nitrophenols and navitoclax

B-Cell Lymphoma 2 (BCL-2), c-MYC and related proteins are arguably amongst the most widely studied in all of biology. Every year there are thousands of papers reporting on different aspects of their biochemistry, cellular and physiological mechanisms and functions. This plethora of literature can be attributed to both proteins playing essential roles in the normal functioning of a cell, and by extension a whole organism, but also due to their central role in disease, most notably, cancer. Many cancers arise due to genetic lesions resulting in deregulation of both proteins, and indeed the development and survival of tumours is often dependent on co-operativity between these protein families. In this review we will discuss the individual roles of both proteins in cancer, describe cancers where co-operativity between them has been well-characterised and finally, some strategies to target these proteins therapeutically.

Topics: Aniline Compounds; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Carcinogenesis; Cell Line, Tumor; Cell Survival; Clinical Trials as Topic; Gene Expression Regulation, Neoplastic; Humans; Morpholinos; Neoplasms; Nitrophenols; Peptide Fragments; Piperazines; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Rituximab; Signal Transduction; Sulfonamides

B-cell lymphoma 2 (Bcl-2) is a regulator protein involved in apoptosis. In the past few decades, this protein has been demonstrated to have high efficacy in cancer therapy, and several approaches targeting Bcl-2 have been tested clinically (e.g., oblimersen, ABT-737, ABT-263, obatoclax mesylate, and AT-101). This review reports potential Bcl-2 inhibitors according to current information on their underlying mechanism and the results of clinical trials. In addition, the function and mechanisms of other potentially valuable Bcl-2 inhibitors that did not show efficacy in clinical studies are also discussed. This summary of the development of Bcl-2 inhibitors provides worthwhile viewpoints on the use of biomedical approaches in future cancer therapy.

Topics: Aniline Compounds; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Gossypol; Humans; Lymphoma, B-Cell; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Thionucleotides

Apoptosis, the process of programmed cell death, occurs normally during development and aging. Members of the B-cell lymphoma 2 (BCL2) family of proteins are central regulators of apoptosis, and resistance to apoptosis is one of the hallmarks of cancer. Targeting the apoptotic pathway via BCL2 inhibitors has been considered a promising treatment strategy in the past decade. Initial efforts with small molecule BH3 mimetics such as ABT-737 and ABT-263 (navitoclax) pioneered the development of the first-in-class Food and Drug Administration (FDA)-approved oral BCL2 inhibitor, venetoclax. Venetoclax was approved for the treatment of chronic lymphocytic leukemia and acute myeloid leukemia, and is now being studied in a number of hematologic malignancies. Several other inhibitors targeting different BCL2 family members are now in early stages of development.

Topics: Aniline Compounds; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Drug Approval; Hematologic Neoplasms; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Molecular Targeted Therapy; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; United States; United States Food and Drug Administration

The B-cell lymphoma/leukemia 2 (BCL-2) family of proteins has attracted the attention of cancer biologists since the cloning of BCL-2 more than 25 years ago. In the intervening decades, the way the BCL-2 family controls commitment to programmed cell death has been greatly elucidated. Several drugs directed at inhibiting BCL-2 and related antiapoptotic proteins have been tested clinically, with some showing considerable promise, particularly in lymphoid malignancies. A better understanding of the BCL-2 family has also provided insight into how conventional chemotherapy selectively kills cancer cells and why some cancers are more chemosensitive than others. Further exploitation of our understanding of the BCL-2 family promises to offer improved predictive biomarkers for oncologists and improved therapies for patients with cancer.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Drug Resistance, Neoplasm; Humans; Indoles; Molecular Targeted Therapy; Neoplasms; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides; Thionucleotides; Treatment Outcome

Blocks in apoptosis are now widely regarded as key pathophysiological maladaptations critical for tumour persistence. Importantly, it has also been recognised that they confer resistance to cytotoxic therapy, and hence often portend an adverse prognosis. The advent of BH3-mimetics represents a nascent clinical capability to directly reverse the evasion of apoptosis, and indeed exploit the very molecular abnormalities which have hitherto posed major obstacles to therapeutic success. Clinical trials with BH3-mimetics have demonstrated clear single agent anti-tumour activity in selected haematological malignancies. These drugs also offer promise as adjuncts to existing or emerging therapies in a broader range of cancers.

Topics: Aniline Compounds; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Hematologic Neoplasms; Humans; Indoles; Molecular Mimicry; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides

A critical regulator of the apoptotic machinery is the Bcl-2 family proteins whose over expression confers a protective effect on malignant cells against death signals of apoptosis. Cancer cells that are resistant to various anti-cancer drugs and treatment regimen are found to over express these Bcl-2 proteins such as Bcl-2, Bcl-X(L), Mcl-1, Bcl-w, and A1/Bfl1. In recent years there has been an exponential growth in the identification as well as synthesis of non-peptidic cell permeable small-molecule inhibitors (SMIs) of protein-protein interaction. The focus of this article is on inhibitors of anti-apoptotic protein Bcl-2. This review summarizes an up to date knowledge of the available SMIs, their mode of action as well as their current status in preclinical as well as clinical development.

Topics: Aniline Compounds; Animals; Apoptosis; Benzamides; Binding Sites; Biphenyl Compounds; Gossypol; Humans; Indoles; Lymphoma; Models, Molecular; Neoplasms; Nitrophenols; Piperazines; Protein Structure, Tertiary; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides; Sulfones

Despite tremendous advances over the last 15 years in understanding fundamental mechanisms of apoptosis, this has failed to translate into improved cancer therapy for patients. However, there may now be light at the end of this long tunnel. Antiapoptotic Bcl-2 family members may be divided into two subclasses, one comprising Bcl-2, Bcl-X(L) and Bcl-w and the other Mcl-1 and Bcl2A1. Neutralization of both subclasses is required for apoptosis induction. Solution of the structure of antiapoptotic Bcl-2 family proteins has led to the design of novel small molecule inhibitors. Although many such molecules have been synthesized, rigorous verification of their specificity has often been lacking. Further studies have revealed that many putative Bcl-2 inhibitors are not specific and have other cellular targets, resulting in non-mechanism based toxicity. Two notable exceptions are ABT-737 and a related orally active derivative, ABT-263, which bind with high affinity to Bcl-2, Bcl-X(L) and Bcl-w and may prove to be useful tools for mechanistic studies. ABT-263 is in early clinical trials in lymphoid malignancies, small-cell lung cancer and chronic lymphocytic leukemia, and some patients have shown promising results. In in vitro studies, primary cells from patients with various B-cell malignancies are exquisitely sensitive to ABT-737, exhibiting novel morphological features of apoptosis including marked outer mitochondrial membrane rupture.

Topics: Aniline Compounds; Apoptosis; Biphenyl Compounds; Cells, Cultured; Humans; Lymphoma, Mantle-Cell; Molecular Structure; Neoplasms; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Defects in apoptotic pathways can promote cancer cell survival and also confer resistance to antineoplastic drugs. One pathway being targeted for antineoplastic therapy is the anti-apoptotic B-cell lymphoma-2 (Bcl-2) family of proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl1/A-1, and Bcl-B) that bind to and inactivate BH3-domain pro-apoptotic proteins. Signals transmitted by cellular damage (including antineoplastic drugs) or cytokine deprivation can initiate apoptosis via the intrinsic apoptotic pathway. It is controversial whether some BH3-domain proteins (Bim or tBid) directly activate multidomain pro-apoptotic proteins (e.g., Bax and Bak) or act via inhibition of those anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl1/A-1, and Bcl-B) that stabilize pro-apoptotic proteins. Overexpression of anti-apoptotic Bcl-2 family members has been associated with chemotherapy resistance in various human cancers, and preclinical studies have shown that agents targeting anti-apoptotic Bcl-2 family members have preclinical activity as single agents and in combination with other antineoplastic agents. Clinical trials of several investigational drugs targeting the Bcl-2 family (oblimersen sodium, AT-101, ABT-263, GX15-070) are ongoing. Here, we review the role of the Bcl-2 family in apoptotic pathways and those agents that are known and/or designed to inhibit the anti-apoptotic Bcl-2 family of proteins.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Gossypol; Humans; Indoles; Mitochondria; Neoplasms; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides; Thionucleotides

Cancer cells show deviant behavior that induces apoptotic signaling. To survive, cancer cells typically acquire changes enabling evasion of death signals. One way they do this is by increasing the expression of anti-apoptotic BCL-2 proteins. Anti-apoptotic BCL-2 family proteins antagonize death signaling by forming heterodimers with pro-death proteins. Heterodimer formation occurs through binding of the pro-apoptotic protein's BH3 domain into the hydrophobic cleft of anti-apoptotic proteins. The BH3 mimetics are small molecule antagonists of the anti-apoptotic BCL-2 members that function as competitive inhibitors by binding to the hydrophobic cleft. Under certain conditions, antagonism of anti-apoptotic BCL-2 family proteins can unleash pro-death molecules in cancer cells. Thus, the BH3 mimetics are a new class of cancer drugs that specifically target a mechanism of cancer cell survival to selectively kill cancer cells.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Apoptosis; Benzamides; Binding, Competitive; Biphenyl Compounds; Clinical Trials as Topic; Dimerization; Drug Delivery Systems; Drug Design; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Humans; Indoles; Mice; Mitochondria; Multigene Family; Neoplasm Proteins; Neoplasms; Nitrophenols; Piperazines; Protein Structure, Tertiary; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Structure-Activity Relationship; Sulfonamides; Sulfones; Thionucleotides

GNA13, encoding one of the G protein alpha subunits of heterotrimeric G proteins that transduce signals of G protein-coupled receptors (GPCR), is frequently mutated in germinal center B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) with poor prognostic outcomes. Due to the "undruggable" nature of GNA13, targeted therapy for these patients is not available. In this study, we found that palmitoylation of GNA13 not only regulates its plasma membrane localization, but also regulates GNA13's stability. It is essential for the tumor suppressor function of GNA13 in GCB-DLBCL cells. Interestingly, GNA13 negatively regulates BCL2 expression in GCB-DLBCL cells in a palmitoylation-dependent manner. Consistently, BCL2 inhibitors were found to be effective in killing GNA13-deficient GCB-DLBCL cells in a cell-based chemical screen. Furthermore, we demonstrate that inactivating GNA13 by targeting its palmitoylation enhanced the sensitivity of GCB-DLBCL to the BCL2 inhibitor. These studies indicate that the loss-of-function mutation of GNA13 is a biomarker for BCL2 inhibitor therapy of GCB-DLBCL and that GNA13 palmitoylation is a potential target for combination therapy with BCL2 inhibitors to treat GCB-DLBCL with wild-type GNA13.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Female; GTP-Binding Protein alpha Subunits, G12-G13; HeLa Cells; Humans; Lipoylation; Lymphoma, Large B-Cell, Diffuse; Male; Mice; Mice, Inbred NOD; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Topics: Allosteric Regulation; Aniline Compounds; Animals; Antineoplastic Agents; Biphenyl Compounds; Brain; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Guinea Pigs; Humans; Male; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Radioligand Assay; Receptors, sigma; Sigma-1 Receptor; Sulfonamides

Mesenchymal-type cancers after epithelial mesenchymal transition (EMT) were recently shown to acquire chemoresistance through expressing EMT specific transcription factors. However, druggable (or actionable) target(s) for chemoresistance in mesenchymal-type lung cancers remain unidentified. Here, we used a public clinical genomic database and mesenchymal lung cancer cells (MLCC) model derived from the A549 lung adenocarcinoma cell line to demonstrate that BCL2 expression, which is highly induced in mesenchymal-type lung cancers, as a predictor of poor prognosis in mesenchymal lung cancer patients and association with acquired chemoradioresistance. Thereby, combination treatment with BH3 mimetics, such as ABT-263 and ABT-737, clearly attenuated chemoresistance in MLCCs. BCL2 expression in MLCCs was induced by ERK1 activity through the upregulation of the MEK1/ERK1 scaffold protein MEK partner-1 (MP1). Interfering with the MEK1/MP1/ERK1 axis using a MEK1 inhibitor or MP1 depletion repressed BCL2 expression and sensitized MLCCs to chemoradiotherapy. Taken together, our results suggest that targeting druggable proteins in the MEK1/MP1/ERK1/BCL2 axis, such as MEK1 or BCL2, with currently available FDA approved drugs is a currently feasible approach to improve clinical outcomes of mesenchymal lung cancer patients.

Topics: A549 Cells; Adaptor Proteins, Signal Transducing; Adenocarcinoma; Adenocarcinoma of Lung; Aniline Compounds; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Biphenyl Compounds; Cell Proliferation; Cell Survival; Chemoradiotherapy; Diphenylamine; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Etoposide; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molecular Mimicry; Nitrophenols; Peptide Fragments; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Radiation Tolerance; RNA Interference; Signal Transduction; Sulfonamides; Transfection; Up-Regulation

Senescent cells may promote tumour progression through the activation of a senescence-associated secretory phenotype (SASP), whether these cells are capable of initiating tumourigenesis in vivo is not known. Expression of oncogenic β-catenin in Sox2+ young adult pituitary stem cells leads to formation of clusters of stem cells and induction of tumours resembling human adamantinomatous craniopharyngioma (ACP), derived from Sox2- cells in a paracrine manner. Here, we uncover the mechanisms underlying this paracrine tumourigenesis. We show that expression of oncogenic β-catenin in Hesx1+ embryonic precursors also results in stem cell clusters and paracrine tumours. We reveal that human and mouse clusters are analogous and share a common signature of senescence and SASP. Finally, we show that mice with reduced senescence and SASP responses exhibit decreased tumour-inducing potential. Together, we provide evidence that senescence and a stem cell-associated SASP drive cell transformation and tumour initiation in vivo in an age-dependent fashion.

Topics: Aniline Compounds; Animals; beta Catenin; Biphenyl Compounds; Cell Transformation, Neoplastic; Cellular Senescence; Child; Craniopharyngioma; Disease Models, Animal; Exome Sequencing; Homeodomain Proteins; Humans; Mice; Neoplastic Stem Cells; Nitrophenols; Oncogenes; Piperazines; Pituitary Gland; Pituitary Neoplasms; Repressor Proteins; SOXB1 Transcription Factors; Sulfonamides; Young Adult

In this work we studied clinically relevant interactions between the BH3 mimetics and the ABCB1 and ABCG2 transporters. We observed that the intracellular levels of ABT-263 and ABT-199, but not ABT-737, might be reduced by ABCB1 or ABCG2. Importantly, this effect was proportional to the transporter expression level. High transporter expression levels decreased the intracellular levels of ABT-263 and ABT-199 substantially. Low transporter expression levels, which are clinically relevant, affected the intracellular level of ABT-263 slightly but significantly, however, they failed to decrease the intracellular level of ABT-199 below the control level in parental cells. Our results further revealed that ABT-263 did not inhibit the ABCB1 mediated transport, however, it partially inhibited the ABCG2 mediated transport at clinically relevant concentrations. In contrast, ABT-199 inhibited partially the ABCB1 mediated transport and it fully inhibited the ABCG2 mediated transport at clinically relevant concentrations. Importantly, cells expressing higher drug transporters levels required higher concentrations of ABT-263 or ABT-199 to achieve certain inhibition of substrate efflux.. Antiproliferative effects of ABT-263 and ABT-199 might be reduced by ABCB1 or ABCG2, however, this effect depends on transporter expression levels. Since the expression levels of ABCB1 and ABCG2 are rarely high in clinical samples, their contribution to the overall resistance to ABT-263 or ABT-199 is probably low. Inhibition study revealed that ABT-199, but not ABT-263, fully inhibited low expression level of ABCG2. Our data suggest that ABT-199 should be evaluated beyond its original application as an inhibitor of the ABCG2 transporter in clinical settings.

Topics: Aniline Compounds; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; Biological Transport; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Gene Expression Regulation; Humans; K562 Cells; Neoplasm Proteins; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Standard chemotherapy is the only systemic treatment for triple-negative breast cancer (TNBC), and despite the good initial response, resistance remains a major therapeutic obstacle. Here, we employed a High-Throughput Screen to identify targeted therapies that overcome chemoresistance in TNBC. We applied short-term paclitaxel treatment and screened 320 small-molecule inhibitors of known targets to identify drugs that preferentially and efficiently target paclitaxel-treated TNBC cells. Among these compounds the SMAC mimetics (BV6, Birinapant) and BH3-mimetics (ABT-737/263) were recognized as potent targeted therapy for multiple paclitaxel-residual TNBC cell lines. However, acquired paclitaxel resistance through repeated paclitaxel pulses result in desensitization to BV6, but not to ABT-263, suggesting that short- and long-term paclitaxel resistance are mediated by distinct mechanisms. Gene expression profiling of paclitaxel-residual, -resistant and naïve MDA-MB-231 cells demonstrated that paclitaxel-residual, as opposed to -resistant cells, were characterized by an apoptotic signature, with downregulation of anti-apoptotic genes (BCL2, BIRC5), induction of apoptosis inducers (IL24, PDCD4), and enrichment of TNFα/NF-κB pathway, including upregulation of TNFSF15, coupled with cell-cycle arrest. BIRC5 and FOXM1 downregulation and IL24 induction was also evident in breast cancer patient datasets following taxane treatment. Exposure of naïve or paclitaxel-resistant cells to supernatants of paclitaxel-residual cells sensitized them to BV6, and treatment with TNFα enhanced BV6 potency, suggesting that sensitization to BV6 is mediated, at least partially, by secreted factor(s). Our results suggest that administration of SMAC or BH3 mimetics following short-term paclitaxel treatment could be an effective therapeutic strategy for TNBC, while only BH3-mimetics could effectively overcome long-term paclitaxel resistance.

Topics: Aniline Compounds; Apoptosis; Apoptosis Regulatory Proteins; Biomimetic Materials; Biphenyl Compounds; Cell Line, Tumor; Dipeptides; Drug Resistance, Neoplasm; Female; Humans; Indoles; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Molecular Targeted Therapy; Nitrophenols; Oligopeptides; Paclitaxel; Peptide Fragments; Piperazines; Proto-Oncogene Proteins; Signal Transduction; Sulfonamides; Triple Negative Breast Neoplasms

Although the ongoing clinical trials of ABT-263 and ABT-199 in chronic lymphocytic leukaemia (CLL) have indicated that BH3 mimetics hold considerable promise, understanding the mechanism of CLL resistance to BH3 mimetics remains a challenge.. The LD50 values of ABT-737, ABT-263 and ABT-199 in a number of primary CLL cells from 40 patients, were determined. The levels of Bcl-2 family proteins, including phosphorylated Bcl-2 (pBcl-2) and their interactions were measured by immunoblotting and co-immunoprecipitation. In vitro binding assays were performed by isothermal titration calorimetry and ELISA. BH3 profiling in isolated mitochondria was analysed.. The ratio of (Mcl-1 + pBcl-2) to Bcl-2 expression provided the most significant predictive marker for the cytotoxic potential of ABT-737, ABT-263 and ABT-199 in the panel of CLL samples. Mechanistically, pBcl-2 inhibited the effects of the ABT compounds on the displacement of Bax and Bim from Bcl-2, thereby suppressing mitochondrial apoptosis. The ABT compounds exhibited 100-300-fold lower binding affinity to the glutamic acid, phosphomimetic, mutant of Bcl-2 (T69E, S70E and S87E; EEE-Bcl-2). BH3 peptides exhibited different rank orders of binding affinities to full-length WT-Bcl-2 and full-length EEE-Bcl-2.. Our study suggested that a structural alteration in the BH3-binding groove was induced by phosphorylation of Bcl-2. Our data also provided a framework to overcome resistance of CLL cells to the ABT compounds by combining pBcl-2 kinase inhibitors with the ABT compounds.

Topics: Aniline Compounds; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cell Survival; Cells, Cultured; Drug Resistance, Neoplasm; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Mitochondria; Nitrophenols; Peptide Fragments; Phosphorylation; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

BH3 mimetic compounds induce tumor cell death through targeted inhibition of anti-apoptotic BCL2 proteins. Resistance to one such compound, ABT-737, is due to increased levels of anti-apoptotic MCL1. Using chemical and genetic approaches, we show that resistance to ABT-737 is abrogated by inhibition of the mitochondrial RING E3 ligase, MARCH5. Mechanistically, this is due to increased expression of pro-apoptotic BCL2 family member, NOXA, and is associated with MARCH5 regulation of MCL1 ubiquitylation and stability in a NOXA-dependent manner. MARCH5 expression contributed to an 8-gene signature that correlates with sensitivity to the preclinical BH3 mimetic, navitoclax. Furthermore, we observed a synthetic lethal interaction between MCL1 and MARCH5 in MCL1-dependent breast cancer cells. Our data uncover a novel level at which the BCL2 family is regulated; furthermore, they suggest targeting MARCH5-dependent signaling will be an effective strategy for treatment of BH3 mimetic-resistant tumors, even in the presence of high MCL1.

Topics: Aniline Compounds; Antineoplastic Agents; Biphenyl Compounds; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Membrane Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Peptide Fragments; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Ubiquitin-Protein Ligases

B-cell lymphoma (Bcl-2) is commonly associated with the progression and preservation of cancer and certain lymphomas; therefore, it is considered as a biological target against cancer. Nevertheless, evidence of all its structural binding sites has been hidden because of the lack of a complete Bcl-2 model, given the presence of a flexible loop domain (FLD), which is responsible for its complex behavior. FLD region has been implicated in phosphorylation, homotrimerization, and heterodimerization associated with Bcl-2 antiapoptotic function. In this contribution, homology modeling, molecular dynamics (MD) simulations in the microsecond (µs) time-scale and docking calculations were combined to explore the conformational complexity of unphosphorylated/phosphorylated monomeric and trimeric Bcl-2 systems. Conformational ensembles generated through MD simulations allowed for identifying the most populated unphosphorylated/phosphorylated monomeric conformations, which were used as starting models to obtain trimeric complexes through protein-protein docking calculations, also submitted to µs MD simulations. Principal component analysis showed that FLD represents the main contributor to total Bcl-2 mobility, and is affected by phosphorylation and oligomerization. Subsequently, based on the most representative unphosphorylated/phosphorylated monomeric and trimeric Bcl-2 conformations, docking studies were initiated to identify the ligand binding site of several known Bcl-2 inhibitors to explain their influence in homo-complex formation and phosphorylation. Docking studies showed that the different conformational states experienced by FLD, such as phosphorylation and oligomerization, play an essential role in the ability to make homo and hetero-complexes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 393-413, 2016.

Topics: Aniline Compounds; Binding Sites; Biphenyl Compounds; Gossypol; Humans; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Nitrophenols; Paclitaxel; Phosphorylation; Piperazines; Principal Component Analysis; Protein Binding; Protein Domains; Protein Multimerization; Protein Structure, Secondary; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Thermodynamics

Inhibition of prosurvival BCL2 family members can induce autophagy, but the mechanism is controversial. We have provided genetic evidence that BCL2 family members block autophagy by inhibiting BAX and BAK1, but others have proposed they instead inhibit BECN1. Here we confirm that small molecule BH3 mimetics can induce BAX- and BAK1-independent MAP1LC3B/LC3B lipidation, but this only occurred at concentrations far greater than required to induce apoptosis and dissociate canonical BH3 domain-containing proteins that bind more tightly than BECN1. Because high concentrations of a less-active enantiomer of ABT-263 also induced BAX- and BAK1-independent LC3B lipidation, induction of this marker of autophagy appears to be an off-target effect. Indeed, robust autophagic flux was not induced by BH3 mimetic compounds in the absence of BAX and BAK1. Therefore at concentrations that are on target and achievable in vivo, BH3 mimetics only induce autophagy in a BAX- and BAK1-dependent manner.

Topics: Aniline Compounds; Animals; Apoptosis; Autophagy; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Mice; Microtubule-Associated Proteins; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Aberrant expression of myeloid cell leukemia-1 (MCL-1) is a major cause of drug resistance in triple-negative breast cancer (TNBC) cells. Mucin 1 (MUC1) is a heterodimeric oncoprotein that is aberrantly overexpressed in most TNBC. The present studies show that targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in TNBC cells with silencing or pharmacologic inhibition with GO-203 is associated with downregulation of MCL-1 levels. Targeting MUC1-C suppresses the MEK → ERK and PI3K → AKT pathways, and in turn destabilizes MCL-1. The small molecules ABT-737 and ABT-263 target BCL-2, BCL-XL and BCL-w, but not MCL-1. We show that treatment with ABT-737 increases reactive oxygen species and thereby MUC1-C expression. In this way, MUC1-C is upregulated in TNBC cells resistant to ABT-737 or ABT-263. We also demonstrate that MUC1-C is necessary for the resistance-associated increases in MCL-1 levels. Significantly, combining GO-203 with ABT-737 is synergistic in inhibiting survival of parental and drug resistant TNBC cells. These findings indicate that targeting MUC1-C is a potential strategy for reversing MCL-1-mediated resistance in TNBC.

Topics: Aniline Compounds; Biphenyl Compounds; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; Mucin-1; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Oxidative Stress; Piperazines; Protein Stability; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various types of cancer cells without damaging normal cells. However, in terms of pancreatic cancer, not all cancer cells are sensitive to TRAIL. In this study, we examined a panel of human pancreatic cancer cell lines for TRAIL sensitivity and investigated the effects of Bcl-2 family inhibitors on their response to TRAIL. Both ABT-263 and ABT-737 inhibited the function of Bcl-2, Bcl-xL, and Bcl-w. Of the nine pancreatic cancer cell lines tested, six showed no or low sensitivity to TRAIL, which correlated with protein expression of Bcl-xL. ABT-263 significantly sensitized four cell lines (AsPC-1, Panc-1, CFPAC-1, and Panc10.05) to TRAIL, with reduced cell viability and increased apoptosis. Knockdown of Bcl-xL, but not Bcl-2, by siRNA transfection increased the sensitivity of AsPC-1 and Panc-1 cells to TRAIL. ABT-263 treatment had no effect on protein expression of Bcl-2, Bcl-xL, or c-FLIPs. In Panc-1 cells, ABT-263 increased the surface expression of death receptor (DR) 5; the NF-κB pathway, but not endoplasmic reticulum stress, participated in the increase. In xenograft mouse models, the combination of TRAIL and ATB-737 suppressed the in vivo tumor growth of AsPC-1 and Panc-1 cells. These results indicate that Bcl-xL is responsible for TRAIL resistance in human pancreatic cancer cells, and that Bcl-2 family inhibitors could represent promising reagents to sensitize human pancreatic cancers in DR-targeting therapy.

Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-X Protein; Biphenyl Compounds; Caspases; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; Humans; Mice, Inbred BALB C; Mice, Nude; Molecular Targeted Therapy; NF-kappa B; Nitrophenols; Pancreatic Neoplasms; Piperazines; Proto-Oncogene Proteins c-bcl-2; Receptors, TNF-Related Apoptosis-Inducing Ligand; RNA Interference; Signal Transduction; Sulfonamides; Time Factors; TNF-Related Apoptosis-Inducing Ligand; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

B-cell leukemia/lymphoma 2 (BCL-2) prevents commitment to programmed cell death at the mitochondrion. It remains a challenge to identify those tumors that are best treated by inhibition of BCL-2. Here, we demonstrate that acute myeloid leukemia (AML) cell lines, primary patient samples, and murine primary xenografts are very sensitive to treatment with the selective BCL-2 antagonist ABT-199. In primary patient cells, the median IC50 was approximately 10 nmol/L, and cell death occurred within 2 hours. Our ex vivo sensitivity results compare favorably with those observed for chronic lymphocytic leukemia, a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, mitochondrial studies using BH3 profiling demonstrate activity at the mitochondrion that correlates well with cytotoxicity, supporting an on-target mitochondrial mechanism of action. Our protein and BH3 profiling studies provide promising tools that can be tested as predictive biomarkers in any clinical trial of ABT-199 in AML.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Biomarkers, Tumor; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Death; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Mice; Mitochondria; Neoplasms, Experimental; Nitrophenols; Peptide Fragments; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Xenograft Model Antitumor Assays

The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these "BH3 mimetics" in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.

Topics: Aniline Compounds; Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-X Protein; Benzamides; Benzopyrans; BH3 Interacting Domain Death Agonist Protein; Biphenyl Compounds; HeLa Cells; Humans; Indoles; Membrane Proteins; Mice; Mitochondria, Liver; Mitochondrial Membranes; Molecular Mimicry; Myeloid Cell Leukemia Sequence 1 Protein; Nitriles; Nitrophenols; Permeability; Piperazines; Protein Interaction Domains and Motifs; Proto-Oncogene Proteins; Pyrroles; Sulfonamides; Sulfones; Unilamellar Liposomes

Docetaxel (DTX) is a useful chemotherapeutic drug for the treatment of hormone-refractory prostate cancer. However, emergence of DTX resistance has been a therapeutic hurdle. In this study, we investigated the effect of combining DTX with Bcl-2 family inhibitors using human prostate cancer cell lines (PC3, LNCaP, and DU145 cells). PC3 cells were less sensitive to DTX than were the other two cell lines. In contrast to ABT-199, which inhibits Bcl-2 and Bcl-w, both ABT-263 and ABT-737, which inhibit Bcl-2, Bcl-xL, and Bcl-w, significantly augmented the antitumor effect of DTX on PC3 cells. ABT-263 also enhanced the antitumor effect of DTX on a DTX-resistant PC3 variant cell line. The antitumor effect of ABT-263 was due mainly to its inhibitory effect on Bcl-xL. In a xenograft mouse model, DTX and ABT-737 combination therapy significantly inhibited PC3 tumor growth. Interestingly, although ABT-263 activated caspase-9 in PC3 cells, inhibition of caspase-9 unexpectedly promoted ABT-263-induced apoptosis in a caspase-8-dependent manner. This augmented apoptosis was also observed in LNCaP cells. These findings indicate that Bcl-xL inhibition can sensitize DTX-resistant prostate cancer cells to DTX, and they reveal a unique apoptotic pathway in which antagonism of Bcl-2 family members in caspase-9-inhibited prostate cancer cells triggers caspase-8-dependent apoptosis.

Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Docetaxel; Drug Synergism; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nitrophenols; Piperazines; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Taxoids; Transfection; Xenograft Model Antitumor Assays

Chondrosarcoma is refractory to conventional chemotherapy. BH-3 mimetics ABT-737 and ABT-263 are synthetic small-molecule inhibitors of anti-apoptotic proteins B-cell lymphoma-2 (Bcl2) and Bcl-xL, which play a critical role in survival of chondrosarcoma cells.. Chondrosarcoma cell lines SW-1353 and CS-1 were used as the disease model. We used immunoblotting to assess the expression of target molecules Bcl2 and Bcl-xL, and the apoptotic inducers Bcl2-associated X (Bax) and Bcl2-antagonist/killer (Bak). In vitro growth inhibition by BH-3 mimetics was confirmed by photomicroscopic cell counting and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Apoptotic induction was confirmed by Enzyme-Linked ImmunoSorbent Assay (ELISA). In vivo growth inhibition was assessed in a non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse model.. Expression of the target and effector molecules was confirmed in chondrosarcoma cell lines. BH3 mimetics significantly inhibited cell growth and induced apoptosis in vitro. Administration of ABT-263 inhibited chondrosarcoma growth and improved survival in a mouse model.. BH3 mimetics represent a novel treatment modality for chondrosarcoma.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Biphenyl Compounds; Blotting, Western; Bone Neoplasms; Cell Proliferation; Chondrosarcoma; Disease Models, Animal; Humans; Male; Mice; Mice, Inbred NOD; Mice, SCID; Middle Aged; Molecular Mimicry; Molecular Targeted Therapy; Nitrophenols; Peptide Fragments; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Synergistic killing was achieved when Small Cell Lung Cancer (SCLC) cell lines were incubated with ABT-263 and an immunotoxin directed to the transferrin receptor. SCLC lines are variably sensitive to the BH-3 only peptide mimetic, ABT-263. To determine their sensitivity to toxin-based reagents, we incubated four representative SCLC lines with a model Pseudomonas exotoxin-based immunotoxin directed to the transferrin receptor. Remarkably in 4-of-4 lines, there was little evidence of immunotoxin-mediated cytotoxicity despite near complete inhibition of protein synthesis. However, when combinations of ABT-263 and immunotoxin were added to the ABT-263-resistant cell lines (H196 and H69AR), there was synergistic killing as evidenced by increased activation of caspase 3/7, annexin V staining, and loss of cell integrity. Synergistic killing was evident at 6 hr and correlated with loss of Mcl-1. This synergy was also noted when the closely related compound ABT-737 was combined with the same immunotoxin. To establish that the synergy seen in tissue culture could be achieved in vivo, H69AR cells were grown as tumors in nude mice and shown to be susceptible to the killing action of an immunotoxin-ABT-737 combination but not to either agent alone. When immunotoxin-ABT combinations were added to ABT-263-sensitive lines (H146 and H1417), killing was additive. Our data support combination approaches for treating ABT-263-resistant SCLC with ABT-263 and a second agent that provides synergistic killing action.

Topics: Aniline Compounds; Animals; Biphenyl Compounds; Caspase 3; Caspase 7; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Humans; Immunotoxins; Mice; Mice, Inbred BALB C; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Receptors, Transferrin; Small Cell Lung Carcinoma; Sulfonamides

Platelet ADP receptor P2Y(12) is well studied and recognized as a key player in platelet activation, hemostasis and thrombosis. However, the role of P2Y(12) in platelet apoptosis remains unknown.. To evaluate the role of the P2Y(12) receptor in platelet apoptosis.. We used flow cytometry and Western blotting to assess apoptotic events in platelets treated with ABT-737 or ABT-263, and stored at 37°C, combined with P2Y(12) receptor antagonists or P2Y(12) -deficient mice..  P2Y(12) activation attenuated apoptosis induced by ABT-737 in human and mouse platelets in vitro, evidenced by reduced phosphatidylserine (PS) exposure, diminished depolarization of mitochondrial inner transmembrane potential (ΔΨm) and decreased caspase-3 activation. Through increasing the phosphorylation level of Akt and Bad, and changing the interaction between different Bcl-2 family proteins, P2Y(12) activation inactivated Bak/Bax. This antiapoptotic effect could be abolished by P2Y(12) antagonism or PI3K inhibition. We also observed the antiapoptotic effect of P2Y(12) activation in platelets stored at 37°C. P2Y(12) activation improved the impaired activation responses of apoptotic platelets stressed by ABT-737. In platelets from mice dosed with ABT-263 in vivo, clopidogrel or deficiency of P2Y(12) receptor enhanced apoptosis along with increased Bak/Bax activation.. This study demonstrates that P2Y(12) activation protects platelets from apoptosis via PI3k-dependent Bak/Bax inactivation, which may be physiologically important to counter the proapoptotic challenge. Our findings that P2Y(12) blockade exaggerates platelet apoptosis induced by ABT-263 (Navitoclax) also imply a novel drug interaction of ABT-263 and P2Y(12) antagonists.

Topics: Aniline Compounds; Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; bcl-Associated Death Protein; Biphenyl Compounds; Blood Platelets; Blotting, Western; Caspase 3; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Phosphatidylinositol 3-Kinase; Phosphatidylserines; Phosphorylation; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Purinergic P2Y Receptor Antagonists; Receptors, Purinergic P2Y12; Signal Transduction; Sulfonamides; Time Factors

The present study demonstrates the important structural features of ceramide required for proper regulation, binding and identification by both pro-apoptotic and anti-apoptotic Bcl-2 family proteins. The C-4=C-5 trans-double bond has little influence on the ability of Bax and Bcl-xL to identify and bind to these channels. The stereochemistry of the headgroup and access to the amide group of ceramide is indispensible for Bax binding, indicating that Bax may interact with the polar portion of the ceramide channel facing the bulk phase. In contrast, Bcl-xL binding to ceramide channels is tolerant of stereochemical changes in the headgroup. The present study also revealed that Bcl-xL has an optimal interaction with long-chain ceramides that are elevated early in apoptosis, whereas short-chain ceramides are not well regulated. Inhibitors specific for the hydrophobic groove of Bcl-xL, including 2-methoxyantimycin A3, ABT-737 and ABT-263 provide insights into the region of Bcl-xL involved in binding to ceramide channels. Molecular docking simulations of the lowest-energy binding poses of ceramides and Bcl-xL inhibitors to Bcl-xL were consistent with the results of our functional studies and propose potential binding modes.

Topics: Aniline Compounds; Animals; Apoptosis; bcl-2-Associated X Protein; Biphenyl Compounds; Caspases; Ceramides; Computer Simulation; Cytochromes c; Ion Channels; Male; Mitochondria, Liver; Models, Molecular; Nitrophenols; Oxidation-Reduction; Piperazines; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; 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

Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed "primed for death" and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O(2) consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O(2) consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by "bioenergetics-based profiling" may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist.

Topics: Aniline Compounds; Animals; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Biphenyl Compounds; Cell Death; Cell Line; Cell Line, Tumor; Cell Respiration; Cytochromes c; Cytological Techniques; Energy Metabolism; Humans; Lymphoma, B-Cell; Mice; Mitochondria; Models, Biological; Nitrophenols; Oxygen Consumption; Phenotype; Piperazines; Proto-Oncogene Proteins c-bcl-2; Rats; Sulfonamides

Increased interactions between pro-apoptotic BH3-only proteins and anti-apoptotic Bcl-2 family proteins at mitochondria result in tumor initiation, progression and resistance to traditional chemotherapy. Drugs that mimic the BH3 region are expected to release BH3-only proteins from anti-apoptotic proteins, inducing apoptosis in some cancer cells and sensitizing others to chemotherapy. Recently, we applied fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer to measure protein:protein interactions for the Bcl-2 family of proteins in live MCF-7 cells using fluorescent fusion proteins. While the BH3-proteins bound to Bcl-XL and Bcl-2, the BH3 mimetic ABT-737 inhibited binding of only Bad and tBid, but not Bim. We have extended our studies by investigating ABT-263, a clinical drug based on ABT-737. We show that the inhibitory effects and pattern of the two drugs are comparable for both Bcl-XL and Bcl-2. Furthermore, we show that mutation of a conserved residue in the BH3 region in Bad and tBid disrupted their interactions with Bcl-XL and Bcl-2, while the corresponding BimEL mutant showed no decrease in binding to these anti-apoptotic proteins. Therefore, in MCF-7 cells, Bim has unique binding properties compared with other BH3-only proteins that resist displacement from Bcl-XL and Bcl-2 by BH3 mimetics.

Topics: Amino Acid Sequence; Aniline Compounds; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; bcl-Associated Death Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Biphenyl Compounds; Cell Survival; Fluorescence Resonance Energy Transfer; Humans; Luminescent Proteins; MCF-7 Cells; Membrane Proteins; Microscopy, Fluorescence; Molecular Sequence Data; Mutation; Nitrophenols; Piperazines; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Red Fluorescent Protein; Sulfonamides

Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using (3)H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.

Topics: Aniline Compounds; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biphenyl Compounds; Cell Line; Dogs; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Substrate Specificity; Sulfonamides

Apoptosis in megakaryocytes results in the formation of platelets. The role of apoptotic pathways in platelet turnover and in the apoptotic-like changes seen after platelet activation is poorly understood. ABT-263 (Navitoclax), a specific inhibitor of antiapoptotic BCL2 proteins, which is currently being evaluated in clinical trials for the treatment of leukemia and other malignancies, induces a dose-limiting thrombocytopenia. In this study, the relationship between BCL2/BCL-X(L) inhibition, apoptosis, and platelet activation was investigated. Exposure to ABT-263 induced apoptosis but repressed platelet activation by physiologic agonists. Notably, ABT-263 induced an immediate calcium response in platelets and the depletion of intracellular calcium stores, indicating that on BCL2/BCL-X(L) inhibition platelet activation is abrogated because of a diminished calcium signaling. By comparing the effects of ABT-263 and its analog ABT-737 on platelets and leukemia cells from the same donor, we show, for the first time, that these BCL2/BCL-X(L) inhibitors do not offer any selective toxicity but induce apoptosis at similar concentrations in leukemia cells and platelets. However, reticulated platelets are less sensitive to apoptosis, supporting the hypothesis that treatment with ABT-263 induces a selective loss of older platelets and providing an explanation for the transient thrombocytopenia observed on ABT-263 treatment.

Topics: Aniline Compounds; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Biphenyl Compounds; Blood Platelets; Calcium Signaling; Gene Expression; Homeostasis; Humans; Kinetics; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Molecular Targeted Therapy; Nitrophenols; Piperazines; Platelet Activation; Platelet Aggregation Inhibitors; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Thrombocytopenia; Thrombopoiesis

BH3 mimetics are a new class of proapo-ptotic anticancer agents that have shown considerable promise in preclinical animal models and early-stage human trials. These agents act by inhibiting the pro-survival function of one or more Bcl-2-related proteins. Agents that inhibit Bcl-x(L) induce rapid platelet death that leads to thrombocytopenia; however, their impact on the function of residual circulating platelets remains unclear. In this study, we demonstrate that the BH3 mimetics, ABT-737 or ABT-263, induce a time- and dose-dependent decrease in platelet adhesive function that correlates with ectodomain shedding of the major platelet adhesion receptors, glycoprotein Ibα and glycoprotein VI, and functional down-regulation of integrin α(IIb)β(3). Analysis of platelets from mice treated with higher doses of BH3 mimetics revealed the presence of a subpopulation of circulating platelets undergoing cell death that have impaired activation responses to soluble agonists. Functional analysis of platelets by intravital microscopy revealed a time-dependent defect in platelet aggregation at sites of vascular injury that correlated with an increase in tail bleeding time. Overall, these studies demonstrate that Bcl-x(L)-inhibitory BH3 mimetics not only induce thrombocytopenia but also a transient thrombocytopathy that can undermine the hemostatic function of platelets.

Topics: Aniline Compounds; Animals; Apoptosis; bcl-X Protein; Biphenyl Compounds; Blood Platelets; Blotting, Western; Collagen; Dose-Response Relationship, Drug; Down-Regulation; Hemostasis; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Nitrophenols; Phosphatidylserines; Piperazines; Platelet Adhesiveness; Platelet Aggregation; Platelet Count; Platelet Glycoprotein GPIb-IX Complex; Platelet Glycoprotein GPIIb-IIIa Complex; Platelet Membrane Glycoproteins; Sulfonamides; Thrombocytopenia; Time Factors

As single agents, ABT-263 and ABT-737 (ABT), molecular antagonists of the Bcl-2 family, bind tightly to Bcl-2, Bcl-xL and Bcl-w, but not to Mcl-1, and induce apoptosis only in limited cell types. The compound 2-deoxyglucose (2DG), in contrast, partially blocks glycolysis, slowing cell growth but rarely causing cell death. Injected into an animal, 2DG accumulates predominantly in tumors but does not harm other tissues. However, when cells that were highly resistant to ABT were pre-treated with 2DG for 3 hours, ABT became a potent inducer of apoptosis, rapidly releasing cytochrome c from the mitochondria and activating caspases at submicromolar concentrations in a Bak/Bax-dependent manner. Bak is normally sequestered in complexes with Mcl-1 and Bcl-xL. 2DG primes cells by interfering with Bak-Mcl-1 association, making it easier for ABT to dissociate Bak from Bcl-xL, freeing Bak to induce apoptosis. A highly active glucose transporter and Bid, as an agent of the mitochondrial apoptotic signal amplification loop, are necessary for efficient apoptosis induction in this system. This combination treatment of cancer-bearing mice was very effective against tumor xenograft from hormone-independent highly metastasized chemo-resistant human prostate cancer cells, suggesting that the combination treatment may provide a safe and effective alternative to genotoxin-based cancer therapies.

Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Blotting, Western; Cell Line, Tumor; Cytochromes c; Deoxyglucose; Dose-Response Relationship, Drug; HeLa Cells; Hep G2 Cells; Humans; Male; Mice; Mice, Knockout; Mice, Nude; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasms; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Survival Analysis; Tumor Burden; Xenograft Model Antitumor Assays

Topics: Aniline Compounds; Animals; Apoptosis; Biphenyl Compounds; HL-60 Cells; Humans; Lung Neoplasms; Lymphoma; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Inhibition of the antiapoptotic BCL2 family is one of the most promising areas of anticancer drug development. However, ABT-737, a specific BCL2 inhibitor, is neither orally bioavailable nor metabolically stable. To overcome these problems, the structurally related molecule ABT-263 was synthesized and recently entered clinical trials in hematologic malignancies, including chronic lymphocytic leukemia (CLL). Almost all laboratory studies have been carried out with ABT-737 rather than ABT-263, the drug being used in clinical trials. Currently there are no published data on the comparative effects of these inhibitors. To gain insight into the potential value or limitations of ABT-263 in the clinic, we assessed its ability to induce apoptosis in clinically relevant cellular models of CLL.. The susceptibility of freshly isolated primary CLL cells to these inhibitors was compared in standard culture conditions and in conditions that more closely mimic in vivo conditions in a whole blood assay system.. ABT-737 was more potent than ABT-263 at inducing apoptosis in CLL cells. In whole blood, approximately 100-fold higher concentrations of both drugs were required to induce apoptosis. We found that ABT-263 was highly bound by albumin and that an increased albumin binding of ABT-263 as compared with ABT-737 accounted for the differential sensitivity of CLL cells.. Our data indicate that the exquisite in vitro sensitivity of CLL cells to BCL2 inhibitors may be lost in vivo due to high cell densities and the albumin binding of ABT-263. Modification of ABT-263 may yield a BCL2 inhibitor with greater bioavailability and more favorable pharmacokinetics.

Topics: Albumins; Aniline Compounds; Antineoplastic Agents; Apoptosis; Biological Availability; Biphenyl Compounds; Cell Line, Tumor; Humans; Immunoprecipitation; Leukemia, Lymphocytic, Chronic, B-Cell; Nitrophenols; Piperazines; Sulfonamides

Topics: Aniline Compounds; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Biphenyl Compounds; Dactinomycin; Drug Synergism; Humans; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasms; Nitrophenols; Peptide Fragments; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides