our data allow us to distinguish a cellular state we call "primed for death," which can be determined by BH3 profiling and which correlates with dependence on antiapoptotic family members for survival.

Topics: Amino Acid Sequence; Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; BH3 Interacting Domain Death Agonist Protein; Biphenyl Compounds; Cell Line, Tumor; Enzyme Activators; Intracellular Membranes; Leukemia; Membrane Proteins; Mice; Mitochondria, Liver; Mitochondrial Membranes; Molecular Sequence Data; Nitrophenols; Peptide Fragments; Piperazines; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sulfonamides

Topics: Alkaline Phosphatase; Cysteamine; Hodgkin Disease; Humans; Infectious Mononucleosis; Intestines; Kinetics; Leukemia; Lymphoma; Lymphoproliferative Disorders; Multiple Myeloma; Nitrophenols; Organophosphorus Compounds; Placenta

Topics: Acid Phosphatase; Blood Platelets; Bone Marrow; Bone Marrow Cells; Chromatography, DEAE-Cellulose; Chromatography, Ion Exchange; Electrophoresis, Disc; Fluorides; Gaucher Disease; Histocytochemistry; Humans; Isoenzymes; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukocytes; Lymphatic Diseases; Lymphocytes; Lymphoma, Non-Hodgkin; Molecular Weight; Monocytes; Nitrophenols; Polycythemia Vera; Staining and Labeling; Tartrates